Shosaku Narumi

Plasmacytoid DCs help lymph node DCs to induce anti-HSV CTLs

Antiviral cell–mediated immunity is initiated by the dendritic cell (DC) network in lymph nodes (LNs). Plasmacytoid DCs (pDCs) are known to migrate to inflamed LNs and produce interferon (IFN)-α, but their other roles in antiviral T cell immunity are unclear. We report that LN-recruited pDCs are activated to create local immune fields that generate antiviral cytotoxic T lymphocytes (CTLs) in association with LNDCs, in a model of cutaneous herpes simplex virus (HSV) infection. Although pDCs alone failed to induce CTLs, in vivo depletion of pDCs impaired CTL-mediated virus eradication. LNDCs from pDC-depleted mice showed impaired cluster formation with T cells and antigen presentation to prime CTLs. Transferring circulating pDC precursors from wild-type, but not CXCR3-deficient, mice to pDC-depleted mice restored CTL induction by impaired LNDCs. In vitro co-culture experiments revealed that pDCs provided help signals that recovered impaired LNDCs in a CD2- and CD40L-dependent manner. pDC-derived IFN-α further stimulated the recovered LNDCs to induce CTLs. Therefore, the help provided by pDCs for LNDCs in primary immune responses seems to be pivotal to optimally inducing anti-HSV CTLs.

Pivotal Role of Dendritic Cell–derived CXCL10 in the Retention of T Helper Cell 1 Lymphocytes in Secondary Lymph Nodes

Various immune diseases are considered to be regulated by the balance of T helper (Th)1 and Th2 subsets. Although Th lymphocytes are believed to be generated in draining lymph nodes (LNs), in vivo Th cell behaviors during Th1/Th2 polarization are largely unexplored. Using a murine granulomatous liver disease model induced by Propionibacterium acnes, we show that retention of Th1 cells in the LNs is controlled by a chemokine, CXCL10/interferon (IFN) inducible protein 10 produced by mature dendritic cells (DCs). Hepatic LN DCs preferentially produced CXCL10 to attract 5′-bromo-2′-deoxyuridine (BrdU)+CD4+ T cells and form clusters with IFN-γ–producing CD4+ T cells by day 7 after antigen challenge. Blockade of CXCL10 dramatically altered the distribution of cluster-forming BrdU+CD4+ T cells. BrdU+CD4+ T cells in the hepatic LNs were selectively diminished while those in the circulation were significantly increased by treatment with anti-CXCL10 monoclonal antibody. This was accompanied by accelerated infiltration of memory T cells into the periphery of hepatic granuloma sites, most of them were in cell cycle and further produced higher amount of IFN-γ leading to exacerbation of liver injury. Thus, mature DC-derived CXCL10 is pivotal to retain Th1 lymphocytes within T cell areas of draining LNs and optimize the Th1-mediated immune responses.

Increase of chemokine interferon‐inducible protein‐10 (IP‐10) in the serum of patients with autoimmune liver diseases and increase of its mRNA expression in hepatocytes

To clarify the role of IP‐10 in autoimmune liver diseases, we studied the serum levels of IP‐10 in 14 patients with autoimmune hepatitis (AIH), 23 patients with primary biliary cirrhosis (PBC), and 65 patients with chronic viral hepatitis (20 type B and 45 type C). The hepatic expression of IP‐10 mRNA and the correlation between the serum levels of IP‐10 and clinical parameters were also evaluated. In addition to 20 healthy controls, 16 rheumatoid arthritis (RA) patients were included as an extrahepatic inflammatory disease. The serum level of IP‐10 was significantly (P < 0·02) higher in patients with AIH, PBC, and chronic hepatitis B and C than in healthy controls, and it was significantly correlated (P < 0·05) with the serum levels of aspartate aminotransferase and alanine aminotransferase in patients with AIH, PBC, and chronic hepatitis B and C. The serum level of IP‐10 was not elevated in RA patients. After successful treatment of AIH and chronic hepatitis C, the serum level of IP‐10 decreased to the same level as in healthy volunteers. As we previously showed in cases with chronic hepatitis B or C, in situ hybridization in both AIH and PBC cases demonstrated the expression of IP‐10 mRNA in hepatocytes around focal or lobular necrosis surrounded by infiltrating mononuclear cells, whereas IP‐10 mRNA was not expressed in areas around the damaged bile ducts in PBC cases. The present results suggest that IP‐10 is specifically produced by hepatocytes in inflammatory areas irrespective of the aetiology of hepatitis, and that IP‐10 may help to recruit T cells to the hepatic lesions in autoimmune liver diseases as well as in chronic viral hepatitis.

Blockade of CXCL10 protects mice from acute colitis and enhances crypt cell survival

Crypt cell renewal is essential for normal intestinal homeostasis as well as mucosal regeneration following injury. However, the factors regulating crypt cell growth in pathological conditionsare not fully understood. We report here that the endogenously produced chemokine CXCL10 regulates crypt cell proliferation. CXCL10 was constitutively expressed by basal crypts in mouse colon, but the expression of CXCL10 as well as CXCR3 was enhanced in the epithelium in the proliferative zone after oral administration of dextran sulfate sodium. Neutralization of CXCL10 protected mice from epithelial ulceration by promoting crypt cell survival without evidence of altered immune cell infiltration. Furthermore, recombinant CXCL10 administration into mice inhibited intestinal epithelial cell proliferation. These findings suggest that CXCL10 regulates crypt cell growth to maintain intestinal homeostasis in an autocrine or paracrine fashion. Thus, CXCL10 can be a new therapeutic target for inflammatory bowel disease by controlling the dynamics of epithelial homeostasis.

Neutralization of IFN‐inducible protein 10/CXCL10 exacerbates experimental autoimmune encephalomyelitis

We examined the effect of a monoclonal antibody (mAb) against interferon (IFN)‐inducible protein 10 (IP‐10)/CXCL10 on the development of experimental autoimmune encephalomyelitis (EAE) in ratsinduced by injecting xenogeneic brain homogenates into footpads. Treatment with neutralizing mAb against CXCL10 exacerbated EAE with increased infiltrating CD4+ cells in the central nervous system. Furthermore, the exacerbation by the mAb treatment was accompanied by less enlarged draining popliteal lymph nodes (LN) in parallel with cell number compared with those of EAE rats treatedwith control mAb, whereas other lymphoid organs such as the spleen and thymus were not significantly different between rats treated with anti‐CXCL10 and the control mAb. Induction of gene expression of CXCL9/Mig and CXCL10 and their receptor CXCR3 was confirmed in the draining LN in EAE rats. Induction of the third CXCR3 ligand, CXCL11/I‐TAC was not seen in the draining LN, whereas all three CXCR3 ligands and CXCR3 itself were markedly detected in the spinal cords following the development of EAE. These findings suggest that CXCL10 produced in the LN plays a specific inhibitory role in the development of Th1‐mediated diseases such as EAE by holding sensitized and activated Th1s expressing CXCR3 in the draining LN.

CXC Chemokine Ligand 10 Neutralization Suppresses the Occurrence of Diabetes in Nonobese Diabetic Mice through Enhanced β Cell Proliferation without Affecting Insulitis

We have shown that neutralization of IFN-inducible protein 10/CXCL10, a chemokine for Th1 cells, breaks Th1 retention in the draining lymph nodes, resulting in exacerbation in Th1-dominant autoimmune disease models induced by immunization with external Ags. However, there have been no studies on the role of CXCL10 neutralization in Th1-dominant disease models induced by constitutive intrinsic self Ags. So, we have examined the effect of CXCL10 neutralization using a type 1 diabetes model initiated by developmentally regulated presentation of β cell Ags. CXCL10 neutralization suppressed the occurrence of diabetes after administration with cyclophosphamide in NOD mice, although CXCL10 neutralization did not significantly inhibit insulitis and gave no influence on the trafficking of effector T cells into the islets. Because both CXCL10 and CXCR3 were, unexpectedly, coexpressed on insulin-producing cells, CXCL10 was considered to affect mature and premature β cells in an autocrine and/or paracrine fashion. In fact, CXCL10 neutralization enhanced proliferative response of β cells and resultantly increased β cell mass without inhibiting insulitis. Thus, CXCL10 neutralization can be a new therapeutic target for β cell survival, not only during the early stage of type 1 diabetes, but also after islet transplantation.

Cross reactivity of three T cell attracting murine chemokines stimulating the CXC chemokine receptor CXCR3 and their induction in cultured cells and during allograft rejection

Recent work identified the murine gene homologous to the human T cell attracting chemokine CXC receptor ligand 11 (CXCL11, also termed I‐TAC, SCYB11, ß‐R1, H174, IP‐9). Here, the biological activity and expression patterns of murine CXCL11 relative to CXCL9 (MIG) and CXCL10 (IP‐10/crg‐2), the other two CXCR3 ligands, were assessed. Calcium mobilization and chemotaxis experiments demonstrated that murine CXCL11 stimulated murine CXCR3 at much lower doses than murine CXCL9 or murine CXCL10. Murine CXCL11 also evoked calcium mobilization in CHO cells transfected with human CXCR3 and was chemotactic for CXCR3‐expressing human T lymphocytes as well as for 300–19 pre‐B cells transfected with human or murine CXCR3. Moreover, murine CXCL11 blocked the chemotactic effect of human CXCL11 on human CXCR3 transfectants. Depending on cell type (macrophage‐like cells RAW264.7, J774A.1, fetal F20 and adult dermal fibroblasts, immature and mature bone marrow‐derived dendritic cells) andstimulus (interferons, LPS, IL‐1β and TNF‐α), an up to 10,000‐fold increase of CXCL9, CXCL10 and CXCL11 mRNA levels, quantified by real‐time PCR, was observed. In vivo, the three chemokines are constitutively expressed in various tissues from healthy BALB/c mice and were strongly up‐regulated during rejection of allogeneic heart transplants. Chemokine mRNA levels exceeded those of CXCR3 and IFN‐γ which were induced with similar kinetics by several orders of magnitude.

Interleukin-1β Induces Tissue- and Cell Type–Specific Expression of Adhesion Molecules In Vivo

We examined the tissue distribution of adhesion molecule gene expression in mice treated intravenously with interleukin (IL)-1 beta. E-selectin mRNA expression was selectively induced in the heart by IL-1 beta, but only slight or no induction was observed in other organs. On the other hand, intercellular adhesion molecule-1 mRNA expression was inducible in all organs examined, although it showed the strongest induction in the lung and the weakest responses in the brain and skin. Vascular cell adhesion molecule-1 mRNA was also inducible in all organs with the exception of the skin, but it was induced most markedly in the lung and the heart. The accessibility of IL-1 beta to the heart was less than that to other organs except the brain. Similar tissue-specific induction of these mRNAs was also seen when tumor necrosis factor (TNF)-alpha or lipopolysaccharide was substituted for IL-1 beta. Analysis of E-selectin mRNA expression in the heart by in situ hybridization indicated that expression was most prominent in microvascular endothelial cells and some other stromal cells, but this transcript was not seen in the lung. Although intercellular adhesion molecule-1 mRNA expression was restricted to the endothelium lining the capillaries and small arteries in the heart, its distribution in the lung covered not only the endothelium but also the cells composing the alveolar septa. In contrast, vascular cell adhesion molecule-1 mRNA expression was most prominent in endothelial cells of larger vessels in both the heart and the lung. Our results demonstrate that expression of adhesion molecules is tissue- and cell type-specific and that endothelial cells differentially express adhesion molecules depending on the size of the blood vessels.

IFN-Inducible Protein-10 Has a Differential Role in Podocyte during Thy 1.1 Glomerulonephritis

IFN-inducible protein-10 (IP-10/CXCL10) is a potent chemoattractant for activated T lymphocytes and was recently reported to have several additional biologic activities. In this study, the expression and the function in normal glomeruli and in Thy1.1 glomerulonephritis (GN) were investigated. The expression of IP-10 was detected in normal rat glomeruli mainly in the podocyte. The expression of IP-10 was also detected on the cultured podocyte. The IP-10 expression was elevated at the early phase of Thy1.1 GN. The double staining immunofluorescence study clearly demonstrated that the elevated expression of IP-10 was mostly detected in the podocyte and very partly in mesangial area. A receptor for IP-10, CXCR3, showed similar expression patterns to that of IP-10. Expressions of neither of IP-10 nor of CXCR3 were detected on the inflammatory cells. For elucidating the role of IP-10, the blocking study was carried out with monoclonal anti-IP-10 antibody. The monoclonal anti-IP-10 antibody treatment decreased the expression of IP-10 and podocyte-associated proteins such as nephrin and podocin that are reported to be essential for maintaining the podocyte function (IP-10, 53.0% to control; nephrin, 43.5%; podocin, 60.4%). The findings indicated that the anti-IP-10 treatment disturbed the podocyte function. The anti-IP-10 treatment given to the rats with Thy1.1 nephritis exacerbated proteinuria, mesangiolysis, and matrix expansion. Collectively, the findings indicated that IP-10 plays a role in maintaining the podocyte function. Also, the findings suggested that anti-IP-10 treatment exacerbated the glomerular alterations in Thy1.1 GN by disturbing the podocyte function.

Clinical significance of elevated serum interferon- inducible protein-10 levels in hepatitis C virus carriers with persistently normal serum transaminase levels.

The aim of this study was to assess the immuno‐ logical profile in hepatitis C virus carriers with persistently normal serum transaminase levels. Forty‐two serum HCV RNA positive patients with persistently normal serum transaminase levels (22 natural ‘asymptomatic HCV carriers’ and 20 biochemical responders to IFN therapy) and 23 complete responders to IFN therapy were enrolled. The HCV genotypes and serum HCV RNA levels were determined before IFN therapy in treatment responders, and at entry in the others. The serum levels of IFN‐inducible protein‐10 (IP‐10) (a protein mainly induced by IFN‐γ), interleukin (IL)‐10, and IL‐4 were measured in all patients while the serum transaminase levels were normal. The serum transaminase levels and platelet counts were then monitored for the next 4 years and the changes in liver fibrosis were assessed. The serum levels of IP‐10 in infected and biochemically normal patients were significantly higher than the levels in complete responders to therapy, whereas the serum levels of IL‐10 and IL‐4 did not vary significantly among the different groups. During the 4‐year follow‐up period, 10/20 (50%) biochemical responders and 12/22 (55%) asymptomatic carriers had an elevation of the serum transaminase levels. A significant (P=0.0370) increase in platelet count after 4 years and improvement in liver fibrosis were noted in treatment responders but not in infected patients. The weak but significant residual immune response as reflected by the increased serum IP‐10 level may underlie the outcome of HCV carriers with persistently normal serum transaminase levels.