IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking

IFN-gamma-inducible protein 10 (IP-10, CXCL10), a chemokine secreted from cells stimulated with type I and II IFNs and LPS, is a chemoattractant for activated T cells. Expression of IP-10 is seen in many Th1-type inflammatory diseases, where it is thought to play an important role in recruiting activated T cells into sites of tissue inflammation. To determine the in vivo function of IP-10, we constructed an IP-10-deficient mouse (IP-10(-/-)) by targeted gene disruption. Immunological analysis revealed that IP-10(-/-) mice had impaired T cell responses. T cell proliferation to allogeneic and antigenic stimulation and IFN-gamma secretion in response to antigenic challenge were impaired in IP-10(-/-) mice. In addition, IP-10(-/-) mice exhibited an impaired contact hypersensitivity response, characterized by decreased ear swelling and reduced inflammatory cell infiltrates. T cells recovered from draining lymph nodes also had a decreased proliferative response to Ag restimulation. Furthermore, IP-10(-/-) mice infected with a neurotropic mouse hepatitis virus had an impaired ability to control viral replication in the brain. This was associated with decreased recruitment of CD4(+) and CD8(+) lymphocytes into the brain, reduced levels of IFN-gamma and the IFN-gamma-induced chemokines monokine induced by IFN-gamma (Mig, CXCL9) and IFN-inducible T cell alpha chemoattractant (I-TAC, CXCL11) in the brain, decreased numbers of virus-specific IFN-gamma-secreting CD8(+) cells in the spleen, and reduced levels of demyelination in the CNS. Taken together, our data suggest a role for IP-10 in both effector T cell generation and trafficking in vivo.     

Role of IP-10/CXCL10 in the progression of pancreatitis-like injury in mice after murine retroviral infection

Exocrinopathy and pancreatitis-like injury were developed in C57BL/6 (B6) mice infected with LP-BM5 murine leukemia virus, which is known to induce murine acquired immunodeficiency syndrome (MAIDS). The role of chemokines, especially CXCL10/interferon (IFN)-gamma-inducible protein 10 (IP-10), a chemokine to attract CXCR3+ T helper 1-type CD4+ T cells, has not been investigated thoroughly in the pathogenesis of pancreatitis. B6 mice were inoculated intraperitoneally with LP-BM5 and then injected every week with either an antibody against IP-10 or a control antibody. Eight weeks after infection, we analyzed the effect of IP-10 neutralization. Anti-IP-10 antibody treatment did not change the generalized lymphadenopathy and hepatosplenomegaly of mice with MAIDS. The treatment significantly reduced the number of IP-10- and CXCR3-positive cells in the mesenteric lymph nodes (mLNs) but not the phenotypes and gross numbers of cells. In contrast, IP-10 neutralization reduced the number of mononuclear cells infiltrating into the pancreas. Anti-IP-10 antibody treatment did not change the numbers of IFN-gamma+ and IL10+ cells in the mLN but significantly reduced their numbers, especially IFN-gamma+ and IL-10+ CD4+ T cells and IFN-gamma+ Mac-1+ cells, in the pancreas. IP-10 neutralization ameliorated the pancreatic lesions of mice with MAIDS probably by blocking the cellular infiltration of CD4+ T cells and IFN-gamma+ Mac-1+ cells into the pancreas at least at 8 wk after infection, suggesting that IP-10 and these cells might play a key role in the development of chronic autoimmune pancreatitis.     

Both CXCR3 and CXCL10/IFN-inducible protein 10 are required for resistance to primary infection by dengue virus

We examined the extent to which CXCR3 mediates resistance to dengue infection. Following intracerebral infection with dengue virus, CXCR3-deficient (CXCR3(-/-)) mice showed significantly higher mortality rates than wild-type (WT) mice; moreover, surviving CXCR3(-/-) mice, but not WT mice, often developed severe hind-limb paralysis. The brains of CXCR3(-/-) mice showed higher viral loads than those of WT mice, and quantitative analysis using real-time PCR, flow cytometry, and immunohistochemistry revealed fewer T cells, CD8(+) T cells in particular, in the brains of CXCR3(-/-) mice. This suggests that recruitment of effector T cells to sites of dengue infection was diminished in CXCR3(-/-) mice, which impaired elimination of the virus from the brain and thus increased the likelihood of paralysis and/or death. These results indicate that CXCR3 plays a protective rather than an immunopathological role in dengue virus infection. In studies to identify critical CXCR3 ligands, CXCL10/IFN-inducible protein 10-deficient (CXCL10/IP-10(-/-)) mice infected with dengue virus showed a higher mortality rate than that of the CXCR3(-/-) mice. Although CXCL10/IP-10, CXCL9/monokine induced by IFN-gamma, and CXCL11/IFN-inducible T cell alpha chemoattractant share a single receptor and all three of these chemokines are induced by dengue virus infection, the latter two could not compensate for the absence of CXCL10/IP-10 in this in vivo model. Our results suggest that both CXCR3 and CXCL10/IP-10 contribute to resistance against primary dengue virus infection and that chemokines that are indistinguishable in in vitro assays differ in their activities in vivo.     

Islet-specific expression of CXCL10 causes spontaneous islet infiltration and accelerates diabetes development

During inflammation, chemokines are conductors of lymphocyte trafficking. The chemokine CXCL10 is expressed early after virus infection. In a virus-induced mouse model for type 1 diabetes, CXCL10 blockade abrogated disease by interfering with trafficking of autoaggressive lymphocytes to the pancreas. We have generated transgenic rat insulin promotor (RIP)-CXCL10 mice expressing CXCL10 in the beta cells of the islets of Langerhans to evaluate how bystander inflammation influences autoimmunity. RIP-CXCL10 mice have islet infiltrations by mononuclear cells and limited impairment of beta cell function, but not spontaneous diabetes. RIP-CXCL10 mice crossed to RIP-nucleoprotein (NP) mice expressing the NP of the lymphocytic choriomeningitis virus in the beta cells had massively accelerated type 1 diabetes after lymphocytic choriomeningitis virus infection. Mechanistically, we found a drastic increase in NP-specific, autoaggressive CD8 T cells in the pancreas after infection. In situ staining with H-2D(b)(NP(396)) tetramers revealed islet infiltration by NP-specific CD8 T cells in RIP-NP-CXCL10 mice early after infection. Our results indicate that CXCL10 expression accelerates the autoimmune process by enhancing the migration of Ag-specific lymphocytes to their target site.     

IFN-inducible protein 10/CXC chemokine ligand 10-independent induction of experimental autoimmune encephalomyelitis

In multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), autoaggressive T cells traffic into the CNS and induce disease. Infiltration of these pathogenic T cells into the CNS has been correlated with the expression of the chemokine IFN-inducible protein (IP)10/CXC chemokine ligand (CXCL)10, a chemoattractant for activated T cells, and its receptor CXCR3, in the CNS of both MS patients and mice with EAE. In the present study, we report that targeted deletion of IP-10 did not diminish the expression, severity, or histopathology of EAE induced by active immunization with 100 micro g of myelin oligodendrocyte glycoprotein peptide (MOG)p35-55. However, we found that IP-10-deficient mice had a lower threshold for expression of disease compared with wild-type littermates. EAE induced by immunization with 5 micro g of MOGp35-55 resulted in more severe disease characterized by a greater number of CNS lesions and infiltrating mononuclear cells in IP-10-deficient mice compared with wild-type controls. IP-10-deficient mice immunized with MOGp35-55 demonstrated increased levels of IFN-inducible T cell alpha-chemokine/CXCL11 mRNA in the CNS and decreased levels of monokine induced by IFN-gamma/CXCL9 mRNA in draining lymph nodes, suggesting differential compensation for loss of IP-10 in lymphoid vs parenchymal tissue compartments. EAE in IP-10-deficient mice induced by low-dose immunization was associated with enhanced Ag-specific Th1 responses in the draining lymph node, which corresponded with diminished lymph node TGF-beta1 expression. Our data demonstrated that IP-10 was not required for the trafficking of pathogenic T cells into the CNS in EAE but played an unexpected role in determining the threshold of disease susceptibility in the periphery.     

The T cell-specific CXC chemokines IP-10, Mig, and I-TAC are expressed by activated human bronchial epithelial cells

Recruitment of activated T cells to mucosal surfaces, such as the airway epithelium, is important in host defense and for the development of inflammatory diseases at these sites. We therefore asked whether the CXC chemokines IFN-induced protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC), which specifically chemoattract activated T cells by signaling through the chemokine receptor CXCR3, were inducible in respiratory epithelial cells. The effects of proinflammatory cytokines, including IFN-gamma (Th1-type cytokine), Th2-type cytokines (IL-4, IL-10, and IL-13), and dexamethasone were studied in normal human bronchial epithelial cells (NHBEC) and in two human respiratory epithelial cell lines, A549 and BEAS-2B. We found that IFN-gamma, but not TNF-alpha or IL-1 beta, strongly induced IP-10, Mig, and I-TAC mRNA accumulation mainly in NHBEC and that TNF-alpha and IL-1 beta synergized with IFN-gamma induction in all three cell types. High levels of IP-10 protein (> 800 ng/ml) were detected in supernatants of IFN-gamma/TNF-alpha-stimulated NHBEC. Neither dexamethasone nor Th2 cytokines modulated IP-10, Mig, or I-TAC expression. Since IFN-gamma is up-regulated in tuberculosis (TB), using in situ hybridization we studied the expression of IP-10 in the airways of TB patients and found that IP-10 mRNA was expressed in the bronchial epithelium. In addition, IP-10-positive cells obtained by bronchoalveolar lavage were significantly increased in TB patients compared with normal controls. These results show that activated bronchial epithelium is an important source of IP-10, Mig, and I-TAC, which may, in pulmonary diseases such as TB (in which IFN-gamma is highly expressed) play an important role in the recruitment of activated T cells.     

Gene expression and production of the monokine induced by IFN-gamma (MIG), IFN-inducible T cell alpha chemoattractant (I-TAC), and IFN-gamma-inducible protein-10 (IP-10) chemokines by human neutrophils

Monokine induced by IFN-gamma (MIG), IFN-inducible T cell alpha chemoattractant (I-TAC), and IFN-gamma-inducible protein of 10 kDa (IP-10) are related members of the CXC chemokine subfamily that bind to a common receptor, CXCR3, and that are produced by different cell types in response to IFN-gamma. We have recently reported that human polymorphonuclear neutrophils (PMN) have the capacity to release IP-10. Herein, we show that PMN also have the ability to produce MIG and to express I-TAC mRNA in response to IFN-gamma in combination with either TNF-alpha or LPS. While IFN-gamma, alone or in association with agonists such as fMLP, IL-8, granulocyte (G)-CSF and granulocyte-macrophage (GM)-CSF, failed to influence MIG, IP-10, and I-TAC gene expression, IFN-alpha, in combination with TNF-alpha, LPS, or IL-1beta, resulted in a considerable induction of IP-10 release by neutrophils. Furthermore, IL-10 and IL-4 significantly suppressed the expression of MIG, IP-10, and I-TAC mRNA and the extracellular production of MIG and IP-10 in neutrophils stimulated with IFN-gamma plus either LPS or TNF-alpha. Finally, supernatants harvested from stimulated PMN induced migration and rapid integrin-dependent adhesion of CXCR3-expressing lymphocytes; these activities were significantly reduced by neutralizing anti-MIG and anti-IP-10 Abs, suggesting that they were mediated by MIG and IP-10 present in the supernatants. Since MIG, IP-10, and I-TAC are potent chemoattractants for NK cells and Th1 lymphocytes, the ability of neutrophils to produce these chemokines might contribute not only to the progression and evolution of the inflammatory response, but also to the regulation of the immune response.     

Peroxisome proliferator-activated receptor-gamma activators inhibit IFN-gamma-induced expression of the T cell-active CXC chemokines IP-10, Mig, and I-TAC in human endothelial cells

Peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of the nuclear hormone receptor superfamily originally shown to play an important role in adipocyte differentiation and glucose homeostasis, is now known to regulate inflammatory responses. Given the importance of endothelial cell (EC)-derived chemokines in regulating leukocyte function and trafficking, we studied the effects of PPARgamma ligands on the expression of chemokines induced in ECs by the Th1 cytokine IFN-gamma. Treatment of ECs with PPARgamma activators significantly inhibited IFN-gamma-induced mRNA and protein expression of the CXC chemokines IFN-inducible protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC), whereas expression of the CC chemokine monocyte chemoattractant protein-1 was not altered. PPARgamma activators decreased IFN-inducible protein of 10 kDa promoter activity and inhibited protein binding to the two NF-kappaB sites but not to the IFN-stimulated response element ISRE site. Furthermore, PPARgamma ligands inhibited the release of chemotactic activity for CXC chemokine receptor 3 (CXCR3)-transfected lymphocytes from IFN-gamma-stimulated ECs. These data suggest that anti-diabetic PPARgamma activators might attenuate the recruitment of activated T cells at sites of Th1-mediated inflammation.     

IL-4 enhances keratinocyte expression of CXCR3 agonistic chemokines

IFN-induced protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC) belong to the non-glutamate-leucine-arginine motif CXC chemokine family and act solely through the CXCR3 receptor for potent attraction of T lymphocytes. In this study, we evaluated the capacity of the T cell-derived cytokines IL-4, IL-10, and IL-17 to modulate IP-10, Mig, and I-TAC in cultured human keratinocytes and CXCR3 expression in T cells from allergic contact dermatitis (ACD). IL-4, but not IL-10 or IL-17, significantly up-regulated IFN-gamma- or TNF-alpha-induced IP-10, Mig, and I-TAC mRNA accumulation in keratinocytes and increased the levels of IP-10 and Mig in keratinocyte supernatants. Immunohistochemistry of skin affected by ACD revealed that >70% of infiltrating cells were reactive for CXCR3 and that CXCR3 staining colocalized in CD4+ and CD8+ T cells. Nickel-specific CD4+ and CD8+ T cell lines established from ACD skin produced IFN-gamma and IL-4 and expressed moderate to high levels of CXCR3. Finally, CXCR3 agonistic chemokines released by stimulated keratinocytes triggered calcium mobilization in skin-derived nickel-specific CD4+ T cells and promoted their migration, with supernatant from keratinocyte cultures stimulated with IFN-gamma and IL-4 attracting more efficaciously than supernatant from keratinocytes activated with IFN-gamma alone. In conclusion, IL-4 exerts a proinflammatory function on keratinocytes by potentiating IFN-gamma and TNF-alpha induction of IP-10, Mig, and I-TAC, which in turn may determine a prominent recruitment of CXCR3+ T lymphocytes at inflammatory reaction sites.     

Fas ligand is responsible for CXCR3 chemokine induction in CD4+ T cell-dependent liver damage

Immune-mediated hepatic damage has been demonstrated in the pathogenesis of hepatitis C virus (HCV) and other hepatotrophic infections. Fas/Fas ligand (FasL) interaction plays a critical role in immune-mediated hepatic damage. To understand the molecular mechanism(s) of FasL-mediated liver inflammation, we examined the effect of CD4(+) T cells expressing high levels of FasL on the initiation of hepatic damage through analysis of chemokine and chemokine receptor expression in HCV core x TCR (DO11.10) double-transgenic mice. In vivo antigenic stimulation triggers a marked influx of core-expressing Ag-specific CD4(+) T cells into the liver of the immunized core(+) TCR mice but not their core(-) TCR littermates. Strikingly, the inflammatory process in the liver of core(+) TCR mice was accompanied by a dramatic increase in IFN-inducible protein 10 and monokine induced by IFN-gamma production. The intrahepatic lymphocytes were primarily CXCR3-positive and anti-CXCR3 Ab treatment abrogates migration of CXCR3(+) lymphocytes into the liver and hepatic damage. Importantly, the blockade of Fas/FasL interaction reduces the expression of IFN-inducible protein 10 and monokine induced by IFN-gamma and cellular infiltration into the liver. These findings suggest that activated CD4(+) T cells with elevated FasL expression are involved in promoting liver inflammation and hepatic damage through the induction of chemokines.     

A second step of chemotaxis after transendothelial migration: keratinocytes undergoing apoptosis release IFN-gamma-inducible protein 10, monokine induced by IFN-gamma,and IFN-gamma-inducible alpha-chemoattractant for T cell chemotaxis toward epidermis in atopic dermatitis

Activation and skin-selective homing of T cells and their effector functions in the skin represent sequential immunological events in the pathogenesis of atopic dermatitis (AD). Apoptosis of keratinocytes, induced mainly by T cells and mediated by IFN-gamma and Fas, is the essential pathogenetic event in eczema formation. Keratinocyte apoptosis appears as activation-induced cell death in AD. By IFN-gamma stimulation, chemokines such as IFN-gamma-inducible protein 10, monokine induced by IFN-gamma, and IFN-gamma-inducible alpha-chemoattractant are strongly up-regulated in keratinocytes. These chemokines attract T cells bearing the specific receptor CXCR3, which is highly expressed on T cells isolated from skin biopsies of AD patients. Accordingly, an increased T cell chemotaxis was observed toward IFN-gamma-treated keratinocytes. Supporting these findings, enhanced IFN-gamma-inducible protein 10, monokine induced by IFN-gamma, and IFN-gamma-inducible alpha-chemoattractant expression was observed in lesional AD skin by immunohistochemical staining. These results indicate a second step of chemotaxis inside the skin after transendothelial migration of the inflammatory cells. Keratinocytes undergoing apoptosis in acute eczematous lesions release chemokines that attract more T cells toward the epidermis, which may further augment the inflammation and keratinocyte apoptosis.     

A functional IFN-gamma-inducible protein-10/CXCL10-specific receptor expressed by epithelial and endothelial cells that is neither CXCR3 nor glycosaminoglycan.

Interferon-gamma-inducible protein-10 (IP-10)/CXCL10 is a CXC chemokine that attracts T lymphocytes and NK cells through activation of CXCR3, the only chemokine receptor identified to date that binds IP-10/CXCL10. We have found that several nonhemopoietic cell types, including epithelial and endothelial cells, have abundant levels of a receptor that binds IP-10/CXCL10 with a Kd of 1-6 nM. Surprisingly, these cells expressed no detectable CXCR3 mRNA. Furthermore, no cell surface expression of CXCR3 was detectable by flow cytometry, and the binding of 125I-labeled IP-10/CXCL10 to these cells was not competed by the other high affinity ligands for CXCR3, monokine induced by IFN-gamma/CXCL9, and I-TAC/CXCL11. Although IP-10/CXCL10 binds to cell surface heparan sulfate glycosaminoglycan (GAG), the receptor expressed by these cells is not GAG, since the affinity of IP-10/CXCL10 for this receptor is much higher than it is for GAG, its binding is not competed by platelet factor 4/CXCL4, and it is present on cells that are genetically incapable of synthesizing GAG. Furthermore, in contrast to IP-10/CXCL10 binding to GAG, IP-10/CXCL10 binding to these cells induces new gene expression and chemotaxis, indicating the ability of this receptor to transduce a signal. These high affinity IP-10/CXCL10-specific receptors on epithelial cells may be involved in cell migration and, perhaps, in the spread of metastatic cells as they exit from the vasculature. (All of the lung cancer cells we examined also expressed CXCR4, which has been shown to play a role in breast cancer metastasis.) CXCR3-negative endothelial cells may also use this receptor to mediate the angiostatic activity of IP-10/CXCL10, which is also expressed by these cells in an autocrine manner.     

IFN-gamma/STAT1 acts as a proinflammatory signal in T cell-mediated hepatitis via induction of multiple chemokines and adhesion molecules: a critical role of IRF-1

We have previously shown that IFN-gamma/STAT1 plays an essential role in concanavalin A (ConA)-induced T cell hepatitis via activation of apoptotic signaling pathways. Here we demonstrate that IFN-gamma/STAT1 also plays a crucial role in leukocyte infiltration into the liver in T cell hepatitis. After injection of ConA, leukocytes were significantly infiltrated into the liver, which was suppressed in IFN-gamma(-/-) and STAT1(-/-) mice. Disruption of the IFN regulatory factor-1 (IRF-1) gene, a downstream target of IFN-gamma/STAT1, abolished ConA-induced liver injury and suppressed leukocyte infiltration into the liver. Additionally, ConA injection induced expression of a wide variety of chemokines and adhesion molecules in the liver. Among them, expression of ICAM-1, VCAM-1, monokine induced by IFN-gamma (Mig), CC chemokine ligand-20, epithelial cell-derived neutrophil-activating peptide (ENA)-78, IFN-inducible T cell-alpha chemoattractant (I-TAC), and IFN-inducible protein-10 (IP-10) was markedly attenuated in IFN-gamma(-/-), STAT1(-/-), and IRF-1(-/-) mice. In primary mouse hepatocytes, Kupffer cells, and endothelial cells, in vitro treatment with IFN-gamma activated STAT1, STAT3, and IRF-1, and induced expression of VCAM-1, ICAM-1, Mig, ENA-78, I-TAC, and IP-10 mRNA. Induction of these chemokines and adhesion molecules was markedly diminished in STAT1(-/-) and IRF-1(-/-) hepatic cells compared with wild-type hepatic cells. These findings suggest that in addition to induction of apoptosis, previously well documented, IFN-gamma also stimulated hepatocytes, sinusoidal endothelial cells, and Kupffer cells partly via an STAT1/IRF-1-dependent mechanism to produce multiple chemokines and adhesive molecules responsible for promoting infiltration of leukocytes and, ultimately, resulting in hepatitis.     

Interferon-independent, human immunodeficiency virus type 1 gp120-mediated induction of CXCL10/IP-10 gene expression by astrocytes in vivo and in vitro.

The CXC chemokine gamma interferon (IFN-gamma)-inducible protein CXCL10/IP-10 is markedly elevated in cerebrospinal fluid and brain of individuals infected with human immunodeficiency virus type 1 (HIV-1) and is implicated in the pathogenesis of HIV-associated dementia (HAD). To explore the possible role of CXCL10/IP-10 in HAD, we examined the expression of this and other chemokines in the central nervous system (CNS) of transgenic mice with astrocyte-targeted expression of HIV gp120 under the control of the glial fibrillary acidic protein (GFAP) promoter, a murine model for HIV-1 encephalopathy. Compared with wild-type controls, CNS expression of the CC chemokine gene CCL2/MCP-1 and the CXC chemokine genes CXCL10/IP-10 and CXCL9/Mig was induced in the GFAP-HIV gp120 mice. CXCL10/IP-10 RNA expression was increased most and overlapped the expression of the transgene-encoded HIV gp120 gene. Astrocytes and to a lesser extent microglia were identified as the major cellular sites for CXCL10/IP-10 gene expression. There was no detectable expression of any class of IFN or their responsive genes. In astrocyte cultures, soluble recombinant HIV gp120 protein was capable of directly inducing CXCL10/IP-10 gene expression a process that was independent of STAT1. These findings highlight a novel IFN- and STAT1-independent mechanism for the regulation of CXCL10/IP-10 expression and directly link expression of HIV gp120 to the induction of CXCL10/IP-10 that is found in HIV infection of the CNS. Finally, one function of IP-10 expression may be the recruitment of leukocytes to the CNS, since the brain of GFAP-HIV gp120 mice had increased numbers of CD3(+) T cells that were found in close proximity to sites of CXCL10/IP-10 RNA expression.     

Dysregulated expression of MIG/CXCL9, IP-10/CXCL10 and CXCL16 and their receptors in systemic sclerosis

Introduction  

Systemic sclerosis (SSc) is characterized by fibrosis and microvascular abnormalities including dysregulated angiogenesis. Chemokines, in addition to their chemoattractant properties, have the ability to modulate angiogenesis. Chemokines lacking the enzyme-linked receptor (ELR) motif, such as monokine induced by interferon-γ (IFN-γ) (MIG/CXCL9) and IFN-inducible protein 10 (IP-10/CXCL10), inhibit angiogenesis by binding CXCR3. In addition, CXCL16 promotes angiogenesis by binding its unique receptor CXCR6. In this study, we determined the expression of these chemokines and receptors in SSc skin and serum.     

CXCR3-dependent recruitment of antigen-specific T lymphocytes to the liver during murine cytomegalovirus infection

Innate inflammatory events promoting antiviral defense in the liver against murine cytomegalovirus (MCMV) infection have been characterized. However, the mechanisms that regulate the selective recruitment of inflammatory T lymphocytes to the liver during MCMV infection have not been defined. The studies presented here demonstrate the expression of monokine induced by gamma interferon (IFN-gamma; Mig/CXCL9) and IFN-gamma-inducible protein 10 (IP-10/CXCL10) in liver leukocytes and correlate their production with the infiltration of MCMV-specific CD8 T cells into the liver. Antibody-mediated neutralization of CXCL9 and CXCL10 and studies using mice deficient in CXCR3, the primary known receptor for these chemokines, revealed that CXCR3-dependent mechanisms promote the infiltration of virus-specific CD8 T cells into the liver during acute infection with MCMV. Furthermore, CXCR3 functions augmented the hepatic accumulation of CD8 T-cell IFN-gamma responses to MCMV. Evaluation of protective functions demonstrated enhanced pathology that overlapped with transient increases in virus titers in CXCR3-deficient mice. However, ultimate viral clearance and survival were not compromised. Thus, CXCR3-mediated signals support the accumulation of MCMV-specific CD8 T cells that contribute to, but are not exclusively required for, protective responses in a virus-infected tissue site.     

Neutralization of the chemokine CXCL10 reduces inflammatory cell invasion and demyelination and improves neurological function in a viral model of multiple sclerosis

Intracerebral infection of mice with mouse hepatitis virus (MHV) results in an acute encephalomyelitis followed by a chronic demyelinating disease with clinical and histological similarities with the human demyelinating disease multiple sclerosis (MS). Following MHV infection, chemokines including CXC chemokine ligand (CXCL)10 (IFN inducible protein 10 kDa), CXCL9 (monokine induced by IFN-gamma), and CC chemokine ligand 5 (RANTES) are expressed during both acute and chronic stages of disease suggesting a role for these molecules in disease exacerbation. Previous studies have shown that during the acute phase of infection, T lymphocytes are recruited into the CNS by the chemokines CXCL10 and CXCL9. In the present study, MHV-infected mice with established demyelination were treated with antisera against these two chemokines, and disease severity was assessed. Treatment with anti-CXCL10 reduced CD4+ T lymphocyte and macrophage invasion, diminished expression of IFN-gamma and CC chemokine ligand 5, inhibited progression of demyelination, and increased remyelination. Anti-CXCL10 treatment also resulted in an impediment of clinical disease progression that was characterized by a dramatic improvement in neurological function. Treatment with antisera against CXCL9 was without effect, demonstrating a critical role for CXCL10 in inflammatory demyelination in this model. These findings document a novel therapeutic strategy using Ab-mediated neutralization of a key chemokine as a possible treatment for chronic human inflammatory demyelinating diseases such as MS.     

The murine chemokine CXCL11 (IFN-inducible T cell alpha chemoattractant) is an IFN-gamma- and lipopolysaccharide-inducible glucocorticoid-attenuated response gene expressed in lung and other tissues during endotoxemia

A new murine chemokine was identified in a search for glucocorticoid-attenuated response genes induced in the lung during endotoxemia. The first 73 residues of the predicted mature peptide are 71% identical and 93% similar to human CXCL11/IFN-inducible T cell alpha chemoattractant (I-TAC) (alias beta-R1, H174, IFN-inducible protein 9 (IP-9), and SCYB9B). The murine chemokine has six additional residues at the carboxyl terminus not present in human I-TAC. Identification of this cDNA as murine CXCL11/I-TAC is supported by phylogenetic analysis and by radiation hybrid mapping of murine I-TAC (gene symbol Scyb11) to mouse chromosome 5 close to the genes for monokine induced by IFN-gamma (MIG) and IP10. Murine I-TAC mRNA is induced in RAW 264.7 macrophages by IFN-gamma or LPS and is weakly induced by IFN-alphabeta. IFN-gamma induction of murine I-TAC is markedly enhanced by costimulation with LPS or IL-1beta in RAW cells and by TNF-alpha in both RAW cells and Swiss 3T3 fibroblasts. Murine I-TAC is induced in multiple tissues during endoxemia, with strongest expression in lung, heart, small intestine, and kidney, a pattern of tissue expression different from those of MIG and IP10. Peak expression of I-TAC message is delayed compared with IP10, both in lung after i.v. LPS and in RAW 264.7 cells treated with LPS or with IFN-gamma. Pretreatment with dexamethasone strongly attenuates both IFN-gamma-induced I-TAC expression in RAW cells and endotoxemia-induced I-TAC expression in lung and small intestine. The structural and regulatory similarities of murine and human I-TAC suggest that mouse models will be useful for investigating the role of this chemokine in human biology and disease.     

Intraallograft chemokine RNA and protein during rejection of MHC-matched/multiple minor histocompatibility-disparate skin grafts

Chemokines direct leukocyte recruitment into sites of tissue inflammation and may facilitate recruitment of leukocytes into allografts following transplantation. Although the expression of chemokines during rejection of MHC-disparate allografts has been examined, chemokine expression in MHC-matched/multiple minor histocompatibility Ag-disparate allografts has not been tested. The intraallograft RNA expression of several C-X-C and C-C chemokines was tested during rejection of full thickness skin grafts from B10. D2 donors on control Ig-, anti-CD4 mAb-, and anti-CD8 mAb-treated BALB/c recipients. In all recipients, two patterns of intragraft chemokine expression were observed during rejection of these grafts: 1) macrophage-inflammatory protein-1alpha, macrophage-inflammatory protein-1beta, GRO-alpha (KC), JE, and IFN-gamma-inducible protein (IP-10) were expressed at equivalent levels in allo- and isografts for 2-4 days posttransplant and then returned to low or undetectable levels; and 2) IP-10 and monokine induced by IFN-gamma (Mig) were expressed in the allografts 3 days before rejection was completed, suggesting a possible role in recruiting primed T cells into the allograft. Three days before completion of rejection, intraallograft IP-10 protein was restricted to the epidermis, whereas Mig was located in the lower dermis and associated with the intense infiltration of mononuclear cells. Treatment of B10.D2 recipients with rabbit antiserum to Mig, but not to IP-10, delayed rejection of the allografts 3-4 days. The results suggest that Mig mediates optimal recruitment of T cells into MHC-matched/multiple minor histocompatibility Ag-disparate allografts during rejection.     

Cutting edge: IFN-inducible ELR- CXC chemokines display defensin-like antimicrobial activity.

Recent reports highlighted the chemotactic activities of antimicrobial peptide defensins whose structure, charge, and size resemble chemokines. By assaying representative members of the four known families of chemokines we explored the obverse: whether some chemokines exert antimicrobial activity. In a radial diffusion assay, only recombinant monokine induced by IFN-gamma (MIG/CXCL9), IFN-gamma-inducible protein of 10 kDa (IP-10/CXCL10), and IFN-inducible T cell alpha chemoattractant (I-TAC/CXCL11), members of the IFN-gamma-inducible tripeptide motif Glu-Leu-Arg (ELR)(-) CXC chemokines, were antimicrobial against Escherichia coli and Listeria monocytogenes. Similar to human defensins, antimicrobial activities of the chemokines were inhibited by 50 and 100 mM NaCl. The concentration of MIG/CXCL9 and IP-10/CXCL10 released from IFN-gamma-stimulated PBMC in 24 h were, respectively, 35- and 28-fold higher than from unstimulated cells. Additionally, the amounts of chemokines released per monocyte suggest that, in tissues with mononuclear cell infiltration, IFN-gamma-inducible chemokines may reach concentrations necessary for microbicidal activity. IFN-gamma-inducible chemokines may directly inactivate microbes before attracting other host defense cells to the area of infection.