**4.2 CY regulates cytokine network released by Mf**

One of the most strongly expressed of CY and their derivatives treatment of macrophages is the activation of IL-6 production (Bryniarski 1996, 2009) . Our results do not address directly the questions by which mechanisms NM modifies the macrophages to produce more IL-6. IL-6 gene expression can be induced by a variety of physiological (cytokines, growth factor, bacterial products) and non-physiological stimuli (certain toxins, medicaments, prostaglandin E1), by at least three different signals pathways (diacyloglycerol, cAMP- and Ca2+- activated pathways). At the DNA level three functional promoter domains were described in conserved region of IL-6 promoter (MRE, NF-IL6 and NFB). Exactly how transduction pathways are assigned seems unknown with the possible exception of protein kinase C signal which seems to focus on MRE region (Bryniarski et al. 1996). Since NM

The Influence of Cyclophosphamide on Immune Function of Murine Macrophages 155

receives a proper signal (e.g. phagocytosis), translocate to form an active enzyme. We propose a possible explanation that under our experimental conditions, ACR, in contrast to NM, does not bind efficiently to important docking proteins to trigger the increased production of ROI. Conflicting results regarding ROI production were also published by other groups. Some reports describe the inhibitory activity of ACR, and others indicate an increased production of radicals. In effect, one can conclude that experimental conditions

Our experimental data showed that untreated and in vivo CY treated populations of peritoneal macrophages produce the similar level of nitrogen oxide (Marcinkiewicz et al. 1994) which does not allow to speculate on its function in the immune regulatory system

Our data show that chemotherapy by CY or its products may activate the immune system by modulating cytokine networks and activation of Mf. This may lead to an enhancement of antigen-specific cell mediated immunity but also to activation of mechanisms of innate immunity mediated by Mf, like the production of ROI. Additionally, in animal models derivatives of different mustards led to decreased secretion of IL-10 and TGF- by tumor cells and to their elimination. These and other similar experiments in humans support the notion that, at a correct dosage, CY and its metabolites can be a promising accessory tool in

The mechanism of CY influences macrophage immune function in as was shown previously in case of peritoneal Mf and TMf and seems to be the effect of network of different related factors. The analysis shows as the most important the influence of CY on the secretory activity of Mf which is the inhibition of IL-10 (in case of peritoneal Mf) and TGF- (mainly in case of TMf) with parallel activation of proinflammatory cytokines secretion mainly IL-6, and to a lesser degree IL-12. Both cytokine signals lead to activation of antigen presentation in Mf. The other important factor mediated by CY treatment is an influence on the activation of a cell surface markers expression responsible for uptaking antigen into APC (FcR I, FcRII, CD23 – FcRII/III) and their following presentation to T lymphocytes

Although our experiments indicate that in the testis – immune privileged organ – some subpopulations of Mf are potentially able to present antigen if they would sneak through the blood-testis barrier, they also suggest how this potential activity is under control of other Mf and Sertoli cells. Our previous observations have shown that TMf are poor producers of oxygen radicals and nitric oxide both involved in the mechanisms of natural immunity which may be an evolutionary adaptation to diminish the risk of DNA mutations during spermatogenesis. Additionally we showed that specific immune responses controlled by the male gonads minimize the risk of development of autoimmune reactions and are potentially deleterious to testicular functions (Bryniarski 2004). Testicular Mf are good producers of TGF-, which allows them to play an important functional population of testicular interstitial tissue cells that preserves state of tolerance in testes an immune privilege organs.

were the key (Bryniarski at al. 2009).

anti-tumor therapy.

mediated by macrophage stimulated with CY.

**4.4 Immunomodulation in chemotherapy with low doses of CY** 

subpopulations (CD80/CD86, MHC class II, CD14-LPS receptor).

**4.5 Influence of CY on testicular macrophages** 

binds to both DNA and proteins, it could stimulate IL-6 production directly by alkylating any domain of promoter region, or indirectly, by the alkylation of cell surfaces or by both mechanisms simultaneously.

IL-6 is one of the major mediators of the immune response, with pleotropic and sometimes opposed effects on many different targets. It has been shown for instance that IL-6 enhances the cytotoxic activity of NK cells, thus may potentially augment the host defenses and contribute to anti-tumor effects of alkylating agents. Nonetheless, in case of the IL-6 dependent tumours like myelomas or plasma cell leukemias increased IL-6 level could be deleterious. The increased production of IL-6 may be responsible for observed paradoxical effects of CY which under certain conditions enhances, rather than suppresses both the humoral and cell mediated immune responses (Bryniarski et al.1996).

Apart from the influence of CY and its metabolites on the IL-6 production the inhibition of IL-10 and TGF- production by macrophages was also observed. The results presented above in Table 4 and Fig.3 clearly suggest that the state of tolerance or unresponsiveness observed after TNP-Mf i.v. injection seems to be mediated by the network of pro- and antiinflammatory cytokines secreted from macrophages and also tentatively delivered by natural regulatory cells. It is highly unlikely that low concentration of cytokine metabolites (10-7 and 10-6M) have a direct cytotoxic effect on the Treg-inducing Mf since the cell viability remains unchanged during 24 h culture. Our interpretation is also supported by finding that shifting the balance between pro- and anti-inflammatory cytokines allows for deliberate manipulation of the outgoing response. IL-10 and TGF-, which are anti-inflammatory cytokines, inhibit the activity of Th1 cells and Mf and down-regulate their function. As shown in Figure 3, administration of anti-IL-10 and/or anti-TGF- mAbs into animals which received non-immunogenic TNP-Mf restores their immune potential although to different degrees. It indicates that the key suppressive cytokine is IL-10, a finding that is supported by other groups (Bryniarski et al. 2009). In a symmetrical situation, as we have shown previously, administration of anti-IL-12 antibodies inhibits the function of immunogenic TNP-Mf *in vivo* (Bryniarski et al. 2009). The increased production of IL-12 and IL-6 by macrophages indicates that the cell surface signal delivered by ACR or NM activates the transcription factor NF-B required for the release of inflammatory cytokines (Bryniarski et al. 2009). As reported by other groups, ACR, when allowed free access to the interior of the cell, can either block or enhance the activity of NF-B in alveolar macrophages depending on the design of cell treatment (Bryniarski et al. 2009).

#### **4.3 CY modulates oxygen radicals formation by macrophages**

Our results indicate that CY upregulates not only the specific immune response, by converting non-immunogenic (tolerogenic) Mf into antigen-presenting cells but also positively influences a typical parameter of innate immunity – production of oxygen radicals. In up-regulating the immune function of Mf, ACR and NM had much the same effect. This was however not the case with regard to the production of ROI's by these cells. Using the low concentrations of metabolites, NM was highly stimulatory while ACR did not influence the formation of oxygen radicals above the level observed in control Mf (high concentration of both metabolites were inhibitory). One possibility is that the ACR and NM bind to different targets on the cell surface. NADPH oxidase catalyzing the generation of ROI is composed of several cytosolic and membrane-bound proteins which, after the cell

binds to both DNA and proteins, it could stimulate IL-6 production directly by alkylating any domain of promoter region, or indirectly, by the alkylation of cell surfaces or by both

IL-6 is one of the major mediators of the immune response, with pleotropic and sometimes opposed effects on many different targets. It has been shown for instance that IL-6 enhances the cytotoxic activity of NK cells, thus may potentially augment the host defenses and contribute to anti-tumor effects of alkylating agents. Nonetheless, in case of the IL-6 dependent tumours like myelomas or plasma cell leukemias increased IL-6 level could be deleterious. The increased production of IL-6 may be responsible for observed paradoxical effects of CY which under certain conditions enhances, rather than suppresses both the

Apart from the influence of CY and its metabolites on the IL-6 production the inhibition of IL-10 and TGF- production by macrophages was also observed. The results presented above in Table 4 and Fig.3 clearly suggest that the state of tolerance or unresponsiveness observed after TNP-Mf i.v. injection seems to be mediated by the network of pro- and antiinflammatory cytokines secreted from macrophages and also tentatively delivered by natural regulatory cells. It is highly unlikely that low concentration of cytokine metabolites (10-7 and 10-6M) have a direct cytotoxic effect on the Treg-inducing Mf since the cell viability remains unchanged during 24 h culture. Our interpretation is also supported by finding that shifting the balance between pro- and anti-inflammatory cytokines allows for deliberate manipulation of the outgoing response. IL-10 and TGF-, which are anti-inflammatory cytokines, inhibit the activity of Th1 cells and Mf and down-regulate their function. As shown in Figure 3, administration of anti-IL-10 and/or anti-TGF- mAbs into animals which received non-immunogenic TNP-Mf restores their immune potential although to different degrees. It indicates that the key suppressive cytokine is IL-10, a finding that is supported by other groups (Bryniarski et al. 2009). In a symmetrical situation, as we have shown previously, administration of anti-IL-12 antibodies inhibits the function of immunogenic TNP-Mf *in vivo* (Bryniarski et al. 2009). The increased production of IL-12 and IL-6 by macrophages indicates that the cell surface signal delivered by ACR or NM activates the transcription factor NF-B required for the release of inflammatory cytokines (Bryniarski et al. 2009). As reported by other groups, ACR, when allowed free access to the interior of the cell, can either block or enhance the activity of NF-B in alveolar macrophages depending

humoral and cell mediated immune responses (Bryniarski et al.1996).

on the design of cell treatment (Bryniarski et al. 2009).

**4.3 CY modulates oxygen radicals formation by macrophages** 

Our results indicate that CY upregulates not only the specific immune response, by converting non-immunogenic (tolerogenic) Mf into antigen-presenting cells but also positively influences a typical parameter of innate immunity – production of oxygen radicals. In up-regulating the immune function of Mf, ACR and NM had much the same effect. This was however not the case with regard to the production of ROI's by these cells. Using the low concentrations of metabolites, NM was highly stimulatory while ACR did not influence the formation of oxygen radicals above the level observed in control Mf (high concentration of both metabolites were inhibitory). One possibility is that the ACR and NM bind to different targets on the cell surface. NADPH oxidase catalyzing the generation of ROI is composed of several cytosolic and membrane-bound proteins which, after the cell

mechanisms simultaneously.

receives a proper signal (e.g. phagocytosis), translocate to form an active enzyme. We propose a possible explanation that under our experimental conditions, ACR, in contrast to NM, does not bind efficiently to important docking proteins to trigger the increased production of ROI. Conflicting results regarding ROI production were also published by other groups. Some reports describe the inhibitory activity of ACR, and others indicate an increased production of radicals. In effect, one can conclude that experimental conditions were the key (Bryniarski at al. 2009).

Our experimental data showed that untreated and in vivo CY treated populations of peritoneal macrophages produce the similar level of nitrogen oxide (Marcinkiewicz et al. 1994) which does not allow to speculate on its function in the immune regulatory system mediated by macrophage stimulated with CY.

#### **4.4 Immunomodulation in chemotherapy with low doses of CY**

Our data show that chemotherapy by CY or its products may activate the immune system by modulating cytokine networks and activation of Mf. This may lead to an enhancement of antigen-specific cell mediated immunity but also to activation of mechanisms of innate immunity mediated by Mf, like the production of ROI. Additionally, in animal models derivatives of different mustards led to decreased secretion of IL-10 and TGF- by tumor cells and to their elimination. These and other similar experiments in humans support the notion that, at a correct dosage, CY and its metabolites can be a promising accessory tool in anti-tumor therapy.

The mechanism of CY influences macrophage immune function in as was shown previously in case of peritoneal Mf and TMf and seems to be the effect of network of different related factors. The analysis shows as the most important the influence of CY on the secretory activity of Mf which is the inhibition of IL-10 (in case of peritoneal Mf) and TGF- (mainly in case of TMf) with parallel activation of proinflammatory cytokines secretion mainly IL-6, and to a lesser degree IL-12. Both cytokine signals lead to activation of antigen presentation in Mf. The other important factor mediated by CY treatment is an influence on the activation of a cell surface markers expression responsible for uptaking antigen into APC (FcR I, FcRII, CD23 – FcRII/III) and their following presentation to T lymphocytes subpopulations (CD80/CD86, MHC class II, CD14-LPS receptor).

#### **4.5 Influence of CY on testicular macrophages**

Although our experiments indicate that in the testis – immune privileged organ – some subpopulations of Mf are potentially able to present antigen if they would sneak through the blood-testis barrier, they also suggest how this potential activity is under control of other Mf and Sertoli cells. Our previous observations have shown that TMf are poor producers of oxygen radicals and nitric oxide both involved in the mechanisms of natural immunity which may be an evolutionary adaptation to diminish the risk of DNA mutations during spermatogenesis. Additionally we showed that specific immune responses controlled by the male gonads minimize the risk of development of autoimmune reactions and are potentially deleterious to testicular functions (Bryniarski 2004). Testicular Mf are good producers of TGF-, which allows them to play an important functional population of testicular interstitial tissue cells that preserves state of tolerance in testes an immune privilege organs.

The Influence of Cyclophosphamide on Immune Function of Murine Macrophages 157

A single administration of low dose of CY (50 mg/kg) into either donors or recipients restores the ability of Mf to induce significant CS reaction as a result of: i.) elimination of suppressive properties of Mf; ii.) and/or depletion of population of regulatory T cells in recipients or iii.) elimination of their suppressive activities. In vitro studies with metabolites of CY in contrast to studies in vivo allow identifying the factors which express direct action on selected populations of cells in contrary to experimental research in vivo which is able to identify parallel with direct also indirect effects of cyclophosphamide action on other than macrophages cell populations ( T reg cells) that may change and modulate the activity of macrophages and their influence on the immune response. We propose the schemes which summarize the influence of low doses of CY on the immune response in mice

Figure 6 a-c. The network of the CY influence on the macrophage and regulatory T cells in

\* Induction of T suppressor factor (see the reference by Bryniarski et al. 2-nd European Congress of

Fig. 6.a. Activation of unresponsiveness in recipients after i.v. injection of TNP substituted

(**Figures 6a-6c**).

Immunology Berlin 2009)

Mf – lack of CHS reaction.

mice.

That state of tolerance eliminates the cellular immune response from the testis and in consequence makes an extremely dangerous the viral infection as well as in malignances taking place in testis. CY treatment often change TMf activity from unresponsiveness into actively antigen presenting cells which in consequence help to undertake anticancer response but also often can leads to activation an autoimmune response and immunological infertility as a consequences of chemotherapy.

#### **5. Conclusions**

The influence of CY on Mf can be summarized as a sum of several different mechanisms mediated by macrophages such as secretion of a specific pattern of cytokines and enhances expression of cell surface markers that can stimulate antigen presenting function by macrophages and last not least production of ROI's. On the other side there are several observations that low dose CY treatment has a direct influence of the different regulatory cells in immune system. One of them is a negative activation of CD8+ T lymphocytes leading to elimination of their effector mediators - suppressor cytokines secretion mainly TGF- and IL-10, which negatively regulate the cellular immune response, but do not express any negative effect of humoral response. That state of abrogation of unresponsiveness is also observed experimentally when the TNP-substituted Mf obtained from oil-induced donors are injected into CY treated recipients of cells (see **Figure 5**) group C. In that case instead of unresponsiveness expressed by control group (group A) strong CHS response appears 24 h after challenge. This phenomenon clearly shows the influence of CY on the suppressor network of T reg cells. This also clearly shows that CY-manipulation leads to manifold effects in which manifold can be described as wiped out or misdirected. In the literature there are two papers suggesting depletion activity of CY on Treg CD4 CD25 T lymphocytes and Treg CD4 CD25 FoxP3+ lymphocytes (Ghiringhelli et al. 2004 & Zhao et al. 2010)

1x106 TNP substituted Mf (groups A and C) or TNP-Mf CY (groups B and D) were injected i.v. into naive (groups A and B) or treated with low dose of CY CBA/J mice. Seven days later the CHS response was measured (see legend to Table 2). Statistical significance (a posteriori Bonferroni test) Group A ve groups B, C and D p<0.001.

Fig. 5. Alleviation of suppression of contact sensitivity response induced by low dose treatment with cyclophosphamide applied either to macrophage donors or recipients.

That state of tolerance eliminates the cellular immune response from the testis and in consequence makes an extremely dangerous the viral infection as well as in malignances taking place in testis. CY treatment often change TMf activity from unresponsiveness into actively antigen presenting cells which in consequence help to undertake anticancer response but also often can leads to activation an autoimmune response and immunological

The influence of CY on Mf can be summarized as a sum of several different mechanisms mediated by macrophages such as secretion of a specific pattern of cytokines and enhances expression of cell surface markers that can stimulate antigen presenting function by macrophages and last not least production of ROI's. On the other side there are several observations that low dose CY treatment has a direct influence of the different regulatory cells in immune system. One of them is a negative activation of CD8+ T lymphocytes leading to elimination of their effector mediators - suppressor cytokines secretion mainly TGF- and IL-10, which negatively regulate the cellular immune response, but do not express any negative effect of humoral response. That state of abrogation of unresponsiveness is also observed experimentally when the TNP-substituted Mf obtained from oil-induced donors are injected into CY treated recipients of cells (see **Figure 5**) group C. In that case instead of unresponsiveness expressed by control group (group A) strong CHS response appears 24 h after challenge. This phenomenon clearly shows the influence of CY on the suppressor network of T reg cells. This also clearly shows that CY-manipulation leads to manifold effects in which manifold can be described as wiped out or misdirected. In the literature there are two papers suggesting depletion activity of CY on Treg CD4 CD25 T lymphocytes

and Treg CD4 CD25 FoxP3+ lymphocytes (Ghiringhelli et al. 2004 & Zhao et al. 2010)

1x106 TNP substituted Mf (groups A and C) or TNP-Mf CY (groups B and D) were injected i.v. into naive (groups A and B) or treated with low dose of CY CBA/J mice. Seven days later the CHS response was measured (see legend to Table 2). Statistical significance (a posteriori Bonferroni test)

Fig. 5. Alleviation of suppression of contact sensitivity response induced by low dose treatment with cyclophosphamide applied either to macrophage donors or recipients.

infertility as a consequences of chemotherapy.

Group A ve groups B, C and D p<0.001.

**5. Conclusions** 

A single administration of low dose of CY (50 mg/kg) into either donors or recipients restores the ability of Mf to induce significant CS reaction as a result of: i.) elimination of suppressive properties of Mf; ii.) and/or depletion of population of regulatory T cells in recipients or iii.) elimination of their suppressive activities. In vitro studies with metabolites of CY in contrast to studies in vivo allow identifying the factors which express direct action on selected populations of cells in contrary to experimental research in vivo which is able to identify parallel with direct also indirect effects of cyclophosphamide action on other than macrophages cell populations ( T reg cells) that may change and modulate the activity of macrophages and their influence on the immune response. We propose the schemes which summarize the influence of low doses of CY on the immune response in mice (**Figures 6a-6c**).

Figure 6 a-c. The network of the CY influence on the macrophage and regulatory T cells in mice.

\* Induction of T suppressor factor (see the reference by Bryniarski et al. 2-nd European Congress of Immunology Berlin 2009)

Fig. 6.a. Activation of unresponsiveness in recipients after i.v. injection of TNP substituted Mf – lack of CHS reaction.

The Influence of Cyclophosphamide on Immune Function of Murine Macrophages 159

The work is supported by grant No K/ZDS/001429 to KB. The author expresses gratitude to Ms. Katarzyna Nazimek M.Sc. for precious help in preparing the Figures and Tables.

Adjalle, R. Plouin, P. F. Pacak, K. & Lehnert, H. (2009). Treatment of malignant

Audia, S. Nicolas, A. Cathelin, D Larmonier N, Ferrand C, Foucher P, Fanton A, Bergoin E,

Ben-Efraim, S. (2001). Immunomodulating anticancer alkylating drugs targets and

Breitfeld, P. P. & Meyer, W. H. (2005). Rhabdomyosarcoma: New windows of opportunity.

Bryniarski, K. Ptak, M. & Ptak, W. (1996). The in vivo and in vitro effects of an alkylating

Bryniarski, K. Szczepanik, M. Maresz, K. Ptak, M. & Ptak, W. (2004). Subpopulations of

Bryniarski, K. Szczepanik, M. Ptak, M. Zemelka, M. & Ptak, W. (2009). Influence of

Bryniarski, K. Ptak, M. Sikora, E. Szczepanik, M. Guerrier-Takada, C. Altman, S. Askenase,

response. Medimond International Proceedings ECI Berlin 2009 pp 183-186. Burger, R. A. (2007). Experience with Bevacizumab in the management of epithelial ovarian

Cole, M. E. Broaddus, R. Thaker, P. Lauden, C. & Freedman, R. S. (2008). Placenta – side of

Czajkowska, B. Ptak, M. Bobek, M. Bryniarski, K. & Szczepanik, M. (1995). Different

Durhan, N. Singh, S. Singh Kadin, Y. Durhan, V. Rajotia & N. Sangwen N. (2009). Primary

Ghiringhelli, F. Larmounier, N. Schmidt, E. Parcellier, A. Cathelin, D. Garrido, C. Chauffert,

macrophages in mice. *Pharmacological Reports*. Vol. 61, pp. 550-557.

cells. *Journal of Clinical Oncology*. Vol. 25 (20) Jul.10, pp. 2902-2908.

agent, mechlormethamine, on IL-6 production in mice and the role of macrophages.

mouse testicular macrophages and their immunoregulatory function. *American* 

cyclophosphamide and its metabolic products on the activity of peritoneal

P.W. Ptak, W. (2009) Role of low molecular weight RNA in contact sensitivity

trophoblastic tumors: case of resistant pulmonary metastasis. *Nature, Clinical* 

isoenzyme patterns of nonspecific esterases and the level of IL-6 production as markers of macrophage functions. *Folia Histologica et Biologica* Vol. 33, (2), pp. 111-

leiomyosarcoma of broad ligament: case report and review of literature. *Arch* 

B. Solary, E. Bonnotte, B. & Martin F. (2004). CD4+CD25+ regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows

Maynadie M, Arnould L, Bateman A, Lorcerie B, Solary E, Chauffert B, Bonnotte B.(2007). Increase of CD4+CD25high regulatory T cells in the peripheral blood of patient with metastatic carcinoma: a phase I clinical trial using cyclophosphamide and immunotherapy to eliminate CD4+CD25+ T lymphocytes. *Clinical and* 

pheochromocytoma. *Horm Metabol Res.* Vol. 41, pp. 687-696.

mechanisms of activity. Curr Drug Targ Vol. 2, pp. 197-212.

*Journal of Reproductive Immunology.* Vol. 52, pp. 27-35.

*Experimental Immunology*. Vol. 150, pp. 532-30.

*The Oncologist*. Vol. 10, pp. 518-527.

*Immunopharmacology.* Vol. 34, pp. 73-78.

*Practice, Oncology.* Vol. 5, (3), pp. 171-175.

*Gynocol Obstet.* Vol. 279, pp. 705-708.

115.

**6. Acknowledgments** 

**7. References** 

Fig. 6.b. The network of the CY influence on the macrophage and regulatory cells in mice. The i.v. injection into naïve recipient of TNP substituted Mf harvested from CY treated donors results in a state of high CHS reaction 24 h after challenge.

Fig. 6.c. The network of the CY influence on the macrophage and regulatory cells in mice. The i.v. injection of TNP substituted Mf harvested from naïve donors into recipients previously treated with low dose of CY results in expression of high CHS reaction 24 h after challenge with PCL hapten. The activation of CHS is the effect of blocking of natural reg T cells or antigen specific Ts cells.

## **6. Acknowledgments**

The work is supported by grant No K/ZDS/001429 to KB. The author expresses gratitude to Ms. Katarzyna Nazimek M.Sc. for precious help in preparing the Figures and Tables.

#### **7. References**

158 Pharmacology

Fig. 6.b. The network of the CY influence on the macrophage and regulatory cells in mice. The i.v. injection into naïve recipient of TNP substituted Mf harvested from CY treated

Fig. 6.c. The network of the CY influence on the macrophage and regulatory cells in mice. The i.v. injection of TNP substituted Mf harvested from naïve donors into recipients

cells or antigen specific Ts cells.

previously treated with low dose of CY results in expression of high CHS reaction 24 h after challenge with PCL hapten. The activation of CHS is the effect of blocking of natural reg T

donors results in a state of high CHS reaction 24 h after challenge.


**8** 

*1Indonesia 2The Netherlands* 

**Modification of Interleukin-10 with Mannose-**

**6-Phosphate Groups Yields a Liver-Specific** 

*2University of Groningen, Dept. of Pharmacokinetics, Toxicology and Targeting,* 

Cytokines and other biological compounds are considered as future drugs and they are of particular interest for the treatment of chronic diseases. These endogenous compounds, that normally mediate local cellular communications, are very promising candidates to generate new drugs because of their high potency (pM-nM concentrations) and their fundamental roles in pathological processes. However, the therapeutic application of cytokines is limited, because several problems are encountered with their application *in* vivo (Schooltink and Rose-John 2002; Standiford 2000; Vilcek and Feldmann 2004). For instance, some cytokines are efficiently degraded in plasma by various enzymes and cytokines are rapidly excreted by the kidneys. Consequently their residence time in the body and thus the exposure to the diseased cells is short (plasma half life is often minutes), which does not favour an optimal biological efficacy. Another major problem is the occurrence of side effects. Because cytokine receptors are ubiquitously expressed in all organs, unusual high plasma concentrations of

To overcome these problems, we use drug targeting techniques to selectively deliver the cytokine to a specific (diseased) cell (Allen and Cullis 2004; Beljaars, Meijer, Poelstra 2002). The challenge is to improve its distribution within the body and direct the cytokine to a cell of interest, while maintaining the biological activity of that particular cytokine after chemical modification. A conventional way to modify proteins is conjugation with polyethylene glycol (PEG) (Jevsevar, Kunstelj, Porekar 2010). The attachment of PEG moieties improves the pharmacokinetics. That is, PEG substitution prevents rapid renal elimination which results in compounds with prolonged plasma concentrations, thereby making a reduced number of doses possible. For instance, PEGasys (PEGylated interferon 2a), an example of a PEGylated cytokine that is now commonly used to treat patients infected with viral hepatitis, is dosed once a week while the unmodified interferon is dosed daily. This leads to an improvement in the compliance and quality of life in patients with chronic

the cytokine can lead to (unwanted) effects in various organs.

diseases. The side effects, however, are not diminished after PEGylation.

**1. Introduction** 

**Cytokine with Antifibrotic Activity in Rats** 

Catharina Reker-Smit2, Klaas Poelstra2 and Leonie Beljaars2

*1Pharmaceutics, Bandung Institute of Technology, Bandung* 

Heni Rachmawati1,2, Adriana Mattos2,

immunotherapy of established tumors to be curative. *European Journal of Immunology*. Vol. 34 pp.336-344.

