Section 3 Cancer and Injuries

**Chapter 6**

**Abstract**

IL-17 Biological Effects and

Leukemia U937 Cells

*Fareed K.N. Arthur, Roland S. Cooper*

*and Joshua C.M. Williams*

ERK1/2 transcriptional factors

**1. Introduction**

**89**

*Samuel Evans Adunyah, Richard Akomeah,*

drugs. This chapter focuses on IL-17A, herein referred to as IL-17.

**Keywords:** IL-17A, leukemia, cytokines, Jak/STAT, PI-3 K/Akt,

IL-17 is a unique cytokine which was initially discovered through differential and subtractive screening of clones from DNA library from murine lymphoid cells and initially called T-lymphocyte associated antigen 8 (CTLA-8) [1]. IL-17 was found to have 50% sequence homology to the open reading frame 13 (ORF-13) in herpes virus Saimiri [1]. Subsequently, the human homolog of CTLA-8 was identified [1] and its incubation with human fibroblast resulted in induction of both IL-6 and IL-8. This led to renaming of the CTLA-8 human equivalent as IL-17 [2]. Human IL-17 production was also found to be limited to particular cellular elements of the immune system and that activated CD4 + T cells of the Th1/Th0 subset and

Signaling Mechanisms in Human

Human Interlekin-17 is produced by memory activated CD4+ T cells and other cells. It was initially considered unique in that its specific receptor is distinct from other cytokine receptors. IL-17 receptor is ubiquitously expressed by different cells including T cells. IL-17 plays a role in regulating growth, immune response and proinflammatory responses. It regulates differentiation of a subset of Th0 cells into Th-17 cells, which produce IL-17-induced cytokines. The IL-17R belongs to type 1 cytokine receptors. IL-17 belongs to a superfamily of its own, which includes IL-17A, IL-17B, IL-17C, IL-17E and IL-17F. These members of IL-17 superfamily have some sequence homology but bind to different receptors. Prior to this investigation, limited information existed on the effects of IL-17A in human leukemia cell lines. Our results show that IL-17A promotes growth, anti-apoptotic effects, chemotaxis, cytokine expression and transcriptional factor activation in leukemia cells. IL-17A activates multiple signaling pathways including PI-3 K, Jak–STAT, Raf-ERK1/2 and SRC kinase pathways, which mediate different biological effects of IL-17A in leukemia cells. Our findings implicate IL-17A in leukemia cell growth and survival, supporting potential leukemia therapy via development of anti-IL-17A

#### **Chapter 6**

## IL-17 Biological Effects and Signaling Mechanisms in Human Leukemia U937 Cells

*Samuel Evans Adunyah, Richard Akomeah, Fareed K.N. Arthur, Roland S. Cooper and Joshua C.M. Williams*

#### **Abstract**

Human Interlekin-17 is produced by memory activated CD4+ T cells and other cells. It was initially considered unique in that its specific receptor is distinct from other cytokine receptors. IL-17 receptor is ubiquitously expressed by different cells including T cells. IL-17 plays a role in regulating growth, immune response and proinflammatory responses. It regulates differentiation of a subset of Th0 cells into Th-17 cells, which produce IL-17-induced cytokines. The IL-17R belongs to type 1 cytokine receptors. IL-17 belongs to a superfamily of its own, which includes IL-17A, IL-17B, IL-17C, IL-17E and IL-17F. These members of IL-17 superfamily have some sequence homology but bind to different receptors. Prior to this investigation, limited information existed on the effects of IL-17A in human leukemia cell lines. Our results show that IL-17A promotes growth, anti-apoptotic effects, chemotaxis, cytokine expression and transcriptional factor activation in leukemia cells. IL-17A activates multiple signaling pathways including PI-3 K, Jak–STAT, Raf-ERK1/2 and SRC kinase pathways, which mediate different biological effects of IL-17A in leukemia cells. Our findings implicate IL-17A in leukemia cell growth and survival, supporting potential leukemia therapy via development of anti-IL-17A drugs. This chapter focuses on IL-17A, herein referred to as IL-17.

**Keywords:** IL-17A, leukemia, cytokines, Jak/STAT, PI-3 K/Akt, ERK1/2 transcriptional factors

#### **1. Introduction**

IL-17 is a unique cytokine which was initially discovered through differential and subtractive screening of clones from DNA library from murine lymphoid cells and initially called T-lymphocyte associated antigen 8 (CTLA-8) [1]. IL-17 was found to have 50% sequence homology to the open reading frame 13 (ORF-13) in herpes virus Saimiri [1]. Subsequently, the human homolog of CTLA-8 was identified [1] and its incubation with human fibroblast resulted in induction of both IL-6 and IL-8. This led to renaming of the CTLA-8 human equivalent as IL-17 [2]. Human IL-17 production was also found to be limited to particular cellular elements of the immune system and that activated CD4 + T cells of the Th1/Th0 subset and

stimulated memory T cells, synthesize IL-17 [3, 4]. IL-17 is a glycosylated homodimeric protein of 30 to 35 kDa also produced by nickel-specific T lymphocytes and it regulates I-CAM-1 expression and chemokine production [5]. Unlike other cytokines, IL-17 was noted to bind to a unique receptor distinct from other cytokine receptors [1–3]. IL-17 is ubiquitously expressed in thymocytes activated by CD3 mAb, CD45R0+ population of T cells, CD8+ splenic cells in mouse cells and synovial fluid of patients with rheumatoid arthritis [6–8]. IL-17 is abundantly produced in CD4+ T cells, now known as Th-17 cells [9–11]. It is also expressed in human peripheral blood mononuclear cells (PBMC) in response to ocular lysate in patients with birdshot chroioretinopathy [12]. IL-17 has biological effects in many cells and tissues [13–15]. IL-17 induces expression and secretion of IL-1-beta, IL-6, IL-8. TNF, GM-CSF, G-CSF, ICAM-1, and PGE2 [16–20]. The molecular characterization of IL-17 receptor (IL-17R) was reported in 1997 [21]. IL-17R is a type 1 transmembrane receptor and it is a single chain, which shares some properties with IL-2R-beta chain, and GM-CSFR, all of which are type 1 membrane receptors [21, 22]. IL-17R is also expressed in synovial endothelial cells and Chondrocytes from arthritis patients [23, 24].

expression and secretion of ICAM-I, IL-2, IL-6, IL-8 [16–20] and other cytokines, which produce different biological effects [45–50]. Also some of the T-cell secreted IL-17 become sequestered and neutralized by a soluble IL-17R (sIL-17R) [51]. Clinically, IL-17 is implicated in numerous diseases including arthritis [52–54], classical Hodgkin lymphomas [55–57], multiple myeloma [58–60], airway diseases including asthma [61, 62], musculoskeletal diseases [63, 64], inflammatory bowel diseases (IBDs) [65] autoimmune diseases [66], and different types of cancer [67–69]. Significantly elevated level of IL-17 and IL-17R are found in these diseases and IL-17 and IL-17R are known to promote anti-apoptotic effects and survival mechanisms in some types of cancer [55–66]. In most cases, IL-17 itself and/or IL-17 dependent cytokines produced downstream of the IL-17R, contribute to various pathological conditions associated with these diseases [55–68]. Furthermore, Hox3/ IL-17R expression ratio has been implicated in poor prognosis in some breast cancer patients undergoing tamoxifen chemotherapy as IL-17 promotes resistance to chemotherapy in breast cancer [67–71]. IL-17 is also implicated in cervical and ovarian cancer [72–74]. Similarly, expression of IL-17 R-like protein has been detected in androgen independent prostate cancer cell lines and it has been implicated in confer-

*IL-17 Biological Effects and Signaling Mechanisms in Human Leukemia U937 Cells*

*DOI: http://dx.doi.org/10.5772/intechopen.96422*

ring resistance to apoptosis and promoting prostate cancer via MM7-induced epithelial-to-mesenchymal transition [75–77]. IL-17 is implicated in CNS and other neurological diseases [78–80], and psoriasis [81–83]. Recent reports suggest potential role for IL-17 in the "cytokine storm event" seen in advance Coronavirus Disease 2019 (COVID-19) infection with inflammation, and pro-thrombic events in severe COVID-19 patients [84–87]. Hence, there is strong interests in understanding IL-17's biology and it roles in COVID-19 patients [85–87]. It is not surprising that IL-17's role in these diseases [60–87], have prompted experts in pharmaceutical industries to develop anti-IL-17 type therapies for diseases in which IL-17 is implicated [88–90]. Earliest report on IL-17 induced activation of MAP kinases and NF-kB pathways was made in chondrocytes [91]. Subsequently, IL-17 was shown to activate Raf-MAPK and Jak/STAT signaling pathways in leukemia cells [92, 93]. These reports show that IL-17 stimulates rapid phosphorylation of RAF, Erk-1/2, Jak1, Jak2, Jak3 and Stat1, Stat2 and Stat3 in human leukemia cells [92, 93]. Currently, IL-17 is known to activate and utilize multiple signaling pathways including the aforementioned as well as JNK, p38 and PI-3 K/Akt pathways to produce diverse biological effects [91–96]. Many reports have confirmed IL-17-induced activation of PI-3 K/Akt signaling mechanisms in both normal and transformed cells [96–99]. IL-17 signaling pathways are implicated in human diseases including inflammation and cancer [100]. Furthermore, TRAF and TGF-beta-1/smad2/3 signaling pathways are activated by IL-17 [99– 102]. Most of the biological effects of IL-17 were initially observed in different variety of cells but to lesser extent in leukemia cells. Therefore, our rationale for initiating this study was to determine the biological effects of IL-17 in leukemia cells and elucidate the various signaling pathways utilized by IL-17 in leukemia cells. Furthermore, we wanted to determine which transcriptional factors are activated by IL-17. Finally, we wanted to determine whether IL-17 protects leukemia cells from undergoing apoptosis since previous report [75] indicated IL-17mediates cancer cell resistance to apoptosis.

**2. Experiment reagents and protocols**

**91**

We purchased human U937 and THP-1 leukemia cell lines from American Tissue

Culture Collection (ATCC) in Manassas, VA, USA. The cells were cultured in Roswell Park Memorial Institute-1640 media, which contained L-Glut (2 mM). Charcoal-filtered and frozen fetal bovine serum (FBS) was purchased from Atlanta Biological, Georgia, USA. Following careful thawing under sterile conditions we

Five different IL-17 ligands are now characterized as members of IL-17 superfamily of cytokines and differ from other cytokines but share some sequence homology with each other [25]. Among the IL-17 super family, IL-17A is most commonly expressed in many tissues as well as in cells of hematopoietic origin including monocytes and macrophages. In addition to IL-17A, there are IL-17B, IL-17C, IL-17E and IL-17F [25, 26]. IL-17B, IL-17C and IL-17E expression are widespread in many tissues including testis, brain and kidney [25, 26]. Each IL-17 family members have their individual specific receptor as these IL-17 family members do not bind to the same receptor type [26–28]. The different members of IL-17 superfamily have different expression patterns but with similar abilities to stimulate cytokine effectors illustrating the potential for the members of the IL-17 superfamily to differentially regulate cellular responses in a wide variety of cells [28–31].

IL-17 regulates hematopoietic cell proliferation, immune response, proinflammatory responses [32–35] and activate specific types of T cells now known as Th-17 cells [9, 10]. These Th-17 cells play a role in host defense against extracellular pathogens by mediating recruitment of neutrophils and macrophages to infected tissues [9–11]. Th-17 cells secrete IL-17 cytokines, which in turn induce expression of IL-17-depedemt cytokines. Hence, aberrant regulation of Th-17 cells may play a role in the pathogenesis of multiple inflammatory and autoimmune disorders [9–11]. IL-17 promotes chemotaxis in human monocytes and regulates angiogenesis and cytokine production in endothelial cells [12, 36–38]. Although the target cells of IL-17-mediated signaling include immune cells such as neutrophils and macrophages [32–38], majority of IL-17's biological effects were seen in cells of either epithelial or mesenchymal origin [39–43]. The role of IL-17 in immunological function was initially examined in vivo in mice by overexpressing IL-17 in the liver of mice where an enhanced granulopoiesis and leukopoiesis led to an 80% increase in splenic mass [38, 39]. IL-17-induced accumulation of neutrophils in the airways requires involvement of GM-CSF [44]. Also, regulation of endogenous stem cell by IL-17 requires both GM-CSF and Stem Cell Factor (SCF) [45]. IL-17 and G-CSF are synergistically involved in the maintenance of normal granulopoiesis [45]. The IL-17R is ubiquitously expressed [12–15] and may explain the ability of IL-17 to stimulate peripheral blood stem cells in in mice [44, 45]. Also, There IL-17 plays active in vivo role in chemoattraction of cells of immune system [46, 47]. In addition, IL-17 exhibits paracrine effects in different cell types [48] whereby secreted IL-17 from T cells binds to its putative receptor on neighboring cells such as fibroblasts and trigger signaling that leads to NF-kB- mediated induction of

#### *IL-17 Biological Effects and Signaling Mechanisms in Human Leukemia U937 Cells DOI: http://dx.doi.org/10.5772/intechopen.96422*

expression and secretion of ICAM-I, IL-2, IL-6, IL-8 [16–20] and other cytokines, which produce different biological effects [45–50]. Also some of the T-cell secreted IL-17 become sequestered and neutralized by a soluble IL-17R (sIL-17R) [51].

Clinically, IL-17 is implicated in numerous diseases including arthritis [52–54], classical Hodgkin lymphomas [55–57], multiple myeloma [58–60], airway diseases including asthma [61, 62], musculoskeletal diseases [63, 64], inflammatory bowel diseases (IBDs) [65] autoimmune diseases [66], and different types of cancer [67–69]. Significantly elevated level of IL-17 and IL-17R are found in these diseases and IL-17 and IL-17R are known to promote anti-apoptotic effects and survival mechanisms in some types of cancer [55–66]. In most cases, IL-17 itself and/or IL-17 dependent cytokines produced downstream of the IL-17R, contribute to various pathological conditions associated with these diseases [55–68]. Furthermore, Hox3/ IL-17R expression ratio has been implicated in poor prognosis in some breast cancer patients undergoing tamoxifen chemotherapy as IL-17 promotes resistance to chemotherapy in breast cancer [67–71]. IL-17 is also implicated in cervical and ovarian cancer [72–74]. Similarly, expression of IL-17 R-like protein has been detected in androgen independent prostate cancer cell lines and it has been implicated in conferring resistance to apoptosis and promoting prostate cancer via MM7-induced epithelial-to-mesenchymal transition [75–77]. IL-17 is implicated in CNS and other neurological diseases [78–80], and psoriasis [81–83]. Recent reports suggest potential role for IL-17 in the "cytokine storm event" seen in advance Coronavirus Disease 2019 (COVID-19) infection with inflammation, and pro-thrombic events in severe COVID-19 patients [84–87]. Hence, there is strong interests in understanding IL-17's biology and it roles in COVID-19 patients [85–87]. It is not surprising that IL-17's role in these diseases [60–87], have prompted experts in pharmaceutical industries to develop anti-IL-17 type therapies for diseases in which IL-17 is implicated [88–90].

Earliest report on IL-17 induced activation of MAP kinases and NF-kB pathways was made in chondrocytes [91]. Subsequently, IL-17 was shown to activate Raf-MAPK and Jak/STAT signaling pathways in leukemia cells [92, 93]. These reports show that IL-17 stimulates rapid phosphorylation of RAF, Erk-1/2, Jak1, Jak2, Jak3 and Stat1, Stat2 and Stat3 in human leukemia cells [92, 93]. Currently, IL-17 is known to activate and utilize multiple signaling pathways including the aforementioned as well as JNK, p38 and PI-3 K/Akt pathways to produce diverse biological effects [91–96]. Many reports have confirmed IL-17-induced activation of PI-3 K/Akt signaling mechanisms in both normal and transformed cells [96–99]. IL-17 signaling pathways are implicated in human diseases including inflammation and cancer [100]. Furthermore, TRAF and TGF-beta-1/smad2/3 signaling pathways are activated by IL-17 [99– 102]. Most of the biological effects of IL-17 were initially observed in different variety of cells but to lesser extent in leukemia cells. Therefore, our rationale for initiating this study was to determine the biological effects of IL-17 in leukemia cells and elucidate the various signaling pathways utilized by IL-17 in leukemia cells. Furthermore, we wanted to determine which transcriptional factors are activated by IL-17. Finally, we wanted to determine whether IL-17 protects leukemia cells from undergoing apoptosis since previous report [75] indicated IL-17mediates cancer cell resistance to apoptosis.

#### **2. Experiment reagents and protocols**

We purchased human U937 and THP-1 leukemia cell lines from American Tissue Culture Collection (ATCC) in Manassas, VA, USA. The cells were cultured in Roswell Park Memorial Institute-1640 media, which contained L-Glut (2 mM). Charcoal-filtered and frozen fetal bovine serum (FBS) was purchased from Atlanta Biological, Georgia, USA. Following careful thawing under sterile conditions we

stimulated memory T cells, synthesize IL-17 [3, 4]. IL-17 is a glycosylated

*Interleukins - The Immune and Non-Immune Systems' Related Cytokines*

from arthritis patients [23, 24].

**90**

homodimeric protein of 30 to 35 kDa also produced by nickel-specific T lymphocytes and it regulates I-CAM-1 expression and chemokine production [5]. Unlike other cytokines, IL-17 was noted to bind to a unique receptor distinct from other cytokine receptors [1–3]. IL-17 is ubiquitously expressed in thymocytes activated by CD3 mAb, CD45R0+ population of T cells, CD8+ splenic cells in mouse cells and synovial fluid of patients with rheumatoid arthritis [6–8]. IL-17 is abundantly produced in CD4+ T cells, now known as Th-17 cells [9–11]. It is also expressed in human peripheral blood mononuclear cells (PBMC) in response to ocular lysate in patients with birdshot chroioretinopathy [12]. IL-17 has biological effects in many cells and tissues [13–15]. IL-17 induces expression and secretion of IL-1-beta, IL-6, IL-8. TNF, GM-CSF, G-CSF, ICAM-1, and PGE2 [16–20]. The molecular characterization of IL-17 receptor (IL-17R) was reported in 1997 [21]. IL-17R is a type 1 transmembrane receptor and it is a single chain, which shares some properties with IL-2R-beta chain, and GM-CSFR, all of which are type 1 membrane receptors [21, 22]. IL-17R is also expressed in synovial endothelial cells and Chondrocytes

Five different IL-17 ligands are now characterized as members of IL-17 super-

family of cytokines and differ from other cytokines but share some sequence homology with each other [25]. Among the IL-17 super family, IL-17A is most commonly expressed in many tissues as well as in cells of hematopoietic origin including monocytes and macrophages. In addition to IL-17A, there are IL-17B, IL-17C, IL-17E and IL-17F [25, 26]. IL-17B, IL-17C and IL-17E expression are widespread in many tissues including testis, brain and kidney [25, 26]. Each IL-17 family members have their individual specific receptor as these IL-17 family members do not bind to the same receptor type [26–28]. The different members of IL-17 superfamily have different expression patterns but with similar abilities to stimulate cytokine effectors illustrating the potential for the members of the IL-17 superfamily to differentially regulate cellular responses in a wide variety of cells [28–31]. IL-17 regulates hematopoietic cell proliferation, immune response, proinflammatory responses [32–35] and activate specific types of T cells now known as Th-17 cells [9, 10]. These Th-17 cells play a role in host defense against extracellular pathogens by mediating recruitment of neutrophils and macrophages to infected tissues [9–11]. Th-17 cells secrete IL-17 cytokines, which in turn induce expression of IL-17-depedemt cytokines. Hence, aberrant regulation of Th-17 cells may play a role in the pathogenesis of multiple inflammatory and autoimmune disorders [9–11]. IL-17 promotes chemotaxis in human monocytes and regulates angiogenesis and cytokine production in endothelial cells [12, 36–38]. Although the target cells of IL-17-mediated signaling include immune cells such as neutrophils and macrophages [32–38], majority of IL-17's biological effects were seen in cells of either epithelial or mesenchymal origin [39–43]. The role of IL-17 in immunological function was initially examined in vivo in mice by overexpressing IL-17 in the liver of mice where an enhanced granulopoiesis and leukopoiesis led to an 80% increase in splenic mass [38, 39]. IL-17-induced accumulation of neutrophils in the airways requires involvement of GM-CSF [44]. Also, regulation of endogenous stem cell by IL-17 requires both GM-CSF and Stem Cell Factor (SCF) [45]. IL-17 and G-CSF are synergistically involved in the maintenance of normal granulopoiesis [45]. The IL-17R is ubiquitously expressed [12–15] and may explain the ability of IL-17 to stimulate peripheral blood stem cells in in mice [44, 45]. Also, There IL-17 plays active in vivo role in chemoattraction of cells of immune system [46, 47]. In addition, IL-17 exhibits paracrine effects in different cell types [48] whereby secreted IL-17 from T cells binds to its putative receptor on neighboring cells such as

fibroblasts and trigger signaling that leads to NF-kB- mediated induction of

heated the serum at 55 °C for 45 minutes for inactivation. After cooling, we vacuum filtered the FBS under the hood and stored 50 ml aliquots at minus 20 °C. Both streptomycin and penicillin were purchased from Invitrogen, Carlsbad, CA. Prior to using the culture media, we added FBS to a final concentration of 10% (v/v). To prevent bacterial growth in the culture media, we added penicillin (50 U/ml) and streptomycin (50 U/ml). We cultured the cells in either 25 ml or 50 ml of complete media in tissue culture flasks in a CO2 incubator set to 5% CO2, 37 °C and 100% humidity. Typically, we passaged the cells 8x before starting a fresh culture. U937 leukemia cells were used in most of the experiments described here.

inhibitors, 0.5% Triton X-100, 50 mM NaF and 2 mM Vanadate) [7]. Total cell lysate protein concentration was determined by Coomassie Blue Protein Assay Kit (Pierce, IL) and 240 ug per sample was solubilized in 50 ul SDS gel sample buffer and resolved by 12% polyacrylamide gel electrophoresis. The protein bands were transferred to membrane and the membrane background blocked in a blocking buffer containing 5% milk. The membranes were incubated with specific antibodies to either total PI-3 K, p-PI-3 K, p-AktSer473, p-AktThre308, total Akt, p-STAT3, total STAT3, p-BAD, p-caspase3 or pGSK3-beta or total actin (for loading control) and protein bands detected [103]. The band intensities were scanned by digital image analyzer for quantitation and the band intensity from IL-17 treated samples com-

*IL-17 Biological Effects and Signaling Mechanisms in Human Leukemia U937 Cells*

Total and phosphorylated Akt can associate with effector proteins [104, 105] via protein–protein interaction [106] and contribute to their regulation. We rationalized that if IL-17 promotes association between p-Akt and any of its downstream effectors, those proteins will be contained in the pulled down p-Akt-antibody complex and can be detected by co-IP/Western blot. To determine whether Akt/p-Akt binds to p-BAD or p-Caspase3 or p-GSK-3 (p-Akt's downstream effectors), we carried out co-IP. Specifically, 240 mg/ml protein from untreated or IL-17 treated cells were suspended in PBS (500 ul) in Eppendorf tubes and incubated with specific antibody that recognizes both total Akt and p-AktSer473 overnight with gentle shaking at 4 °C. Next, protein A agarose slurry (500 ul) was added to the complex in each tube and incubated for 2 hours at 4 °C with gentle shaking to allow protein A agarose to capture all

the phosphoproteins bound to Akt/p-Akt-antibody complex. The tubes were

to the bands in the untreated cells. Representative results are presented.

tated using digital image analyzer.

**2.6 Transcription factor array**

**93**

**2.5 Detection of IL-17RA in U937 leukemia cells and THP-1 leukemia cells**

Specific antibody to IL-17AR was purchased from Santa Cruz, CA and goat antirabbit IgG-HRP antibody was from Amersham, CA. In order to detect IL-17AR, 240 ug of total cell lysate protein from 40 million cells was separated on 15% polyacrylamide gel [106]. The protein bands were transferred to nitrocellulose membrane followed by incubation in a blocking buffer containing 5% filtered non-fat milk to block non-specific sites on the membrane. Both the IL-17AR antibody and the goatanti-rabbit antibody were used at dilutions of 1:1000. The rest of the Western blot protocols were performed as described [103, 106]. The band intensity was quanti-

To detect transcriptional factors regulated by IL-17 stimulation in leukemia cells,

we examined the profile of 54 transcriptional factors (TFs) using Panomics

centrifuged at 10,000 x g for 10 minutes at 4 °C, the supernatants carefully removed and the pellets were washed 5x with lysis buffer (see above). After the final wash, the phosphoprotein complexes in each tube was solubilized in SDS-gel sample buffer (50 ul) and boiled for 3 minutes to dissociate the phosphoproteins in each complex. The dissociated phosphoproteins were separated on 12% SDS-polyacrylamide gel as described above. The phosphoprotein bands were transferred to membrane and Western blotted for either p-BAD or p-Caspase3 p-GSK-3 or total Akt using specific antibodies. The band intensity for p-BAD, p-Caspase3 and p-GSK-3 were scanned with digital image analyzer. The old levels in the IL-17 treated cells calculated relative

pared to the intensity in the untreated sample.

*DOI: http://dx.doi.org/10.5772/intechopen.96422*

**2.4 Co-immunoprecipitation (co-IP) assay/Western blot**

#### **2.1 Monocyte isolation**

We purchased de-identified human blood samples from New York Blood Center, Long Island, NY and Percoll gradients from GR Health Care, Piscataway, NJ [103]. To isolate monocytes for chemotaxis assays, we isolated peripheral blood mononuclear cells (PBMC) as previously described [103]. After centrifugation to remove all red blood cells, the white blood cells were carefully retrieved and suspended in 10 ml of complete media and spread across the surface of plastic dishes. The plastic dishes were incubated in the incubator for 1.5 hours to allow monocytes to attach to the surface of the dishes. Subsequently, all non-adherent cells were carefully aspirated off and discarded. The attached monocytes were carefully scraped from the dishes and suspended in culture media. The monocytes were about 95% pure based on positive staining for CD14 marker.

#### **2.2 Detection of cytokine expression by cytokine antibody array**

To determine the effect of IL-17 on cytokine expression in leukemia cells, we performed cytokine antibody array using tissue media from untreated and IL-17 treated leukemia cells [103]. Specifically, 20million cells were either untreated or treated with IL-17 (100 ng/ml) alone or with IL-17 (100 ng/ml) plus the PI-3 K inhibitor LY20094 (20 uM) or with LY20094 (20 uM) alone for 24 hours. The tissue culture media were filtered to remove debris and their protein concentration determined by Coomassie Blue Protein Assay Kit (Pierce, IL). The culture media containing 50 ug protein from untreated and treated cells were spotted onto each of the cytokine/antibody array membranes containing antibodies for over 42 cytokines (RayBiotech, Corners, GA, USA), and incubated with gentle shaking for 2 hours at 30 °C. This allowed hybridization of each cytokine in the media to its respective cytokine antibody on the array. Next, the media was carefully removed and each membrane was washed 5x with wash buffer (provided by kit) to remove all nonspecific binding. Each membrane was incubated for a specified time in the color development solution provided with kit and air dried. The dark spots representing various cytokines were visualized and quantitated by digital image scanning. The spot intensities were converted to fold change relative to the corresponding spots on the membrane of the untreated cells, which was set as 1-fold.

#### **2.3 Western blotting detection of proteins and phosphoproteins**

One million leukemia cells per ml media were either untreated (control) or treated with [92, 93] IL-17 (1 ng/ml) for 2, 5, 15, 30, 60 min or with IL-17 (100 ng/ ml) at 30 °C treatments up to 48 hours. Next, the cells were rapidly pelleted by micro-centrifugation at 1,800 x g for 3 minutes. The pelleted cells were washed 3x with PBS. The final pellets were collected by centrifugation at 1800 rpm for 3 min and each pellet was lysed in 500 ul of cell lysis buffer A (containing protease

*IL-17 Biological Effects and Signaling Mechanisms in Human Leukemia U937 Cells DOI: http://dx.doi.org/10.5772/intechopen.96422*

inhibitors, 0.5% Triton X-100, 50 mM NaF and 2 mM Vanadate) [7]. Total cell lysate protein concentration was determined by Coomassie Blue Protein Assay Kit (Pierce, IL) and 240 ug per sample was solubilized in 50 ul SDS gel sample buffer and resolved by 12% polyacrylamide gel electrophoresis. The protein bands were transferred to membrane and the membrane background blocked in a blocking buffer containing 5% milk. The membranes were incubated with specific antibodies to either total PI-3 K, p-PI-3 K, p-AktSer473, p-AktThre308, total Akt, p-STAT3, total STAT3, p-BAD, p-caspase3 or pGSK3-beta or total actin (for loading control) and protein bands detected [103]. The band intensities were scanned by digital image analyzer for quantitation and the band intensity from IL-17 treated samples compared to the intensity in the untreated sample.

#### **2.4 Co-immunoprecipitation (co-IP) assay/Western blot**

Total and phosphorylated Akt can associate with effector proteins [104, 105] via protein–protein interaction [106] and contribute to their regulation. We rationalized that if IL-17 promotes association between p-Akt and any of its downstream effectors, those proteins will be contained in the pulled down p-Akt-antibody complex and can be detected by co-IP/Western blot. To determine whether Akt/p-Akt binds to p-BAD or p-Caspase3 or p-GSK-3 (p-Akt's downstream effectors), we carried out co-IP. Specifically, 240 mg/ml protein from untreated or IL-17 treated cells were suspended in PBS (500 ul) in Eppendorf tubes and incubated with specific antibody that recognizes both total Akt and p-AktSer473 overnight with gentle shaking at 4 °C. Next, protein A agarose slurry (500 ul) was added to the complex in each tube and incubated for 2 hours at 4 °C with gentle shaking to allow protein A agarose to capture all the phosphoproteins bound to Akt/p-Akt-antibody complex. The tubes were centrifuged at 10,000 x g for 10 minutes at 4 °C, the supernatants carefully removed and the pellets were washed 5x with lysis buffer (see above). After the final wash, the phosphoprotein complexes in each tube was solubilized in SDS-gel sample buffer (50 ul) and boiled for 3 minutes to dissociate the phosphoproteins in each complex. The dissociated phosphoproteins were separated on 12% SDS-polyacrylamide gel as described above. The phosphoprotein bands were transferred to membrane and Western blotted for either p-BAD or p-Caspase3 p-GSK-3 or total Akt using specific antibodies. The band intensity for p-BAD, p-Caspase3 and p-GSK-3 were scanned with digital image analyzer. The old levels in the IL-17 treated cells calculated relative to the bands in the untreated cells. Representative results are presented.

#### **2.5 Detection of IL-17RA in U937 leukemia cells and THP-1 leukemia cells**

Specific antibody to IL-17AR was purchased from Santa Cruz, CA and goat antirabbit IgG-HRP antibody was from Amersham, CA. In order to detect IL-17AR, 240 ug of total cell lysate protein from 40 million cells was separated on 15% polyacrylamide gel [106]. The protein bands were transferred to nitrocellulose membrane followed by incubation in a blocking buffer containing 5% filtered non-fat milk to block non-specific sites on the membrane. Both the IL-17AR antibody and the goatanti-rabbit antibody were used at dilutions of 1:1000. The rest of the Western blot protocols were performed as described [103, 106]. The band intensity was quantitated using digital image analyzer.

#### **2.6 Transcription factor array**

To detect transcriptional factors regulated by IL-17 stimulation in leukemia cells, we examined the profile of 54 transcriptional factors (TFs) using Panomics

heated the serum at 55 °C for 45 minutes for inactivation. After cooling, we vacuum filtered the FBS under the hood and stored 50 ml aliquots at minus 20 °C. Both streptomycin and penicillin were purchased from Invitrogen, Carlsbad, CA. Prior to using the culture media, we added FBS to a final concentration of 10% (v/v). To prevent bacterial growth in the culture media, we added penicillin (50 U/ml) and streptomycin (50 U/ml). We cultured the cells in either 25 ml or 50 ml of complete media in tissue culture flasks in a CO2 incubator set to 5% CO2, 37 °C and 100% humidity. Typically, we passaged the cells 8x before starting a fresh culture. U937

We purchased de-identified human blood samples from New York Blood Center, Long Island, NY and Percoll gradients from GR Health Care, Piscataway, NJ [103]. To isolate monocytes for chemotaxis assays, we isolated peripheral blood mononuclear cells (PBMC) as previously described [103]. After centrifugation to remove all red blood cells, the white blood cells were carefully retrieved and suspended in 10 ml of complete media and spread across the surface of plastic dishes. The plastic dishes were incubated in the incubator for 1.5 hours to allow monocytes to attach to the surface of the dishes. Subsequently, all non-adherent cells were carefully aspirated off and discarded. The attached monocytes were carefully scraped from the dishes and suspended in culture media. The monocytes

To determine the effect of IL-17 on cytokine expression in leukemia cells, we performed cytokine antibody array using tissue media from untreated and IL-17 treated leukemia cells [103]. Specifically, 20million cells were either untreated or treated with IL-17 (100 ng/ml) alone or with IL-17 (100 ng/ml) plus the PI-3 K inhibitor LY20094 (20 uM) or with LY20094 (20 uM) alone for 24 hours. The tissue culture media were filtered to remove debris and their protein concentration determined by Coomassie Blue Protein Assay Kit (Pierce, IL). The culture media containing 50 ug protein from untreated and treated cells were spotted onto each of the cytokine/antibody array membranes containing antibodies for over 42 cytokines (RayBiotech, Corners, GA, USA), and incubated with gentle shaking for 2 hours at 30 °C. This allowed hybridization of each cytokine in the media to its respective cytokine antibody on the array. Next, the media was carefully removed and each membrane was washed 5x with wash buffer (provided by kit) to remove all nonspecific binding. Each membrane was incubated for a specified time in the color development solution provided with kit and air dried. The dark spots representing various cytokines were visualized and quantitated by digital image scanning. The spot intensities were converted to fold change relative to the corresponding spots on

leukemia cells were used in most of the experiments described here.

*Interleukins - The Immune and Non-Immune Systems' Related Cytokines*

were about 95% pure based on positive staining for CD14 marker.

**2.2 Detection of cytokine expression by cytokine antibody array**

the membrane of the untreated cells, which was set as 1-fold.

**2.3 Western blotting detection of proteins and phosphoproteins**

One million leukemia cells per ml media were either untreated (control) or treated with [92, 93] IL-17 (1 ng/ml) for 2, 5, 15, 30, 60 min or with IL-17 (100 ng/ ml) at 30 °C treatments up to 48 hours. Next, the cells were rapidly pelleted by micro-centrifugation at 1,800 x g for 3 minutes. The pelleted cells were washed 3x with PBS. The final pellets were collected by centrifugation at 1800 rpm for 3 min and each pellet was lysed in 500 ul of cell lysis buffer A (containing protease

**2.1 Monocyte isolation**

**92**

Transcriptional Factor Array (1) Kit. Panomics TranSignal™ Protein/DNA Arrays simplifies the functional analysis of eukaryotic TFs and can be used to study TF activation in a variety of biological processes, including cell proliferation, differentiation, transformation, apoptosis and drug treatment [107]. The array membranes were spotted with 54 different consensus-binding sequences (oligos) and enable one to detect over 54 TFs at once in one treatment. Twenty million leukemia cells/ ml were pretreated with vanadate (5 mM) for 30 minutes to inhibit endogenous phosphatases. Next, 5 million of the cells we either untreated or stimulated with IL-17 (100 ng/ml) for 4 hours in the incubator. The cells were packed by centrifugation at 1,800 x g for 3 minutes, washed 3x with PBS and gently lysed in a lysing buffer (see Western blot protocols above) without detergents by repeated aspiration through a 22-gauge needle to prevent rupturing of the nuclei. Intact nuclei were isolated by layering the cell lysate over 50% glycerol solution in Eppendorf tubes followed by centrifugation at 1,000 x g for 5 minutes. The supernatants were carefully aspirated, the nuclei pellet harvested and washed 2x with PBS. Next, the nuclei were disrupted in a nuclei lysing buffer (provided by the kit) and protein concentration determined as described above. In a slightly modified version, 12 ug of nuclei proteins in 200 ul of incubation buffer were incubated with each array membrane containing the oligos for hybridization of each oligo to its specific transcriptional factor in the nuclei extract. The membranes were washed several times and the oligo/transcriptional factor complexes (DNA/protein complexes) were detected by detection per the kit. The spots representing the various transcription factors were identified based on the charts provided by the kit. The intensities of the spots were scanned by digital image analyzer and the fold stimulation by IL-17 compared to the intensities in the untreated cells.

untreated or NaB treated cells. Specifically, the cells were either untreated or treated with NaB (5 mM) alone, or treated with NaB (5 mM) plus IL-17 (100 ng/ml) or with IL-17 (100 ng/ml) alone in tissue culture media for up to 48 hours. At the end of the incubation, aliquot of cells were counted prior to lysing in RIPA buffer (provided in the caspase3 assay kit) and total lysate protein concentration was determined as indicated above. To measure caspase3 activity in the cell lysates, 30 ug total lysate protein from each sample was added to each experimental assay well.

*IL-17 Biological Effects and Signaling Mechanisms in Human Leukemia U937 Cells*

The enzymatic activity was measured in triplicate in microtiter plate reader

**2.10 Assessment of chemotactic effects of media from IL-17 treated cells**

**2.11 Assessing IL-17-induced cytokine expression by cytokine ELISA**

**3.1 Effects of IL-17 on cytokine expression in human leukemia cells**

In order to ascertain if media from IL-17 treated cells has chemotactic effects towards monocytes [103, 113], we employed the Boden Chamber chemotaxis assay method as previously described [103]. Specifically, 2000 monocytes/ml in fresh complete media were placed in the upper portion of the Boden Chamber. Equal volume of media from either untreated or IL-17 stimulated cells was put in the lower chamber to serve as a source of chemotaxis. The setup was incubated for 2 hours. Next, estimation of number of monocytes which crossed the membrane barrier to the lower chamber was performed according to the kit. Experiments were

In order to validate IL-17-induced cytokine expression observed in the cytokine antibody array, we performed cytokine ELISA assay as specified in the cytokine ELISA Kits (Ray Biotech, Norcross, GA) using equal amount of tissue culture proteins from untreated or IL-17 treated cells [103]. The ELISA monitored expression of IL-2, IL-3 and IL-8. In addition, to determine whether IL-17-induced IL-2 expression was mediated by either PI-3 K/Akt or by Jak2, in some experiments, we pre-incubated some of the cells with PI-3 K inhibitor LY20094 (20 nM) or Jak2 inhibitor AG490 (15 nM) for 30 minutes prior to stimulating the cells with IL-17

To determine whether IL-17 stimulates cytokine expression in leukemia cells, we performed cytokine antibody array using tissue culture media from untreated and treated cell. As seen in **Figure 1**, within 24 hours IL-17 stimulated several fold differential expression of various cytokines in the ranking order of IL-2 > IL-3 > GRO > IL-10 > RANTES>IL-15 > IL-1. However, IL-17 failed to simulate IL-8 expression. Stimulation of cytokine expression by IL-17 was significantly inhibited by the PI-3 K inhibitor LY20094 (**Figure 1**), suggesting a role for PI-3 K in the mechanism by which IL-17 stimulates cytokine expression. Similar results were observed in THP-1 human leukemia cell line (data not shown). Also, a neutralizing antibody against of IL-17 blocked IL-17 from stimulating cytokine expression (data not shown), confirming that the observed stimulation of cytokine expression is attributed to L-17. Using ELISA assay, we confirmed stimulation of IL-2 and IL-3 expression by IL-17 without effect on IL-8 expression (**Figure 2**). The lack of effect of IL-17 on IL-8 expression seen in leukemia cells are in contrast to IL-17-induced

according to instructions provided by the kit.

*DOI: http://dx.doi.org/10.5772/intechopen.96422*

conducted in triplicate.

(100 ng/ml) for 24 hours**.**

**3. Results**

**95**

#### **2.7 NF-kB/DNA and STAT3/DNA binding assays for detection of NF-kB and STAT3 activation by IL-17**

To study the effect of IL-17 on NF-kB and STAT3 DNA binding functions [108, 109], 4 million leukemia cells/ml were untreated or IL-17 in time course experiments. The cells were used for specific NF-kB/DNA binding or STAT3/DNA binding assays using the NF-kB and STAT transcription factor assay kits (Active Motif, Chemicon). The kit enabled us to monitor the activation or repression of NF-kB or STAT3 proteins. The experiments were performed in triplicate.

#### **2.8 Cell proliferation assays**

To determine whether IL-17 stimulates cell proliferation in leukemia cells, 6 x 10<sup>5</sup> cells were either untreated or stimulated with IL-17 (100 ng/ml) for 48 hours [110]. The cells were harvested and aliquots were diluted into 0.4% trypan blue/ PBS solution at a ratio of 1:10. The cells were counted in triplicate and the average viable cell count was recorded from each sample. We also performed MTT proliferation assay using 4 x 10<sup>5</sup> cells untreated or cells treated with IL-17 (100 ug/ml) for 48 hours in 96 well plates in triplicate. The rest of the details of the MTT assay protocols were as previously described [110].

#### **2.9 Caspase3 activity assays as evidence for apoptosis**

We purchased caspase3 assay kit from MBL International, Woburn, MA, US [111]. Sodium butyrate (NaB) is a strong inducer of apoptosis in cancer cells [112]. To determine the effects of IL-17 on NaB-induced apoptosis in leukemia cells we performed caspases3 enzymatic (colorimetric) assays in lysates from 15 x 10<sup>6</sup>

*IL-17 Biological Effects and Signaling Mechanisms in Human Leukemia U937 Cells DOI: http://dx.doi.org/10.5772/intechopen.96422*

untreated or NaB treated cells. Specifically, the cells were either untreated or treated with NaB (5 mM) alone, or treated with NaB (5 mM) plus IL-17 (100 ng/ml) or with IL-17 (100 ng/ml) alone in tissue culture media for up to 48 hours. At the end of the incubation, aliquot of cells were counted prior to lysing in RIPA buffer (provided in the caspase3 assay kit) and total lysate protein concentration was determined as indicated above. To measure caspase3 activity in the cell lysates, 30 ug total lysate protein from each sample was added to each experimental assay well. The enzymatic activity was measured in triplicate in microtiter plate reader according to instructions provided by the kit.

#### **2.10 Assessment of chemotactic effects of media from IL-17 treated cells**

In order to ascertain if media from IL-17 treated cells has chemotactic effects towards monocytes [103, 113], we employed the Boden Chamber chemotaxis assay method as previously described [103]. Specifically, 2000 monocytes/ml in fresh complete media were placed in the upper portion of the Boden Chamber. Equal volume of media from either untreated or IL-17 stimulated cells was put in the lower chamber to serve as a source of chemotaxis. The setup was incubated for 2 hours. Next, estimation of number of monocytes which crossed the membrane barrier to the lower chamber was performed according to the kit. Experiments were conducted in triplicate.

#### **2.11 Assessing IL-17-induced cytokine expression by cytokine ELISA**

In order to validate IL-17-induced cytokine expression observed in the cytokine antibody array, we performed cytokine ELISA assay as specified in the cytokine ELISA Kits (Ray Biotech, Norcross, GA) using equal amount of tissue culture proteins from untreated or IL-17 treated cells [103]. The ELISA monitored expression of IL-2, IL-3 and IL-8. In addition, to determine whether IL-17-induced IL-2 expression was mediated by either PI-3 K/Akt or by Jak2, in some experiments, we pre-incubated some of the cells with PI-3 K inhibitor LY20094 (20 nM) or Jak2 inhibitor AG490 (15 nM) for 30 minutes prior to stimulating the cells with IL-17 (100 ng/ml) for 24 hours**.**

#### **3. Results**

Transcriptional Factor Array (1) Kit. Panomics TranSignal™ Protein/DNA Arrays simplifies the functional analysis of eukaryotic TFs and can be used to study TF activation in a variety of biological processes, including cell proliferation, differentiation, transformation, apoptosis and drug treatment [107]. The array membranes were spotted with 54 different consensus-binding sequences (oligos) and enable one to detect over 54 TFs at once in one treatment. Twenty million leukemia cells/ ml were pretreated with vanadate (5 mM) for 30 minutes to inhibit endogenous phosphatases. Next, 5 million of the cells we either untreated or stimulated with IL-17 (100 ng/ml) for 4 hours in the incubator. The cells were packed by centrifugation at 1,800 x g for 3 minutes, washed 3x with PBS and gently lysed in a lysing buffer (see Western blot protocols above) without detergents by repeated aspiration through a 22-gauge needle to prevent rupturing of the nuclei. Intact nuclei were isolated by layering the cell lysate over 50% glycerol solution in Eppendorf tubes followed by centrifugation at 1,000 x g for 5 minutes. The supernatants were carefully aspirated, the nuclei pellet harvested and washed 2x with PBS. Next, the nuclei were disrupted in a nuclei lysing buffer (provided by the kit) and protein concentration determined as described above. In a slightly modified version, 12 ug of nuclei proteins in 200 ul of incubation buffer were incubated with each array membrane containing the oligos for hybridization of each oligo to its specific transcriptional factor in the nuclei extract. The membranes were washed several times and the oligo/transcriptional factor complexes (DNA/protein complexes) were detected by detection per the kit. The spots representing the various transcription factors were identified based on the charts provided by the kit. The intensities of the spots were scanned by digital image analyzer and the fold stimulation by IL-17

*Interleukins - The Immune and Non-Immune Systems' Related Cytokines*

**2.7 NF-kB/DNA and STAT3/DNA binding assays for detection of NF-kB and**

To study the effect of IL-17 on NF-kB and STAT3 DNA binding functions [108, 109], 4 million leukemia cells/ml were untreated or IL-17 in time course experiments. The cells were used for specific NF-kB/DNA binding or STAT3/DNA binding assays using the NF-kB and STAT transcription factor assay kits (Active Motif, Chemicon). The kit enabled us to monitor the activation or repression of NF-kB or STAT3 proteins. The experiments were performed in triplicate.

To determine whether IL-17 stimulates cell proliferation in leukemia cells, 6 x 10<sup>5</sup> cells were either untreated or stimulated with IL-17 (100 ng/ml) for 48 hours [110]. The cells were harvested and aliquots were diluted into 0.4% trypan blue/ PBS solution at a ratio of 1:10. The cells were counted in triplicate and the average viable cell count was recorded from each sample. We also performed MTT proliferation assay using 4 x 10<sup>5</sup> cells untreated or cells treated with IL-17 (100 ug/ml) for 48 hours in 96 well plates in triplicate. The rest of the details of the MTT assay

We purchased caspase3 assay kit from MBL International, Woburn, MA, US [111]. Sodium butyrate (NaB) is a strong inducer of apoptosis in cancer cells [112]. To determine the effects of IL-17 on NaB-induced apoptosis in leukemia cells we performed caspases3 enzymatic (colorimetric) assays in lysates from 15 x 10<sup>6</sup>

compared to the intensities in the untreated cells.

**STAT3 activation by IL-17**

**2.8 Cell proliferation assays**

**94**

protocols were as previously described [110].

**2.9 Caspase3 activity assays as evidence for apoptosis**

#### **3.1 Effects of IL-17 on cytokine expression in human leukemia cells**

To determine whether IL-17 stimulates cytokine expression in leukemia cells, we performed cytokine antibody array using tissue culture media from untreated and treated cell. As seen in **Figure 1**, within 24 hours IL-17 stimulated several fold differential expression of various cytokines in the ranking order of IL-2 > IL-3 > GRO > IL-10 > RANTES>IL-15 > IL-1. However, IL-17 failed to simulate IL-8 expression. Stimulation of cytokine expression by IL-17 was significantly inhibited by the PI-3 K inhibitor LY20094 (**Figure 1**), suggesting a role for PI-3 K in the mechanism by which IL-17 stimulates cytokine expression. Similar results were observed in THP-1 human leukemia cell line (data not shown). Also, a neutralizing antibody against of IL-17 blocked IL-17 from stimulating cytokine expression (data not shown), confirming that the observed stimulation of cytokine expression is attributed to L-17. Using ELISA assay, we confirmed stimulation of IL-2 and IL-3 expression by IL-17 without effect on IL-8 expression (**Figure 2**). The lack of effect of IL-17 on IL-8 expression seen in leukemia cells are in contrast to IL-17-induced

#### **Figure 1.**

*IL-17 Stimulation of differential expression of cytokines: Inhibition by PI-3K Inhibitor (LY20094). Tissue culture media from untreated and treated cells were assayed for cytokine expression by cytokine-antibody array. Data is an average of two experiments.*

**Figure 4**, as compared to culture media from untreated cells, culture media from IL-17 treated cells exhibited significant time-dependent chemotaxis towards monocytes, confirming that culture media from IL-17 treated cells contains secreted

*Inhibition of IL-17-induced IL-2 expression by PI-3K inhibitors (LY and WM) and Jak2 inhibitor (AG). Asterisk (\*) indicates significant differences between IL-17 alone and IL-17 plus PI-3K Inhibitors (LY and*

*IL-17 Biological Effects and Signaling Mechanisms in Human Leukemia U937 Cells*

*DOI: http://dx.doi.org/10.5772/intechopen.96422*

**3.3 IL-17 stimulates leukemia cell growth and protection from apoptosis**

Next, we investigated the effect of IL-17 on leukemia cell proliferation. Untreated or IL-17 stimulated cells were assessed for cell growth using trypan blue exclusion and MTT assays. As shown in **Table 1**, IL-17 exhibited a time-dependent stimulation of leukemia cell growth by 3.3-fold within 48 hours. Similar results were seen in MMT assays (data not shown). Next, we examined whether IL-17 promotes leukemia cell survival and anti-apoptotic effects in leukemia cells by protecting the leukemia cells from apoptosis. The results in **Table 2** indicates that NaB alone causes significant reduction in leukemia cell survival from 100% to 52% in 24 hours. However, in the presence of IL-17, Na-induced decline in cell survival is

*IL-17 induced Chemotaxis. Tissue culture media from untreated IL-17 cells were assayed for chemotaxis in a*

chemotactic chemokines that induced chemotaxis [46].

**Figure 3.**

**Figure 4.**

**97**

*Boyden Chamber with monocytes on the upper chamber.*

*WM) or Jak2 inhibitor (AG490).*

#### **Figure 2.**

*Effects of IL-17 on IL-2, IL-3 and IL-8 expression. Asterisk (\*) indicates significant differences between IL-17 treated and untreated cells.*

IL-8 expression reported in different cell types [16–20]. As a follow up to our results in **Figure 1**, and our previous report that IL-17 activates Jak/STAT pathway [93], we determined whether both the PI-3 K and Jak2 mediate stimulation of specific cytokine expression by IL-17. To do so, we examined the effects of PI-3 K inhibitors LY20094 (LY) and wortmannin (WM) and Jak2 inhibitor AG490 (AG)) on IL-17 induced IL-2 expression. The ELISA array results in **Figure 3** indicate that individually LY20094 and wortmannin partially inhibited IL-17 stimulated IL-2 expression. The Jak2 inhibitor AG490 also exhibited similar inhibitory effect on ability of IL-17 to stimulate IL-2 expression. A combination of both LY20094 and AG490 completely blocked stimulation of IL-2 expression by IL-17 (not shown). These results confirmed roles for both PI-3 K and Jak2 in the mechanisms by which IL-17 stimulates IL-2 expression.

#### **3.2 Media from IL-17 treated leukemia cells produce chemotaxis**

Given that IL-17 stimulated significant expression of two chemokines (GRO and RANTES), we examined whether the culture media from IL-17 treated leukemia cells could serve as chemoattractant to human monocytes from PBMC. As seen in

*IL-17 Biological Effects and Signaling Mechanisms in Human Leukemia U937 Cells DOI: http://dx.doi.org/10.5772/intechopen.96422*

**Figure 3.**

*Inhibition of IL-17-induced IL-2 expression by PI-3K inhibitors (LY and WM) and Jak2 inhibitor (AG). Asterisk (\*) indicates significant differences between IL-17 alone and IL-17 plus PI-3K Inhibitors (LY and WM) or Jak2 inhibitor (AG490).*

**Figure 4**, as compared to culture media from untreated cells, culture media from IL-17 treated cells exhibited significant time-dependent chemotaxis towards monocytes, confirming that culture media from IL-17 treated cells contains secreted chemotactic chemokines that induced chemotaxis [46].

#### **3.3 IL-17 stimulates leukemia cell growth and protection from apoptosis**

Next, we investigated the effect of IL-17 on leukemia cell proliferation. Untreated or IL-17 stimulated cells were assessed for cell growth using trypan blue exclusion and MTT assays. As shown in **Table 1**, IL-17 exhibited a time-dependent stimulation of leukemia cell growth by 3.3-fold within 48 hours. Similar results were seen in MMT assays (data not shown). Next, we examined whether IL-17 promotes leukemia cell survival and anti-apoptotic effects in leukemia cells by protecting the leukemia cells from apoptosis. The results in **Table 2** indicates that NaB alone causes significant reduction in leukemia cell survival from 100% to 52% in 24 hours. However, in the presence of IL-17, Na-induced decline in cell survival is

#### **Figure 4.**

*IL-17 induced Chemotaxis. Tissue culture media from untreated IL-17 cells were assayed for chemotaxis in a Boyden Chamber with monocytes on the upper chamber.*

IL-8 expression reported in different cell types [16–20]. As a follow up to our results in **Figure 1**, and our previous report that IL-17 activates Jak/STAT pathway [93], we determined whether both the PI-3 K and Jak2 mediate stimulation of specific cytokine expression by IL-17. To do so, we examined the effects of PI-3 K inhibitors LY20094 (LY) and wortmannin (WM) and Jak2 inhibitor AG490 (AG)) on IL-17 induced IL-2 expression. The ELISA array results in **Figure 3** indicate that individually LY20094 and wortmannin partially inhibited IL-17 stimulated IL-2 expression. The Jak2 inhibitor AG490 also exhibited similar inhibitory effect on ability of IL-17

*Effects of IL-17 on IL-2, IL-3 and IL-8 expression. Asterisk (\*) indicates significant differences between IL-17*

*IL-17 Stimulation of differential expression of cytokines: Inhibition by PI-3K Inhibitor (LY20094). Tissue culture media from untreated and treated cells were assayed for cytokine expression by cytokine-antibody array.*

*Interleukins - The Immune and Non-Immune Systems' Related Cytokines*

Given that IL-17 stimulated significant expression of two chemokines (GRO and RANTES), we examined whether the culture media from IL-17 treated leukemia cells could serve as chemoattractant to human monocytes from PBMC. As seen in

to stimulate IL-2 expression. A combination of both LY20094 and AG490 completely blocked stimulation of IL-2 expression by IL-17 (not shown). These results confirmed roles for both PI-3 K and Jak2 in the mechanisms by which IL-17

**3.2 Media from IL-17 treated leukemia cells produce chemotaxis**

stimulates IL-2 expression.

**Figure 1.**

**Figure 2.**

**96**

*treated and untreated cells.*

*Data is an average of two experiments.*

#### *Interleukins - The Immune and Non-Immune Systems' Related Cytokines*


**Table 1.**

*Effects of IL-17 on cell growth, cell survival and caspase3 activity—IL-17 promotes cell growth.*


#### **Table 2.**

*Effects of IL-17 on cell growth, cell survival and caspase3 activity—IL-17 protects cells from butyrate-induced apoptosis.*


#### **Table 3.**

*Effects of IL-17 on cell growth, cell survival and caspase3 activity—IL-17 inhibits butyrate-induced caspase3 activation. Data represent mean plus/minus SD.*

> is already highly constitutively expressed in active from in these leukemia cells. This could explain why IL-17 failed to stimulate NF-kB activation further in these cells.

> Because the PI-3 K inhibitor Ly20094 inhibited IL-17-induced cytokine expression, we examined the direct effects of IL-17 on PI-3 K and Akt phosphorylation and activation. As shown in **Figure 7a** and **7b**, in as early as 0.5 minutes, IL-17 stimulated PI-3 K tyrosine phosphorylation by 4.5-fold. PI-3 K phosphorylation and activation usually lead to downstream Akt (PKB) activation [113]. Therefore, we next examined the effects of IL-17 on Akt (PKB) phosphorylation and activation. Akt can be phosphorylated on Serine 473 (Ser473) and/or Threonine 308 (Thr308), which is in the activation domain. The western blot results in **Figure 8a** show that IL-17 stimulated Akt phosphorylation on Serine<sup>473</sup> to 5-fold within 10 min in these cells. The results in **Figure 8b** show that IL-17 stimulates rapid phosphorylation of Akt on Thr<sup>308</sup> with maximum effect noted at 5 minutes. Stimulation of Akt phosphorylation on Serine<sup>473</sup> by IL-17 was inhibited by the PI-3 K inhibitor wortmannin (WM) (no shown). These results imply that stimulation of Akt phosphorylation by IL-17 is mediated by PI-3 K. Once Akt is activated, it phosphorylates a host of downstream effectors including BAD, Caspase3, forkhead transcription factor (FKHR), glycogen synthase kinase-3 (GSK3-beta), AFX, eNOS,TSC2, MDM2, P21/ CIP1 and other downstream effectors as shown in **Figure 9a** Dephosphorylated BAD, caspase3 and GSK3-beta play vital roles in induction of apoptosis [114].

**3.5 Direct evidence that IL-17 activates PI-3 K/Akt signaling pathway in**

*IL-17 Biological Effects and Signaling Mechanisms in Human Leukemia U937 Cells*

**leukemia cells**

**Figure 5.**

**Figure 6.**

**99**

*Activation of STAT3 Transcriptional Activity by IL-17.*

*DOI: http://dx.doi.org/10.5772/intechopen.96422*

*Lack of IL-17 stimulation of NF-kB DNA Binding Activity.*

markedly inhibited cell survival improved from 52% to 83%. As shown in **Table 3**, NaB alone stimulated activation of caspase3 activity from 1-fold in untreated cells to 3.2 fold in 24 hours and 4.3-fold in 48 hours, indicating NaB-induced apoptosis in cells in the absence of IL-17. However, in the presence of IL-17, NaB-induced caspase3 activation is markedly reduced from 3.2-fold to 1.7-fold in 24 hours and from 4.3-fold to 2.0-fold in 48 hours. IL-17 also upgrades Bcl2 in the presence of NaB (not shown). Thus, IL-17 protects leukemia cells from undergoing apoptosis and enhances their survival. The results suggest that IL-17 may be inducing inactivation of pro-apoptotic signals while partially restoring the anti-apoptotic protein Bcl-2 expression.

#### **3.4 IL-17 stimulates differential activation of transcription factors in leukemia cells**

Within 4 hours IL-17 stimulated significant and differential activation of several transcription factors in the order of c-Myb (5.5-fold) > EGR-1 (5.0-fold) > STAT3 (4.0-fold)> > Smad3/4 (3.4-fold) > SRE (3.0 fold>CDP (2.5-.fold). IL-17 failed to activate NF-kB. Using individual transcription factor/DNA binding assays, we confirmed that STAT3/DNA binding activity is significantly enhanced by IL-17 (**Figure 5).** In contrast, IL-17 did not stimulate NF-kB/DNA binding activity in these leukemia cells (**Figure 6**). Together these results show that IL-17 differentially activates several transcriptional factors associated with regulation of cell growth, cell differentiation and apoptosis but failed to stimulate NF-kB in these cells even though IL-17 is known to stimulate NF-kB in many cell types [50]. Of note, NF-kB

*IL-17 Biological Effects and Signaling Mechanisms in Human Leukemia U937 Cells DOI: http://dx.doi.org/10.5772/intechopen.96422*

**Figure 5.** *Activation of STAT3 Transcriptional Activity by IL-17.*

**Figure 6.** *Lack of IL-17 stimulation of NF-kB DNA Binding Activity.*

is already highly constitutively expressed in active from in these leukemia cells. This could explain why IL-17 failed to stimulate NF-kB activation further in these cells.

#### **3.5 Direct evidence that IL-17 activates PI-3 K/Akt signaling pathway in leukemia cells**

Because the PI-3 K inhibitor Ly20094 inhibited IL-17-induced cytokine expression, we examined the direct effects of IL-17 on PI-3 K and Akt phosphorylation and activation. As shown in **Figure 7a** and **7b**, in as early as 0.5 minutes, IL-17 stimulated PI-3 K tyrosine phosphorylation by 4.5-fold. PI-3 K phosphorylation and activation usually lead to downstream Akt (PKB) activation [113]. Therefore, we next examined the effects of IL-17 on Akt (PKB) phosphorylation and activation. Akt can be phosphorylated on Serine 473 (Ser473) and/or Threonine 308 (Thr308), which is in the activation domain. The western blot results in **Figure 8a** show that IL-17 stimulated Akt phosphorylation on Serine<sup>473</sup> to 5-fold within 10 min in these cells. The results in **Figure 8b** show that IL-17 stimulates rapid phosphorylation of Akt on Thr<sup>308</sup> with maximum effect noted at 5 minutes. Stimulation of Akt phosphorylation on Serine<sup>473</sup> by IL-17 was inhibited by the PI-3 K inhibitor wortmannin (WM) (no shown). These results imply that stimulation of Akt phosphorylation by IL-17 is mediated by PI-3 K. Once Akt is activated, it phosphorylates a host of downstream effectors including BAD, Caspase3, forkhead transcription factor (FKHR), glycogen synthase kinase-3 (GSK3-beta), AFX, eNOS,TSC2, MDM2, P21/ CIP1 and other downstream effectors as shown in **Figure 9a** Dephosphorylated BAD, caspase3 and GSK3-beta play vital roles in induction of apoptosis [114].

markedly inhibited cell survival improved from 52% to 83%. As shown in **Table 3**, NaB alone stimulated activation of caspase3 activity from 1-fold in untreated cells to 3.2 fold in 24 hours and 4.3-fold in 48 hours, indicating NaB-induced apoptosis in cells in the absence of IL-17. However, in the presence of IL-17, NaB-induced caspase3 activation is markedly reduced from 3.2-fold to 1.7-fold in 24 hours and from 4.3-fold to 2.0-fold in 48 hours. IL-17 also upgrades Bcl2 in the presence of NaB (not shown). Thus, IL-17 protects leukemia cells from undergoing apoptosis and enhances their survival. The results suggest that IL-17 may be inducing inactivation of pro-apoptotic signals while partially restoring the anti-apoptotic

*Effects of IL-17 on cell growth, cell survival and caspase3 activity—IL-17 inhibits butyrate-induced caspase3*

*Effects of IL-17 on cell growth, cell survival and caspase3 activity—IL-17 protects cells from butyrate-induced*

**48 hours Relative Caspase 3 Activity (Fold)**

**Treatment 24 hours Cell Growth (Fold) 48 hours Cell Growth (Fold)**

Cells 1.0 1.0 Cells + IL-17 2.3.0 3.3 0.2

*Interleukins - The Immune and Non-Immune Systems' Related Cytokines*

*Effects of IL-17 on cell growth, cell survival and caspase3 activity—IL-17 promotes cell growth.*

**Treatment 24 hours Cell Survival (%)**

**Activity (Fold)**

Cells alone 1.0 1.0 Cells + NaB 3.2 0.4 4.3 0.1 Cells + IL-17 + NaB 1.7 0.2 2.0 0.3

Cells alone 100 Cells + NaB 52 2.2 Cells + IL-17 + NaB 83 1.3

**Treatment 24 hour; Relative Caspase 3**

*activation. Data represent mean plus/minus SD.*

**3.4 IL-17 stimulates differential activation of transcription factors in**

Within 4 hours IL-17 stimulated significant and differential activation of several transcription factors in the order of c-Myb (5.5-fold) > EGR-1 (5.0-fold) > STAT3 (4.0-fold)> > Smad3/4 (3.4-fold) > SRE (3.0 fold>CDP (2.5-.fold). IL-17 failed to activate NF-kB. Using individual transcription factor/DNA binding assays, we confirmed that STAT3/DNA binding activity is significantly enhanced by IL-17 (**Figure 5).** In contrast, IL-17 did not stimulate NF-kB/DNA binding activity in these leukemia cells (**Figure 6**). Together these results show that IL-17 differentially activates several transcriptional factors associated with regulation of cell growth, cell differentiation and apoptosis but failed to stimulate NF-kB in these cells even though IL-17 is known to stimulate NF-kB in many cell types [50]. Of note, NF-kB

protein Bcl-2 expression.

**Table 1.**

**Table 2.**

*apoptosis.*

**Table 3.**

**leukemia cells**

**98**

p-Caspase3 and p-GSK3-beta were contained in Akt pulled down complex from IL-17 treated cells. Phosphorylation of caspse3, BAD, GSK3-beta and STAT3 are associated with enhanced cell survival [115, 116] and could explain in part how IL-17 promotes cell survival. Also, IL-17 stimulated Akt-dependent phosphorylation of mammalian target of rapamycin (mTor) on serine 2448 (motor Ser 2448) [117],

*IL-17 Biological Effects and Signaling Mechanisms in Human Leukemia U937 Cells*

We have provided strong evidence that IL-17 stimulates significance and differential expression of IL-2, IL-3, IL-10, IL-15, GRO, and RANTES in human leukemia cells. The stimulatory effect of IL-17 on cytokine expression in these cells is similar to previous reports in non-hematopoietic cells by IL-17 [16–20]. However, IL-17 does not stimulate IL-8 expression in these cells, which contradicts early reports that IL-17-induces IL-8 expression in different cell types [17–20]. Induction of cytokine expression by IL-17 in these leukemia cells could have strong biological relevance in vivo because increases in IL-17 level in a tumor microenvironment can trigger induction of other cytokines including chemokines that could generate combination of proinflammatory, anti-inflammatory and chemotactic responses [16–20, 54]; [79–81]. IL-2 is a proinflammatory cytokine [118], which also regulates helper T cell differentiation [119]. IL-3 stimulates regulation of multipotent hematopoietic stem cell function and differentiation of all lineages as well as promote proliferation of myeloid progenitor cells [120, 121]. IL-10 is a master regulator of immunity to infection and an anti-inflammatory cytokine that can counteract the pro-inflammatory effects of IL-2 [122]. Secondly, IL-10 is known to synergize with IL-2 to promote CD8 <sup>+</sup> T cell cytotoxicity [123]. IL-15 is known to suppress apoptosis in T-lymphocytes by inducing Bcl2 and/or Bcl-xl in humans [124]. Perhaps, IL-15 contributes to the anti-apoptotic effect of IL-17 in these cells. Both IL-2 and IL-15 have structural and functional similarities, share the common gamma chain of their

receptors and promote immune response [125]. Both GRO and RANTES are chemokines and are associated with induction of chemotaxis and recruitment of neutrophils and macrophages to sites of infection [37]. Thus, IL-17- induced GRO and RANTES expression and secretion from leukemia cells into the culture media,

These leukemia cells express receptors for some of the cytokines secreted to the culture media in response to IL-17. Therefore, some of the cytokines secreted into culture media can promote both autocrine and paracrine effects on the leukemia cells. We have provided evidence that IL-17 stimulates phosphorylation of the proapoptotic proteins BAD, caspase3 and GSK3-beta, thus negating their functions. In addition, IL-17 promotes inhibition of Caspase3 activity in these leukemia cells. Furthermore, IL-17 enhances Akt phosphorylation and activation, which are associated with cell survival [126]. The ability of IL-17 to enhance protection of the leukemia cells from apoptosis implies that elevated IL-17 levels in a tumor microenvironment could lead to promotion of leukemia cell proliferation and survival, both of which could potentially produce poor prognosis in leukemia patients. Another interesting outcome of this study is that IL-17 stimulates activation of several transcriptional factors including cMyb, EGR-1, STAT3, Smad3/4, SRE, CDP, which are known to regulate proliferation, differentiation and survival [115, 127, 128]. This effect of IL-17 could in part contribute to the mechanism growth promotion and survival in these leukemia cells. Stimulation of smad3/4 transcriptional factors of the TGF-beta signaling pathway [102, 115] by IL-17 may point to potential cross talk between IL-17 and TGF-beta-induced signaling pathways to synergize

could account for the chemotactic effect IL-17 seen in our studies.

which was inhibited by the Akt inhibitor SH5 (not shown).

*DOI: http://dx.doi.org/10.5772/intechopen.96422*

**4. Discussions**

**101**

#### **Figure 7.**

*Time course of IL-17-induced PI-3K phosphorylation detected by Western blot using either specific antibody for tyrosine phosphorylated PI-3K or total PI-3K as loading control. Scanned values represent ptyr-PI-3K/PI-3K ratios from 3 experiments (b). Asterisk (\*) indicates significant differences between IL-17 treated and untreated cells. Results are representation form several experiments.*

#### **Figure 8.**

*Time course of IL-17 stimulation of AktSer473 phosphorylation (a) and (b) AktThr308 Specific antibody to either AktSer473 or AktThr308 was used to monitor Akt phosphorylation by Western blot. Blots were stripped and reprobed for total Akt for loading control. The blots from 3 experiments were scanned and results are presented. Asterisk (\*) indicates significant differences between IL-17 treated and untreated cells.*

However, upon their phosphorylation, these pro-apoptotic proteins lose their proapoptotic activities [114] as phosphorylation of both BAD, caspase3 and GSK-3 leads to their inactivation. The results in **Figure 9b** and **c** show that IL-17 stimulates Akt-mediated BAD, Caspase3 and GSK-3-beta phosphorylation as p-BAD,

#### **Figure 9.**

*Model showing activated Akt phosphorylation of its downstream targets (a). Effect of IL-17 on Caspase3 and BAD phosphorylation (b), GSK-3-beta phosphorylation (c). In (b) and (c) cells were untreated or stimulated with IL-17 and total cell lysates were monitored for Caspase3, BAD and GSK-3-beta phosphorylation by Western blot using specific phosphoprotein antibody to each protein. Total Akt (b) or total GSK-3-beta (c) was probed for loading control. Results are representation of several experiments.*

p-Caspase3 and p-GSK3-beta were contained in Akt pulled down complex from IL-17 treated cells. Phosphorylation of caspse3, BAD, GSK3-beta and STAT3 are associated with enhanced cell survival [115, 116] and could explain in part how IL-17 promotes cell survival. Also, IL-17 stimulated Akt-dependent phosphorylation of mammalian target of rapamycin (mTor) on serine 2448 (motor Ser 2448) [117], which was inhibited by the Akt inhibitor SH5 (not shown).

#### **4. Discussions**

We have provided strong evidence that IL-17 stimulates significance and differential expression of IL-2, IL-3, IL-10, IL-15, GRO, and RANTES in human leukemia cells. The stimulatory effect of IL-17 on cytokine expression in these cells is similar to previous reports in non-hematopoietic cells by IL-17 [16–20]. However, IL-17 does not stimulate IL-8 expression in these cells, which contradicts early reports that IL-17-induces IL-8 expression in different cell types [17–20]. Induction of cytokine expression by IL-17 in these leukemia cells could have strong biological relevance in vivo because increases in IL-17 level in a tumor microenvironment can trigger induction of other cytokines including chemokines that could generate combination of proinflammatory, anti-inflammatory and chemotactic responses [16–20, 54]; [79–81]. IL-2 is a proinflammatory cytokine [118], which also regulates helper T cell differentiation [119]. IL-3 stimulates regulation of multipotent hematopoietic stem cell function and differentiation of all lineages as well as promote proliferation of myeloid progenitor cells [120, 121]. IL-10 is a master regulator of immunity to infection and an anti-inflammatory cytokine that can counteract the pro-inflammatory effects of IL-2 [122]. Secondly, IL-10 is known to synergize with IL-2 to promote CD8 <sup>+</sup> T cell cytotoxicity [123]. IL-15 is known to suppress apoptosis in T-lymphocytes by inducing Bcl2 and/or Bcl-xl in humans [124]. Perhaps, IL-15 contributes to the anti-apoptotic effect of IL-17 in these cells. Both IL-2 and IL-15 have structural and functional similarities, share the common gamma chain of their receptors and promote immune response [125]. Both GRO and RANTES are chemokines and are associated with induction of chemotaxis and recruitment of neutrophils and macrophages to sites of infection [37]. Thus, IL-17- induced GRO and RANTES expression and secretion from leukemia cells into the culture media, could account for the chemotactic effect IL-17 seen in our studies.

These leukemia cells express receptors for some of the cytokines secreted to the culture media in response to IL-17. Therefore, some of the cytokines secreted into culture media can promote both autocrine and paracrine effects on the leukemia cells. We have provided evidence that IL-17 stimulates phosphorylation of the proapoptotic proteins BAD, caspase3 and GSK3-beta, thus negating their functions. In addition, IL-17 promotes inhibition of Caspase3 activity in these leukemia cells. Furthermore, IL-17 enhances Akt phosphorylation and activation, which are associated with cell survival [126]. The ability of IL-17 to enhance protection of the leukemia cells from apoptosis implies that elevated IL-17 levels in a tumor microenvironment could lead to promotion of leukemia cell proliferation and survival, both of which could potentially produce poor prognosis in leukemia patients. Another interesting outcome of this study is that IL-17 stimulates activation of several transcriptional factors including cMyb, EGR-1, STAT3, Smad3/4, SRE, CDP, which are known to regulate proliferation, differentiation and survival [115, 127, 128]. This effect of IL-17 could in part contribute to the mechanism growth promotion and survival in these leukemia cells. Stimulation of smad3/4 transcriptional factors of the TGF-beta signaling pathway [102, 115] by IL-17 may point to potential cross talk between IL-17 and TGF-beta-induced signaling pathways to synergize

However, upon their phosphorylation, these pro-apoptotic proteins lose their proapoptotic activities [114] as phosphorylation of both BAD, caspase3 and GSK-3 leads to their inactivation. The results in **Figure 9b** and **c** show that IL-17 stimulates

*Model showing activated Akt phosphorylation of its downstream targets (a). Effect of IL-17 on Caspase3 and BAD phosphorylation (b), GSK-3-beta phosphorylation (c). In (b) and (c) cells were untreated or stimulated with IL-17 and total cell lysates were monitored for Caspase3, BAD and GSK-3-beta phosphorylation by Western blot using specific phosphoprotein antibody to each protein. Total Akt (b) or total GSK-3-beta (c) was*

*probed for loading control. Results are representation of several experiments.*

*Time course of IL-17 stimulation of AktSer473 phosphorylation (a) and (b) AktThr308 Specific antibody to either AktSer473 or AktThr308 was used to monitor Akt phosphorylation by Western blot. Blots were stripped and reprobed for total Akt for loading control. The blots from 3 experiments were scanned and results are presented.*

*Time course of IL-17-induced PI-3K phosphorylation detected by Western blot using either specific antibody for tyrosine phosphorylated PI-3K or total PI-3K as loading control. Scanned values represent ptyr-PI-3K/PI-3K ratios from 3 experiments (b). Asterisk (\*) indicates significant differences between IL-17 treated and*

Akt-mediated BAD, Caspase3 and GSK-3-beta phosphorylation as p-BAD,

*Asterisk (\*) indicates significant differences between IL-17 treated and untreated cells.*

*untreated cells. Results are representation form several experiments.*

*Interleukins - The Immune and Non-Immune Systems' Related Cytokines*

**Figure 8.**

**Figure 9.**

**100**

**Figure 7.**

their biological effects [127, 128]. The lack of activation of NF-kB by IL-17 in these leukemia cells is not surprising since typically these leukemia cells constitutively express high levels of active NF-kB, which could explain the apparent lack of NF-kB response to IL-17. Lack of NF-kB activation by IL-17 in these cells is in contrast to IL-17-induced NF-kB activation reported in many cells [50].

conclusion, there are calls for development of anti-IL-17 drugs as adjunct therapy for diseases in which IL-17 plays an active role [87, 131]. IL-17-enhanced leukemia cell growth, survival and anti-apoptosis strengthens the argument in favor of inclusion of leukemia in the list of human diseases for which anti-IL-17 adjunct therapy should be considered. Our model in **Figure 10** explains the paracrine role of T-cell secreted IL-17 in leukemia cells. Elucidation of the multiple signaling mechanisms of IL-17 in leukemia cells in our study and illustrated in **Figure 11** further enrich our

*IL-17 Biological Effects and Signaling Mechanisms in Human Leukemia U937 Cells*

Our studies on effects and mechanisms of IL-17 in human U937 leukemia cells revealed that these cells express IL-17A receptor and IL-17 stimulates cell growth, survival, chemotaxis and differential expression of cytokines. These results suggest that IL-17 could trigger expression and secretion of various cytokines including chemotactic chemokines in leukemia patients. Also, IL-17 promotes anti-apoptotic effects in these cells. If these biological effects of IL-17 described here, were to occur in leukemia patients, IL-17 could promote poor prognosis in the patients. Furthermore, IL-17 stimulates differential activation of several transcriptional factors including c-Myb, EGR-1, STAT3, smad3/4 CDP and SRE but not NF-kB in these cells. Lastly, multiple signaling pathways including PI-3 K/Akt, Jak/STAT,

Raf–MEK-ERK-1/2 and Lck signaling pathways differentially mediate the biological effects of IL-17 in the U937 leukemia cells. Any of these pathways could serve as a

This work was partially supported by NIAMS/NIH R03 grant, U54 cancer part-

Samuel Evans Adunyah\*, Richard Akomeah, Fareed K.N. Arthur, Roland S. Cooper

Department of Biochemistry, Cancer Biology, Neuroscience and Pharmacology,

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

nership NCI grant U54CA091408 and NIGMS/NIH SCORE grant to Professor Adunyah, who was also supported by cancer partnership grant U54CA163069/NCI during preparation of this chapter. Professor Arthur was partially supported by Biochemistry Department, KNUST, Kumasi, Ghana during his sabbatical. We thank Dr. S. V. Subramaniam and W. Williams for their contribution in the initial stages

knowledge on the biological effects and mechanisms of IL-17.

*DOI: http://dx.doi.org/10.5772/intechopen.96422*

**5. Conclusion**

target for anti-IL-17 drugs.

**Acknowledgements**

of this work.

**Author details**

**103**

and Joshua C.M. Williams

Meharry Medical College, Nashville, TN, USA

provided the original work is properly cited.

\*Address all correspondence to: sadunyah@mmc.edu

We have provided ample evidence that IL-17 activates and utilizes the Jak/STAT signaling pathway in these leukemia cells. In this pathway, IL-17 stimulates phosphorylation of Jak1, Jak2 and Jak3, STAT1, STAT2, and STAT3 [55, 93, 97]. We have also shown that Jak2 partially mediates IL-17-induced IL-2 expression. Furthermore, IL-17 strongly stimulates phosphorylation and activation of PI-3 K/ Akt pathway and promoting Akt-mediated phosphorylation of its downstream effectors. Another interesting observation is that Akt-partially mediates stimulation of IL-2 expression and secretion by IL-17. Also, IL-17 promotes phospho-Akt's association with BAD, caspase3 and GSK3-beta, supporting Akt-mediated phosphorylation of these proteins in IL-17 treated cells. These observations could in part explain how IL-17 promotes anti-apoptosis and survival in these leukemia cells [75–77, 126].

IL-17 stimulates activation of Raf–MEK–ERK1/2 pathway [92–95, 101], which could partially account for the growth promoting effects of IL-17 in leukemia cells. Previous thesis research in our laboratory revealed that IL-17 stimulates activation of LCK [129] and PKC [130]. Also, IL-17 promotes association between LCK and the p85 subunit of the PI-3 K, thus providing another mechanism for PI-3 K activation by IL-17 via LCK, a member of the Src kinases family [129]. Activation of PKC by IL-17 is associated with enhanced PKC ability to regulate cell cycle progression in leukemia cells [130]. As indicated earlier, IL-17 is profoundly implicated in many human diseases [55–74], thus supporting the suggestion that design and production of anti-IL-17 drugs could lead to better strategies for development of new therapies for those diseases [88–90]. Although the recent reports implicating IL-17 in the mechanism of the "cytokine storm" event in COVID-19 infection is far from

#### **Figure 10.**

*Model showing activated memory T cell secreted IL-17: Paracrine mechanism of how secreted IL-17 activates cytokine expression and secretion in leukemia cells.*

**Figure 11.** *Model showing multiple signaling mechanisms used by IL-17 in Leukemia Cells.*

*IL-17 Biological Effects and Signaling Mechanisms in Human Leukemia U937 Cells DOI: http://dx.doi.org/10.5772/intechopen.96422*

conclusion, there are calls for development of anti-IL-17 drugs as adjunct therapy for diseases in which IL-17 plays an active role [87, 131]. IL-17-enhanced leukemia cell growth, survival and anti-apoptosis strengthens the argument in favor of inclusion of leukemia in the list of human diseases for which anti-IL-17 adjunct therapy should be considered. Our model in **Figure 10** explains the paracrine role of T-cell secreted IL-17 in leukemia cells. Elucidation of the multiple signaling mechanisms of IL-17 in leukemia cells in our study and illustrated in **Figure 11** further enrich our knowledge on the biological effects and mechanisms of IL-17.

#### **5. Conclusion**

their biological effects [127, 128]. The lack of activation of NF-kB by IL-17 in these leukemia cells is not surprising since typically these leukemia cells constitutively express high levels of active NF-kB, which could explain the apparent lack of NF-kB response to IL-17. Lack of NF-kB activation by IL-17 in these cells is in contrast to

We have provided ample evidence that IL-17 activates and utilizes the Jak/STAT signaling pathway in these leukemia cells. In this pathway, IL-17 stimulates phosphorylation of Jak1, Jak2 and Jak3, STAT1, STAT2, and STAT3 [55, 93, 97]. We have also shown that Jak2 partially mediates IL-17-induced IL-2 expression. Furthermore, IL-17 strongly stimulates phosphorylation and activation of PI-3 K/ Akt pathway and promoting Akt-mediated phosphorylation of its downstream effectors. Another interesting observation is that Akt-partially mediates stimulation of IL-2 expression and secretion by IL-17. Also, IL-17 promotes phospho-Akt's association with BAD, caspase3 and GSK3-beta, supporting Akt-mediated phosphorylation of these proteins in IL-17 treated cells. These observations could in part explain how IL-17 promotes anti-apoptosis and survival in these leukemia cells [75–77, 126].

IL-17 stimulates activation of Raf–MEK–ERK1/2 pathway [92–95, 101], which could partially account for the growth promoting effects of IL-17 in leukemia cells. Previous thesis research in our laboratory revealed that IL-17 stimulates activation of LCK [129] and PKC [130]. Also, IL-17 promotes association between LCK and the p85 subunit of the PI-3 K, thus providing another mechanism for PI-3 K activation by IL-17 via LCK, a member of the Src kinases family [129]. Activation of PKC by IL-17 is associated with enhanced PKC ability to regulate cell cycle progression in leukemia cells [130]. As indicated earlier, IL-17 is profoundly implicated in many human diseases [55–74], thus supporting the suggestion that design and production of anti-IL-17 drugs could lead to better strategies for development of new therapies for those diseases [88–90]. Although the recent reports implicating IL-17 in the mechanism of the "cytokine storm" event in COVID-19 infection is far from

*Model showing activated memory T cell secreted IL-17: Paracrine mechanism of how secreted IL-17 activates*

IL-17-induced NF-kB activation reported in many cells [50].

*Interleukins - The Immune and Non-Immune Systems' Related Cytokines*

**Figure 10.**

**Figure 11.**

**102**

*cytokine expression and secretion in leukemia cells.*

*Model showing multiple signaling mechanisms used by IL-17 in Leukemia Cells.*

Our studies on effects and mechanisms of IL-17 in human U937 leukemia cells revealed that these cells express IL-17A receptor and IL-17 stimulates cell growth, survival, chemotaxis and differential expression of cytokines. These results suggest that IL-17 could trigger expression and secretion of various cytokines including chemotactic chemokines in leukemia patients. Also, IL-17 promotes anti-apoptotic effects in these cells. If these biological effects of IL-17 described here, were to occur in leukemia patients, IL-17 could promote poor prognosis in the patients. Furthermore, IL-17 stimulates differential activation of several transcriptional factors including c-Myb, EGR-1, STAT3, smad3/4 CDP and SRE but not NF-kB in these cells. Lastly, multiple signaling pathways including PI-3 K/Akt, Jak/STAT, Raf–MEK-ERK-1/2 and Lck signaling pathways differentially mediate the biological effects of IL-17 in the U937 leukemia cells. Any of these pathways could serve as a target for anti-IL-17 drugs.

#### **Acknowledgements**

This work was partially supported by NIAMS/NIH R03 grant, U54 cancer partnership NCI grant U54CA091408 and NIGMS/NIH SCORE grant to Professor Adunyah, who was also supported by cancer partnership grant U54CA163069/NCI during preparation of this chapter. Professor Arthur was partially supported by Biochemistry Department, KNUST, Kumasi, Ghana during his sabbatical. We thank Dr. S. V. Subramaniam and W. Williams for their contribution in the initial stages of this work.

#### **Author details**

Samuel Evans Adunyah\*, Richard Akomeah, Fareed K.N. Arthur, Roland S. Cooper and Joshua C.M. Williams Department of Biochemistry, Cancer Biology, Neuroscience and Pharmacology, Meharry Medical College, Nashville, TN, USA

\*Address all correspondence to: sadunyah@mmc.edu

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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immunopathology. Journal Immunol.2017;doi:10.4049/ jimmunol.1601366:1-19

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**113**

**1. Introduction**

**Chapter 7**

**Abstract**

The Role of Interleukins after

*Daniel J. Hellenbrand, Rylie M. Roddick, Sophia M. Mauney,* 

In skin wound healing the injured tissue goes through a normal progression, inflammation subsides and remodeling occurs. However after spinal cord injury inflammation persists and there is less progression into a regenerative/rebuilding phase. This inflammatory process after spinal cord injury is orchestrated by many cell types and numerous cytokines. Although there are several positive effects of inflammation after spinal cord injury, such as the removal of debris, the substantial upregulation of immune cells has been shown to contribute to neural degeneration. Several chemokines and cytokines including many interleukins are involved in guiding these immune cells to the lesion. While there are many inflammatory cytokines acting on these immune cells after SCI, there are also several anti-inflammatory interleukins that have shown beneficial effects in reducing inflammation. After SCI in a rat model, interleukin-10 and interleukin-19 have been shown to downregulate the synthesis of pro-inflammatory species including interleukin-1β and tumor necrosis factor-α, which resulted in a significant improvement in rat hind limb function. Also, interleukin-4 and interleukin-13 are related anti-inflammatory cytokines that regulate many aspects of inflammation and have also been shown to induce alternative macrophage activation. The differing and complex roles interleukins play, highlight their importance on the inflammation that persists after spinal cord injury. Here we review both the positive effects and negative effects that interleukins have during the multifaceted inflammation process following spinal cord injury.

**Keywords:** interleukins, spinal cord injury, inflammation, macrophages, microglia

Spinal Cord Injury (SCI) is a devastating trauma and according to the National Spinal Cord Injury Statistical Center (NSCISC) there are approximately 294,000 people living with SCI in the United States [1]. After spinal SCI there is immediate cell death caused directly from the insult followed by a cascade of inflammation that leads to additional cell death and a much larger scar formation that impedes axonal regeneration [2, 3]. Although there are several positive effects of inflammation after SCI, the extensive infiltration of immune cells is a principal contributor to neural degeneration [4, 5]. These immune cells are guided to the lesion site from the periphery via many signaling cues including several interleukins (ILs) released by

microglia, astrocytes, and peripheral macrophages within the lesion [5, 6].

*Ryan T. Elder, Carolyn N. Morehouse and Amgad S. Hanna*

Spinal Cord Injury

#### **Chapter 7**

[126] Brunet A, Bonni A, Zigmond MJ, et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead Transcription Factor. Cell.

*Interleukins - The Immune and Non-Immune Systems' Related Cytokines*

[127] Trizzino M, Zucco A, Deliard S, Wang F, et al. EGR-1 is a gatekeeper of inflammatory enhancers in human macrophages. Science Advances. 2021;7 (3):eaaz8836 doi.10.1126/sciadv,

[128] Yang H, Wang L, Zhao J, et al. TGF-beta-activated smad3/4 complex transcriptionally upregulates Ncadherin expression in non-small cell lung cancer. Lung Cancer. 2015;87(3):

[129] Williams JCM. Evidence of the involvement of specific Src kinases and phospholipase D in IL-17A signaling mechanisms in U937 leukemia cells [thesis]. Biochemistry and Cancer Biology Department: Mehary Medical

College, Nashville, TN; 2004

[130] Davis WL, Jr. An in vitro examination of the effects of Interleukin-17 in proliferation and differentiation of U937 leukemia cells [thesis]. Biochemistry and Cancer Biology Department: Mehary Medical

College, Nashville, TN; 2004

Infect. 2020;53:368–370

**112**

[131] Wu D, Yang XO. TH17 responses in cytokine storm of COVID-19: An emerging target of Jak2 inhibitor Fedratinib. J. MicroBiol. Immunol. &

1999;96:857–868

aaz8836

249–257

## The Role of Interleukins after Spinal Cord Injury

*Daniel J. Hellenbrand, Rylie M. Roddick, Sophia M. Mauney, Ryan T. Elder, Carolyn N. Morehouse and Amgad S. Hanna*

#### **Abstract**

In skin wound healing the injured tissue goes through a normal progression, inflammation subsides and remodeling occurs. However after spinal cord injury inflammation persists and there is less progression into a regenerative/rebuilding phase. This inflammatory process after spinal cord injury is orchestrated by many cell types and numerous cytokines. Although there are several positive effects of inflammation after spinal cord injury, such as the removal of debris, the substantial upregulation of immune cells has been shown to contribute to neural degeneration. Several chemokines and cytokines including many interleukins are involved in guiding these immune cells to the lesion. While there are many inflammatory cytokines acting on these immune cells after SCI, there are also several anti-inflammatory interleukins that have shown beneficial effects in reducing inflammation. After SCI in a rat model, interleukin-10 and interleukin-19 have been shown to downregulate the synthesis of pro-inflammatory species including interleukin-1β and tumor necrosis factor-α, which resulted in a significant improvement in rat hind limb function. Also, interleukin-4 and interleukin-13 are related anti-inflammatory cytokines that regulate many aspects of inflammation and have also been shown to induce alternative macrophage activation. The differing and complex roles interleukins play, highlight their importance on the inflammation that persists after spinal cord injury. Here we review both the positive effects and negative effects that interleukins have during the multifaceted inflammation process following spinal cord injury.

**Keywords:** interleukins, spinal cord injury, inflammation, macrophages, microglia

#### **1. Introduction**

Spinal Cord Injury (SCI) is a devastating trauma and according to the National Spinal Cord Injury Statistical Center (NSCISC) there are approximately 294,000 people living with SCI in the United States [1]. After spinal SCI there is immediate cell death caused directly from the insult followed by a cascade of inflammation that leads to additional cell death and a much larger scar formation that impedes axonal regeneration [2, 3]. Although there are several positive effects of inflammation after SCI, the extensive infiltration of immune cells is a principal contributor to neural degeneration [4, 5]. These immune cells are guided to the lesion site from the periphery via many signaling cues including several interleukins (ILs) released by microglia, astrocytes, and peripheral macrophages within the lesion [5, 6].

Throughout the first hours after injury, polymorphonuclear leukocytes are the predominant infiltrating cells and over-activation of these cells causes tissue destruction through the release of significant amounts of neurotoxins including reactive oxygen species (ROS), reactive nitrogen species (RNS), chemokines, and enzymes [5, 7, 8]. Microglia, the resident macrophages, are also activated and migrate to the site of injury, proliferate, and transform from the ramified phenotype to amoeboid phagocytic cells [9]. These activated microglia and peripheral macrophages make up the majority of inflammatory cells present at the site of the lesion. Although in normal wound healing macrophages sequentially change and reduce inflammation, after SCI macrophages persist in an inflammatory state for prolonged periods resulting in progressive tissue degeneration [10, 11]. However these microglia/macrophages can be activated toward an anti-inflammatory phenotype and ILs are important signaling cues in the extracellular environment that help dictate this contrasting phenotype. The goal of this chapter is to examine the role ILs have on the dynamic inflammatory process that occurs after SCI.

#### **2. Interleukins involved in inflammation after SCI**

There are numerous known ILs and several of these ILs are shown to be involved in inflammation after SCI. Throughout this chapter we will discuss the ILs that have been investigated after SCI and whether their role is predominately inflammatory or anti-inflammatory. It is important to note, the role an IL plays after injury is not as simple as just inflammatory or anti-inflammatory. For many of the ILs there are multiple factors that determine whether they will have a beneficial role or a detrimental role, including the extent of initial injury, concentration of IL, other associated molecules in the injury, and the response of immune cells and glial cells [12, 13].

Although there is a broad spectrum of signaling molecules including cytokines, chemokines, and other reactive species after SCI, this chapter will just focus on ILs and only the ILs that are known to play a role in inflammation after SCI [14]. These ILs will be discussed in terms of the cell types that produce them, receptors they bind, cell types they target, timeline of upregulation, and ultimately their effect on inflammation after SCI.

#### **2.1 Interleukin-1 family cytokines**

IL-1α, IL-1β, and IL-33 are members of the IL-1 family that have been studied after SCI and all are predominantly inflammatory [15]. IL-1 is released via activated macrophages and microglia largely in response to disease, infection, or inflammatory events. IL-1 has two structurally and biologically similar isoforms, IL-1α and IL-1β [13]. These two isoforms share roughly 30% amino acid sequence homology and although they perform similar biological functions, IL-1β plays a more substantial role post-SCI [16, 17]. IL-1β has been shown to contribute to the exaggerated neuroinflammation following SCI that leads to secondary neural degeneration and cell death [16]. IL-1 signaling following SCI is diverse and complex, resulting in a recruitment of neurotoxins or immune system molecules that contribute to the inflammatory response [13].

The primary receptor for signaling of both IL-1 isoforms is the type-I interleukin-1 receptor (IL-1RI). IL-1 signaling is further regulated by a decoy receptor (IL-1RII) and a receptor antagonist (IL-1ra) [13]. The expression of these receptors mediates the inflammatory response to SCI and their mechanisms have been widely studied following SCI. After SCI in rats, IL-1R1 expression is elevated as

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comparison to wild-type mice [18].

logic and neurologic damage or inflammation [22].

made up of four α helixes and is produced mainly by CD4<sup>+</sup>

macrophage/microglia [21].

**2.2 Interleukin-2 family cytokines**

within the same microenvironments [25].

understood.

early as 4 hours, peaks at 8 hours to 1-day and remains elevated for 7 days postinjury [13]. Another study tested the role of IL-1ra as a regulatory molecule following SCI and observed that increased expression of IL-1ra suppressed IL-1β levels and increased locomotor function following SCI, suggesting that IL-1β and IL-1RI play critical roles in secondary tissue damage and impaired functional recovery post-SCI [16]. Likewise, administration of IL-1β suppressed the expression of IL-1ra following SCI indicating the regulatory nature of IL-1β interactions with its receptor antagonist [16]. Similarly, a study using IL-1 knockout mice observed a significantly smaller lesion area and improved locomotor function after SCI in

The signaling cascade of the IL-1β/IL-1RI pathway is complex and yet to be completely understood, however it is widely understood that it stimulates the production of toxic intermediates that cause neural degeneration and cell death [13]. These toxic inflammatory mediators include prostaglandins, cyclooxygenase 2, and phospholipase A2 [13]. However, there are studies showing benefits of IL-1, where IL-1β null mice failed to remyelinate as rapidly as wild-type mice [19]. These different roles IL-1 plays are likely due to several factors including extent of injury as well as IL-1 concentration and timing of upregulation, but at present are not well

Another member of the IL-1 family, IL-33, predominantly induces type-2 immune responses against allergens and infectious diseases [20]. IL-33 is upregulated in response to SCI and tends to localize in spinal cord astrocytes to reduce T cell infiltration and overexaggerated inflammation that leads to neuronal cell death [21]. IL-33 is classified as an alarm signal (alarmin) and is released by epithelial cells upon signals of cell or tissue death, but the exact *in vivo* mechanism of release is not fully understood [22]. After its release, IL-33 binds to ST2 receptors (IL-1RL1) that are present on multiple immune cells as an alert signal for immuno-

One study that treated SCI injury in mice with administration of recombinant IL-33 indicated an attenuation of spinal cord encephalomyelitis progression and a significant decrease in neural tissue death, decrease in demyelination, and an overexaggerated astrocyte infiltration at the lesion site of the contused spinal cord [21]. These results yielded a significant increase in functional recovery and a dramatic decrease of the expression of TNF-α in the spinal cord for as long as 42 days post-SCI. In addition to suppression of pro-inflammatory cytokine release, IL-33 administration promoted the activation of anti-inflammatory M2

Cytokines from the IL-2 family, IL-2, IL-4, IL-7, IL-15, and IL-21, all share a common receptor subunit (gammac), which plays a major role in promoting and maintaining T lymphocyte populations [23]. IL-2 is a pro-inflammatory cytokine

At an mRNA level, signals from T-cell receptor (TCR) and CD28 closely regulate the production of IL-2 [24]. After synthesis, IL-2 binds to a receptor complex, which consists of three subunits, IL-2Rα, IL-2Rβ, and the common γ-chain [24]. All three subunits are needed to achieve high affinity binding. These receptors are located on regulatory T cells and antigen-activated T lymphocytes [25]. To produce an IL-2-dependent response, IL-2 must be produced and IL-2R must be expressed

IL-2 and its receptor, IL-2R, are crucial to maintaining the balance of the timing and adequacy of an immune response [26]. The primary role of IL-2 is to perpetuate

cells when activated.

#### *The Role of Interleukins after Spinal Cord Injury DOI: http://dx.doi.org/10.5772/intechopen.96293*

*Interleukins - The Immune and Non-Immune Systems' Related Cytokines*

ILs have on the dynamic inflammatory process that occurs after SCI.

There are numerous known ILs and several of these ILs are shown to be involved in inflammation after SCI. Throughout this chapter we will discuss the ILs that have been investigated after SCI and whether their role is predominately inflammatory or anti-inflammatory. It is important to note, the role an IL plays after injury is not as simple as just inflammatory or anti-inflammatory. For many of the ILs there are multiple factors that determine whether they will have a beneficial role or a detrimental role, including the extent of initial injury, concentration of IL, other associated molecules in the injury, and the response of immune

Although there is a broad spectrum of signaling molecules including cytokines, chemokines, and other reactive species after SCI, this chapter will just focus on ILs and only the ILs that are known to play a role in inflammation after SCI [14]. These ILs will be discussed in terms of the cell types that produce them, receptors they bind, cell types they target, timeline of upregulation, and ultimately their effect on

IL-1α, IL-1β, and IL-33 are members of the IL-1 family that have been studied after SCI and all are predominantly inflammatory [15]. IL-1 is released via activated macrophages and microglia largely in response to disease, infection, or inflammatory events. IL-1 has two structurally and biologically similar isoforms, IL-1α and IL-1β [13]. These two isoforms share roughly 30% amino acid sequence homology and although they perform similar biological functions, IL-1β plays a more substantial role post-SCI [16, 17]. IL-1β has been shown to contribute to the exaggerated neuroinflammation following SCI that leads to secondary neural degeneration and cell death [16]. IL-1 signaling following SCI is diverse and complex, resulting in a recruitment of neurotoxins or immune system molecules that contribute to the

The primary receptor for signaling of both IL-1 isoforms is the type-I interleukin-1 receptor (IL-1RI). IL-1 signaling is further regulated by a decoy receptor (IL-1RII) and a receptor antagonist (IL-1ra) [13]. The expression of these receptors mediates the inflammatory response to SCI and their mechanisms have been widely studied following SCI. After SCI in rats, IL-1R1 expression is elevated as

**2. Interleukins involved in inflammation after SCI**

cells and glial cells [12, 13].

inflammation after SCI.

inflammatory response [13].

**2.1 Interleukin-1 family cytokines**

Throughout the first hours after injury, polymorphonuclear leukocytes are the predominant infiltrating cells and over-activation of these cells causes tissue destruction through the release of significant amounts of neurotoxins including reactive oxygen species (ROS), reactive nitrogen species (RNS), chemokines, and enzymes [5, 7, 8]. Microglia, the resident macrophages, are also activated and migrate to the site of injury, proliferate, and transform from the ramified phenotype to amoeboid phagocytic cells [9]. These activated microglia and peripheral macrophages make up the majority of inflammatory cells present at the site of the lesion. Although in normal wound healing macrophages sequentially change and reduce inflammation, after SCI macrophages persist in an inflammatory state for prolonged periods resulting in progressive tissue degeneration [10, 11]. However these microglia/macrophages can be activated toward an anti-inflammatory phenotype and ILs are important signaling cues in the extracellular environment that help dictate this contrasting phenotype. The goal of this chapter is to examine the role

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early as 4 hours, peaks at 8 hours to 1-day and remains elevated for 7 days postinjury [13]. Another study tested the role of IL-1ra as a regulatory molecule following SCI and observed that increased expression of IL-1ra suppressed IL-1β levels and increased locomotor function following SCI, suggesting that IL-1β and IL-1RI play critical roles in secondary tissue damage and impaired functional recovery post-SCI [16]. Likewise, administration of IL-1β suppressed the expression of IL-1ra following SCI indicating the regulatory nature of IL-1β interactions with its receptor antagonist [16]. Similarly, a study using IL-1 knockout mice observed a significantly smaller lesion area and improved locomotor function after SCI in comparison to wild-type mice [18].

The signaling cascade of the IL-1β/IL-1RI pathway is complex and yet to be completely understood, however it is widely understood that it stimulates the production of toxic intermediates that cause neural degeneration and cell death [13]. These toxic inflammatory mediators include prostaglandins, cyclooxygenase 2, and phospholipase A2 [13]. However, there are studies showing benefits of IL-1, where IL-1β null mice failed to remyelinate as rapidly as wild-type mice [19]. These different roles IL-1 plays are likely due to several factors including extent of injury as well as IL-1 concentration and timing of upregulation, but at present are not well understood.

Another member of the IL-1 family, IL-33, predominantly induces type-2 immune responses against allergens and infectious diseases [20]. IL-33 is upregulated in response to SCI and tends to localize in spinal cord astrocytes to reduce T cell infiltration and overexaggerated inflammation that leads to neuronal cell death [21]. IL-33 is classified as an alarm signal (alarmin) and is released by epithelial cells upon signals of cell or tissue death, but the exact *in vivo* mechanism of release is not fully understood [22]. After its release, IL-33 binds to ST2 receptors (IL-1RL1) that are present on multiple immune cells as an alert signal for immunologic and neurologic damage or inflammation [22].

One study that treated SCI injury in mice with administration of recombinant IL-33 indicated an attenuation of spinal cord encephalomyelitis progression and a significant decrease in neural tissue death, decrease in demyelination, and an overexaggerated astrocyte infiltration at the lesion site of the contused spinal cord [21]. These results yielded a significant increase in functional recovery and a dramatic decrease of the expression of TNF-α in the spinal cord for as long as 42 days post-SCI. In addition to suppression of pro-inflammatory cytokine release, IL-33 administration promoted the activation of anti-inflammatory M2 macrophage/microglia [21].

#### **2.2 Interleukin-2 family cytokines**

Cytokines from the IL-2 family, IL-2, IL-4, IL-7, IL-15, and IL-21, all share a common receptor subunit (gammac), which plays a major role in promoting and maintaining T lymphocyte populations [23]. IL-2 is a pro-inflammatory cytokine made up of four α helixes and is produced mainly by CD4<sup>+</sup> cells when activated. At an mRNA level, signals from T-cell receptor (TCR) and CD28 closely regulate the production of IL-2 [24]. After synthesis, IL-2 binds to a receptor complex, which consists of three subunits, IL-2Rα, IL-2Rβ, and the common γ-chain [24]. All three subunits are needed to achieve high affinity binding. These receptors are located on regulatory T cells and antigen-activated T lymphocytes [25]. To produce an IL-2-dependent response, IL-2 must be produced and IL-2R must be expressed within the same microenvironments [25].

IL-2 and its receptor, IL-2R, are crucial to maintaining the balance of the timing and adequacy of an immune response [26]. The primary role of IL-2 is to perpetuate the proper response of memory T-cells to invading pathogens [27]. In addition, IL-2 is vital to the survival, as well as death, of lymphocytes, which has an effect on the development of the immune system. By properly maintaining the life of regulatory T cells (T reg) and activation-induced cell death, IL-2 is able to eliminate selfreactive T cells as a preventative measure against autoimmune diseases [27]. After SCI in a rat, IL-2 levels were significantly lower than intact controls from 3 days to 2 weeks post-SCI [14]. In addition, the interaction of IL-2 with its receptor after SCI contributes to the proliferation of T-helpers, which also have an effect on the proliferation of cytotoxic T cells, natural killer cells, lymphokine-activated killers, B cells, and macrophages [14].

IL-4 and IL-13 are related anti-inflammatory cytokines that regulate many aspects of inflammation and have also been shown to induce alternative macrophage activation (**Figure 1**) [28]. IL-4 is a cytokine that is involved in regulating immunity, and is secreted by Th2 cells, eosinophils, basophils, and mast cells [29]. IL-4 is also involved in allergic inflammation by utilizing Th2 lymphocytes, differentiated from Th cells, which can then be used in the production of effector cytokines [30]. IL-4 binds to its receptor IL-4Rα, and will dimerize with either γc (the common cytokine-receptor γ-chain) and produce the type-1 signaling complex, or with IL-13Rα1 and produce the type-2 signaling complex (**Figure 1**) [29, 31]. Although IL-4 has a major impact on immunity, it also affects cognition based on T-cells mediated by IL-4. When administered within a short period post injury, IL-4 exhibits anti-inflammatory effects; however, it can exert a pro-inflammatory response when macrophages possessing IL-4 are undergoing pro-inflammatory stimulation [29].

Lima et al. (2017) performed a study to understand the effect of the acute and sub-acute treatment using IL-4 on various populations of neural cells and on functional recovery *in vivo*. In the injured spinal cord, treatment using a systemic delivery of IL-4 (0.35 μg/kg) for 7 days, led to an upregulation of the anti-inflammatory

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ARG-1-expressing macrophages [37].

after SCI.

periphery [39].

treatment for SCI [38].

IL-10 and a reduction in area of macrophage/microglia expressing inflammation markers CD11b and inducible nitric oxide synthase (iNOS) [32]. After systemic IL-4 treatment, they also observed an increase in the number of O4-positive cells (a marker for both type I and type II oligodendrocytes) and neuronal markers βIII-tubulin and NeuN, suggesting that IL-4 has a role in neuroprotection. This overall reduction in inflammation resulted in improved hind limb function in rats after SCI. Although they observed several positive effects, systemic IL-4 did not have an effect on the number of astrocytes or lesion size [32]. In another study a delayed intraspinal injection of IL-4 (100 ng of recombinant IL-4, 48 hours after injury) was given after a spinal cord contusion in mice [33]. The intraspinal injection of IL-4 resulted in an increase in microglia/macrophages expressing antigens characteristic of an anti-inflammatory M2 phenotype, reduced tissue damage, and improved hind limb function in mice after SCI [33]. These studies suggest that therapies using IL-4 could be a valuable treatment for improving function

IL-13 is produced by different T cell subsets, dendritic cells, and activated Th2 cells [34]. Although the IL-13α2 receptor was originally thought to be a "decoy" receptor that serves as a neutralizer (**Figure 1**) [35], Fichtner-Feigl et al. showed a role for IL-13Rα2-mediated signaling that required the cytoplasmic tail of IL-13Rα2 in the production of transforming growth factor beta (TGF-β), an anti-inflammatory shown to down-regulate inflammatory cytokines, providing evidence for IL -13Ra2 mediated signaling (**Figure 1**) [31, 36]. Furthermore, after SCI it was shown that transplanted mesenchymal stem cells continuously expressing IL-13 improved functional recovery and decreased lesion size. In addition, IL-13 increased the amount of

IL-7 is a homeostatic cytokine that plays a key role in the survival of multiple immune cells and acts on lymphocytes [38]. The IL-7 receptor complex is composed of two chains, IL-7Rα and γc (the common cytokine-receptor γ-chain), which signal downstream to the JAK/STAT5 pathway, and assists in regulating the survival and development of immune cells [38]. IL-7 is produced by stromal cells in lymphoid organs and is necessary for T-cell development and their survival in the

After SCI in mice, IL-7 is promptly upregulated and displays as a strong chemotactic property for macrophages [40]. An intraspinal injection of IL-7 after SCI in mice, resulted in an increase in pro-inflammatory cytokines IL-1β, IL-6, and TNFα, and a decrease in the anti-inflammatory cytokine IL-10 [38]. The increase in IL-7 also led to an increase in apoptosis, macrophage infiltration, and a decrease in hind limb function in mice after SCI [38]. Moreover, blocking the IL-7 receptor after SCI in mice, resulted in suppression of pro-inflammatory cytokines IFN-γ and TNF-α, an increase in IL-4 and IL-13, more macrophages expressing antigens characteristic of an anti-inflammatory M2 phenotype, an increase in spared white matter, and an improvement in hind limb function [40]. During SCI, the JAK/STAT5 pathway is activated, and IL-7 post-SCI also contributes to the activation of the JAK/STAT5 pathway, which upholds a crucial role in the inflammatory response and secondary damage [38]. When the JAK/STAT5 pathway was inhibited by pimozide, the effects of IL-7 discontinued, which emphasizes the relationship between the JAK/STAT5 pathway and IL-7 function [38]. Therefore, Yuan et al. (2019) concluded that the IL-7/JAK/STAT5 axis targeted by antagonists may represent a potential therapeutic

Similar to IL-2, IL-15 is a pro-inflammatory cytokine that is also part of the four α helix cytokine family. The main function of IL-15 is to provide a long-term immune response to invading pathogens by contributing to the homeostasis of natural killer cells and CD8+ memory T cells that express IL-2/IL-15Rβ and γc [27].

**Figure 1.**

*IL-4, IL-13, and IL-10 pathways and effects.*

#### *The Role of Interleukins after Spinal Cord Injury DOI: http://dx.doi.org/10.5772/intechopen.96293*

*Interleukins - The Immune and Non-Immune Systems' Related Cytokines*

cells, and macrophages [14].

pro-inflammatory stimulation [29].

the proper response of memory T-cells to invading pathogens [27]. In addition, IL-2 is vital to the survival, as well as death, of lymphocytes, which has an effect on the development of the immune system. By properly maintaining the life of regulatory T cells (T reg) and activation-induced cell death, IL-2 is able to eliminate selfreactive T cells as a preventative measure against autoimmune diseases [27]. After SCI in a rat, IL-2 levels were significantly lower than intact controls from 3 days to 2 weeks post-SCI [14]. In addition, the interaction of IL-2 with its receptor after SCI contributes to the proliferation of T-helpers, which also have an effect on the proliferation of cytotoxic T cells, natural killer cells, lymphokine-activated killers, B

IL-4 and IL-13 are related anti-inflammatory cytokines that regulate many aspects of inflammation and have also been shown to induce alternative macrophage activation (**Figure 1**) [28]. IL-4 is a cytokine that is involved in regulating immunity, and is secreted by Th2 cells, eosinophils, basophils, and mast cells [29]. IL-4 is also involved in allergic inflammation by utilizing Th2 lymphocytes, differentiated from Th cells, which can then be used in the production of effector cytokines [30]. IL-4 binds to its receptor IL-4Rα, and will dimerize with either γc (the common cytokine-receptor γ-chain) and produce the type-1 signaling complex, or with IL-13Rα1 and produce the type-2 signaling complex (**Figure 1**) [29, 31]. Although IL-4 has a major impact on immunity, it also affects cognition based on T-cells mediated by IL-4. When administered within a short period post injury, IL-4 exhibits anti-inflammatory effects; however, it can exert a pro-inflammatory response when macrophages possessing IL-4 are undergoing

Lima et al. (2017) performed a study to understand the effect of the acute and sub-acute treatment using IL-4 on various populations of neural cells and on functional recovery *in vivo*. In the injured spinal cord, treatment using a systemic delivery of IL-4 (0.35 μg/kg) for 7 days, led to an upregulation of the anti-inflammatory

**116**

**Figure 1.**

*IL-4, IL-13, and IL-10 pathways and effects.*

IL-10 and a reduction in area of macrophage/microglia expressing inflammation markers CD11b and inducible nitric oxide synthase (iNOS) [32]. After systemic IL-4 treatment, they also observed an increase in the number of O4-positive cells (a marker for both type I and type II oligodendrocytes) and neuronal markers βIII-tubulin and NeuN, suggesting that IL-4 has a role in neuroprotection. This overall reduction in inflammation resulted in improved hind limb function in rats after SCI. Although they observed several positive effects, systemic IL-4 did not have an effect on the number of astrocytes or lesion size [32]. In another study a delayed intraspinal injection of IL-4 (100 ng of recombinant IL-4, 48 hours after injury) was given after a spinal cord contusion in mice [33]. The intraspinal injection of IL-4 resulted in an increase in microglia/macrophages expressing antigens characteristic of an anti-inflammatory M2 phenotype, reduced tissue damage, and improved hind limb function in mice after SCI [33]. These studies suggest that therapies using IL-4 could be a valuable treatment for improving function after SCI.

IL-13 is produced by different T cell subsets, dendritic cells, and activated Th2 cells [34]. Although the IL-13α2 receptor was originally thought to be a "decoy" receptor that serves as a neutralizer (**Figure 1**) [35], Fichtner-Feigl et al. showed a role for IL-13Rα2-mediated signaling that required the cytoplasmic tail of IL-13Rα2 in the production of transforming growth factor beta (TGF-β), an anti-inflammatory shown to down-regulate inflammatory cytokines, providing evidence for IL -13Ra2 mediated signaling (**Figure 1**) [31, 36]. Furthermore, after SCI it was shown that transplanted mesenchymal stem cells continuously expressing IL-13 improved functional recovery and decreased lesion size. In addition, IL-13 increased the amount of ARG-1-expressing macrophages [37].

IL-7 is a homeostatic cytokine that plays a key role in the survival of multiple immune cells and acts on lymphocytes [38]. The IL-7 receptor complex is composed of two chains, IL-7Rα and γc (the common cytokine-receptor γ-chain), which signal downstream to the JAK/STAT5 pathway, and assists in regulating the survival and development of immune cells [38]. IL-7 is produced by stromal cells in lymphoid organs and is necessary for T-cell development and their survival in the periphery [39].

After SCI in mice, IL-7 is promptly upregulated and displays as a strong chemotactic property for macrophages [40]. An intraspinal injection of IL-7 after SCI in mice, resulted in an increase in pro-inflammatory cytokines IL-1β, IL-6, and TNFα, and a decrease in the anti-inflammatory cytokine IL-10 [38]. The increase in IL-7 also led to an increase in apoptosis, macrophage infiltration, and a decrease in hind limb function in mice after SCI [38]. Moreover, blocking the IL-7 receptor after SCI in mice, resulted in suppression of pro-inflammatory cytokines IFN-γ and TNF-α, an increase in IL-4 and IL-13, more macrophages expressing antigens characteristic of an anti-inflammatory M2 phenotype, an increase in spared white matter, and an improvement in hind limb function [40]. During SCI, the JAK/STAT5 pathway is activated, and IL-7 post-SCI also contributes to the activation of the JAK/STAT5 pathway, which upholds a crucial role in the inflammatory response and secondary damage [38]. When the JAK/STAT5 pathway was inhibited by pimozide, the effects of IL-7 discontinued, which emphasizes the relationship between the JAK/STAT5 pathway and IL-7 function [38]. Therefore, Yuan et al. (2019) concluded that the IL-7/JAK/STAT5 axis targeted by antagonists may represent a potential therapeutic treatment for SCI [38].

Similar to IL-2, IL-15 is a pro-inflammatory cytokine that is also part of the four α helix cytokine family. The main function of IL-15 is to provide a long-term immune response to invading pathogens by contributing to the homeostasis of natural killer cells and CD8+ memory T cells that express IL-2/IL-15Rβ and γc [27]. IL-15 has three receptors, IL-15Rα, IL-2Rβ, and γc, and shares two of the receptors with IL-2 (IL-2Rβ and γc) [41]. Although IL-15 has not been well studied after SCI, it has been shown to be involved in the development of neuropathic pain from nerve injury [42]. After sciatic nerve injury, IL-15 expression was observed in the spinal cord in astrocytes and microglia, and it is also present in neurons located in the dorsal and ventral horn [42].

IL-21 is a pleiotropic cytokine expressed by many immune cells including natural killer T cells and activated CD4+ T cells [43]. Similar to other inflammatory mediators, IL-21 is upregulated after SCI [44, 45]. Fu et al. (2017) studied peripheral blood-derived mesenchymal stem cells (PBMSCs) as a therapy for SCI and their role in the lesion microenvironment by analyzing the neuroprotection, differentiation, and immunoregulation of PBMSCs that were engrafted. When IL-21 was inhibited, a decrease in the secretion of IL-23a and IL-22 occurred [44]. When investigating the potential Th17/Treg-relative mechanism of PBMSCs therapy after SCI, Fu et al. (2017) discovered that the M1 macrophage migrated to lesion site and resulted in the pro-inflammatory secretion of IL-6 and IL-21, which led to CD4 + T cells differentiating into CD4 + IL17 + Th17 cells [44]. Furthermore it has been shown that IL-17 production is stimulated by the combination of IL-21 and TGF-β [45].

#### **2.3 β common chain cytokines IL-3, and IL-5**

The β common cytokine family, including IL-3 and IL-5, is defined by a shared receptor structure, comprising of a specific α chain and a common β chain that is essential for cytokine-specific receptor signaling [46]. IL-3 is a cytokine that is produced by activated T cell lymphocytes, which then induces the production of various hematopoietic cell types that are crucial to the immune response [47]. IL-5 is cytokine that is produced by hematopoietic and non-hematopoietic cells, including granulocytes, T cells, and natural helper cells [48]. IL-5 is also a mediator for eosinophilic inflammation by providing stimulation, differentiation, recruitment and activity of eosinophils. Due to their roles with eosinophils, IL-3 and IL-5 have been primarily studied in asthma, and their roles after SCI are not clear. However, both of them co-express in TH2 cells, which is a subset of CD4+ cells. These TH2 cells are characterized by the production of IL-4, IL-5, IL-10 and IL-13 and thus, may be beneficial in exerting anti-inflammatory effects after SCI [49, 50].

#### **2.4 IL-6 and IL-11**

IL-6 and IL-11 are grouped into one cytokine family because the receptor complex of each cytokine contains two of the signaling receptor subunit gp130 (**Figure 2**) [51]. For both IL-6 and IL-11 there are membrane bound receptors as well as soluble receptors and after ligand binding to either the membrane bound receptor or the soluble receptor, they form a complex with two gp130 receptors leading to Jak/ STAT pathway signaling (**Figure 2**) [52, 53].

IL-6 is predominantly an inflammatory cytokine. After SCI in a mouse model, Pineau et al. observed IL-6 mRNA expression in astrocytes, microglia/ macrophages, and neurons, starting at 3 hours post-injury, peaking at 12 hours and continuing for 4 days post-injury [54]. Similarly after SCI in humans IL-6 is strongly upregulated. IL-6 levels in cerebrospinal fluid of SCI patients changed from undetectable (<4 pg./ml) in non-injured controls to an average of almost 30,000 pg./mL in the subset of patients with complete SCI [55]. Furthermore, the cerebrospinal levels of IL-6 correlated with the extent of spinal cord damage in humans, which demonstrates the importance of IL-6 after SCI.

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functional recovery.

**Figure 2.**

*The Role of Interleukins after Spinal Cord Injury DOI: http://dx.doi.org/10.5772/intechopen.96293*

IL-6 leads to recruitment of immune cells. Delivery of IL-6/sIL-6 receptor fusion protein to injury sites induced a sixfold increase in neutrophils and a twofold increase of macrophages/microglial [56]. Mice treated with an antibody against IL-6 receptor showed a reduction in neutrophil and monocyte/macrophage invasion [57, 58]. It was also shown that blocking IL-6 signaling after SCI reduces the damaging inflammatory activity by promoting the formation of alternatively activated M2 macrophages [59]. Taken together these data suggest IL-6 signaling is an activator of inflammation and a strong recruiter of immune cells after SCI. After SCI astrocytes proliferate and migrate to the injury leading to a dense astroglial scar surrounding the lesion. It has been shown *in vitro* that IL-6 signaling acts on neural stem cells to induce their differentiation into astrocytes [60]. This was supported by several *in vivo* studies including, IL-6 knockout mice that showed suppression of astrogliosis following SCI [61], mice with an excessive expression of IL-6 and IL-6R showed abundant astrogliosis suggesting that astrocytes were selectively affected in these mice [62], and the development of astrogliosis was inhibited

*Classic and trans-signaling. Cells that express the IL-6R or IL-11R will undergo classic signaling when IL-6 or IL-11 bind to the corresponding receptor, inducing gp130 dimerization and initiating intracellular signal transduction. Trans-signaling occurs when the ligand and soluble receptor complex (IL-6/sIL-6R or IL-11/sIL-11R)* 

*associate with gp130 inducing gp130 dimerization and initiating intracellular signal transduction.*

Several studies have shown that blocking IL-6 signaling improves functional recovery after SCI [57–59]. It has also been shown that delivery of IL-6/IL-6 receptor resulted in a four fold decrease in axon growth [56]. However there are studies showing that IL-6 is neuroprotective and aids in axonal regeneration [63, 64]. The differences in IL-6 effect may depend on the level of expression and timeline of IL-6 upregulation. The studies using an IL-6 blocker were performed in the sub-acute timeframe after SCI. In the sub-acute SCI, any neurotrophic effects of IL-6 appear to be overwhelmed by its proinflammatory features. Taken together, the aforementioned data demonstrates the importance of IL-6 after SCI. IL-6 upregulates inflammatory cytokines, recruits immune cells, effects macrophage phenotype, effects astrocyte activation, effects axonal regeneration, and effects

IL-11 has been shown to be primarily anti-inflammatory. Recombinant IL-11 administered to activated macrophages inhibited the production of proinflammatory cytokines TNF-α, IL-1β, IL-12, and nitric oxide production [65–67]. Furthermore, IL-11 has been shown to play an anti-inflammatory role in the airways for asthma [68], play a role in decreasing mucosal damage in inflammatory bowel disease [69], and importantly IL-11 has a neuroprotective role in multiple sclerosis [70]. Due to these anti-inflammatory roles, Cho et al. analyzed the role of IL-11 after SCI using IL-11Rα knockout mice [71]. In wild type mice, they observed

in mice given an IL-6 receptor blocker after SCI [58].

**Figure 2.**

*Interleukins - The Immune and Non-Immune Systems' Related Cytokines*

dorsal and ventral horn [42].

and TGF-β [45].

**2.4 IL-6 and IL-11**

**2.3 β common chain cytokines IL-3, and IL-5**

STAT pathway signaling (**Figure 2**) [52, 53].

demonstrates the importance of IL-6 after SCI.

IL-15 has three receptors, IL-15Rα, IL-2Rβ, and γc, and shares two of the receptors with IL-2 (IL-2Rβ and γc) [41]. Although IL-15 has not been well studied after SCI, it has been shown to be involved in the development of neuropathic pain from nerve injury [42]. After sciatic nerve injury, IL-15 expression was observed in the spinal cord in astrocytes and microglia, and it is also present in neurons located in the

IL-21 is a pleiotropic cytokine expressed by many immune cells including natural killer T cells and activated CD4+ T cells [43]. Similar to other inflammatory mediators, IL-21 is upregulated after SCI [44, 45]. Fu et al. (2017) studied peripheral blood-derived mesenchymal stem cells (PBMSCs) as a therapy for SCI and their role in the lesion microenvironment by analyzing the neuroprotection, differentiation, and immunoregulation of PBMSCs that were engrafted. When IL-21 was inhibited, a decrease in the secretion of IL-23a and IL-22 occurred [44]. When investigating the potential Th17/Treg-relative mechanism of PBMSCs therapy after SCI, Fu et al. (2017) discovered that the M1 macrophage migrated to lesion site and resulted in the pro-inflammatory secretion of IL-6 and IL-21, which led to CD4 + T cells differentiating into CD4 + IL17 + Th17 cells [44]. Furthermore it has been shown that IL-17 production is stimulated by the combination of IL-21

The β common cytokine family, including IL-3 and IL-5, is defined by a shared receptor structure, comprising of a specific α chain and a common β chain that is essential for cytokine-specific receptor signaling [46]. IL-3 is a cytokine that is produced by activated T cell lymphocytes, which then induces the production of various hematopoietic cell types that are crucial to the immune response [47]. IL-5 is cytokine that is produced by hematopoietic and non-hematopoietic cells, including granulocytes, T cells, and natural helper cells [48]. IL-5 is also a mediator for eosinophilic inflammation by providing stimulation, differentiation, recruitment and activity of eosinophils. Due to their roles with eosinophils, IL-3 and IL-5 have been primarily studied in asthma, and their roles after SCI are not clear. However, both of them co-express in TH2 cells, which is a subset of CD4+ cells. These TH2 cells are characterized by the production of IL-4, IL-5, IL-10 and IL-13 and thus, may be beneficial in exerting anti-inflammatory effects after SCI [49, 50].

IL-6 and IL-11 are grouped into one cytokine family because the receptor complex of each cytokine contains two of the signaling receptor subunit gp130 (**Figure 2**) [51]. For both IL-6 and IL-11 there are membrane bound receptors as well as soluble receptors and after ligand binding to either the membrane bound receptor or the soluble receptor, they form a complex with two gp130 receptors leading to Jak/

IL-6 is predominantly an inflammatory cytokine. After SCI in a mouse model, Pineau et al. observed IL-6 mRNA expression in astrocytes, microglia/ macrophages, and neurons, starting at 3 hours post-injury, peaking at 12 hours and continuing for 4 days post-injury [54]. Similarly after SCI in humans IL-6 is strongly upregulated. IL-6 levels in cerebrospinal fluid of SCI patients changed from undetectable (<4 pg./ml) in non-injured controls to an average of almost 30,000 pg./mL in the subset of patients with complete SCI [55]. Furthermore, the cerebrospinal levels of IL-6 correlated with the extent of spinal cord damage in humans, which

**118**

*Classic and trans-signaling. Cells that express the IL-6R or IL-11R will undergo classic signaling when IL-6 or IL-11 bind to the corresponding receptor, inducing gp130 dimerization and initiating intracellular signal transduction. Trans-signaling occurs when the ligand and soluble receptor complex (IL-6/sIL-6R or IL-11/sIL-11R) associate with gp130 inducing gp130 dimerization and initiating intracellular signal transduction.*

IL-6 leads to recruitment of immune cells. Delivery of IL-6/sIL-6 receptor fusion protein to injury sites induced a sixfold increase in neutrophils and a twofold increase of macrophages/microglial [56]. Mice treated with an antibody against IL-6 receptor showed a reduction in neutrophil and monocyte/macrophage invasion [57, 58]. It was also shown that blocking IL-6 signaling after SCI reduces the damaging inflammatory activity by promoting the formation of alternatively activated M2 macrophages [59]. Taken together these data suggest IL-6 signaling is an activator of inflammation and a strong recruiter of immune cells after SCI.

After SCI astrocytes proliferate and migrate to the injury leading to a dense astroglial scar surrounding the lesion. It has been shown *in vitro* that IL-6 signaling acts on neural stem cells to induce their differentiation into astrocytes [60]. This was supported by several *in vivo* studies including, IL-6 knockout mice that showed suppression of astrogliosis following SCI [61], mice with an excessive expression of IL-6 and IL-6R showed abundant astrogliosis suggesting that astrocytes were selectively affected in these mice [62], and the development of astrogliosis was inhibited in mice given an IL-6 receptor blocker after SCI [58].

Several studies have shown that blocking IL-6 signaling improves functional recovery after SCI [57–59]. It has also been shown that delivery of IL-6/IL-6 receptor resulted in a four fold decrease in axon growth [56]. However there are studies showing that IL-6 is neuroprotective and aids in axonal regeneration [63, 64]. The differences in IL-6 effect may depend on the level of expression and timeline of IL-6 upregulation. The studies using an IL-6 blocker were performed in the sub-acute timeframe after SCI. In the sub-acute SCI, any neurotrophic effects of IL-6 appear to be overwhelmed by its proinflammatory features. Taken together, the aforementioned data demonstrates the importance of IL-6 after SCI. IL-6 upregulates inflammatory cytokines, recruits immune cells, effects macrophage phenotype, effects astrocyte activation, effects axonal regeneration, and effects functional recovery.

IL-11 has been shown to be primarily anti-inflammatory. Recombinant IL-11 administered to activated macrophages inhibited the production of proinflammatory cytokines TNF-α, IL-1β, IL-12, and nitric oxide production [65–67]. Furthermore, IL-11 has been shown to play an anti-inflammatory role in the airways for asthma [68], play a role in decreasing mucosal damage in inflammatory bowel disease [69], and importantly IL-11 has a neuroprotective role in multiple sclerosis [70]. Due to these anti-inflammatory roles, Cho et al. analyzed the role of IL-11 after SCI using IL-11Rα knockout mice [71]. In wild type mice, they observed a significant upregulation in IL-11 with a peak gene expression 24 hours after injury and a significant upregulation of IL-11Rα at 3 and 7 days after SCI. Somewhat surprisingly, they did not observe significant differences in functional recovery or histopathology in IL-11Rα knockout mice as compared to wild type mice after SCI. The authors speculate that since "the peak in IL-11Rα expression is on the order of days after SCI, suggests that IL-11 signaling may not play as significant a role in the acute inflammatory response after injury, but more in the long-term sequelae such as oligodendrocyte survival". Maheshwari et al. used a cuprizone induced mouse model of demyelination in the central nervous system to analyze the effects of overexpression of IL-11 on demyelination/remyelination [72]. Overexpression of IL-11 was able to limit cuprizone-induced demyelination by reducing oligodendrocyte cell death, decrease microglial activation, and enhance spontaneous remyelination. Maheshwari's results further suggest that IL-11 likely plays a role in the long-term remyelination efforts after SCI and is not as involved in the sub-acute stage [72].

#### **2.5 Interleukin-8 and interleukin-16**

IL-8, also known as neutrophil chemotactic factor or CXCL8, primarily induces chemotaxis in neutrophils and granulocytes. IL-8 is a member of the chemokine family that acts on CXCR1 and CXCR2 receptors (il8ra and il8rb, respectively), which have been primarily studied on polymorphonuclear leukocytes. However many other cell types express these receptors including neurons [73]. Several studies have shown that IL-8 can be released by a wide variety of cells including monocytes endothelial cells, T lymphocytes, and macrophages [73]. After SCI in rat, GRO, the rat analogue of human IL-8, is strongly upregulated for at least 14 days and the upregulation of GRO strongly correlates with the extent of injury [14, 74, 75]. Furthermore IL-8 is upregulated in the cerebrospinal fluid of dogs and humans after SCI, and for humans the IL-8 levels are also shown to correlate with the extent of damage [55, 76, 77]. Although IL-8 clearly plays a role in neutrophil infiltration and overall inflammation after SCI, as shown by its significant upregulation, it has not been extensively studied after SCI.

IL-16 is a proinflammatory cytokine that is produced by mast and leukemic cells, fibroblasts, endothelial cells, granulocytes, dendritic cells, CD4+ and CD8+ T lymphocytes, monocytes, and microglial cells. IL-16 plays a role in the release of other proinflammatory cytokines (IL-1β, IL-6, IL-15, and TNFα), the increase of intracellular Ca++ or inositol-(1,4,5)-triphosphatase, and the translocation of protein kinase C [78]. These processes occur after IL-16 binds to the signal-transducing CD4 receptor molecule [79]. Moreover, IL-16 promotes lymphocyte migration and modulates apoptosis [80].

Following spinal cord injury, IL-16 plays a role in recruiting and activating inflammatory cells. Microglia that produce IL-16 migrate to the lesion site and other areas of significant neuronal damage [78]. Following neuroinflammation, it is suggested that IL-16 microglia are one of the first cells to respond [80]. In addition, macrophages with IL-16 remained present at the injury site for up to thirty days post injury, indicating long-term IL-16 function [78]. One study found that expression of IL-16 in microglia and macrophages is induced by the IL-12 p40 homodimer through IL-12Rβ1, but not IL-12 p70 [80]. Overall, the ability of IL-16 to quickly recruit microglia/macrophages to the lesion site following SCI results in increased neuronal damage and microvessel clustering [78].

#### **2.6 Interleukin-10 family cytokines**

Members of the IL-10 family of cytokines that have been studied after SCI include, IL-10, IL-19, IL-20, and IL-22 [81]. IL-10 is an anti-inflammatory cytokine that is produced by monocytes, B cells, dendritic cells, natural killer cells,

**121**

region [87].

*The Role of Interleukins after Spinal Cord Injury DOI: http://dx.doi.org/10.5772/intechopen.96293*

regulate each other [82].

limb function [11, 84].

and T cells [82]. In leukocytes, IL-10 acts on both innate and adaptive immune cells with a wide range of immunomodulatory activities that suppress proliferation, cytokine secretion, and costimulatory molecule expression of proinflammatory immune cells. The IL-10 receptor consists of heterotetramer complex made of two IL-10R1 molecules, encoded by the IL10ra gene, and two IL-10R2 molecules, encoded by the IL10rb gene (**Figure 1**) [83]. IL-10 downregulates several pro-inflammatory cytokines and inflammatory species [11]. In addition, IL-10 can affect T cell and natural killer cell function indirectly and directly through connection with monocytes and macrophages. The overall impact of IL-10 is determined by the timing and site of its production, which are both affected by which cells are making IL-10. Since IL-10 production by one cell type affects the ability of other cells to make IL-10, IL-10-producing cells show potential to

Following SCI, IL-10 downregulates pro-inflammatory molecules IL-1β, IL-2, IL-6, TNF-α, IFN-γ, matrix metalloproteinase-9, nitric oxide synthase, myeloperoxidase, and reactive oxygen species. IL-10 also provides trophic support to neurons through downregulation of pro-apoptotic factors cytochrome c, caspase 3, and Bax, as well as upregulation of anti-apoptotic factors B cell lymphoma 2 (Bcl-2) and Bcl-2-associated X, B-cell lymphoma-extra large (Bcl-xl) (**Figure 1**) [11]. There have been several studies performed to test IL-10's therapeutic value as a treatment for SCI. Although these studies used a variety of different systemic and local methods to administer IL-10 after SCI, the majority of results showed strong positive effects from the IL-10. These positive effects after SCI include a reduction in pro-inflammatory molecules, macrophages expressing more antigens characteristic of an anti-inflammatory M2 phenotype, reduced lesion size, and an improvement in hind

IL-19 is produced by monocytes and microglia, and binds to the IL-20 receptor complex, which consists of IL-20R1 and IL-20R2 chains [85]. Activated microglia upregulate IL-19 and express the IL-20 receptor complex [86]. It has also been shown that ablation of IL-19 in activated microglia increased the production of pro-inflammatory cytokines IL-6 and TNF-α, which demonstrates that IL-19 is predominately an anti-inflammatory cytokine in the central nervous system [86]. After SCI in mice, IL-19, IL-20R1 and IL-20R2 are upregulated [87]. In a series of four different experiments, mice with spinal cord injuries were treated with IL-19 [87]. As a result, Th2 cytokine synthesis was promoted, which polarized spinal microglial cells to an M2 phenotype. This helped resolve the inflammation, preserving myelin, neurons, and neuronal function. Overall, IL-19 attenuated macrophage accumulation, reduced protein levels of TNF-α and CCl2, promoted Th2 response and M2 macrophage activation, promoted angiogenesis by upregulating VEGF, upregulated HO-1 expression, and decreased oxidative stress in the injured

IL-20 is a proinflammatory cytokine that is predominately produced by monocytes and skin keratinocytes. IL-20 signals through both the IL-20R1/IL-20R2 heterodimer complex and the receptor complex composed of IL-22R1 and IL-20R2 [85]. Following spinal cord injury, IL-20 and its receptors are expressed in neurons, astrocytes, oligodendrocytes, and microglia in large amounts. IL-20 upregulates glial fibrillary acidic protein (GFAP), TGF-β1, TNF-α, MCP-1, and IL-6 expression, which stimulates astrocyte reactivation and migration [88]. As a result, glial scar border formation is enhanced. Moreover, IL-20 inhibits neuron outgrowth through upregulation of Sema3A/NRP-1 in PC-12 cells [88]. The overall result is irreversible neuronal loss and glial scar formation post-SCI. *In vivo*, anti-IL-20 mAb reduces the IL-20 inflammatory response, which improves motor and sensory functions, spinal

cord tissue preservation, and reduces glial scar formation [88].

*Interleukins - The Immune and Non-Immune Systems' Related Cytokines*

**2.5 Interleukin-8 and interleukin-16**

a significant upregulation in IL-11 with a peak gene expression 24 hours after injury and a significant upregulation of IL-11Rα at 3 and 7 days after SCI. Somewhat surprisingly, they did not observe significant differences in functional recovery or histopathology in IL-11Rα knockout mice as compared to wild type mice after SCI. The authors speculate that since "the peak in IL-11Rα expression is on the order of days after SCI, suggests that IL-11 signaling may not play as significant a role in the acute inflammatory response after injury, but more in the long-term sequelae such as oligodendrocyte survival". Maheshwari et al. used a cuprizone induced mouse model of demyelination in the central nervous system to analyze the effects of overexpression of IL-11 on demyelination/remyelination [72]. Overexpression of IL-11 was able to limit cuprizone-induced demyelination by reducing oligodendrocyte cell death, decrease microglial activation, and enhance spontaneous remyelination. Maheshwari's results further suggest that IL-11 likely plays a role in the long-term remyelination efforts after SCI and is not as involved in the sub-acute stage [72].

IL-8, also known as neutrophil chemotactic factor or CXCL8, primarily induces chemotaxis in neutrophils and granulocytes. IL-8 is a member of the chemokine family that acts on CXCR1 and CXCR2 receptors (il8ra and il8rb, respectively), which have been primarily studied on polymorphonuclear leukocytes. However many other cell types express these receptors including neurons [73]. Several studies have shown that IL-8 can be released by a wide variety of cells including monocytes endothelial cells, T lymphocytes, and macrophages [73]. After SCI in rat, GRO, the rat analogue of human IL-8, is strongly upregulated for at least 14 days and the upregulation of GRO strongly correlates with the extent of injury [14, 74, 75]. Furthermore IL-8 is upregulated in the cerebrospinal fluid of dogs and humans after SCI, and for humans the IL-8 levels are also shown to correlate with the extent of damage [55, 76, 77]. Although IL-8 clearly plays a role in neutrophil infiltration and overall inflammation after SCI, as shown by its significant upregulation, it has not been extensively studied after SCI. IL-16 is a proinflammatory cytokine that is produced by mast and leukemic cells, fibroblasts, endothelial cells, granulocytes, dendritic cells, CD4+ and CD8+ T lymphocytes, monocytes, and microglial cells. IL-16 plays a role in the release of other proinflammatory cytokines (IL-1β, IL-6, IL-15, and TNFα), the increase of intracellular Ca++ or inositol-(1,4,5)-triphosphatase, and the translocation of protein kinase C [78]. These processes occur after IL-16 binds to the signal-transducing CD4 receptor molecule [79].

Moreover, IL-16 promotes lymphocyte migration and modulates apoptosis [80]. Following spinal cord injury, IL-16 plays a role in recruiting and activating inflammatory cells. Microglia that produce IL-16 migrate to the lesion site and other areas of significant neuronal damage [78]. Following neuroinflammation, it is suggested that IL-16 microglia are one of the first cells to respond [80]. In addition, macrophages with IL-16 remained present at the injury site for up to thirty days post injury, indicating long-term IL-16 function [78]. One study found that expression of IL-16 in microglia and macrophages is induced by the IL-12 p40 homodimer through IL-12Rβ1, but not IL-12 p70 [80]. Overall, the ability of IL-16 to quickly recruit microglia/macrophages to the lesion site following SCI results in increased

Members of the IL-10 family of cytokines that have been studied after SCI include, IL-10, IL-19, IL-20, and IL-22 [81]. IL-10 is an anti-inflammatory cytokine that is produced by monocytes, B cells, dendritic cells, natural killer cells,

neuronal damage and microvessel clustering [78].

**2.6 Interleukin-10 family cytokines**

**120**

and T cells [82]. In leukocytes, IL-10 acts on both innate and adaptive immune cells with a wide range of immunomodulatory activities that suppress proliferation, cytokine secretion, and costimulatory molecule expression of proinflammatory immune cells. The IL-10 receptor consists of heterotetramer complex made of two IL-10R1 molecules, encoded by the IL10ra gene, and two IL-10R2 molecules, encoded by the IL10rb gene (**Figure 1**) [83]. IL-10 downregulates several pro-inflammatory cytokines and inflammatory species [11]. In addition, IL-10 can affect T cell and natural killer cell function indirectly and directly through connection with monocytes and macrophages. The overall impact of IL-10 is determined by the timing and site of its production, which are both affected by which cells are making IL-10. Since IL-10 production by one cell type affects the ability of other cells to make IL-10, IL-10-producing cells show potential to regulate each other [82].

Following SCI, IL-10 downregulates pro-inflammatory molecules IL-1β, IL-2, IL-6, TNF-α, IFN-γ, matrix metalloproteinase-9, nitric oxide synthase, myeloperoxidase, and reactive oxygen species. IL-10 also provides trophic support to neurons through downregulation of pro-apoptotic factors cytochrome c, caspase 3, and Bax, as well as upregulation of anti-apoptotic factors B cell lymphoma 2 (Bcl-2) and Bcl-2-associated X, B-cell lymphoma-extra large (Bcl-xl) (**Figure 1**) [11]. There have been several studies performed to test IL-10's therapeutic value as a treatment for SCI. Although these studies used a variety of different systemic and local methods to administer IL-10 after SCI, the majority of results showed strong positive effects from the IL-10. These positive effects after SCI include a reduction in pro-inflammatory molecules, macrophages expressing more antigens characteristic of an anti-inflammatory M2 phenotype, reduced lesion size, and an improvement in hind limb function [11, 84].

IL-19 is produced by monocytes and microglia, and binds to the IL-20 receptor complex, which consists of IL-20R1 and IL-20R2 chains [85]. Activated microglia upregulate IL-19 and express the IL-20 receptor complex [86]. It has also been shown that ablation of IL-19 in activated microglia increased the production of pro-inflammatory cytokines IL-6 and TNF-α, which demonstrates that IL-19 is predominately an anti-inflammatory cytokine in the central nervous system [86].

After SCI in mice, IL-19, IL-20R1 and IL-20R2 are upregulated [87]. In a series of four different experiments, mice with spinal cord injuries were treated with IL-19 [87]. As a result, Th2 cytokine synthesis was promoted, which polarized spinal microglial cells to an M2 phenotype. This helped resolve the inflammation, preserving myelin, neurons, and neuronal function. Overall, IL-19 attenuated macrophage accumulation, reduced protein levels of TNF-α and CCl2, promoted Th2 response and M2 macrophage activation, promoted angiogenesis by upregulating VEGF, upregulated HO-1 expression, and decreased oxidative stress in the injured region [87].

IL-20 is a proinflammatory cytokine that is predominately produced by monocytes and skin keratinocytes. IL-20 signals through both the IL-20R1/IL-20R2 heterodimer complex and the receptor complex composed of IL-22R1 and IL-20R2 [85]. Following spinal cord injury, IL-20 and its receptors are expressed in neurons, astrocytes, oligodendrocytes, and microglia in large amounts. IL-20 upregulates glial fibrillary acidic protein (GFAP), TGF-β1, TNF-α, MCP-1, and IL-6 expression, which stimulates astrocyte reactivation and migration [88]. As a result, glial scar border formation is enhanced. Moreover, IL-20 inhibits neuron outgrowth through upregulation of Sema3A/NRP-1 in PC-12 cells [88]. The overall result is irreversible neuronal loss and glial scar formation post-SCI. *In vivo*, anti-IL-20 mAb reduces the IL-20 inflammatory response, which improves motor and sensory functions, spinal cord tissue preservation, and reduces glial scar formation [88].

#### **2.7 Interleukin-12 family cytokines**

The IL-12 family is comprised of 4 members, IL-12, IL-23, IL-27 and IL-35 and each member is composed of α-subunit with a helical structure similar to type 1 cytokines and a β-subunit structurally related to the extracellular regions of Type 1 cytokine receptors [89]. However from this family of 4 cytokines, only the proinflammatory cytokine IL-12 has been assessed after SCI. IL-12 is produced by dendritic cells, macrophages, monocytes, neutrophils, microglia cells, and B cells [90]. The IL-12 receptor is made up of IL-12Rβ1 and IL-12Rβ2 chains [91]. IL-12 is a heterodimeric molecule, p70, formed from p40 and p35 chains. IL-12p70 is considered to be the biologically active cytokine that expresses nitric oxide synthase and TNF-α in microglia and macrophages. In T cells, p70 interacts with both IL-12Rβ1 and IL-12Rβ2. However, p70 treatment results in IL-16 mRNA inhibition due to inability to induce IL-16 promoter [80].

Yaguchi et al., administered IL-12 after SCI in mice and observed an increase in the number of activated macrophages and dendritic cells surrounding the lesion site and an increase in the expression of brain-derived neurotrophic factor adjacent to the injury. After IL-12 treatment, immunohistochemical analyses revealed that *de novo* neurogenesis and remyelination occurred. The mice treated with IL-12 also had a significant improvement in hind limb function [92].

#### **2.8 Interleukin-17 family cytokines**

IL-17 cytokines play important roles in both innate and adaptive immunity. IL-17A to IL-17F are highly conserved at the C terminus, and contain five spatially conserved cysteine residues that mediate dimerization [93, 94]. IL-17A and IL-17E have been identified and studied for the roles they play after SCI.

IL-17A is an important cytokine in regard to protective mechanisms against infectious diseases and inflammatory pathology within the immune system [95]. IL-17A is secreted by a multitude of cells including T cells, dendritic cells, and macrophages among others and binds to the A and C subunits of the IL-17 receptor to initiate signaling [95]. After SCI in rats, IL-17A is upregulated as early as 1 hour after injury, peaks at 24 hours, and remains above normal levels for at least 72 hours after injury [45]. This upregulation of IL-17 does appear to play a degenerative role on SCI recovery after a study was conducted using IL-17 knockout mice. IL-17 knockout mice showed increased locomotor function and decreased lesion size after SCI, which suggests that IL-17 expression regulates secondary degeneration of the neural tissue at the lesion site [96]. Recruitment of immune cells such as B cells, neutrophils, and dendritic cells were downregulated at 6 weeks following SCI [96].

Interleukin-25, also known as Interleukin-17E, is in the IL-17 family and binds to the heterodimer complex of IL-17A and IL-17-B receptor subunits. IL-25 has primarily been understood as a systemic type-2 inflammatory mediator that triggers significant helper T-cell expression and proinflammatory cytokine suppression, however its response following spinal cord injury is largely unknown. IL-25 is primarily derived from epithelial cells and macrophages in response to infection or inflammation and contributes to type-2 helper T cell (Th-2) activation [97]. Th2 cells are responsible for the release of anti-inflammatory cytokines IL-4, IL-5, and IL-13 which play a role in neural protection and regeneration against inflammation and neurotoxins [97].

The trafficking mechanism and inflammatory response of IL-25 post-SCI remains relatively unclear, but local injection of IL-25 into the lesion site post-SCI yields interesting and contradictory results. The local administration of IL-25 following spinal cord injury in 10-week old mice results in decreased locomotor

**123**

**Author details**

after SCI.

Daniel J. Hellenbrand, Rylie M. Roddick, Sophia M. Mauney, Ryan T. Elder,

\*Address all correspondence to: hanna@neurosurgery.wisc.edu

Department of Neurological Surgery, University of Wisconsin, Madison, WI, USA

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Carolyn N. Morehouse and Amgad S. Hanna\*

provided the original work is properly cited.

*The Role of Interleukins after Spinal Cord Injury DOI: http://dx.doi.org/10.5772/intechopen.96293*

interventions using IL-25.

**3. Conclusions**

involved after SCI.

function, an increase in lesion size, and neuronal demyelination which contradicts the systemic immune response upon an IL-25 presence [97]. Interestingly, the systemic administration of IL-25 show ineffective results in regard to improved functional mobility following spinal cord injury. Microglia and astrocytes survival are also unaffected upon injection of IL-25 suggesting that IL-25 indirectly activates inflammatory molecules associated with these immune events [97]. These results raise questions about the precise role of IL-25 after SCI and possible therapeutic

Although significant progress has been made in terms of spinal stabilization and medical care of patients after SCI, there has not been much progress made in terms of treatments for SCI to retain or regain the function that is lost. In order to design treatments for SCI, a better understanding of the inflammation process is crucial. As outlined in this chapter ILs are an intricate player in inflammation after SCI. For some of these ILs, there timeline of involvement and roles they play in inflammation has been defined. However, there is still much more research that needs to be completed to understand the roles many of these ILs play. Along with understanding the current ILs, there will assuredly be more signaling cues discovered that are

Could inflammation be modulated to retain or regain a significant amount of function after SCI? This is a fundamental question that needs to be addressed. As highlighted in rodent models, such as what is observed in IL-17 knockout mice or treatments with anti-inflammatory cytokines, modulating inflammation is a promising approach for treating SCI. However it is important to realize that all variables including age, sex, level of injury, and force to cause the trauma, are controlled in these rodent models, and thus treating human SCI will be more challenging. These facts highlight the essential need to conduct more research on inflammation

*The Role of Interleukins after Spinal Cord Injury DOI: http://dx.doi.org/10.5772/intechopen.96293*

function, an increase in lesion size, and neuronal demyelination which contradicts the systemic immune response upon an IL-25 presence [97]. Interestingly, the systemic administration of IL-25 show ineffective results in regard to improved functional mobility following spinal cord injury. Microglia and astrocytes survival are also unaffected upon injection of IL-25 suggesting that IL-25 indirectly activates inflammatory molecules associated with these immune events [97]. These results raise questions about the precise role of IL-25 after SCI and possible therapeutic interventions using IL-25.

#### **3. Conclusions**

*Interleukins - The Immune and Non-Immune Systems' Related Cytokines*

The IL-12 family is comprised of 4 members, IL-12, IL-23, IL-27 and IL-35 and each member is composed of α-subunit with a helical structure similar to type 1 cytokines and a β-subunit structurally related to the extracellular regions of Type 1 cytokine receptors [89]. However from this family of 4 cytokines, only the proinflammatory cytokine IL-12 has been assessed after SCI. IL-12 is produced by dendritic cells, macrophages, monocytes, neutrophils, microglia cells, and B cells [90]. The IL-12 receptor is made up of IL-12Rβ1 and IL-12Rβ2 chains [91]. IL-12 is a heterodimeric molecule, p70, formed from p40 and p35 chains. IL-12p70 is considered to be the biologically active cytokine that expresses nitric oxide synthase and TNF-α in microglia and macrophages. In T cells, p70 interacts with both IL-12Rβ1 and IL-12Rβ2. However, p70 treatment results in IL-16 mRNA inhibition due to

Yaguchi et al., administered IL-12 after SCI in mice and observed an increase in the number of activated macrophages and dendritic cells surrounding the lesion site and an increase in the expression of brain-derived neurotrophic factor adjacent to the injury. After IL-12 treatment, immunohistochemical analyses revealed that *de novo* neurogenesis and remyelination occurred. The mice treated with IL-12 also

IL-17 cytokines play important roles in both innate and adaptive immunity. IL-17A to IL-17F are highly conserved at the C terminus, and contain five spatially conserved cysteine residues that mediate dimerization [93, 94]. IL-17A and IL-17E

IL-17A is an important cytokine in regard to protective mechanisms against infectious diseases and inflammatory pathology within the immune system [95]. IL-17A is secreted by a multitude of cells including T cells, dendritic cells, and macrophages among others and binds to the A and C subunits of the IL-17 receptor to initiate signaling [95]. After SCI in rats, IL-17A is upregulated as early as 1 hour after injury, peaks at 24 hours, and remains above normal levels for at least 72 hours after injury [45]. This upregulation of IL-17 does appear to play a degenerative role on SCI recovery after a study was conducted using IL-17 knockout mice. IL-17 knockout mice showed increased locomotor function and decreased lesion size after SCI, which suggests that IL-17 expression regulates secondary degeneration of the neural tissue at the lesion site [96]. Recruitment of immune cells such as B cells, neutrophils, and dendritic cells were downregulated at 6 weeks following SCI [96]. Interleukin-25, also known as Interleukin-17E, is in the IL-17 family and binds to the heterodimer complex of IL-17A and IL-17-B receptor subunits. IL-25 has primarily been understood as a systemic type-2 inflammatory mediator that triggers significant helper T-cell expression and proinflammatory cytokine suppression, however its response following spinal cord injury is largely unknown. IL-25 is primarily derived from epithelial cells and macrophages in response to infection or inflammation and contributes to type-2 helper T cell (Th-2) activation [97]. Th2 cells are responsible for the release of anti-inflammatory cytokines IL-4, IL-5, and IL-13 which play a role in neural protection and regeneration against inflammation

The trafficking mechanism and inflammatory response of IL-25 post-SCI remains relatively unclear, but local injection of IL-25 into the lesion site post-SCI yields interesting and contradictory results. The local administration of IL-25 following spinal cord injury in 10-week old mice results in decreased locomotor

**2.7 Interleukin-12 family cytokines**

inability to induce IL-16 promoter [80].

**2.8 Interleukin-17 family cytokines**

had a significant improvement in hind limb function [92].

have been identified and studied for the roles they play after SCI.

**122**

and neurotoxins [97].

Although significant progress has been made in terms of spinal stabilization and medical care of patients after SCI, there has not been much progress made in terms of treatments for SCI to retain or regain the function that is lost. In order to design treatments for SCI, a better understanding of the inflammation process is crucial. As outlined in this chapter ILs are an intricate player in inflammation after SCI. For some of these ILs, there timeline of involvement and roles they play in inflammation has been defined. However, there is still much more research that needs to be completed to understand the roles many of these ILs play. Along with understanding the current ILs, there will assuredly be more signaling cues discovered that are involved after SCI.

Could inflammation be modulated to retain or regain a significant amount of function after SCI? This is a fundamental question that needs to be addressed. As highlighted in rodent models, such as what is observed in IL-17 knockout mice or treatments with anti-inflammatory cytokines, modulating inflammation is a promising approach for treating SCI. However it is important to realize that all variables including age, sex, level of injury, and force to cause the trauma, are controlled in these rodent models, and thus treating human SCI will be more challenging. These facts highlight the essential need to conduct more research on inflammation after SCI.

#### **Author details**

Daniel J. Hellenbrand, Rylie M. Roddick, Sophia M. Mauney, Ryan T. Elder, Carolyn N. Morehouse and Amgad S. Hanna\* Department of Neurological Surgery, University of Wisconsin, Madison, WI, USA

\*Address all correspondence to: hanna@neurosurgery.wisc.edu

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### *Edited by Payam Behzadi*

The immune system recruits a wide range of molecule groups and categories, each of which has its own function, property, and structure. Among these, interleukins play a pivotal role in supporting the immune and non-immune systems of the human body. Interleukins as effective cytokines participate in different conditions such as homeostasis, infectious diseases, autoimmune diseases, and cancers. This unique property of interleukins makes them invaluable biomarkers that can be used as important biosensors. This book is divided into three sections: "Interleukins' Classification and Evolutionary Features", "Autoimmune Diseases and Low Immune System", and "Cancer and Injuries". Chapters examine the role of various interleukins in conditions such as leukemia, rheumatoid arthritis, and allergic and autoimmune diseases.

Published in London, UK © 2021 IntechOpen © selvanegra / iStock

Interleukins - The Immune and Non-Immune Systems' Related Cytokines

Interleukins

The Immune and Non-Immune Systems'

Related Cytokines

*Edited by Payam Behzadi*