TNF-like weak inducer of apoptosis (TWEAK) is a TNF family member with pleiotropic effects on a variety of cell types, one of which is the induction of proinflammatory cytokines by synovial fibroblasts derived from rheumatoid arthritis (RA) patients. In this study, we report that the serum TWEAK level was dramatically elevated during mouse collagen-induced arthritis (CIA) and blocking TWEAK by a neutralizing mAb significantly reduced the clinical severity of CIA. Histological analyses also revealed that TWEAK inhibition diminished joint inflammation, synovial angiogenesis, as well as cartilage and bone erosion. Anti-TWEAK treatment proved efficacious when administered just before the disease onset but not during the priming phase of CIA. Consistent with this, TWEAK inhibition did not affect either cellular or humoral responses to collagen. In contrast, TWEAK inhibition significantly reduced serum levels of a panel of arthritogenic mediators, including chemokines such as MIP-1β (CCL-4), lymphotactin (XCL-1), IFN-γ-inducible protein 10 (IP-10) (CXCL-10), MCP-1 (CCL-2), and RANTES (CCL-5), as well as the matrix metalloprotease-9. Exploring the possible role of the TWEAK/Fn14 pathway in human RA pathogenesis, we showed that TWEAK can target human primary chondrocytes and osteoblast-like cells, in addition to synovial fibroblasts. We further demonstrated that TWEAK induced the production of matrix metalloproteases in human chondrocytes and potently inhibited chondrogenesis and osteogenesis using in vitro models. These results provide evidence for a novel cytokine pathway that contributes to joint tissue inflammation, angiogenesis, and damage, as well as may inhibit endogenous repair, suggesting that TWEAK may be a new therapeutic target for human RA.

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in folding and stabilizing multiple intracellular proteins that have roles in cell activation and proliferation. Many Hsp90 client proteins in tumor cells are mutated or overexpressed oncogenic proteins driving cancer cell growth, leading to the acceptance of Hsp90 as a potential therapeutic target for cancer. Because several signal transduction molecules that are dependent on Hsp90 function are also involved in activation of innate and adaptive cells of the immune system, we investigated the mechanism by which inhibiting Hsp90 leads to therapeutic efficacy in rodent models of inflammation and autoimmunity. EC144, a synthetic Hsp90 inhibitor, blocked LPS-induced TLR4 signaling in RAW 264.7 cells by inhibiting activation of ERK1/2, MEK1/2, JNK, and p38 MAPK but not NF-κB. Ex vivo LPS-stimulated CD11b+ peritoneal exudate cells from EC144-treated mice were blocked from phosphorylating tumor progression locus 2, MEK1/2, and ERK1/2. Consequently, EC144-treated mice were resistant to LPS administration and had suppressed systemic TNF-α release. Inhibiting Hsp90 also blocked in vitro CD4+ T cell proliferation in mouse and human MLRs. In vivo, semitherapeutic administration of EC144 blocked disease development in rat collagen-induced arthritis by suppressing the inflammatory response. In a mouse collagen-induced arthritis model, EC144 also suppressed disease development, which correlated with a suppressed Ag-specific Ab response and a block in activation of Ag-specific CD4+ T cells. Our results describe mechanisms by which blocking Hsp90 function may be applicable to treatment of autoimmune diseases involving inflammation and activation of the adaptive immune response.

Integrin ␣ 4 ␤ 1 plays an important role in inflammatory processes by regulating the migration of lymphocytes into inflamed tissues. Here we evaluated the biochemical, pharmacological, and pharmacodynamic properties and efficacy in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, of two types of ␣ 4 ␤ 1 inhibitors, the anti-rat ␣ 4 monoclonal antibody TA-2 and the small molecule inhibitor. TA-2 has been extensively studied in rats and provides a benchmark for assessing function. BIO5192 is a highly selective and potent (K D of Ͻ10 pM) inhibitor of ␣ 4 ␤ 1 . Dosing regimens were identified for both inhibitors, which provided full receptor occupancy during the duration of the study.Both inhibitors induced leukocytosis, an effect that was used as a pharmacodynamic marker of activity, and both were efficacious in the EAE model. Treatment with TA-2 caused a decrease in ␣ 4 integrin expression on the cell surface, which resulted from internalization of ␣ 4 integrin/TA-2 complexes. In contrast, BIO5192 did not modulate cell surface ␣ 4 ␤ 1 . Our results with BIO5192 indicate that ␣ 4 ␤ 7 does not play a role in this model and that blockade of ␣ 4 ␤ 1 /ligand interactions without down-modulation is sufficient for efficacy in rat EAE. BIO5192 is highly selective and binds with high affinity to ␣ 4 ␤ 1 from four of four species tested. These studies demonstrate that BIO5192, a novel, potent, and selective inhibitor of ␣ 4 ␤ 1 integrin, will be a valuable reagent for assessing ␣ 4 ␤ 1 biology and may provide a new therapeutic for treatment of human inflammatory diseases.Integrins are a large family of cell surface receptors that mediate cell/cell and cell/matrix interactions and signal transduction. They exist as noncovalent ␣␤ heterodimers of different combinations of ␣ and ␤ chains and share extensive structural homology. The leukocyte integrin ␣ 4 ␤ 1 regulates normal lymphocyte trafficking (Lobb and Hemler, 1994) and provides a key costimulatory signal supporting cell activation (Clark and Brugge, 1995). During inflammatory responses, it regulates lymphocyte migration into the damaged tissues and thus has been recognized as an attractive therapeutic target. In vivo studies using blocking monoclonal antibodies (Lobb and Hemler, 1994) and inhibitory peptides (Molossi et al., 1995) have verified the critical role of ␣ 4 ␤ 1 integrins in leukocyte-mediated inflammation. Numerous EAE models of multiple sclerosis have been designed to recapitulate important aspects of the disease and are responsive to ␣ 4 inhibitors (Yednock et al., 1992). Recent positive phase II data using the anti-␣ 4 antibody 1 Current address: Wyeth, Cambridge, MA.

Integrin ␣4␤1 plays an important role in inflammatory processes by regulating the migration of leukocytes into inflamed tissues. Previously, we identified BIO5192 [2(S)-{[1-(3,5-dichloro-benzenesulfonyl)-pyrrolidine-2(S)-carbonyl]-amino}-4-[4-methyl-2(S)- (methyl-{2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl}-amino)-pentanoylamino]-butyric acid], a highly selective and potent (K D of 9 pM) small molecule inhibitor of ␣4␤1. Although BIO5192 is efficacious in various animal models of inflammatory disease, high doses and daily treatment of the compound are needed to achieve a therapeutic effect because of its relatively short serum half-life. To address this issue, polyethylene glycol modification (PEGylation) was used as an approach to improve systemic exposure. BIO5192 was PEGylated by a targeted approach in which derivatizable amino groups were incorporated into the molecule. Two sites were identified that could be modified, and from these, five PEGylated compounds were synthesized and characterized. One compound, 2a-PEG (K D of 19 pM), was selected for in vivo studies. The pharmacokinetic and pharmacodynamic properties of 2a-PEG were dramatically improved relative to the unmodified compound. The PEGylated compound was efficacious in a rat model of experimental autoimmune encephalomyelitis at a 30-fold lower molar dose than the parent compound and required only a once-aweek dosing regimen compared with a daily treatment for BIO5192. Compound 2a-PEG was highly selective for ␣4␤1. These studies demonstrate the feasibility of PEGylation of ␣4␤1-targeted small molecules with retention of activity in vitro and in vivo. 2a-PEG, and related compounds, will be valuable reagents for assessing ␣4␤1 biology and may provide a new therapeutic approach to treatment of human inflammatory diseases.Integrins are a large family of cell surface receptors that mediate cell-cell and cell-matrix interaction. They exist as noncovalent ␣␤ heterodimers of different combinations of ␣ and ␤ chains and share extensive structural homology. Integrins mediate a wide variety of physiological processes and are relevant to a wide variety of pathological conditions. The integrin ␣4␤1 regulates normal leukocyte trafficking (Lobb and Hemler, 1994) and provides a key costimulatory signal supporting cell activation (Clark and Brugge, 1995). During inflammatory responses, ␣4␤1 regulates leukocyte migration into the damaged tissues and has been recognized as an attractive therapeutic target. In vivo studies using blocking monoclonal antibodies (Lobb and Hemler, 1994;Issekutz et al., 1996;Enders et al., 1998;Hojo et al., 1998;Schneider et al., 1999;Ramos-Barbon et al., 2001), inhibitory peptides (Molossi et al., 1995;Abraham, 1997;van der Laan et al., 2002), and small molecule antagonists (Lin et al., 1999;Kudlacz et al., 2002) have verified the critical role of ␣4␤1 integrins in leukocyte-mediated inflammation and have implicated ␣4␤1 inhibitors as potential treatments for diseases such as asthma and arthritis. Numerous EAE models that recapitulate i...

In studies using genetically deficient mice, a role for the lymphotoxin (LT) system in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) has remained controversial. Here, we have reassessed this conclusion by using a fusion protein decoy that blocks the LT pathway in vivo without evoking the developmental defects inherent in LT-deficient mice. We have found that inhibition of the LT pathway prevented disease in two models of EAE that do not rely on the administration of pertussis toxin. Surprisingly, disease attenuation was due to specific blockade of LTαβ binding rather than the binding of LIGHT to its receptors. In a third system that requires pertussis toxin, LT inhibition did not affect disease, as was observed when the same model was used with LT-deficient mice. Disease prevention in pertussis toxin-free models was associated with defects in T cell responses and migration. When the DO11.10 T cell transgenic system was used, inhibition of the LT pathway was shown to uncouple T cell priming from T cell recall responses. Therefore, it is hypothesized that the LT pathway and its ability to maintain lymphoid microenvironments is critical for sustaining late-phase T cell responses in multiple sclerosis.

In studies using genetically deficient mice, a role for the lymphotoxin (LT) system in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) has remained controversial. Here, we have reassessed this conclusion by using a fusion protein decoy that blocks the LT pathway in vivo without evoking the developmental defects inherent in LT-deficient mice. We have found that inhibition of the LT pathway prevented disease in two models of EAE that do not rely on the administration of pertussis toxin. Surprisingly, disease attenuation was due to specific blockade of LTαβ binding rather than the binding of LIGHT to its receptors. In a third system that requires pertussis toxin, LT inhibition did not affect disease, as was observed when the same model was used with LT-deficient mice. Disease prevention in pertussis toxin–free models was associated with defects in T cell responses and migration. When the DO11.10 T cell transgenic system was used, inhibition of the LT pathway was shown to uncouple T cell priming from T cell recall responses. Therefore, it is hypothesized that the LT pathway and its ability to maintain lymphoid microenvironments is critical for sustaining late-phase T cell responses in multiple sclerosis

No abstract