93 PGE2-mediated chronic inflammation: A role for IFN-γ?

Abstracts / Cytokine 43 (2008) 243–262 Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), is a group of immune mediated disorders of the intestine and/or colon. These diseases are characterized by a dysregulated T cell activity, an aberrant cytokine production and cellular inflammation. This uncontrolled immune responses results in the sustained overproduction of reactive metabolites of oxygen and nitrogen. Nitric oxide (NO) is an important mediator of inflammation and cytotoxic responses. This leads to intestinal tissue damage in IBD. The aim of this study was to evaluate in vivo and in vitro nitric oxide production in IBD Algerian patients with different degrees of colonic mucosal injury. Moreover, we investigated whether Th1 and Th2 cytokines (IFN-c, IL-4, and IL-13) could modulate nitric oxide production by PBMC of UC (n = 27) and CD (n = 29) patients with active and inactive phases of disease. Our in vivo results show a significantly higher production of NO in IBD patients compared to healthy controls. This production was related to clinical outcome of disease. The in vitro experiments show the involvement of IFN-c in the production of nitric oxide during active and inactive phases of IBD. Furthermore, Th2 cytokines (IL-4, and IL-13) suppressed NO production by PBMC of UC and CD patients. In conclusion, the results reported here, show the implication of NO production in physiopathology of IBD disease. This production was modulated by Th1 and Th2 cytokines. doi:10.1016/j.cyto.2008.07.129

89 Suppression of pro-inflammatory cytokines and mediators expression by brown algae Sargassum micracanthum extracts in murine macrophage RAW 264.7 cells Weon-Jong Yoon 1,2, Kil-Nam Kim 1, Ji-Young Kim 1, Hee-Jung Kim 1, Soo-Yeong Park 1, Wook Jae Lee 1, Chang-Gu Hyun 1, 1 Jeju Biodiversity Research Institute, Jeju Hi-Tech Industry Development Institute, Jeju 690-121, Republic of Korea, 2 Department of Pharmacology, College of Medicine, Cheju National University, Jeju 690-756, Republic of Korea This study describes a preliminary evaluation of the anti-inflammatory activity of Sargassum micracanthum extracts. Sargassum micracanthum was extracted using 80% ethanol and then fractionated sequentially with n-hexane, dichloromethane, ethylacetate, and butanol. To screen for anti-inflammatory agents effectively, we first examined the inhibitory effect of the S. micracanthum extracts on the production of pro-inflammatory cytokines (TNF-a, IL-1b and IL-6) activated with lipopolysaccharide (LPS). In addition, we examined the inhibitory effect of S. micracanthum extracts on pro-inflammatory factors (NO, iNOS, COX-2 and PGE2) in murine macrophage RAW 264.7 cells. Protein levels were quantified using immunoblotting. Of the sequential solvent fractions of S. micracanthum, the n-hexane and dichloromethane fractions inhibited the mRNA expression of pro-inflammatory cytokines, production of NO and PGE2, and the protein level of iNOS and COX-2. These results suggest that S. micracanthum has significant effects on inflammatory factors and is a possible antiinflammatory therapeutic marine algae. Keywords: Pro-inflammatory cytokines, Brown algae, Sargassum micracanthum, RAW 264.7 cells. doi:10.1016/j.cyto.2008.07.130

90 Modulation of macrophage infiltration and inflammatory activity by the phosphatase SHP-1 in virus-induced demyelinating disease George P. Christophi 1,2, Chad A. Hudson 1,2, Ross Gruber 1, Michael Panos 1, Paul T. Massa 1,2, 1 Department of Neurology and SUNY Upstate Medical University, Syracuse, NY, USA, 2 Department of Microbiology & Immunology, SUNY Upstate Medical University, Syracuse, NY, USA The protein tyrosine phosphatase SHP-1 is a crucial negative regulator of cytokine signaling and inflammatory gene expression both in the immune and central nervous system (CNS). Mice genetically lacking SHP-1 (me/me) display severe inflammatory demyelinating disease following inoculation with the Theiler’s murine encephalomyelitis virus (TMEV) compared to infected wild type mice. Therefore, it became essential to investigate the mechanisms of TMEV-induced inflammation in the CNS of SHP-1 deficient mice. Herein, we show that several genes relevant to inflammatory demyelination are elevated in the CNS of infected me/me mice compared to wild type mice. Furthermore, SHP-1 deficiency led to a dramatic and exclusive increase in infiltrating CD45hi CD11b+ Ly-6Chi macrophages in the CNS of me/me mice in concert with the development of paralysis. Isolated CNS infiltrating macrophages from infected me/me mice showed increased amounts of viral RNA and enhanced inflammatory profile compared to wild type macrophages. Importantly, application of clodronate liposomes effectively depleted splenic and CNS infiltrating macrophages and significantly delayed the onset of TMEV-induced paralysis. Furthermore, macrophage depletion resulted in lower viral loads and inflammatory gene expression in the spinal cords of me/ me mice. Finally, me/me macrophages were more responsive to chemoattractive

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stimuli secreted by me/me glia serving as a mechanism for the increased numbers of infiltrating macrophages seen in the CNS of me/me mice. Taken together, infiltrating macrophages in SHP-1-deficient mice play a crucial role in promoting viral replication by providing abundant viral targets and contributing to increased pro-inflammatory gene expression relevant to the effector mechanisms of macrophage-mediated demyelination. doi:10.1016/j.cyto.2008.07.131

91 The use of cytokine response profiles to inform viral vaccine development Stacie Lambert, Kathy Wang, Jennifer Woo, George Kemble, MedImmune, LLC, Mountain View, CA, USA Viral respiratory diseases such as seasonal and pandemic influenza cause considerable morbidity and mortality in the human population, particularly the old and the young. Vaccination against these viruses can minimize their detrimental effects. Cytokine response profiles could provide a valuable tool for understanding the immune response mechanisms associated with vaccine responses. The development of systems to compare cytokine responses to viral vaccines in animal models such as ferret and mouse and their application to humans are in progress. In the ferret model, the recent publication of the sequences of several key cytokines has allowed the use of Taqman-based quantitative PCR (qPCR) methods to track these responses. We have applied qPCR to measure the induction of ferret cytokines such as TNF-a and IFN-c by vaccine components in both in vitro and in vivo studies. Correlating cytokine response profiles with protective vaccine responses should allow the development of even more effective vaccines against respiratory viruses. doi:10.1016/j.cyto.2008.07.132

92 IFNc modulation by IL 18, IL 4 and IL 10 during BEHCET disease evolution Houda Belguendouz 1, Djamel Messaoudene 1, Mohammed L. Ahmedi 1, Karima Lahmar 1, Fatma Z. Ider 1, Oussama Medjber 1, Dahbia Hartani 2, Chafia TouilBoukoffa 1, 1 ‘‘Cytokines & NO Synthases” group, MCB laboratory, FSB, USTHB, Algiers, Algeria, 2 Department of Ophtalmology, CHU Mustapha Bacha, Algiers, Algeria Behçet disease (BD) is an inflammatory, chronic, systemic affection with uncertain etiology. We and others showed both in vivo and in vitro significantly higher cytokine production during BD when compared to controls. In order to investigate the implication of Th1, Th2 and T regulatory (T reg) cytokines in the modulation of IFNc production in vitro, PBMC from venous blood of 21 patients with BD and 15 controls were cultured with or without IL18 (125 pg/ml), anti –IL4 (1/100) and anti- IL10 (1/100) (Sigma-Aldrich, Germany) in RPMI 1640 supplemented by 5% FBS, 100 UI/ml of streptomycin, 100 lg/ml penicillin and 2 mM of glutamine. After incubation in humidified chamber at 37 °C in 5% CO2 for 24 h, culture supernatants were harvested and IFNc was measured by ELISA Sandwich (Immunotech, Coulter Beckman, UK). Statistical study was performed by the non parametrical Mann Whitney U test. Our results showed higher IFNc production in BD patients when compared to controls (p < 0.05). We observed different IFNc production profile depending on disease duration. IFNc levels were significantly higher in culture supernatants of patients with disease duration 65 years (p < 0.05). These patients showed a Th1/T reg profile with significant IFNc rate elevation in IL18 and anti- IL10 treated culture supernatants (p < 0.01) while no significant responses where observed in anti-IL4 treated culture ones when compared to untreated culture supernatants (p = 0.2). Conversely, patients with BD duration >5 years showed significant elevation in IFNc production in IL18, anti-IL4 and anti-IL10 treated culture supernatants when compared to untreated culture supernatants (p < 0.05) reflecting a Th1/Th2/Treg cytokine profile. In conclusion, our results showed differences in patients PBMC responses depending on disease duration. These results suggest time developing Th2 profile probably induced by long administration of corticoids to patients with BD. doi:10.1016/j.cyto.2008.07.133

93 PGE2-mediated chronic inflammation: A role for IFN-c? Alex G. Therien, Simon Lord-Dufour, Virginie Bernier, Yves Boie, Jason D. Burch, Patsy Clark, Danielle Denis, Yongxin Han, James R. Mortimer, Marie-Claude Mathieu, Merck Frosst Center for Therapeutic Research, Kirkland, Canada It is well established that cox-2 derived prostanoids, in particular PGE2, play an important role in chronic inflammatory diseases such as rheumatoid arthritis (RA).

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Indeed, not only are cox-2 inhibitors (coxibs) clinically efficacious in RA, we and others have shown that genetic or pharmacological blockade of the PGE2 receptor EP4 has coxib-like efficacy in pre-clinical models of inflammation and inflammatory pain. Here we probed the PGE2/EP4 activation pathway to identify potential downstream mechanisms that may play a role in inflammation. In an initial gene expression analysis of human blood-derived leukocytes, subsequently confirmed at the protein level, we found that activation of EP4 is associated with inhibition of IFN-c and IFN-c-associated genes. This effect of the PGE2/EP4 axis on IFN-c is a reciprocal phenomenon since IFN-c blocks PGE2 release and blocks EP receptor expression. The mutually antagonistic relationship between IFN-c and PGE2 extends to downstream cytokine- and chemokine-release as well, since PGE2 counters the effects of IFN-c on the release of IP-10, IL-8, TNFa and IL-1b. To gain a better understanding of local (i.e. in the joint), rather than systemic RA pathology, we extended our analysis of PGE2 and IFN-c mediated events to primary human fibroblast-like synoviocytes. Overall, we observed an antagonistic effect of these two mediators on expression of neutrophil and macrophage-attracting chemokines as well as of metalloproteases. These results suggest the existence of a mutually antagonistic relationship between PGE2 and IFN-c which may represent a fundamental mechanism of immune control in diseases such as RA. doi:10.1016/j.cyto.2008.07.134

94 Leucine-rich diet and ascorbic acid affect the proteolysis-inducing factor level and the muscle antioxidant stress response in tumor-bearing rats T.C. Marcondes, B.L.G. Cruz, E.M. Salomão, M.C.C. Gomes-Marcondes, Departament of Physiology and Biophysics, Biology Institute, State University of Campinas, UNICAMP, 13083970 Campinas, SP, Brazil Cancer induces several changes in host cancer, increasing the oxidative stress and protein waste, and leading the cancer-cachexia state. Branched-chain amino acid leucine improves muscle protein synthesis and cell signalling. The ascorbic acid acts improving the antioxidant response. This work evaluated the effects of Walker 256 tumour development on the serum proteolysis-inducing factor (PIF), a-fetoprotein (AFP), and TNFa and the muscle‘s protein and oxidative stress in young rats when submitted to leucine-rich diet and vitamin C. Weanling Wistar rats were distributed into 8 groups: C, control; L, leucine; CC, control with vitamin C in drink water; LC, leucine and vitamin C; W, tumour-bearing; WL, tumour-bearing fed leucine diet; WC, tumour-bearing received vitamin C in drink water; WCL, tumour-bearing fed leucine-rich diet and vitamin C. The skeletal muscle protein and alkaline phosphatase activity was decreased and associated to increase on muscle malondialdehyde (MDA) content in W, WL and WC groups; in addition the leucine and vitamin C (WLC rats) maintained the MDA level and the glutathione-S-transferase activity in gastrocnemius. Although, tumour weight was similar in all groups, the leucine and vitamin C affected positively the survival time: WC and WCL rats survived 2–3 days more than the other groups (14 days tumour evolution). The cytokines levels were altered in tumour-bearing rats: the PIF was increased in all groups, especially in WC and WCL, even though these groups presented similar muscle protein content compared to the control group; the TNFa was increased in all tumour-bearing groups and AFP was similar among the controls and tumour-bearing groups. Although the leucine-rich diet associated to vitamin C enhanced the survival time, this nutritional supplementation did not prevent the increase on proteolysis-inducing factor level, even though could improved the muscle antioxidant response and protein content, especially in this tissue which was mainly wasted in cachexia state. Financial support FAPESP, CNPq, CAPES, FAEP-UNICAMP doi:10.1016/j.cyto.2008.07.135

95 Plasma derived HBsAG inhibits toll-like receptor-2 ligand induced expression of interleukin-12 and CD80 in monocytic cell line THP-1 Chen Zhiao 1,2, Cheng Yuming 1,2, Hu Yuwen 2, Yuan Zhenghong 1,2, 1 Key Laboratory of Medical Molecular Virology, Ministry of Education and Health Shanghai Medical College, Fudan University, Shanghai, China, 2 Shanghai Public Health Clinical Center, Fudan University, Shanghai, China Previous studies have shown that hepatitis B virus suppresses human immune system and HBsAg inhibits the release of LPS-induced cytokines in human monocytes. However, the mechanism by which HBsAg affects the function of monocyte remains unknown. In this study, we examined if HBsAg influences the toll like receptor 2 (TLR2) ligand-induced gene expression and related signaling pathways in macrophage-like cells derived from monocytic cell line THP-1. The macrophage-like cells were pretreated with plasma-derived HBsAg and then stimulated with TLR1/2 ligand (pam3csk4). Expression of cytokines and costimulatory molecules was detected to assess the activation of macrophage-like cells. Results showed that plasma-derived

HBsAg inhibited pam3csk4 induced production of IL-12 and expression of CD80 in a dose dependent manner. In order to further study how HBsAg affect TLR signaling pathway, NF-jB pathway which is downstream of TLR signaling was detected by phosflow. Results showed that the number of the cells positive for phosflow NFjBp65 was significant lower in HBsAg-treated cells than that in the control cells after the pam3csk4 stimulation. In conclusion, the alteration of TLR-mediated signals by plasma-derived HBsAg may be one of the mechanisms of HBV-induced immune modulation, which may contribute to pathogenesis of HBV chronic infection. doi:10.1016/j.cyto.2008.07.136

96 Injectable, thermo-reversible and complex coacervated combination gels containing IL-1 receptor antagonis inhibits IL-1 b-induced MMP-3 expression in human osteoarthritis chondrocytes Jae-Bum Jun 1, Young-In Na 2, Choong Hyeok Choi 3, Jang Kyoung Kim 4, Yong-Hee Kim 4, 1 Department of Rheumatology, The Hospital for Rheumatic Diseases, Hanyang University, Seoul, Republic of Korea, 2 Institute of Rheumatism, Hanyang University, Seoul, Republic of Korea, 3 Department of Surgery for Rheumatism, The Hospital for Rheumatic Diseases, Hanyang University, Seoul, Republic of Korea, 4 Department of Bioengineering, College of Engineering, Hanyang University, Seoul, Republic of Korea IL-1 has been implicated in degradation of articular cartilage in various arthritides including osteoarthritis (OA). Therefore, blocking of IL-1 by IL-1 receptor antagonist (IL-1Ra) may be the pathogenesis-based strategy for inhibiting degradation of cartilage matrix. We investigated the role of injectable, thermo-reversible and complex coacervated combination gels containing IL-1Ra for matrix degradation in OA.The combination gels, injectable to the joint, were formed in aqueous solution by preparing complex coacervate with IL-1Ra and cationic macromolecules, and co-formulating with methylcellulose. The gels containing IL-1Ra were positioned in upper insert of transwell system, and human OA chondrocytes were placed in lower compartment and stimulated with IL-1b. Expression of matrix metalloproteinase 3 (MMP-3) was examined by real time PCR and ELISA.Release profile of IL-1Ra from the gels showed a sustained release pattern through 6 days with minimal initial bursts. IL-1Ra-loaded gels after 5 days of formation were used. IL-1b markedly induced the expression of MMP-3 gene and protein. However, loading of combination gels containing IL-1Ra profoundly inhibited IL-1b-induced MMP-3 expression in human OA chondrocytes. We are planning to examine the effectiveness of 30 day-matured gels in degradation of extracellular matrix with articular chondrocytes and cartilage explants.We applied our optimized novel in situ gel depot system containing IL-1Ra to human OA chondrocytes in transwell system. According to our results, intraarticular injection of our gels containing IL-1Ra into OA knee will provide a prolonged therapeutic option based on the pathophysiology in the treatment of knee OA. doi:10.1016/j.cyto.2008.07.137

97 Estimation of cytokine levels and its ratio for autoimmune pathology, adenoma and cancer of thyroid glands Sergei P. Kazakov 1, Nikolai Ev. Kushlinsky 2, 1 Research Scientific Center, Burdenko Main Military Clinical Hospital, DM, Moscow, Russia, 2 Biochemical Laboratory, Blokhin Oncology Research Center, RAMS, Moscow, Russia Cytokines investigation of thyroid autoimmune pathology, adenoma and cancer could be useful for careful diagnostics pathology and research pathogenesis habits.We had been investigated c-INF, IL-4, IL-6, IL-10, TNF-b levels and its ratio (index) c-INF/IL-10, c-INF/IL-4, b-INF/TNF-b, IL-6/c-INF from 12 patients with autoimmune pathology (Hoshimoto), 15 patients with adenoma, 24 patients with papillary cancer of thyroid glands. We used for determine cytokines levels Beckman Coulter XL-MCL flow cytometry and multiplex kits Bender Medsystems. We have got results which showed that IL-4, IL-10, TNF-b levels had increased and had been more higher from patients with papillary cancer. The level of c-INF had been decreased and had been minimal from patients with thyroid cancer. IL-6 level were minimal from patients with thyroid adenoma.Most interesting results had been got when we researched ratio (index) c-INF and others cytokines. Index c-INF/IL-10, c-INF/IL-4, c-INF/TNF-b had decreased and were most low from patients with thyroid cancer. In that time IL-6/c-INF ratio were higher by comparison donors from patients with thyroid autoimmune pathology, more low from patients with adenoma and significant increase from patients with thyroid cancer.So, the usage cytokines levels and cytokines ratio (index) particularly IL-6/c-INF permit to improve diagnostics thyroid autoimmune pathology, adenoma and cancer. doi:10.1016/j.cyto.2008.07.138