**5.1 Peritoneal macrophages in endometriosis**

Macrophages are key cellular constituents of the immune response in peritoneal cavity because macrophages comprise up to 90% of peritoneal fluid cells (Tariverdian et al., 2007). It is well known that macrophages are ubiquitous immune cells that play important role in both innate and acquired immunity (Wu et al., 2004). In addition to protection the body from foreign organisms and antigens, macrophages maintain homeostasis in many tissues and in peritoneal cavity as well through their cytokine production and remodeling capabilities. With regard to female reproduction macrophages contribute to the regulation of the pituitary-gonadal axis and are found throughout female reproductive tissues including the ovary, uterus, oviduct and mammary glands (Wu et al., 2004). Macrophages derive from bone-marrow precursors which when mature enter the bloodstream as monocytes and subsequent migrate into tissues and to various body cavities, where they function primarily

cells from the peritoneal cavity and the stimulatory effect of humoral peritoneal factors on the synthesis of proteolytic enzymes by endometrial cells might be considered as the

Fig. 2. Invasion of eutopic endometrium stromal cells of women with endometriosis. Holes represent the 8-μm pore in the filter. A- spontaneous invasion of endometrial stromal cells in

supernatants of 24-h culture of macrophages; C - invasion of endometrial stromal cells in the presence of the autologous peritoneal fluid. Arrows indicate cells. (Original magnification

As we can see from our results and from number of literature data, behavior of endometrial cells, floating in peritoneal fluid, is directly regulated by the peritoneal fluid microenvironment. Deficiency of immune response in peritoneal cavity supposedly might prevent clearance of the retrograde menstrual debris from the peritoneal environment and permits the implantation of misplaced endometrial cells that result in the development of endometriosis (Dmowski & Braun, 2004). To define more precisely the mechanisms attributing to the impairment of immune response in peritoneal fluid, we have studied the functional state of peritoneal macrophages and peritoneal fluid cytokine profile in

Macrophages are key cellular constituents of the immune response in peritoneal cavity because macrophages comprise up to 90% of peritoneal fluid cells (Tariverdian et al., 2007). It is well known that macrophages are ubiquitous immune cells that play important role in both innate and acquired immunity (Wu et al., 2004). In addition to protection the body from foreign organisms and antigens, macrophages maintain homeostasis in many tissues and in peritoneal cavity as well through their cytokine production and remodeling capabilities. With regard to female reproduction macrophages contribute to the regulation of the pituitary-gonadal axis and are found throughout female reproductive tissues including the ovary, uterus, oviduct and mammary glands (Wu et al., 2004). Macrophages derive from bone-marrow precursors which when mature enter the bloodstream as monocytes and subsequent migrate into tissues and to various body cavities, where they function primarily

RPMI 1640 medium; B - invasion of endometrial stromal cells in the presence of the

**5. Immune response in peritoneal cavity in endometriosis** 

**5.1 Peritoneal macrophages in endometriosis** 

x200)

endometriosis.

important pathogenetic mechanisms of endometriotic tissue formation and growth.

as phagocytes when activated. Within tissues, differentiation of monocytes into macrophages occurs in response to the surrounding microenvironmental context, which directs the acquisition of tissue-specific phenotype. Within most organs macrophages are involved in tissue homeostasis via their ability to execute diverse functional activity, including (i) phagocytosis and degradation of foreign antigens, (ii) matrix dissolution and tissue remodeling, and (iii) production and secretion of growth factors, cytokines and chemokines (Wu et al., 2004). These effector functions allow macrophages to regulate local immune and inflammatory responses as well as influence normal tissue function.

Macrophages are identified in tissues by their expression of specific proteins markers which are predominantly cell surface receptors. The proteins considered most exclusively restricted to macrophages are CD68 molecules, class II MHC antigens, receptors, that are involved in phagocytosis, including Fc receptors, complement receptors, integrins, mannose receptor, sialoadhesin and scavenger receptors (Wu et al., 2004). Macrophages are considered "professional phagocytes" and can internalize particles much more rapidly and efficiently then other cells due to their expression of specific cell surface receptors, which initiates actin polymerization and internalization of the foreign molecule or organism into a phagosome. Macrophages phagocytose endogenous and exogenous substances, such as cell debris, bacteria and viruses. Macrophages in vivo recognize and internalize apoptotic and necrotic cells (Wu et al., 2004). Macrophages sequestered in the peritoneal cavity remove red blood cells, damaged tissue fragments, apoptotic cells, and probably endometrial cells that gain access to the peritoneal cavity through the fallopian tubes (Dmowski & Braun, 2004).

In endometriotic peritoneal fluid the concentration and number of peritoneal macrophages are significantly increased as compared to healthy controls (Tariverdian et al., 2007). These are large, activated macrophages that produce high levels of smooth-muscle-contracting prostaglandins, such as PGE2 and PGF2α (Gazvani & Templeton, 2002; Dmowski & Braun, 2004). Elevated PGE2 in the peritoneal fluid of endometriosis patients due to macrophages activation have been proposed to subsequently aggravate endometriosis-associated pain by altering uterine and tubal contractility and cause infertility due to a delayed ovum transport (Tariverdian et al., 2007). But despite of activated status macrophages evidently are not capable to effectively control the growth of endometrial tissue in peritoneal cavity of women with endometriosis. Molecular basis for this phenomenon isn't completely elucidated yet.

One of the mechanisms by which abnormally functioning macrophages could contribute to the growth of ectopic endometrial cells is through defective scavenging activity (Sidell et al., 2002). Important function of macrophages in the face of an invading "foreign" material or when encountering cellular debris and apoptotic cells is the scavenger function (Sidell et al., 2002). A family of specific scavenger receptors (SRs) is involved in this activity. This family was aptly named because these receptors have been found to bind and "scavenge" a broad array of modified self and nonself ligands, including apoptotic cells, anionic phospholipids and amyloid and pathogen components (Moore & Freeman, 2006). The SRs are believed to be members of the group of pattern recognition receptors (PRR) that mediate the innate immune host response through recognition of highly conserved pathogen-associated molecular patterns (PAMP). This evolutionarily ancient but highly effective system of host defense enables the immune system to discriminate between "noninfectious self" and "infectious nonself". However, there is a growing body of evidence to suggest that SRs may recognize endogenous neoantigens and apoptotic cells through molecular mimicry of

The Local Immune Mechanisms Involved in the Formation of Endometriotic Lesions 231

peritoneal macrophages in endometriosis independently from the stage of the disease are characterized by the low level of expression of functional receptors, such as integrins,

This aberrant expression of the surface receptors might lead to the impairment of peritoneal macrophages function in endometriosis which in turn can contribute to the rescue of endometrial cells from immunosurveillance in peritoneal cavity. It is known that CD11b and CD49e molecules belong to the class of integrins, superfamily of cell adhesion receptors that bind to extracellular matrix ligands, cell-surface ligands and soluble ligands (Takakda et al., 2007). It is known that upon binding extracellular ligands, integrins generate an intracellular signal and, conversely, their functioning can be regulated by signals from within the cell. They serve as transmembrane links between extracellular contacts (other cells or the extracellular matrix) and the actin microfilaments of the cytoskeleton, whose behavior integrins also regulate and modulate. Extracellular ligation of integrins triggers a large variety of signal transduction events that modulate cell behaviors such as adhesion, proliferation survival or apoptosis, shape, polarity, motility and differentiation (Takakda et al., 2007). The low level expression of integrins by peritoneal macrophages might lead to the impairment of the interaction of macrophages with ECM and other cells, including the cells of reflux menstrual endometrium. More other, it is known, that if the peritoneal macrophages are not attached to extracellular matrix components, despite their differentiated status may not be competent scavengers (Sidell et al., 2002). Our results about the simultaneously diminishment of the expression of integrins and scavenger receptors A and B type by peritoneal macrophages of women with endometriosis completely confirmed the hypothesis that the defective scavenger function plays an important role in the immune mechanisms of endometriosis

Today the A class of SRs has grown to include 5 members that share common collagenlike domains and homotrimeric structure. SR-A receptors bind oxidized low-density lipoproteins, apoptotic cells, β-amyloid peptide, anionic phospholipids and advanced glycation end-products (Murphy et al., 2005). These receptors have also been implicated in both innate and adaptive immune responses through their recognition of pathogens and pathogen-associated molecules (Moore & Freeman, 2006). The B class of SRs was established with the identification of CD36 as a receptor for oxidized low-density lipoproteins. Unlike the SR-A family, CD36 is a type III (multiple transmembrane domains) receptor that traverses the membrane twice to form a heavily glycosylated extracellular loop with 2 short intracellular tails (Murphy et al., 2005). CD36 has a wider cellular distribution, including monocytes, macrophages, adipocytes, microvascular endothelium, platelets and erythroid precursors. CD36 bind several ligands common to SR-A (β-amyloid, anionic phospholipids, apoptotic cells, advanced glycation endproducts), however, it is distinct from SR-A in its ability to bind native lipoproteins and very low-density lipoprotein as well as thrombospondin-I, collagen, fatty acids and pathogen-derived ligands (Moore & Freeman, 2006). As a result of its broad specificity, CD36 has been reported to contribute to a varied list of normal and pathologic processes such as apoptotic cell clearance, fatty acid transport, adhesion, angiogenesis and microbial defense (Murphy et al., 2005). Decrease of SR-AI and SR-B molecules on the surface of peritoneal macrophages of women with endometriosis likely might to contribute to the

scavenger receptors and apoptosis-inducing molecules.

development.

microbial pathogen ligands (Moore & Freeman, 2006). In the last several years it was found that SRs initiate signaling cascades that regulate macrophages activation, lipid metabolism, and inflammatory programs. In addition, these receptors play role in the induction of apoptosis and apoptotic cell clearance. So, it is possible that SRs are directly involved in the clearance of endometrial debris from the peritoneal cavity. And it is also possible that peritoneal macrophages from women with endometriosis do not express fully functional scavenger receptors. But, now we know very little about the character of scavenger receptors expression by peritoneal macrophages of women with endometriosis. So, the study of the scavenger function of peritoneal macrophages in endometriosis has a special interest.

We have analyzed the expression of some surface functional receptors, such as integrin molecules CD11b and CD49e, HLA-DR molecules, CD95 and ligand for CD95 molecules (CD95L) and scavenger receptors SR-AI (CD204) and SR-B (CD36) as well by peritoneal macrophages of healthy women and women with endometriosis. It was found that peritoneal macrophages of women with endometriosis were characterized by the diminished level of the expression of scavenger receptors A and B type compared to that in healthy women (Table 8). More other, in women with endometriosis the amount of CD11b+, CD49e+, HLA-DR+ and CD95L+ macrophages in peritoneal fluid was also lower than that in healthy women (Table 8). We checked out the correlation between the character of receptors expression and the stage of endometriosis. All women with endometriosis were divided into two subgroups: women with mild and severe endometriosis. In subgroup with mild endometriosis the 1-2 stage of the disease was diagnosed during laparoscopy. Women with severe endometriosis have diagnosed of 3-4 stage of endometriosis during laparoscopic investigation.


Table 8. Membrane expression of functional receptors by peritoneal macrophages of women with endometriosis (\* - given in comparison to the control group, \* - p<0.05, \*\*- p<-0.01, \*\*\* p<0.001)

Differential analysis of surface macrophages phenotype had shown that the expression of SRs, HLA-DR, CD11b and FasL molecules was significantly decreased both in mild and severe endometriosis compared to analogous parameters in control group (Table 8). The only association between the level of receptor expression and the stage of endometriosis was noted for CD49e molecules. The low expression of CD49e was seen only in women with 1-2 stage of the endometriosis, and in women with 3-4 stage of the disease the amount of CD49+ peritoneal macrophages was completely corresponded to that in healthy women. So,

microbial pathogen ligands (Moore & Freeman, 2006). In the last several years it was found that SRs initiate signaling cascades that regulate macrophages activation, lipid metabolism, and inflammatory programs. In addition, these receptors play role in the induction of apoptosis and apoptotic cell clearance. So, it is possible that SRs are directly involved in the clearance of endometrial debris from the peritoneal cavity. And it is also possible that peritoneal macrophages from women with endometriosis do not express fully functional scavenger receptors. But, now we know very little about the character of scavenger receptors expression by peritoneal macrophages of women with endometriosis. So, the study of the scavenger function of peritoneal macrophages in endometriosis has a special

We have analyzed the expression of some surface functional receptors, such as integrin molecules CD11b and CD49e, HLA-DR molecules, CD95 and ligand for CD95 molecules (CD95L) and scavenger receptors SR-AI (CD204) and SR-B (CD36) as well by peritoneal macrophages of healthy women and women with endometriosis. It was found that peritoneal macrophages of women with endometriosis were characterized by the diminished level of the expression of scavenger receptors A and B type compared to that in healthy women (Table 8). More other, in women with endometriosis the amount of CD11b+, CD49e+, HLA-DR+ and CD95L+ macrophages in peritoneal fluid was also lower than that in healthy women (Table 8). We checked out the correlation between the character of receptors expression and the stage of endometriosis. All women with endometriosis were divided into two subgroups: women with mild and severe endometriosis. In subgroup with mild endometriosis the 1-2 stage of the disease was diagnosed during laparoscopy. Women with severe endometriosis have diagnosed of 3-4 stage of endometriosis during laparoscopic

> Endometriosis (n=30)

CD204 (SR-AI)+ 68.31±5.37 53.20±2.36\* 50.88±3.20\*\* 56.10±2.15\* CD36 (SR-B)+ 70.86±9.22 57.20±2.36\* 56.48±2.96\* 59.53±2.00\* CD11b+ 85.14±1.57 71.97±2.30\*\*\* 71.58±2.91\*\*\* 73.10±3.19\*\* CD49e+ 87.91±2.19 80.16±2.34\* 78.03±2.55\*\* 87.6±4.12 HLA-DR+ 87.35±2.28 78.35±2.43\* 80.59±2.37\* 71.97±6.23\* CD95+ 48.12±3.16 51.39±2.54 47.46±4.32 54.02±3.05 CD95L+ 60.62±2.42 46.37±2.65\*\*\* 45.62±3.42\*\*\* 48.50±3.41\*\* Table 8. Membrane expression of functional receptors by peritoneal macrophages of women with endometriosis (\* - given in comparison to the control group, \* - p<0.05, \*\*- p<-0.01, \*\*\*-

Differential analysis of surface macrophages phenotype had shown that the expression of SRs, HLA-DR, CD11b and FasL molecules was significantly decreased both in mild and severe endometriosis compared to analogous parameters in control group (Table 8). The only association between the level of receptor expression and the stage of endometriosis was noted for CD49e molecules. The low expression of CD49e was seen only in women with 1-2 stage of the endometriosis, and in women with 3-4 stage of the disease the amount of CD49+ peritoneal macrophages was completely corresponded to that in healthy women. So,

1-2 stage of endometriosis (n=20)

3-4 stage of endometriosis

(n=10)

interest.

investigation.

p<0.001)

Parameter,% Control group

(n=10)

peritoneal macrophages in endometriosis independently from the stage of the disease are characterized by the low level of expression of functional receptors, such as integrins, scavenger receptors and apoptosis-inducing molecules.

This aberrant expression of the surface receptors might lead to the impairment of peritoneal macrophages function in endometriosis which in turn can contribute to the rescue of endometrial cells from immunosurveillance in peritoneal cavity. It is known that CD11b and CD49e molecules belong to the class of integrins, superfamily of cell adhesion receptors that bind to extracellular matrix ligands, cell-surface ligands and soluble ligands (Takakda et al., 2007). It is known that upon binding extracellular ligands, integrins generate an intracellular signal and, conversely, their functioning can be regulated by signals from within the cell. They serve as transmembrane links between extracellular contacts (other cells or the extracellular matrix) and the actin microfilaments of the cytoskeleton, whose behavior integrins also regulate and modulate. Extracellular ligation of integrins triggers a large variety of signal transduction events that modulate cell behaviors such as adhesion, proliferation survival or apoptosis, shape, polarity, motility and differentiation (Takakda et al., 2007). The low level expression of integrins by peritoneal macrophages might lead to the impairment of the interaction of macrophages with ECM and other cells, including the cells of reflux menstrual endometrium. More other, it is known, that if the peritoneal macrophages are not attached to extracellular matrix components, despite their differentiated status may not be competent scavengers (Sidell et al., 2002). Our results about the simultaneously diminishment of the expression of integrins and scavenger receptors A and B type by peritoneal macrophages of women with endometriosis completely confirmed the hypothesis that the defective scavenger function plays an important role in the immune mechanisms of endometriosis development.

Today the A class of SRs has grown to include 5 members that share common collagenlike domains and homotrimeric structure. SR-A receptors bind oxidized low-density lipoproteins, apoptotic cells, β-amyloid peptide, anionic phospholipids and advanced glycation end-products (Murphy et al., 2005). These receptors have also been implicated in both innate and adaptive immune responses through their recognition of pathogens and pathogen-associated molecules (Moore & Freeman, 2006). The B class of SRs was established with the identification of CD36 as a receptor for oxidized low-density lipoproteins. Unlike the SR-A family, CD36 is a type III (multiple transmembrane domains) receptor that traverses the membrane twice to form a heavily glycosylated extracellular loop with 2 short intracellular tails (Murphy et al., 2005). CD36 has a wider cellular distribution, including monocytes, macrophages, adipocytes, microvascular endothelium, platelets and erythroid precursors. CD36 bind several ligands common to SR-A (β-amyloid, anionic phospholipids, apoptotic cells, advanced glycation endproducts), however, it is distinct from SR-A in its ability to bind native lipoproteins and very low-density lipoprotein as well as thrombospondin-I, collagen, fatty acids and pathogen-derived ligands (Moore & Freeman, 2006). As a result of its broad specificity, CD36 has been reported to contribute to a varied list of normal and pathologic processes such as apoptotic cell clearance, fatty acid transport, adhesion, angiogenesis and microbial defense (Murphy et al., 2005). Decrease of SR-AI and SR-B molecules on the surface of peritoneal macrophages of women with endometriosis likely might to contribute to the

The Local Immune Mechanisms Involved in the Formation of Endometriotic Lesions 233

autologous endometrial cells. We tried to prove this hypothesis and have done series of experiments in vitro for co-cultivation of peritoneal fluid macrophages with explants of autologous endometrial tissue with subsequent estimation of the surface membrane receptors by macrophages. We found that in healthy women the significant elevation of the scavenger receptors A and B type upon macrophages after their incubation with endometrial explants was seen (Fig.3). These results confirmed the suggestion about the direct involvement of SRs in normal response of peritoneal macrophages on the ectopic

On the contrary, in endometriosis women the stimulation of peritoneal macrophages by autologous endometrial cells didn't change the expression of scavenger receptors by macrophages (Fig.4). Moreover, after co-cultivation of macrophages from endometriosis women with eutopic endometrial the significant diminishment of the number of FasL+,

Fig. 4. Changes in the expression of functional molecules and scavenger receptors by peritoneal macrophages of women with endometriosis after its incubation in vitro with autologous eutopic endometrium (\* - given in comparison to macrophages incubating only

endometrial cells which, in turn escape the immunosurveillance in peritoneal cavity.

**5.2 Peritoneal fluid cytokine production in endometriosis** 

Supposedly, the initial aberrant expression of functional receptors by peritoneal macrophages results in inadequate response of macrophages to stimulation of self

The majority of studies up to now have shown significantly increase of the content of cytokines and growth factors in peritoneal fluid of women with endometriosis (Dmowski & Braun, 2004). It is known that cytokines and growth factors are proteins or glycoproteins produced by leukocytes or other cells, and secreted to the extracellular environment. These molecules exert their effects on the same (autocrine) or nearby cells (paracrine activity). They are key mediators of intercellular communication within immune system. Cytokines may have proliferative, cytostatic, chemo atractant or differetiative effects (Berkkanoglu & Arici, 2003). Cytokines possess pleiotropic function

misplaced endometrium.

CD11b+ and CD49e+ macrophages was seen (Fig.4).

in the presence of RPMI 1640, \* - p<0.05).

impairment of clearance of endometrial cells from peritoneal cavity and facilitate the implantation and growth of ectopic endometrium.

The low expression of FasL molecules by peritoneal macrophages might be attributed to the decrease of apoptosis-inducting function of macrophages in endometriosis. As we have demonstrated above macrophages of women with endometriosis were incapable to induce apoptosis in autologous eutopic endometrium. The observed low expression of FasL molecules on the macrophages surface likely is probably one of the possible mechanisms which impair the apoptosis-inducing function of macrophages in endometriosis. It is known that FasL or Fas ligand is one of the main transmembrane receptors initiating extrinsic pathway of apoptosis (Elmore 2007). The binding of FasL to its receptor Fas on the surface of cell-target results in the binding of the adapter protein FADD, activation of pro-caspase-8 and formation of death-inducing signaling complex (DISC) (Elmore 2007). So, the FasLpositive cells are powerful inducers of apoptosis and impairment of FasL expression on the surface of peritoneal macrophages might be an important factor contributing to inhibition of endometrial apoptosis.

We shown that the expression of HLA-DR molecules by peritoneal macrophages in endometriosis also was decreased comparing to that in healthy women. The same results were received Yamamoto et al. (2008), which observed low HLA expression and particularly reduced HLA-DR in the lipid raft. It is known that HLA-DR molecules are involved in antigen presentation by macrophages (Wu et al., 2004). So, impairment of HA-DR expression might compromise antigen presentation in women with endometriosis, limiting the immune response to peritoneal cavity antigens such as implanted or metaplastic endometrial cells.

Fig. 3. Changes in the expression of functional molecules and scavenger receptors by peritoneal macrophages of healthy women after its incubation in vitro with autologous eutopic endometrium (\* - given in comparison to macrophages incubating only in the

presence of RPMI 1640, \* - p<0.05).

Limited expression of the surface functional receptors by macrophages in endometriosis can lead to the incapability of macrophages to correctly response upon stimulation by

impairment of clearance of endometrial cells from peritoneal cavity and facilitate the

The low expression of FasL molecules by peritoneal macrophages might be attributed to the decrease of apoptosis-inducting function of macrophages in endometriosis. As we have demonstrated above macrophages of women with endometriosis were incapable to induce apoptosis in autologous eutopic endometrium. The observed low expression of FasL molecules on the macrophages surface likely is probably one of the possible mechanisms which impair the apoptosis-inducing function of macrophages in endometriosis. It is known that FasL or Fas ligand is one of the main transmembrane receptors initiating extrinsic pathway of apoptosis (Elmore 2007). The binding of FasL to its receptor Fas on the surface of cell-target results in the binding of the adapter protein FADD, activation of pro-caspase-8 and formation of death-inducing signaling complex (DISC) (Elmore 2007). So, the FasLpositive cells are powerful inducers of apoptosis and impairment of FasL expression on the surface of peritoneal macrophages might be an important factor contributing to inhibition of

We shown that the expression of HLA-DR molecules by peritoneal macrophages in endometriosis also was decreased comparing to that in healthy women. The same results were received Yamamoto et al. (2008), which observed low HLA expression and particularly reduced HLA-DR in the lipid raft. It is known that HLA-DR molecules are involved in antigen presentation by macrophages (Wu et al., 2004). So, impairment of HA-DR expression might compromise antigen presentation in women with endometriosis, limiting the immune response to peritoneal cavity antigens such as implanted or metaplastic

Fig. 3. Changes in the expression of functional molecules and scavenger receptors by peritoneal macrophages of healthy women after its incubation in vitro with autologous eutopic endometrium (\* - given in comparison to macrophages incubating only in the

Limited expression of the surface functional receptors by macrophages in endometriosis can lead to the incapability of macrophages to correctly response upon stimulation by

implantation and growth of ectopic endometrium.

endometrial apoptosis.

endometrial cells.

presence of RPMI 1640, \* - p<0.05).

autologous endometrial cells. We tried to prove this hypothesis and have done series of experiments in vitro for co-cultivation of peritoneal fluid macrophages with explants of autologous endometrial tissue with subsequent estimation of the surface membrane receptors by macrophages. We found that in healthy women the significant elevation of the scavenger receptors A and B type upon macrophages after their incubation with endometrial explants was seen (Fig.3). These results confirmed the suggestion about the direct involvement of SRs in normal response of peritoneal macrophages on the ectopic misplaced endometrium.

On the contrary, in endometriosis women the stimulation of peritoneal macrophages by autologous endometrial cells didn't change the expression of scavenger receptors by macrophages (Fig.4). Moreover, after co-cultivation of macrophages from endometriosis women with eutopic endometrial the significant diminishment of the number of FasL+, CD11b+ and CD49e+ macrophages was seen (Fig.4).

Fig. 4. Changes in the expression of functional molecules and scavenger receptors by peritoneal macrophages of women with endometriosis after its incubation in vitro with autologous eutopic endometrium (\* - given in comparison to macrophages incubating only in the presence of RPMI 1640, \* - p<0.05).

Supposedly, the initial aberrant expression of functional receptors by peritoneal macrophages results in inadequate response of macrophages to stimulation of self endometrial cells which, in turn escape the immunosurveillance in peritoneal cavity.

#### **5.2 Peritoneal fluid cytokine production in endometriosis**

The majority of studies up to now have shown significantly increase of the content of cytokines and growth factors in peritoneal fluid of women with endometriosis (Dmowski & Braun, 2004). It is known that cytokines and growth factors are proteins or glycoproteins produced by leukocytes or other cells, and secreted to the extracellular environment. These molecules exert their effects on the same (autocrine) or nearby cells (paracrine activity). They are key mediators of intercellular communication within immune system. Cytokines may have proliferative, cytostatic, chemo atractant or differetiative effects (Berkkanoglu & Arici, 2003). Cytokines possess pleiotropic function

The Local Immune Mechanisms Involved in the Formation of Endometriotic Lesions 235

culture (Mulayim et al., 2004). Calprotektin, comprise the heterodimeric complex from C2a+-binding proteins S100A8 and S100 A9, also participates in cells invasiveness regulation. It was found that enhanced expression of S100A8/A9 gene is the marker of metastatic potential of epithelial tumor cells (Moon et al., 2008). It was also demonstrated that calprotektin is involved in the activation of gene MMP-2 transcription in tumor cell line SNU484 (Yong & Moon, 2007). Chemokine MCP-1 acts as paracrine and autocrine regulator of growth and invasion of prostate tumor (Lu et al., 2009). So, all these cytokines can be stimulators of the eutopic endometrial cells invasiveness in peritoneal fluid. But, the significant elevation of studied cytokines content was noted only in women with severe endometriosis. In cases of mild endometriosis the cytokines concentrations didn't differ

Endometriosis

1-2 stage of endometriosis

1-2 stage of endometriosis

(n=18)

3-4 stage of endometriosis

3-4 stage of endometriosis

(n=10)

(n=17)

(n=18)

0.44±0.17 1.28±0.38\* 0.62±0.26 1.86±0.65\*

(n=35)

IL-8, pg/ml 6.19±3.41 19.09±5.00\* 10.83±4.18 27.73±9.12\* MCP-1, pg/ml 308.12±29.87 528.40±51.45\*\* 376.56±50.32 689.18±74.79\*\*\*ххх

Table 9. Cytokine content in peritoneal fluid of women with endometriosis (\* - given in comparison to the control group, \* - p<0.05, \*\*- p<-0.01, \*\*\*- p<0.001; х - given in comparison

Endometriosis

IL-1β+ 64.51±3.40 74.69±2.90\* 73.47±3.09 80.53±3.46\*\*\* IL-8+ 67.10±2.96 75.13±2.03\* 68.42±4.14 78.17±1.70\*\* TNFα+ 63.11±3.37 74.77±1.95\*\* 74.72±2.96\*\* 76.24±2.38\* IL-6+ 52.74±4.18 66.63±2.87 58.41±3.36 66.25±2.70\* IL-12+ 67.35±3.39 63.64±2.56 63.268±3.32 65.53±0.95 Table 10. Intracellular production of cytokines by peritoneal macrophages of women with endometriosis (\* - given in comparison to the control group, \* - p<0.05, \*\*- p<-0.01, \*\*\*-

(n=28)

It is known, that peritoneal macrophages are the major producers of cytokines in peritoneal fluid (Wu & Ho, 2003). We assessed the production of several proinflammatory cytokines (IL-1β, IL-8, TNFα, IL-6, and IL-12) by peritoneal macrophages in endometriosis. It was found that that character of intracellular production of proinflammatory cytokines by macrophage was correspondent to cytokines content in peritoneal fluid (Table 10). The level of cytokines production directly correlated with the severity of endometriosis and significantly increased only in group of women with 3-4 stages of endometriosis. The only exception we noted for TNFα. We found that that the intracellular production of TNFα by peritoneal macrophages was significantly elevated both in women with mild and severe

to the group of women with 1-2 stage of endometriosis, ххх- p<0.001)

from that in healthy women.

Calprotektin, μg/ml

endometriosis (Table 10).

p<0.001)

Parameter,% Control

group (n=10)

Parameter Control group

(n=10)

and act in cascade manner. The involvements of cytokines in the pathogenesis of many diseases, connected with inflammation and cells proliferation, such as autoimmune pathology, rheumatoid arthritis, cancer etc, have been proved. The role of cytokines system in pathogenesis of endometriosis was studied very intensively. The increased leukocyte number found in peritoneal fluid of women with endometriosis is likely to be attributable to the enhanced synthesis of various cytokines and growth factors (Dmowski & Braun, 2004). Cytokines also might be produced by endothelial cells, misplaced endometrial cells and ectopic tissue cells. The detailed analysis of the cytokines profile of peritoneal fluid of endometriosis women is above scope of this paper. To receive more information we recommend to direct reader's attention to some detailed reviews (Harada et al., 2001; Gazvani & Templeton, 2002; Kyama et al., 2003; Wu & Ho, 2003; Dmowski & Braun, 2004). Briefly, significantly elevated concentration of numerous proinflammatory cytokines such as IL-1β, TNFα, IL-6, IL-8, IL-12, IL-16, RANTES, EGF, TGF β, CSG, IFG, HGF was found in peritoneal fluid of endometriosis women and practically in all cases investigators conclude that this local sterile inflammation possibly "plays a decisive role in the pathogenesis if the endometriosis". This conclusion is based on the experimental data, showing capability of several proinflammatory cytokines increase the proliferation and growth endometrial cells. For example, experiments in vitro demonstrated that TNFα and IL-8 collaborate in the proliferation of stromal cells from ectopic endometrium and endometriomata (Harada et al., 2001). It was also shown that TNFα as the factor of peritoneal fluid from women with endometriosis promotes proliferation of eutopic and ectopic endometrial cells (Braun et al., 2002). IL-8 stimulates the adhesion of endometrial cells to fibronectin (Berkkanoglu & Arici, 2003). IL-1β promotes angiogenesis in endometriotic lesions by inducing the angiogenic factors in endometriotic stromal cells but not in normal endometrial stromal cells (Lebovis et al., 2000). But the majority of works are only descriptive and the functional involvement of cytokines in the development or progression of endometriosis has not been proven. Up to now we don't know exactly: is this peritoneal inflammation cause or consequence of the disease. From one side high concentration of growth factors and some cytokines, influencing upon cells proliferation, evidently might promote of endometrium implantation and growth in peritoneal cavity. Bur from another side, as ectopic endometrial lesions are a valuable source of cytokines itself, so peritoneal inflammation may be only the consequence of the presence of endometrial lesions in peritoneal cavity. Solutions of this problem evidently will be very helpful as to the endometriosis pathogenesis comprehension and search of new approaches to the medical treatment of endometriosis as well.

We attempted to trace the connection between cytokine profile of peritoneal fluid of women with endometriosis and possible mechanisms of peritoneal fluid action upon invasiveness of endometrial cells. To this purpose we estimated the concentration of few cytokines with possible action upon cells invasiveness, such as chemokines IL-8 and MCP and protein calprotektin, belonging to the family S100 proteins. We found the significant elevation all above mentioned cytokines in the peritoneal fluid of patients with endometriosis (Table 9). When we analyzed these cytokines content in subgroups of women with mild and severe endometriosis we found, that concentration of these cytokines correlated with the degree of endometriosis: maximal levels of IL-8, MCP-1 and calprotektin were seen in women with severe endometriosis (Table 9).

All of these cytokines are powerful stimulators of cells invasiveness. Earlier it was found that IL-8 directly increases the MMPs activity and invasive potential of endometrial cells in

and act in cascade manner. The involvements of cytokines in the pathogenesis of many diseases, connected with inflammation and cells proliferation, such as autoimmune pathology, rheumatoid arthritis, cancer etc, have been proved. The role of cytokines system in pathogenesis of endometriosis was studied very intensively. The increased leukocyte number found in peritoneal fluid of women with endometriosis is likely to be attributable to the enhanced synthesis of various cytokines and growth factors (Dmowski & Braun, 2004). Cytokines also might be produced by endothelial cells, misplaced endometrial cells and ectopic tissue cells. The detailed analysis of the cytokines profile of peritoneal fluid of endometriosis women is above scope of this paper. To receive more information we recommend to direct reader's attention to some detailed reviews (Harada et al., 2001; Gazvani & Templeton, 2002; Kyama et al., 2003; Wu & Ho, 2003; Dmowski & Braun, 2004). Briefly, significantly elevated concentration of numerous proinflammatory cytokines such as IL-1β, TNFα, IL-6, IL-8, IL-12, IL-16, RANTES, EGF, TGF β, CSG, IFG, HGF was found in peritoneal fluid of endometriosis women and practically in all cases investigators conclude that this local sterile inflammation possibly "plays a decisive role in the pathogenesis if the endometriosis". This conclusion is based on the experimental data, showing capability of several proinflammatory cytokines increase the proliferation and growth endometrial cells. For example, experiments in vitro demonstrated that TNFα and IL-8 collaborate in the proliferation of stromal cells from ectopic endometrium and endometriomata (Harada et al., 2001). It was also shown that TNFα as the factor of peritoneal fluid from women with endometriosis promotes proliferation of eutopic and ectopic endometrial cells (Braun et al., 2002). IL-8 stimulates the adhesion of endometrial cells to fibronectin (Berkkanoglu & Arici, 2003). IL-1β promotes angiogenesis in endometriotic lesions by inducing the angiogenic factors in endometriotic stromal cells but not in normal endometrial stromal cells (Lebovis et al., 2000). But the majority of works are only descriptive and the functional involvement of cytokines in the development or progression of endometriosis has not been proven. Up to now we don't know exactly: is this peritoneal inflammation cause or consequence of the disease. From one side high concentration of growth factors and some cytokines, influencing upon cells proliferation, evidently might promote of endometrium implantation and growth in peritoneal cavity. Bur from another side, as ectopic endometrial lesions are a valuable source of cytokines itself, so peritoneal inflammation may be only the consequence of the presence of endometrial lesions in peritoneal cavity. Solutions of this problem evidently will be very helpful as to the endometriosis pathogenesis comprehension and search of

new approaches to the medical treatment of endometriosis as well.

severe endometriosis (Table 9).

We attempted to trace the connection between cytokine profile of peritoneal fluid of women with endometriosis and possible mechanisms of peritoneal fluid action upon invasiveness of endometrial cells. To this purpose we estimated the concentration of few cytokines with possible action upon cells invasiveness, such as chemokines IL-8 and MCP and protein calprotektin, belonging to the family S100 proteins. We found the significant elevation all above mentioned cytokines in the peritoneal fluid of patients with endometriosis (Table 9). When we analyzed these cytokines content in subgroups of women with mild and severe endometriosis we found, that concentration of these cytokines correlated with the degree of endometriosis: maximal levels of IL-8, MCP-1 and calprotektin were seen in women with

All of these cytokines are powerful stimulators of cells invasiveness. Earlier it was found that IL-8 directly increases the MMPs activity and invasive potential of endometrial cells in culture (Mulayim et al., 2004). Calprotektin, comprise the heterodimeric complex from C2a+-binding proteins S100A8 and S100 A9, also participates in cells invasiveness regulation. It was found that enhanced expression of S100A8/A9 gene is the marker of metastatic potential of epithelial tumor cells (Moon et al., 2008). It was also demonstrated that calprotektin is involved in the activation of gene MMP-2 transcription in tumor cell line SNU484 (Yong & Moon, 2007). Chemokine MCP-1 acts as paracrine and autocrine regulator of growth and invasion of prostate tumor (Lu et al., 2009). So, all these cytokines can be stimulators of the eutopic endometrial cells invasiveness in peritoneal fluid. But, the significant elevation of studied cytokines content was noted only in women with severe endometriosis. In cases of mild endometriosis the cytokines concentrations didn't differ from that in healthy women.


Table 9. Cytokine content in peritoneal fluid of women with endometriosis (\* - given in comparison to the control group, \* - p<0.05, \*\*- p<-0.01, \*\*\*- p<0.001; х - given in comparison to the group of women with 1-2 stage of endometriosis, ххх- p<0.001)

It is known, that peritoneal macrophages are the major producers of cytokines in peritoneal fluid (Wu & Ho, 2003). We assessed the production of several proinflammatory cytokines (IL-1β, IL-8, TNFα, IL-6, and IL-12) by peritoneal macrophages in endometriosis. It was found that that character of intracellular production of proinflammatory cytokines by macrophage was correspondent to cytokines content in peritoneal fluid (Table 10). The level of cytokines production directly correlated with the severity of endometriosis and significantly increased only in group of women with 3-4 stages of endometriosis. The only exception we noted for TNFα. We found that that the intracellular production of TNFα by peritoneal macrophages was significantly elevated both in women with mild and severe endometriosis (Table 10).


Table 10. Intracellular production of cytokines by peritoneal macrophages of women with endometriosis (\* - given in comparison to the control group, \* - p<0.05, \*\*- p<-0.01, \*\*\* p<0.001)

The Local Immune Mechanisms Involved in the Formation of Endometriotic Lesions 237

anti-apoptotic genes was seen. Likely, this phenomenon might provide the ectopic but not malignant growth of endometrium in endometriosis. The development and growth of already formed ectopic lesions but not the eutopic endometrium of women with endometriosis is under the control of proteolytic enzymes from metalloproteinases family. We found that some of these differences between eutopic and ectopic endometrium might be due to the impairment of immunosurveillance in peritoneal cavity. Both peritoneal macrophages and humoral factors of peritoneal fluid directly influenced upon apoptosis and invasiveness of endometrial cells. This action might be connected with the changes of functional activity of peritoneal macrophages and local cytokine production. The impairment of the scavenger function of peritoneal macrophages due to the decreased level of membrane expression of integrins, scavenger receptors and apoptosis-inducing molecules FasL is possibly one of the fundamental defects of immune response in peritoneal cavity, which allow endometrial cell to live, proliferate and be implanted. Altered cytokine profile of peritoneal fluid in endometriosis likely can promote the invasiveness of ectopic lesions. Though it can be said that now we don't fully understand the fine immune mechanisms providing the development of endometriosis. More studies into the macrophage functioning and cytokines production as well as clinical experiments on the animal model may improve our understanding of endometriosis pathogenesis and results in the novel therapeutic

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**7. References** 

TNFα or tumor necrosis factor α initially was identified for its ability to kill certain cell lines, but now it is known that TNFα have the ability to initiate the cascade of other cytokines and factors associated with inflammatory responses. Earlier it was shown that TNFα increased the adherence of cultured endometrial stromal cells to mesothelial cells (Zang et al., 1993). This finding suggests that the presence of TNFα in peritoneal fluid may facilitate the adherence of ectopic endometrial tissue to the peritoneum and allow implants to develop. There are some experimental data, also evidenced about implication of TNFα in the pathogenesis of endometriosis (Agic et al., 2006). In baboons with laparoscopically confirmed endometriosis, TNFα blockade with p55 soluble TNFαreceptors results in inhibition of the development and growth of endometriotic implants (D'Hooghe et al., 2006). In rats with ectopically transplanted endometrial tissue, the administration of recombinant human TNFα- binding protein-1 (r-hTBP-1) resulted in defective development of implants compared with controls (D'Antonio et al., 2000). All these data and our own results let us to suggest the possible involvement of TNFα in initial mechanisms of ectopic endometrium implantation and growth. But it must be special noted that the macrophage's production and peritoneal fluid content of the majority of proinflammatory cytokines significantly elevated only at advantage stages of endometriosis.

Thus, we must very careful to speculate about possible involvement of cytokines in the peritoneal fluid in regulation of the behavior of endometrial cells in peritoneal cavity, because the prominent action of peritoneal fluid cytokines generally is seen only in advantage stages of endometriosis. We thought that the impairment of peritoneal macrophages function plays the decisive role in immune mechanisms participating in implantation and growth of ectopic endometrium. Incapability of peritoneal macrophages to act as effective scavengers and adequately to eliminate the misplaced endometrial cells from peritoneal cavity possibly is one of the primary immunological disorders participating in endometriosis pathogenesis. Elevation of proinflammatory cytokines production in peritoneal cavity likely depends on the presence of ectopic lesions and can be stimulated by viable endometrial cells proliferating and growing in peritoneal cavity. Invasion of already implanted ectopic endometrial cells is evidently under the control of proinflammatory cytokines of peritoneal fluid.
