Alpha-galactosylceramide-induced liver injury in mice is mediated by TNF-alpha but independent of Kupffer cells

NKT cells expressing phenotypic markers of both T and NK cells seem to be pivotal in murine models of immune-mediated liver injury, e.g., in Con A-induced hepatitis. Also alpha-galactosylceramide (alpha-GalCer), a specific ligand for invariant Valpha14 NKT cells, induces hepatic injury. To improve the comprehension of NKT-cell mediated liver injury, we investigated concomitants and prerequisites of alpha-GalCer-induced hepatitis in mice. Liver injury induced by alpha-GalCer injection into C57BL/6 mice was accompanied by intrahepatic caspase-3 activity but appeared independent thereof. alpha-GalCer injection also induces pronounced cytokine responses, including TNF-alpha, IFN-gamma, IL-2, IL-4, and IL-6. We provide a detailed time course for the expression of these cytokines, both in liver and plasma. Cytokine neutralization revealed that, unlike Con A-induced hepatitis, IFN-gamma is not only dispensable for alpha-GalCer-induced hepatotoxicity but even appears to exert protective effects. In contrast, TNF-alpha was clearly identified as an important mediator for hepatic injury in this model that increased Fas ligand expression on NKT cells. Whereas intrahepatic Kupffer cells are known as a pivotal source for TNF-alpha in Con A-induced hepatitis, they were nonessential for alpha-GalCer-mediated hepatotoxicity. In alpha-GalCer-treated mice, TNF-alpha was produced by intrahepatic lymphocytes, in particular NKT cells. BALB/c mice were significantly less susceptible to alpha-GalCer-induced liver injury than C57BL/6 mice, in particular upon pretreatment with d-galactosamine, a hepatocyte-specific sensitizer to TNF-alpha-mediated injury. Finally, we demonstrate resemblance of murine alpha-GalCer-induced hepatitis to human autoimmune-like liver disorders. The particular features of this model compared with other immune-mediated hepatitis models may enhance comprehension of basic mechanisms in the etiopathogenesis of NKT cell-comprising liver disorders.     

IL-6 prevents T cell-mediated hepatitis via inhibition of NKT cells in CD4+ T cell- and STAT3-dependent manners

The hepatoprotective effect of IL-6 on various forms of liver injury including T cell-mediated hepatitis has been well documented, and it is believed that induction of antiapoptotic proteins is an important mechanism. In this study, we provide evidence suggesting an additional mechanism involved in the protective role of IL-6 in T cell-mediated hepatitis. In NKT cell-depleted mice, Con A-induced liver injury is diminished; this can be restored by the adoptive transfer of liver mononuclear cells or NKT cells from wild-type mice, but not from IL-6-treated mice. In vitro IL-6 treatment inhibits the ability of mononuclear cells to restore Con A-induced liver injury in NKT-depleted mice, whereas the same treatment does not inhibit purified NKT cells from restoring the injury. The addition of CD3(+) T cells or CD4(+) T cells can restore the inhibitory effect of IL-6 on purified NKT cells, whereas the addition of CD3(+) T cells from CD4-deficient mice fails to restore this inhibitory effect. The expression of IL-6R was detected in 52.6% of hepatic CD3(+) T cells and 32.7% of hepatic CD4(+) T cells, but only in 3.9% of hepatic NK and 1.5% of hepatic NKT cells. Finally, treatment with IL-6 induces STAT3 activation in hepatic lymphocytes and hepatic T cells, and blocking such activation abolishes the inhibitory effect of IL-6 on hepatic lymphocytes to restore liver injury. Taken together, these findings suggest that in addition to its antiapoptotic abilities, as previously well documented, IL-6/STAT3 inhibits NKT cells via targeting CD4(+) T cells and consequently prevents T cell-mediated hepatitis.     

Hyperproduction of proinflammatory cytokines by WSX-1-deficient NKT cells in concanavalin A-induced hepatitis

Administration of Con A induces liver injury that is considered to be an experimental model for human autoimmune or viral hepatitis, where immunopathology plays roles mediated by activated lymphocytes, especially NK1.1+ CD3+ NKT cells, and inflammatory cytokines, including IFN-gamma and IL-4. In the present study we investigated the role of WSX-1, a component of IL-27R, in Con A-induced hepatitis by taking advantage of WSX-1 knockout mice. WSX-1-deficient mice were more susceptible to Con A treatment than wild-type mice, showing serum alanine aminotransferase elevation and massive necrosis in the liver. Although the development of NKT cells appeared normal in WSX-1 knockout mice, purified NKT cells from the knockout mice produced more IFN-gamma and IL-4 than those from wild-type mice in response to stimulation with Con A both in vitro and in vivo. In addition, hyperproduction of proinflammatory cytokines, including IL-1, IL-6, and TNF-alpha, was observed in the knockout mice after Con A administration. These data revealed a novel role for WSX-1 as an inhibitory regulator of cytokine production and inflammation in Con A-induced hepatitis.     

Crucial role of IL-4/STAT6 in T cell-mediated hepatitis: up-regulating eotaxins and IL-5 and recruiting leukocytes

T cell-mediated immune responses are implicated in the pathogenesis of a variety of liver disorders; however, the underlying mechanism remains obscure. Con A injection is a widely accepted mouse model to study T cell-mediated liver injury, in which STAT6 is rapidly activated. Disruption of the IL-4 and STAT6 gene by way of genetic knockout abolishes Con A-mediated liver injury without affecting IFN-gamma/STAT1, IL-6/STAT3, or TNF-alpha/NF-kappaB signaling or affecting NKT cell activation. Infiltration of neutrophils and eosinophils in Con A-induced hepatitis is markedly suppressed in IL-4 (-/-) and STAT6(-/-) mice compared with wild-type mice. IL-4 treatment induces expression of eotaxins in hepatocytes and sinusoidal endothelial cells isolated from wild-type mice but not from STAT6(-/-) mice. Con A injection induces expression of eotaxins in the liver and elevates serum levels of IL-5 and eotaxins; such induction is markedly attenuated in IL-4(-/-) and STAT6(-/-) mice. Finally, eotaxin blockade attenuates Con A-induced liver injury and leukocyte infiltration. Taken together, these findings suggest that IL-4/STAT6 plays a critical role in Con A-induced hepatitis, via enhancing expression of eotaxins in hepatocytes and sinusoidal endothelial cells, and induces IL-5 expression, thereby facilitating recruitment of eosinophils and neutrophils into the liver and resulting in hepatitis.     

Differential roles of CC chemokine ligand 2/monocyte chemotactic protein-1 and CCR2 in the development of T1 immunity

CCR2 and its major ligand, chemokine ligand 2 (CCL2)/monocyte chemotactic protein-1, have been found to influence T1/T2 immune response polarization. Our objective was to directly compare the roles of CCR2 and CCL2 in T1/T2 immune response polarization using a model of pulmonary Cryptococcus neoformans infection. Either deletion of CCR2 or treatment of wild-type mice with CCL2 neutralizing Ab produced significant and comparable reductions in macrophage and T cell recruitment into the lungs following infection. Both CCL2 neutralization and CCR2 deficiency resulted in significantly diminished IFN-gamma production, and increased IL-4 and IL-5 production by lung leukocytes (T1 to T2 switch), but only CCR2 deficiency promoted pulmonary eotaxin production and eosinophilia. In the lung-associated lymph nodes (LALN), CCL2-neutralized mice developed Ag-specific IFN-gamma-producing cells, while CCR2 knockout mice did not. LALN from CCR2 knockout mice also had fewer MHCII(+)CD11c(+) and MHCII(+)CD11b(+) cells, and produced significantly less IL-12p70 and TNF-alpha when stimulated with heat-killed yeast than LALN from wild-type or CCL2-neutralized mice, consistent with a defect in APC trafficking in CCR2 knockout mice. Neutralization of CCL2 in CCR2 knockout mice did not alter immune response development, demonstrating that the high levels of CCL2 in these mice did not play a role in T2 polarization. Therefore, CCR2 (but not CCL2) is required for afferent T1 development in the lymph nodes. In the absence of CCL2, T1 cells polarize in the LALN, but do not traffic from the lymph nodes to the lungs, resulting in a pulmonary T2 response.     

Sphingosine-1-phosphate receptor agonists suppress concanavalin A-induced hepatic injury in mice

T cell-mediated immune responses play a critical role in a variety of liver injuries including autoimmune hepatitis. Injection of concanavalin A (Con A) into mice mimics the histological and pathological phenotype of T cell-mediated hepatitis. Recent advances in host immune control of organ transplantation include the development of sphingosine-1-phosphate (S1P) receptor agonists such as FTY720, which alter lymphocyte homing but do not suppress host general immunity. Herein we examined the effect of the new S1P receptor agonist KRP-203 on the Con A-induced liver damage model. In normal liver lymphocytes of BALB/c mice, both FTY720 and KRP203 promoted lymphocyte sequestering from the liver to secondary lymph nodes and significantly reduced the number of liver lymphocytes (p<0.05). Based on this observation, KRP203 was employed in the Con A-induced hepatitis model. KRP203 markedly reduced the number of CD4(+) lymphocytes that infiltrate Con A-treated liver (p<0.05) and successfully reduced serum transaminase elevation (p=0.017), therefore protecting mice from Con A-induced liver injury. Interestingly this homing modulation less occurs in natural hepatic T cell homing through the chemokine receptor, CXCR4. Therefore, S1P receptor agonists preferentially target CXCR4(+)CD4(+) peripheral blood T lymphocytes and suppress the occurrence of Con A-induced hepatitis, suggesting their therapeutic usefulness against T cell-mediated hepatic injury.     

Blockade of Retinol Metabolism Protects T Cell-Induced Hepatitis by Increasing Migration of Regulatory T Cells

Retinols are metabolized into retinoic acids by alcohol dehydrogenase (ADH) and retinaldehyde dehydrogenase (Raldh). However, their roles have yet to be clarified in hepatitis despite enriched retinols in hepatic stellate cells (HSCs). Therefore, we investigated the effects of retinols on Concanavalin A (Con A)-mediated hepatitis.Con A was injected into wild type (WT), Raldh1 knock-out (Raldh1^−/−), CCL2^−/− and CCR2^−/− mice. For migration study of regulatory T cells (Tregs), we used in vivo and ex vivo adoptive transfer systems. Blockade of retinol metabolism in mice given 4-methylpyrazole, an inhibitor of ADH, and ablated Raldh1 gene manifested increased migration of Tregs, eventually protected against Con A-mediated hepatitis by decreasing interferon-γ in T cells. Moreover, interferon-γ treatment increased the expression of ADH3 and Raldh1, but it suppressed that of CCL2 and IL-6 in HSCs. However, the expression of CCL2 and IL-6 was inversely increased upon the pharmacologic or genetic ablation of ADH3 and Raldh1 in HSCs. Indeed, IL-6 treatment increased CCR2 expression of Tregs. In migration assay, ablated CCR2 in Tregs showed reduced migration to HSCs. In adoptive transfer of Tregs in vivo and ex vivo, Raldh1-deficient mice showed more increased migration of Tregs than WT mice. Furthermore, inhibited retinol metabolism increased survival rate (75%) compared with that of the controls (25%) in Con A-induced hepatitis.These results suggest that blockade of retinol metabolism protects against acute liver injury by increased Treg migration, and it may represent a novel therapeutic strategy to control T cell-mediated acute hepatitis.     

Monocyte chemoattractant protein-1 and CCR2 interactions are required for IFN-alpha/beta-induced inflammatory responses and antiviral defense in liver

IFN-alpha/beta-mediated functions promote production of MIP-1alpha (or CCL3) by mediating the recruitment of MIP-1alpha-producing macrophages to the liver during early infection with murine CMV. These responses are essential for induction of NK cell inflammation and IFN-gamma delivery to support effective control of local infection. Nevertheless, it remains to be established if additional chemokine functions are regulated by IFN-alpha/beta and/or play intermediary roles in supporting macrophage trafficking. The chemokine MCP-1 (or CCL2) plays a distinctive role in the recruitment of macrophages by predominantly stimulating the CCR2 chemokine receptor. Here, we examine the roles of MCP-1 and CCR2 during murine CMV infection in liver. MCP-1 production preceded that of MIP-1alpha during infection and was dependent on IFN-alpha/beta effects for induction. Resident F4/80(+) liver leukocytes were identified as primary IFN-alpha/beta responders and major producers of MCP-1. Moreover, MCP-1 deficiency was associated with a dramatic reduction in the accumulation of macrophages and NK cells, as well as decreased production of MIP-1alpha and IFN-gamma in liver. These responses were also markedly impaired in mice with a targeted disruption of CCR2. Furthermore, MCP-1- and CCR2-deficient mice exhibited increased viral titers and elevated expression of the liver enzyme alanine aminotransferase in serum. These mice also had widespread virus-induced liver pathology and succumbed to infection. Collectively, these results establish MCP-1 and CCR2 interactions as factors promoting early liver inflammatory responses and define a mechanism for innate cytokines in regulation of chemokine functions critical for effective localized antiviral defenses.     

A selective adenosine A2A receptor agonist, ATL-146e, prevents concanavalin A-induced acute liver injury in mice

BACKGROUND AND AIMS: Concanavalin A (Con A) activates T lymphocytes and induces CD4+ T cell-mediated hepatic injury in mice. Pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and interleukin-6 (IL-6), are critical mediators in this experimental model. Activation of adenosine A2A receptors reduces the production of various pro-inflammatory cytokines and suppresses T cell activation. A selective adenosine A2A receptor agonist (ATL-146e) has been shown to be a potent inhibitor of inflammation by increasing intracellular cyclic AMP (cAMP) in leukocytes. The aim of the present study was to determine whether ATL-146e could ameliorate Con A-induced hepatic injury, reduction of pro-inflammatory cytokine production. METHODS: Balb/c mice were injected with 25mg/kg Con A with or without a single injection of ATL-146e (0.5-50 microg/kg), 5 min prior to Con A administration. Liver enzymes, histology, and serum levels of tumor necrosis factor-alpha, interferon-gamma, and interleukin-6 were examined. We also assessed the effects of ATL-146e on pro-inflammatory cytokine production with CD4+ T cell. RESULTS: Pretreatment with ATL-146e significantly reduced serum levels of liver enzymes (P<0.001). The serum pro-inflammatory cytokines were all increased after Con A administration and reduced to near normal levels by ATL-146e. ATL-146e also inhibited CD4+ T cell pro-inflammatory cytokine production. CONCLUSION: A selective adenosine A2A receptor agonist, ATL-146e, can prevent concanavalin A-induced hepatic injury that is presumably mediated by its anti-inflammatory properties.     

Critical role of interleukin-17/interleukin-17 receptor axis in mediating Con A-induced hepatitis

Concanavalin A (Con A)-induced hepatitis is thought to be a T-cell-mediated disease with active destruction of liver cells. Interleukin (IL)-17 is a cytokine produced principally by CD4(+) T cells. However, whether IL-17/IL-17 receptor (IL-17/IL-17R)-mediated responses are involved in T-cell-mediated Con A-induced liver injury remains unclear. In this study, we found that IL-17 expression was highly elevated in liver tissues during Con A-induced hepatitis. The increased levels of IL-17 were paralleled with the severity of liver injury reflected by Alanine aminotransaminase and histological assay as well as the secretion of tumor necrosis factor (TNF)-α and IL-6. Blockage of IL-17 significantly ameliorated Con A-induced hepatitis, while overexpression of IL-17 systemically resulted in massive hepatocyte necrosis in mice. Furthermore, overexpression of an IL-17R immunoglobulin G1 fusion protein significantly attenuated liver inflammation after acute Con A treatment. High expression of IL-17R on Kupffer cells was also observed along with the production of cytokines including TNF-α and IL-6. Inhibition of Kupffer cells by gadolinium chloride completely prevented Con A-induced liver injury and cytokine release. Finally, IL-17-expressing CD4(+) T and natural killer T cells were greatly increased in Con A-injected mice compared with that in controls. Overall, our results indicate that IL-17R signaling is critically involved in the pathogenesis in Con A-induced hepatitis, and blockade of IL-17/IL-17R signaling pathway may represent a novel therapeutic intervention in human autoimmune-related hepatitis.     

Differentiation of human gamma-delta T cells towards distinct memory phenotypes

Vgamma9/Vdelta2 T cells comprise a small population of peripheral T cells responding towards the low molecular weight antigen, (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMB-PP). HMB-PP-stimulated Vgamma9/Vdelta2 T cells proliferated, expressed CCL5/RANTES, and upregulated markers like CD16, CD25, CD69, and CD94, in the presence of either IL-15 or IL-21. Vgamma9/Vdelta2 T cells grown in the presence of IL-15 differentiated into an effector/memory population characterized by production of TNF-alpha, expression of CD45RO and CCR5, and lack of CD62L, CD81, and CCR7. In contrast, Vgamma9/Vdelta2 T cells grown with IL-21 differentiated into putative central memory CD45RO(+) T cells that did not produce TNF-alpha, IFN-gamma, or IL-4, and maintained expression of CD62L, CD81, and CCR7.     

A novel accessory role of neutrophils in concanavalin A-induced hepatitis

Concanavalin A (Con A)-induced hepatitis has been investigated as a model of T cell-mediated liver injury, in which IFN-gamma plays an essential role by inducing apoptosis of liver cells. Since a large number of neutrophils infiltrate into the liver in the model, the role of neutrophils was investigated in this study. Con A hardly caused liver injury in neutrophil-depleted mice, as assessed as to the plasma alanine aminotransferase level as well as histochemistry. Neutrophil-depleted mice also failed to produce IFN-gamma. Intracellular IFN-gamma staining revealed that, among liver leukocytes, T and NK cells but not neutrophils are the main producers of IFN-gamma. Nylon wool-purified "T cells", however, failed to produce IFN-gamma in response to Con A in vitro, while the production was restored by the addition of neutrophils. Overall, this study suggests that neutrophils play a novel accessory role in IFN-gamma production in Con A-induced hepatitis.     

Protective effect of NK1.1(+) T cells as well as NK cells against intraperitoneal tumors in mice

Peritoneal resident cells of mice normally contain small populations of NK cells and NK1.1(+) alphabetaT cells. These populations increased after either 3LL or EL4 tumor inoculations into the peritoneal cavity. In vivo depletion of NK cell alone by anti-asialo GM1 (ASGM1) Ab significantly decreased survival time of tumor-injected mice, while depletion of both NK cells and NK1.1(+) T cells by anti-NK 1.1 Ab greatly shortened mouse survival time. NK1. 1(+) T cells in peritoneal cavity consist of a larger proportion of double-negative T cells and smaller populations of CD4(+) T cells and Vbeta8(+) T cells compared with liver NK1.1(+) T cells and normally lack Vbeta2(+) T cells. Tumor inoculation induced rapid IL-12 and IFN-gamma mRNA in tumor-infiltrating mononuclear cells (TIM). Although anti-NK1 Ab pretreatment in vivo abrogated IFN-gamma mRNA expression and IFN-gamma production of TIM, NK cell depletion alone by anti-ASGM1 Ab pretreatment retained IFN-gamma mRNA expression and partly inhibited IFN-gamma production of TIM. Peritoneal NK cells as well as NK1.1(+) T cells but not NK1.1(-) T cells of 3LL cell- or EL4 cell-injected mice showed cytotoxicities against the same tumor cells. Further, either anti-IL-12 Ab or anti-IFN-gamma Ab ip injection significantly shortened EL4 cell-inoculated mouse survival time. Our findings suggest that peritoneal macrophages activated by tumors produce IL-12 which activates NK cells and NK1.1(+) T cells to produce IFN-gamma and both NK cells and NK1.1(+) T cells are important in suppressing the growth of the intraperitoneal tumors.     

Lack of chemokine receptor CCR5 promotes murine fulminant liver failure by preventing the apoptosis of activated CD1d-restricted NKT cells

Fulminant liver failure (FLF) consists of a cascade of events beginning with a presumed uncontrolled systemic activation of the immune system. The etiology of FLF remains undefined. In this study, we demonstrate that CCR5 deficiency promotes the development of acute FLF in mice following Con A administration by preventing activated hepatic CD1d-restricted NKT cells (but not conventional T cells) from dying from activation-induced apoptosis. The resistance of CCR5-deficient NKT cells from activation-induced apoptosis following Con A administration is not due to a defective Fas-driven death pathway. Moreover, FLF in CCR5-deficient mice also correlated with hepatic CCR5-deficient NKT cells, producing more IL-4, but not IFN-gamma, relative to wild-type NKT cells. Furthermore, FLF in these mice was abolished by IL-4 mAb or NK1.1 mAb treatment. We propose that CCR5 deficiency may predispose individuals to the development of FLF by preventing hepatic NKT cell apoptosis and by regulating NKT cell function, establishing a novel role for CCR5 in the development of this catastrophic liver disease that is independent of leukocyte recruitment.     

Chronic alcohol consumption accelerates liver injury in T cell-mediated hepatitis: alcohol disregulation of NF-kappaB and STAT3 signaling pathways

Alcohol consumption is a major risk factor accelerating the progression of liver disease in patients with chronic hepatitis virus infection. However, the mechanism underlying the enhanced susceptibility of alcoholics to liver injury is not fully understood. Here, we demonstrate that chronic ethanol consumption increases the susceptibility of C57BL/6 mice to concanavalin A (Con A)-induced T cell-mediated hepatitis. Injection of a low dose of Con A (5 microg/g) causes severe liver damage in ethanol-fed mice as evidenced by a significant elevation of serum alanine aminotransaminase levels, massive necrosis, and infiltration of leukocytes but only slightly induces liver injury in control pair-fed mice. In ethanol-fed mice, the activation and cytotoxicity of natural killer T cells, cells that play key roles in Con A-induced T cell hepatitis, are not significantly enhanced relative to pair-fed mice. Moreover, Con A-induced activation of hepatic NF-kappaB is increased, whereas activation of STAT1 and STAT3 is attenuated in ethanol-fed mice. Consistent with this result, the expression of chemokines and adhesion molecules [such as ICAM-1, macrophage inflammatory protein (MIP)-1, MIP-2, and MCP-1] controlled by NF-kappaB is upregulated, whereas STAT1-controlled expression of chemokines (such as MIG and IP-10) is downregulated in ethanol-fed mice compared with pair-fed mice. In conclusion, chronic alcohol consumption accelerates T cell-mediated hepatitis via upregulation of the NF-kappaB signaling pathway and subsequently enhances expression of chemokines/adhesive molecules and recruitment of leukocytes into the liver. Downregulation of the antiapoptotic STAT3 signal may also contribute to alcohol potentiation of T cell hepatitis.     

Phenotype and effector function of CC chemokine receptor 9-expressing lymphocytes in small intestinal Crohn's disease

CCL25/CCR9 chemokine ligand/receptor pair has been reported to play an important role in small bowel (SB) immunity and inflammation. We have previously reported an aberrant SB expression of CCL25 in Crohn's disease (CD) and an increased frequency of CCR9(+) T cells in the peripheral blood of patients with SB inflammatory diseases such as CD and celiac disease. In this study, we have characterized the phenotype and effector function of CCR9(+) T cells in mucosal lymphoid tissues in CD. We show that CCR9(+) T cells isolated from mesenteric lymph nodes (MLN) draining CD SB express a more activated phenotype compared with MLN draining normal SB. Stimulation of CCR9(+) T cells isolated from CD SB lamina propria produced more IFN-gamma and IL-17 in response to anti-CD3 or IL-12/IL-18 stimulation compared with those isolated from normal SB. The addition of TL1A to the cytokine combination markedly augmented the secretion of IFN-gamma, but not IL-17, by CD lamina propria CCR9(+) T cells. CCL25 incubation of CD SB lamina propria lymphocytes and MLN lymphocytes increased their adhesion to VCAM-1/Fc in vitro. Finally, the TCRVbeta analysis of CCR9(+) T cells revealed a diverse TCRVbeta repertoire among MLN CCR9(+) T cells in patients with SB CD. Our data indicate that CCR9(+) T cells in SB CD are proinflammatory and support the rationale for the use of CCR9 antagonists for the treatment of human SB CD.     

Blockade of virus infection by human CD4+ T cells via a cytokine relay network

CD4(+) T cells directly participate in bacterial clearance through secretion of proinflammatory cytokines. Although viral clearance relies heavily on CD8(+) T cell functions, we sought to determine whether human CD4(+) T cells could also directly influence viral clearance through cytokine secretion. We found that IFN-gamma and TNF-alpha, secreted by IL-12-polarized Th1 cells, displayed potent antiviral effects against a variety of viruses. IFN-gamma and TNF-alpha acted directly to inhibit hepatitis C virus replication in an in vitro replicon system, and neutralization of both cytokines was required to block the antiviral activity that was secreted by Th1 cells. IFN-gamma and TNF-alpha also exerted antiviral effects against vesicular stomatitis virus infection, but in this case, functional type I IFN receptor activity was required. Thus, in cases of vesicular stomatitis virus infection, the combination of IFN-gamma and TNF-alpha secreted by human Th1 cells acted indirectly through the IFN-alpha/beta receptor. These results highlight the importance of CD4(+) T cells in directly regulating antiviral responses through proinflammatory cytokines acting in both a direct and indirect manner.     

Activated T lymphocytes regulate hyaluronan binding to monocyte CD44 via production of IL-2 and IFN-gamma

Interactions of the cell surface proteoglycan CD44 with the extracellular matrix glycosaminoglycan hyaluronan (HA) are important during inflammatory immune responses. Our previous studies indicated that monocyte HA binding could be induced by TNF-alpha. Moreover, monocyte HA binding could be markedly up-regulated by culturing PBMC with anti-CD3 (TCR complex) mAbs. The present study was undertaken to identify soluble factors and/or cell surface molecules of activated T lymphocytes that might regulate HA binding to monocytes. Abs to IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-10, IL-15, GM-CSF, IFN-gamma, and TNF-alpha were tested for their effects on anti-CD3 mAb-, Con A-, and PMA/ionomycin-mediated monocyte HA binding in PBMC cultures. Anti-TNF-alpha, anti-IL-2, and anti-IFN-gamma Abs, when added together to PBMC cultures, completely blocked Con A- and partially blocked anti-CD3- and PMA/ionomycin-induced monocyte HA binding. Furthermore, when added together to PBMC cultures, IL-2 and TNF-alpha induced high levels of monocyte HA binding. Likewise, IFN-gamma augmented TNF-alpha-induced monocyte HA binding. To investigate the role of T cell-monocyte direct contact in induction of monocyte HA binding, we studied PMA/ionomycin-activated, paraformaldehyde-fixed CD4(+) T cells in these assays. Fixed, PMA/ionomycin-activated CD4(+) T lymphocytes induced monocyte HA binding, but direct T cell-monocyte contact was not required. Moreover, anti-IFN-gamma and anti-TNF-alpha Abs blocked fixed PMA/ionomycin-activated CD4(+) T cell-induced monocyte HA binding. Taken together, these studies indicate roles for soluble T lymphocyte-derived factor(s), such as IL-2 and IFN-gamma, and a role for monocyte-derived TNF-alpha in Con A-, TCR complex-, and PMA/ionomycin-induced HA binding to monocyte CD44.     

Eotaxin-3/CC chemokine ligand 26 is a functional ligand for CX3CR1

Eotaxin-3/CCL26 is a functional ligand for CCR3 and abundantly produced by IL-4-/IL-13-stimulated vascular endothelial cells. CCL26 also functions as a natural antagonist for CCR1, CCR2, and CCR5. In this study, we report that CCL26 is yet a functional ligand for CX3CR1, the receptor for fractalkine/CX3CL1, which is expressed by CD16(+) NK cells, cytotoxic effector CD8(+) T cells, and CD14(low)CD16(high) monocytes. Albeit at relatively high concentrations, CCL26 induced calcium flux and chemotaxis in mouse L1.2 cells expressing human CX3CR1 but not mouse CX3CR1 and competed with CX3CL1 for binding to CX3CR1. In chemotaxis assays using human PBMCs, CCL26 attracted not only eosinophils but also CD16(+) NK cells, CD45RA(+)CD27(-)CD8(+) T cells, and CD14(low)CD16(high) monocytes. Intraperitoneal injection of CCL26 into mice rapidly recruited mouse eosinophils and intravenously transferred human CD16(+) NK cells into the peritoneal cavity. IL-4-stimulated HUVECs produced CCL26 and efficiently induced adhesion of cells expressing CX3CR1. Real-time PCR showed that skin lesions of psoriasis consistently contained CX3CL1 mRNA but not CCL26 mRNA, whereas those of atopic dermatitis contained CCL26 mRNA in all samples but CX3CL1 mRNA in only about half of the samples. Nevertheless, the skin lesions from both diseases consistently contained CX3CR1 mRNA at high levels. Thus, CCL26 may be partly responsible for the recruitment of cells expressing CX3CR1 in atopic dermatitis particularly when the expression of CX3CL1 is low. Collectively, CCL26 is another agonist for CX3CR1 and may play a dual role in allergic diseases by attracting eosinophils via CCR3 and killer lymphocytes and resident monocytes via CX3CR1.