Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid–polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29(+/−)/MxCre(+/−) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.

MIF is a chemokine-like inflammatory mediator that triggers leukocyte recruitment by binding to CXCR2 and CXCR4. MIF also interacts with CD74/invariant chain, a single-pass membrane-receptor. We identified complexes between CD74 and CXCR2 with a role in leukocyte recruitment. It is unknown whether CD74 also binds to CXCR4. We demonstrate that CD74/CXCR4 complexes formed when CD74 was expressed with CXCR4 in HEK293 cells. Expression of CD74-variants lacking an ER-retention signal showed CD74/CXCR4 complexes at the cell surface. Importantly, endogenous CD74/CXCR4 complexes were isolated by co-immunoprecipitation from monocytes. Finally, MIF-stimulated CD74-dependent AKT activation was blocked by anti-CXCR4 and anti-CD74 antibodies and AMD3100, whereas CXCL12-stimulated AKT activation was not reduced by anti-CD74. Thus, CD74 forms functional complexes with CXCR4 that mediate MIF-specific signaling.

CD73-deficient mice are valuable for evaluating the ability of CD73-generated adenosine to modulate adenosine receptor-mediated responses. Here we report the role of CD73 in regulating lymphocyte migration across two distinct barriers. In the first case, CD73-generated adenosine restricts the migration of lymphocytes across high endothelial venules (HEV) into draining lymph nodes after an inflammatory stimulus, apparently by triggering A2B receptors on HEV. Secondly, CD73 promotes the migration of pathogenic T cells into the central nervous system during experimental autoimmune encephalomyelitis. Experiments are in progress to determine whether this effect is also adenosine receptor-mediated and to identify the relevant adenosine receptor.

After an inflammatory stimulus, lymphocyte migration into draining lymph nodes increases dramatically to facilitate the encounter of naïve T cells with antigen-loaded dendritic cells. Here we show that CD73 (ecto-5′-nucleotidase) plays an important role in regulating this process. CD73 produces adenosine from AMP and is expressed on high endothelial venules (HEV) and subsets of lymphocytes. Cd73-/- mice have normal sized lymphoid organs in the steady state, but approximately 1.5-fold larger draining lymph nodes and 2.5-fold increased rates of L-selectin-dependent lymphocyte migration from the blood through HEV compared to wild type mice 24 hours after LPS administration. Migration rates of cd73+/+ and cd73-/- lymphocytes into lymph nodes of wild type mice are equal, suggesting that it is CD73 on HEV that regulates lymphocyte migration into draining lymph nodes. The A2B receptor is a likely target of CD73-generated adenosine, as it is the only adenosine receptor expressed on the HEV-like cell line KOP2.16 and it is up regulated by TNFα. Furthermore, increased lymphocyte migration into draining lymph nodes of cd73-/- mice is largely normalized by pretreatment with the selective A2B receptor agonist BAY 60-6583. Adenosine receptor signaling to restrict lymphocyte migration across HEV may be an important mechanism to control the magnitude of an inflammatory response.

Lymphocyte trafficking to lymph nodes (LNs) is initiated by the interaction between lymphocyte L-selectin and certain sialomucins, collectively termed peripheral node addressin (PNAd), carrying specific carbohydrates expressed by LN high endothelial venules (HEVs). Here, we identified a novel HEV-associated sialomucin, nepmucin (mucin not expressed in Peyer's patches [PPs]), that is expressed in LN HEVs but not detectable in PP HEVs at the protein level. Unlike conventional sialomucins, nepmucin contains a single V-type immunoglobulin (Ig) domain and a mucin-like domain. Using materials affinity-purified from LN lysates with soluble L-selectin, we found that two higher molecular weight species of nepmucin (75 and 95 kD) were decorated with oligosaccharides that bind L-selectin as well as an HEV-specific MECA-79 monoclonal antibody. Electron microscopic analysis showed that nepmucin accumulates in the extended luminal microvillus processes of LN HEVs. Upon appropriate glycosylation, nepmucin supported lymphocyte rolling via its mucin-like domain under physiological flow conditions. Furthermore, unlike most other sialomucins, nepmucin bound lymphocytes via its Ig domain, apparently independently of lymphocyte function–associated antigen 1 and very late antigen 4, and promoted shear-resistant lymphocyte binding in combination with intercellular adhesion molecule 1. Collectively, these results suggest that nepmucin may serve as a dual-functioning PNAd in LN HEVs, mediating both lymphocyte rolling and binding via different functional domains.

Nurse-like stromal cell lines from the synovial tissue of patients with rheumatoid arthritis (RA-SNC) produce, on coculture with lymphocytes, large amounts of proinflammatory cytokines. In the present paper, we analyze the molecular events necessary for the induction of cytokine release from RA-SNC cells, and particularly the roles played by cell adhesion and the transmigration (also known as pseudoemperipolesis) of lymphocytes. For this purpose, the effects of various mAbs on the binding and transmigration of a human B-cell line, MC/car, were examined using a cloned RA-SNC line, RA-SNC77. To analyze the role of lymphocyte binding and transmigration on upregulated cytokine production by the RA-SNC77 cells, we used C3 exoenzyme-treated MC/car cells, which could bind to RA-SNC77 cells but could not transmigrate. Treatment with anti-CD29 or anti-CD49d mAb significantly reduced binding and transmigration of the MC/car cells. In contrast, the neutralizing anti-CD106/vascular cell adhesion molecule 1 mAb did not show any inhibitory effect. Likewise, none of the neutralizing mAbs against CD11a, CD18, CD44, CD49e, or CD54 showed significant effects. Binding of C3-treated or untreated MC/car cells to RA-SNC77 cells induced comparable levels of IL-6 and IL-8 production. In addition, the enhanced cytokine production by RA-SNC77 cells required direct lymphocyte contact via a very late antigen-4 (VLA-4)-independent adhesion pathway. These results indicate that, although both the VLA-4-dependent/vascular cell adhesion molecule 1-independent and the VLA4-independent adhesion pathways are involved in MC/car binding and subsequent transmigration, only the VLA4-independent adhesion pathway is necessary and sufficient for the enhanced proinflammatory cytokine production by RA-SNC77 cells. The transmigration process, which is dependent on Rho-GTPase, is not a prerequisite for this phenomenon.

The effects of monoclonal antibodies (mAbs) to cell-surface molecules, divalent cations, and various cell-signaling and metabolic inhibitors on the binding of thymocytes to rat thymic dendritic cells (TDC) were studied using a rosette assay. It was found that TDC/thymocyte adhesion was stronger and faster at 37°C than at 4°C. Flow cytometric analysis demonstrated that bound thymocytes were predominantly CD4+CD8+ and CD4+CD8-, but in comparison to the phenotype of whole thymocytes, they were enriched in the mature TCRαβhi subset. The binding of thymocytes to TDC at 37°C was almost completely dependent on Ca2+ and Mg2+ and partly on an intact cytoskeleton and calmodulin-dependent protein kinase. The adhesion was independent of new protein synthesis and the activities of protein kinases A and C, tyrosine kinases, as well as phosphotyrosine protein phosphatases. The TDC/thymocyte adhesion at 37°C was partly blocked by anti-LFA-1 (WT.1), anti-CD18 (WT.3), and anti-ICAM-1 (1A29) mAb. MAbs to class II MHC (OX-3 and OX-6), CD4 (W3/25), CD8 (OX-8), and αβTCR (R73) stimulated the adhesion via an LFA-1-dependent pathway, whereas an anti-CD45 mAb (G3C5) stimulated the rosette formation independently of LFA-1. MAbs to CD2 (OX-34), CD11b (ED7), CD11b/c (OX-42), and class I MHC (OX-18) were without significant effects on the adhesion process.

A role of β2 integrins and one of their ligands, ICAM-1, in thymic macrophage (TMF)/thymocyte interactions was studied. TMF were isolated as adherent cells from 4-day old culture of thymic-cell suspensions either from normal or hydrocortisone-treated rats. Adherent cells were 94-98% positive with ED1 (a pan-macrophage marker). The majority of them (75-95%) expressed the CD11b and CD18 molecules, and 60-70% expressed CD54 (ICAM-1). A low proportion of TMF (10-20%) expressed CDlla (LFA-1). The expression of all these antigens was upregulated by IFN-α and TNF-α. The effect of these mAbs on TMF/thymocyte binding was studied using a simple rosette assay by incubating unstimulated or IFN-γ or TNF-α stimulated TMF, grown on microscopic slides with resting or ConA +IL-2 activated thymocytes. It was found that LFA-1/CD18 and ICAM-1 play a significant role in the TMF/thymocyte adhesion. In addition, a LFA-l-dependent/ICAM- 1-independent adhesion pathway was observed, suggesting that LFA-1 might use another ligand. The inhibitory effect of anti-CD18 mAb (WT-3) was higher than the effect of anti-LFA-1 mAb (WT-1) and was a consequence of blocking the CD18 chain both on thymocytes and TMF. No significant difference in the expression and function of adhesion molecules was found between TMF obtained from normal or hydrocortisone-treated rats. The involvement of CD1 1b in these processes was of lesser importance than the role of the CD11a molecule. By using mAbs to different epitopes of the CD11b molecule, such as OX-42 (anti-CD11b/CD11c), ED7, and ED8 (anti-CD11b), it was found that they were either slightly or moderately inhibitory under certain experimental conditions or did not significantly modulate TMF/thymocyte binding. Oχ-42 was slightly stimulatory in some experiments. Cumulatively, these results show that 2 integrins play a significant role in TMF/thymocyte interactions and probably contribute to T-cell development in vivo.