CXCR3 axis: role in inflammatory bowel disease and its therapeutic implication.

There is a great need for new intervention and prevention strategies against Crohn's disease (CD), a chronic, relapsing tissue-destructive inflammatory bowel disease (IBD). Estimates indicate more than 1 million cases of IBD in the United States occur annually, with 50% involving CD. The clinical features of CD correlate with certain mouse models of colitis, including the spontaneous colitis observed in interleukin-10 deficient (IL-10(-/-)), senescence accelerated mice (SAMP1/Yit) and trinitrobenzene sulfonic acid (TNBS)-treated mice. Chemokines undoubtedly play a pivotal role in the regulation (i.e., initiation, maintenance, and suppression) of mucosal inflammation and tissue destruction. A number of key advances have led to greater understanding of the steps responsible for colitis and the roles played by chemokines. In fact, CXCR3 and the ligands for this chemokine receptor, monokine-induced by interferon-gamma (IFN-gamma) (MIG/CXCL9), IFN-gamma-inducible 10 kDa protein (IP-10/CXCL10), and IFN-gamma-inducible T cell alpha-chemoattractant (I-TAC/CXCL11) are differentially expressed at sites of colitis in IL-10(-/-) mice and in clinical cases of CD. While we have demonstrated that antibodies directed against CXCL10 could both prevent the onset and cure of pre-existing colitis in IL-10(-/-) mice, studies by other investigators have shown the efficacy of CXCR3 blockade to mitigate colitis and other inflammatory diseases. This review describes the hallmarks of IBD, CXCL9-11, and CXCR3 expression during murine colitis and IBD, gives an overview of the antagonist therapies targeting the CXCR3 axis, details current and pending bio-therapies for IBD, and discusses what is known about the cellular and CXCR3-mediated mechanisms of colitis.

Death receptor-6 regulates the development of pulmonary eosinophilia and airway inflammation in a mouse model of asthma.

Death receptor-6 (DR6), a member of the death domain-containing TNFR superfamily, is highly expressed in lymphoid tissues and regulated upon lymphocyte activation. Targeted disruption of DR6 results in enhanced CD4(+) T cell proliferation and T helper 2 (Th2) differentiation in vitro, whereas the in vivo role of DR6 in regulating Th2 cell differentiation and effector function remains largely unknown. In the current study, we used a Th2-skewed allergic airway inflammation model induced by ovalbumin (OVA) sensitization and challenge to compare the inflammatory response in the lung of both wild type (WT) and DR6(-/-) mice. DR6(-/-) mice were protected from the development of airway inflammation as evidenced by attenuated eosinophil accumulation and reduced mucus-producing cells in the lining airways of allergen-challenged animals. Consistent with these observations, a profound reduction of Th2 cytokine production (IL-5 and IL-13) was detected in the bronchoalveolar lavage fluid (BAL). Furthermore, a significant increase in the frequency of IFN-gamma secreting cells was observed in the DR6(-/-) mouse lungs after OVA challenge, which may account for the reduced pulmonary Th2 cytokine production. These data point to a critical role of DR6 in regulating airway inflammation in the OVA-induced mouse model of asthma.

Selective role of PKCbeta enzymatic function in regulating cell survival mediated by B cell antigen receptor cross-linking.

Cross-linking of the B cell antigen receptor (BCR) results in the activation of several protein tyrosine kinases leading to phospholipase C-gamma2-dependent phospholipid hydrolysis and Ca2+ mobilization, followed by activation of the protein kinase C (PKC) family members. Sustained Ca2+ release in B lymphocytes is dependent on the membrane localization and activation of the protein tyrosine kinase BTK. Ca2+ release is a tightly regulated process involving BTK membrane localization through its phosphorylation by PKCbeta. A selective role of PKCbeta in B cell signaling was first revealed by the characterization of PKCbeta knockout mice, which displayed decreased B cell proliferation in response to various mitogenic stimuli. However, it is not clear whether the B cell defects displayed by the PKCbeta knockout mice are due a B cell developmental defect or the scaffolding function of PKCbeta, resulting in a defect in the recruitment or formation of signal transducing complex molecules. Thus, in this report we investigated the effects of pharmacologic inhibition of the catalytic function of PKCbeta on B cell survival and growth. Treatment of Daudi B lymphoma cell line with a selective PKCbeta inhibitor, LY333531, inhibited anti-IgM-induced phosphorylation of BTK on Ser180 in a concentration-dependent manner, which was concomitant with an increase in BTK activation, and Ca2+ mobilization. In primary splenic B cells, LY333531 inhibited BCR-induced B cell proliferation, but did not affect basal or LPS-induced proliferation. Finally, LY333531 treatment resulted in the induction of apoptosis of anti-IgM-activated B cells, which corroborated with their inability to up-regulate pro-survival factors, Bcl-X(L) and Bcl-2. These results support the important and selective role of the PKCbeta enzymatic function in controlling Ca2+ release during BCR signaling leading to B lymphocyte survival and growth.

Cloning and functional characterization of the rabbit C-C chemokine receptor 2.

BACKGROUND: CC-family chemokine receptor 2 (CCR2) is implicated in the trafficking of blood-borne monocytes to sites of inflammation and is implicated in the pathogenesis of several inflammatory diseases such as rheumatoid arthritis, multiple sclerosis and atherosclerosis. The major challenge in the development of small molecule chemokine receptor antagonists is the lack of cross-species activity to the receptor in the preclinical species. Rabbit models have been widely used to study the role of various inflammatory molecules in the development of inflammatory processes. Therefore, in this study, we report the cloning and characterization of rabbit CCR2. Data regarding the activity of the CCR2 antagonist will provide valuable tools to perform toxicology and efficacy studies in the rabbit model. RESULTS: Sequence alignment indicated that rabbit CCR2 shares 80 % identity to human CCR2b. Tissue distribution indicated that rabbit CCR2 is abundantly expressed in spleen and lung. Recombinant rabbit CCR2 expressed as stable transfectants in U-937 cells binds radiolabeled 125I-mouse JE (murine MCP-1) with a calculated Kd of 0.1 nM. In competition binding assays, binding of radiolabeled mouse JE to rabbit CCR2 is differentially competed by human MCP-1, -2, -3 and -4, but not by RANTES, MIP-1alpha or MIP-1beta. U-937/rabbit CCR2 stable transfectants undergo chemotaxis in response to both human MCP-1 and mouse JE with potencies comparable to those reported for human CCR2b. Finally, TAK-779, a dual CCR2/CCR5 antagonist effectively inhibits the binding of 125I-mouse JE (IC50 = 2.3 nM) to rabbit CCR2 and effectively blocks CCR2-mediated chemotaxis. CONCLUSION: In this study, we report the cloning of rabbit CCR2 and demonstrate that this receptor is a functional chemotactic receptor for MCP-1.

The G-protein coupled receptor, GPR84 regulates IL-4 production by T lymphocytes in response to CD3 crosslinking.

The orphan G-protein coupled receptor, GPR84 is highly expressed in the bone marrow, and in splenic T cells and B cells. In this study, GPR84-deficient mice were generated to understand the biological function of this orphan receptor. The proliferation of T and B cells in response to various mitogens was normal in GPR84-deficient mice. Interestingly, primary stimulation of T cells with anti-CD3 resulted in increased IL-4 but not IL-2 or IFN-gamma production in GPR84(-/-) mice compared to wild-type mice. Augmented IL-4 production in GPR84-deficient T cells was not related to increased frequency of IL-4-secreting cells in response to anti-CD3 stimulation. In fact, stimulation with anti-CD3 and anti-CD28 resulted in increased levels of IL-4 but not IFN-gamma steady-state mRNA in GPR84(-/-) T cells. In addition, Th2 effector cells generated in vitro from GPR84(-/-) mice produced higher levels of IL-4, IL-5 and IL-13 compared to wild-type mice. However, there was no detectable difference in the extent of IL-4 and IL-5 production between the two groups of mice in response to antigen stimulation of spleen cells, isolated from mice previously immunized with OVA in alum. These studies reveal a novel role for GPR84 in regulating early IL-4 gene expression in activated T cells.

An essential role for CCL3 in the development of collagen antibody-induced arthritis.

CCL3 is a C-C family chemokine detected at high levels in the synovial tissue and fluids of rheumatoid arthritis (RA) patients. CCL3 binds to the chemokine receptors CCR1 and CCR5, which are expressed by inflammatory leukocytes such as macrophages and T cells present in the affected joints of RA patients. The present study was undertaken to investigate whether absence of CCL3 prevented development of inflammation and joint destruction in anti-type II collagen monoclonal antibody (anti-CII mAb)-induced arthritis. "CCL3 null mice were different from wild-type control mice in terms of body weight loss". In addition, CCL3 null mice exhibited milder clinical and histopathological scores following administration of anti-CII mAb and endotoxin. Moreover, the release of TNF in response to systemic administration of endotoxin was not affected in CCL3 null mice compared to wild-type mice, indicating that the phenotype was not attributable to a defect in endotoxin response. These results indicate that CCL3 plays an essential role in the development of inflammation and joint destruction induced by anti-CII mAb.

CCR8 is not essential for the development of inflammation in a mouse model of allergic airway disease.

Chemokine receptors play an important role in the trafficking of various immune cell types to sites of inflammation. Several chemokine receptors are differentially expressed in Th1 and Th2 effector populations. Th2 cells selectively express CCR3, CCR4, and CCR8, which could direct their trafficking to sites of allergic inflammation. Additionally, increased expression of the CCR8 ligand, TCA-3, has been detected in affected lungs in a mouse model of asthma. In this study, CCR8-deficient mice were generated to address the biological role of CCR8 in a model of allergic airway disease. Using two different protocols of allergen challenge, we demonstrate that absence of CCR8 does not affect the development of pulmonary eosinophilia and Th2 cytokine responses. In addition, administration of anti-TCA-3-neutralizing Ab during allergen sensitization and rechallenge failed to inhibit airway allergic inflammation. These results suggest that CCR8 does not play an essential role in the pathogenesis of inflammation in this mouse model of allergic airway disease.

Defective CD19-dependent signaling in B-1a and B-1b B lymphocyte subpopulations.

Peritoneal and pleural cavities in mice and humans contain a unique population of B-lymphocytes called B-1 cells that are defective in B cell antigen receptor (BCR) signaling but have an increased propensity to produce autoantibodies. Several molecules such as Btk, Vav, and CD19 known to be important for BCR signaling have been shown to be critical for the development of B-1 cells from undefined precursors. Here we demonstrate that B-1 cell unresponsiveness to BCR cross-linking is in part due to defective signaling through CD19, a molecule known to modulate signaling thresholds in B cells. The defective CD19 signaling is manifested in reduced synergy between mIgM and CD19 to stimulate calcium mobilization in B-1 cells. BCR induced tyrosine phosphorylation of CD19 was transient in B-1 cells while it was prolonged in splenic B-2 cells. In both B-1 and B-2 cells BCR cross-linking induced a modest increase of CD19 associated Lyn, a Src family protein tyrosine kinase (PTK) thought to be important for CD19 phosphorylation. However, the tyrosine phosphorylated CD19 in B-1 cells binds less phosphatidylinositol 3-kinase (PI3-K) compared to B-2 cells. Most interestingly, we find that Vav-1 and Vav-2, proteins thought to be critical for CD19 signal transduction, are severely reduced in B-1 cells resulting in a complete absence of any CD19 associated Vav. Also we showed that both B-1a and B-1b B cells failed to proliferate in response to BCR cross-linking which in part appears to be due to defects in CD19 mediated amplification of BCR induced calcium mobilization.