Involvement of CXCR3-associated chemokines in MHV-3 induced fulminant hepatic failure

The role of chemokines in murine hepatitis virus strain 3 (MHV-3) induced fulminant hepatic failure (FHF) is not well defined. In this study, we investigated the role of the CXC chemokine receptor 3 (CXCR3)-associated chemokine [monokine induced by IFN-gamma (Mig/CXCL9) and interferon-gamma-inducible protein 10 (IP-10/CXCL10)] in the recruitment of intrahepatic lymphocytes and subsequent fulminant hepatic failure induced by MHV-3. Balb/cJ mice (6–8 weeks, female) were intraperitioneally injected with 100 PFU MHV-3.The proportions and numbers of T cells and NK cells as well as the expression of CXCR3 on T cells and NK cells in the liver, spleen and blood were analyzed by flow cytometry. The hepatic mRNA level of the CXCR3-associated chemokines (CXCL9 and CXCL10) was detected by realtime PCR. A transwell migration assay was used to assess the chemotactic effect of MHV-3-infected hepatocytes on the splenic lymphocytes. Following MHV-3 infection, the number of hepatic NK cells and T cells and the frequencies of hepatic NK cells and T cells expressing CXCR3 increased markedly; however, in the spleen and peripheral blood, they both decreased significantly. Moreover, the hepatic mRNAs levels of CXCL9 and CXCL10 were significantly elevated post infection. The transwell migration assay demonstrated that MHV-3-infected hepatocytes have the capacity to attract and recruit the splenic NK cells and T cells, and CXCL10 plays a key role in lymphocyte mobilization from the spleen. These results suggest that the CXCR3-associated chemokines (CXCL9 and CXCL10) may play an important role in the recruitment of intrahepatic lymphocytes and subsequent necroinflammation and hepatic failure in MHV-3 infection.     

Chemokine and chemokine receptor interactions provide a mechanism for selective T cell recruitment to specific liver compartments within hepatitis C-infected liver

The role played by chemokines in regulating the selective recruitment of lymphocytes to different tissue compartments in disease is poorly characterized. In hepatitis C infection, inflammation confined to portal areas is associated with a less aggressive course, whereas T cell infiltration of the liver parenchyma is associated with progressive liver injury and cirrhosis. We propose a mechanism to explain how lymphocytes are recruited to hepatic lobules during bursts of necroinflammatory activity in chronic hepatitis C infection. We report here that lymphocytes infiltrating hepatitis C-infected liver express high levels of the chemokine receptors CCR5 and CXCR3. However, whereas the CCR5 ligands macrophage inflammatory protein-1alpha and -1beta were largely confined to vessels within portal tracts, the CXCR3 ligands IFN-inducible protein-10 and monokine-induced by IFN-gamma were selectively up-regulated on sinusoidal endothelium. In vitro, human hepatic sinusoidal endothelial cells secreted IFN-inducible protein-10 and monokine-induced by IFN-gamma in response to stimulation with IFN-gamma in combination with either IL-1 or TNF-alpha. This suggests that intrahepatic Th1 cytokines drive the increased expression of IFN-inducible protein-10 and monokine-induced by IFN-gamma and thereby promote the continuing recruitment of CXCR3-expressing T cells into the hepatic lobule in chronic hepatitis C infection.     

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.     

CXC chemokine receptor 4 expression and function in human astroglioma cells

Chemokines constitute a superfamily of proteins that function as chemoattractants and activators of leukocytes. Astrocytes, the major glial cell type in the CNS, are a source of chemokines within the diseased brain. Specifically, we have shown that primary human astrocytes and human astroglioma cell lines produce the CXC chemokines IFN-gamma-inducible protein-10 and IL-8 and the CC chemokines monocyte chemoattractant protein-1 and RANTES in response to stimuli such as TNF-alpha, IL-1beta, and IFN-gamma. In this study, we investigated chemokine receptor expression and function on human astroglioma cells. Enhancement of CXC chemokine receptor 4 (CXCR4) mRNA expression was observed upon treatment with the cytokines TNF-alpha and IL-1beta. The peak of CXCR4 expression in response to TNF-alpha and IL-1beta was 8 and 4 h, respectively. CXCR4 protein expression was also enhanced upon treatment with TNF-alpha and IL-1beta (2- to 3-fold). To study the functional relevance of CXCR4 expression, stable astroglioma transfectants expressing high levels of CXCR4 were generated. Stimulation of cells with the ligand for CXCR4, stromal cell-derived factor-1alpha (SDF-1alpha), resulted in an elevation in intracellular Ca(2+) concentration and activation of the mitogen-activated protein kinase cascade, specifically, extracellular signal-regulated kinase 2 (ERK2) mitogen-activated protein kinase. Of most interest, SDF-1alpha treatment induced expression of the chemokines monocyte chemoattractant protein-1, IL-8, and IFN-gamma-inducible protein-10. SDF-1alpha-induced chemokine expression was abrogated upon inclusion of U0126, a pharmacological inhibitor of ERK1/2, indicating that the ERK signaling cascade is involved in this response. Collectively, these data suggest that CXCR4-mediated signaling pathways in astroglioma cells may be another mechanism for these cells to express chemokines involved in angiogenesis and inflammation.     

Critical role for CXCR3 chemokine biology in the pathogenesis of bronchiolitis obliterans syndrome

Bronchiolitis obliterans syndrome (BOS) is the major limitation to survival post-lung transplantation and is characterized by a persistent peribronchiolar inflammation that eventually gives way to airway fibrosis/obliteration. Acute rejection is the main risk factor for the development of BOS and is characterized by a perivascular/bronchiolar leukocyte infiltration. The specific mechanism(s) by which these leukocytes are recruited have not been elucidated. The CXC chemokines (monokine induced by IFN-gamma (MIG)/CXC chemokine ligand (CXCL)9, IP-10/CXCL10, and IFN-inducible T cell alpha chemoattractant (ITAC)/CXCL11) act through their shared receptor, CXCR3. Because they are potent leukocyte chemoattractants and are involved in other inflammation/fibroproliferative diseases, we hypothesized that the expression of these chemokines during an allogeneic response promotes the persistent recruitment of mononuclear cells, leading to chronic lung rejection. We found that elevated levels of MIG/CXCL9, IFN-inducible protein 10 (IP-10)/CXCL10, and ITAC/CXCL11 in human bronchoalveolar lavage fluid were associated with the continuum from acute to chronic rejection. Translational studies in a murine model demonstrated increased expression of MIG/CXCL9, IP-10/CXCL10, and ITAC/CXCL11 paralleling the recruitment of CXCR3-expressing mononuclear cells. In vivo neutralization of CXCR3 or its ligands MIG/CXCL9 and IP-10/CXCL10 decreased intragraft recruitment of CXCR3-expressing mononuclear cells and attenuated BOS. This supports the notion that ligand/CXCR3 biology plays an important role in the recruitment of mononuclear cells, a pivotal event in the pathogenesis of BOS.     

Astrocytes modulate the chemokine network in a pathogen-specific manner

Immune responses in the central nervous system (CNS) are carefully regulated. Despite the absence of most immune processes and a substantive blood brain barrier, potent immune responses form during infection and autoimmunity. Astrocytes are innate immune sentinels that ensheath parenchymal blood vessels and sit at the gateway to the CNS parenchyma. Viral and bacterial infections trigger the influx of distinct leukocyte subsets. We show that astrocytes alone are sufficient for distinguishing between these two main types of infection and triggers release of relevant chemokines that relate to the microbe recognised. Bacterial-associated molecules induced the preferential expression of CCL2, CXCL1, CCL20 and CCL3 whilst a virus-associated dsRNA analogue preferentially up-regulated CXCL10 and CCL5. Thus, astrocytes can respond to infection in a distinct and appropriate manner suggesting they have the capacity to attract appropriate sets of leukocytes into the brain parenchyma. Astrocytes themselves are unable to respond to these chemokines since they were devoid of most chemokine receptors but expressed CXCR4, CXCR7 and CXCR6 at rest. Stimulation with TGF-β specifically up-regulated CXCR6 expression and may explain how TGF-β/CXCL16-expressing gliomas are so effective at attracting astroglial cells.     

Regulation of cockroach antigen-induced allergic airway hyperreactivity by the CXCR3 ligand CXCL9

Allergic airway disease is characterized by a robust lymphocytic infiltrate, elaboration of Th2-type inflammatory mediators, pulmonary eosinophil accumulation, and airway hyperreactivity. The CXCR3 ligands, CXCL9 (monokine induced by IFN-gamma) and CXCL10 (IFN-inducible protein, 10 kDa), are IFN-gamma-inducible, Th1-type chemokines. As CXCL10 has been previously shown to participate in the modulation of allergic inflammation, we were interested in investigating the possible role that CXCL9 may play in this inflammatory response. Expression of CXCL9 was primarily identified in airway epithelial cells by immunohistochemical staining. Airway neutralization of CXCL9 at the time of allergen challenge significantly increased airway hyperreactivity, airway eosinophil accumulation, and IL-4 levels in the bronchoalveolar lavage while significantly decreasing airway levels of IL-12. In contrast, introduction of exogenous CXCL9 into the airway at the time of allergen challenge dramatically reduced airway hyper-reactivity and eosinophil accumulation. Moreover, pulmonary levels of IL-4 were significantly reduced, whereas levels of IL-12 were significantly increased, with exogenous CXCL9 treatment. In lymphocytes restimulated with CXCL9 and allergen in vitro, CXCL9 down-regulated IL-4 expression and up-regulated IFN-gamma expression, suggesting that CXCL9 is able to direct activated lymphocytes toward a Th1-type phenotype. Additionally, CXCL9 was shown to inhibit CC chemokine ligand 11-induced eosinophil chemotaxis in in vitro assays. Taken together, our results demonstrate that the CXCR3 ligand CXCL9 is involved in regulation of the allergic response in the lung by regulation of lymphocyte activation and eosinophil recruitment.     

A CXC chemokine sequence isolated from the rainbow trout Oncorhynchus mykiss resembles the closely related interferon-gamma-inducible chemokines CXCL9, CXCL10 and CXCL11

A sequence encoding a CXC - type chemokine from rainbow trout was found to most resemble members of the CXCL9/CXCL10/CXCL11 sub-family. In mammals, all 3 chemokines are regulated by IFN-gamma and are chemotactic for activated T lymphocytes. The trout chemokine (gammaIP1), with a message of 787 nucleotides, contains 100 amino acids in a typical non-ELR CXC chemokine arrangement. A second sequence (gammaIP2), with 6 nucleotide differences in the coding region when compared to the first, was also identified although it is not known whether this is a second functional gene or a second allele. The gene is separated onto 4 exons, and the introns intervene in conserved positions according to the mammalian equivalents. The sequence encoded by the second exon shares the highest amino acid identity (37%) with CXCL10, with lower values of identity to other CXC chemokines (17-31%). Furthermore, phylogenetic analysis groups the trout chemokine with mammalian CXCL9, CXCL10 and CXCL11 peptides. Constitutive expression of gammaIP is seen in trout gill and low level expression in spleen, head kidney and liver. In RTS-11 cells, gammaIP expression can be induced with poly I:C, but not by LPS, suggesting virus-mediated regulation of gammaIP. Intraperitoneal injection of recombinant trout TNF-alpha caused elevation in gammaIP mRNA levels in trout head kidney.     

CXCR2 deficiency confers impaired neutrophil recruitment and increased susceptibility during Toxoplasma gondii infection.

Neutrophil migration to the site of infection is a critical early step in host immunity to microbial pathogens, in which chemokines and their receptors play an important role. In this work, mice deficient in expression of the chemokine receptor CXCR2 were infected with Toxoplasma gondii and the outcome was monitored. Gene-deleted animals displayed completely defective neutrophil recruitment, which was apparent at 4 h and sustained for at least 36 h. Kit(W)/Kit(W-v) animals also displayed defective polymorphonuclear leukocyte migration, suggesting mast cells as one source of chemokines driving the response. Tachyzoite infection and replication were accelerated in CXCR2(-/-) animals, resulting in establishment of higher cyst numbers in the brain relative to wild-type controls. Furthermore, serum and spleen cell IFN-gamma levels in infected, gene-deleted mice were reduced 60-75% relative to infected normal animals, and spleen cell TNF-alpha was likewise reduced by approximately 50%. These results highlight an important role for CXCR2 in neutrophil migration, which may be important for early control of infection and induction of immunity during Toxoplasma infection.     

TNF influences chemokine expression of macrophages in vitro and that of CD11b+ cells in vivo during Mycobacterium tuberculosis infection

Granulomas, focal accumulations of immune cells, form in the lung during Mycobacterium tuberculosis infection. Chemokines, chemotactic cytokines, are logical candidates for inducing migration of T lymphocytes and monocytes to and within the lung. TNF influences chemokine expression in some models. TNF-deficient mice infected with M. tuberculosis are highly susceptible to disease, and granuloma formation is inhibited. Through in vitro assays, we demonstrate that neutralization of TNF in M. tuberculosis-infected macrophages led to a reduction in many inflammatory chemokines, such as C-C chemokine ligand 5, CXC ligand 9 (CXCL9), and CXCL10. In TNF-deficient mice, immune cells migrated to the lungs early after infection, but did not organize to form granulomas within the lung. Although chemokine expression, as measured in whole lung tissue, was not different, the expression of chemokines in the CD11b(+) subset of cells isolated ex vivo from the lungs of TNF-deficient mice had reduced expression of C-C chemokine ligand 5, CXCL9, and CXCL10 at early time points after TNF neutralization. Local expression of CXCR3-binding chemokines within the lungs, as determined by in situ hybridization, was also affected by TNF. Therefore, TNF affects the expression of chemokines by macrophages in vitro and CD11b(+) cells in vivo, which probably influences the local chemokine gradients and granuloma formation.     

Colon carcinoma cells induce CXCL11-dependent migration of CXCR3-expressing cytotoxic T lymphocytes in organotypic culture

Adoptive immunotherapy of cancer patients with cytolytic T lymphocytes (CTL) has been hampered by the inability of the CTL to home into tumors in vivo. Chemokines can attract T lymphocytes to the tumor site, as demonstrated in animal models, but the role of chemokines in T-lymphocyte trafficking toward human tumor cells is relatively unexplored. In the present study, the role of chemokines and their receptors in the migration of a colon carcinoma (CC) patient’s CTL toward autologous tumor cells has been studied in a novel three-dimensional organotypic CC culture. CTL migration was mediated by chemokine receptor CXCR3 expressed by the CTL and CXCL11 chemokine secreted by the tumor cells. Excess CXCL11 or antibodies to CXCL11 or CXCR3 inhibited migration of CTL to tumor cells. T cell and tumor cell analyses for CXCR3 and CXCL11 expression, respectively, in ten additional CC samples, may suggest their involvement in other CC patients. Our studies, together with previous studies indicating angiostatic activity of CXCL11, suggest that CXCL11 may be useful as an immunotherapeutic agent for cancer patients when transduced into tumor cells or fused to tumor antigen-specific Ab.     

The expression and possible roles of chemokine CXCL11 and its receptor CXCR3 in the human endometrium

IFN-gamma secreted by a human embryo and trophoblast cells during implantation is suggested to play an important role in implantation and pregnancy. In the present study, we explored expression and possible functions of CXCL11, a CXC chemokine strongly induced by IFN-gamma, and its receptor CXCR3 in the human endometrium. Secreted CXCL11 protein was not detected in cultured endometrial stromal cells (ESC) but was detected in cultured endometrial epithelial cells (EEC). IFN-gamma stimulated the protein levels of CXCL11 in a dose-dependent manner in EEC and ESC. CXCL11 secreted from EEC with 100 ng/ml IFN-gamma was 220-fold of the control, and 100-fold as compared with that secreted from ESC with the same dose of IFN-gamma. CXCR3 was expressed in EEC, ESC, and trophoblast cells. Addition of IFN-gamma to EEC increased the chemotactic activity of its culture medium to trophoblast cells and T cells, and the effect was suppressed by immunoneutralization with Abs of three CXCR3 ligands, including anti-CXCL11 Ab. CXCL11 significantly increased BrdU incorporation of ESC, which was inhibited by a p42/44 MAPK pathway inhibitor PD98059. In contrast, CXCL11 significantly decreased BrdU incorporation and increased the release of lactate dehydrogenase and the positive staining of annexin V in EEC. These findings suggest that IFN-gamma promotes implantation by stimulating EEC to produce CXCL11, which induces migration of trophoblast cells and T cells, proliferation of ESC, and apoptosis of EEC.     

The chemokine and scavenger receptor CXCL16/SR-PSOX is expressed in human vascular smooth muscle cells and is induced by interferon gamma

Atherosclerosis is an inflammatory disease that is characterised by the involvement of chemokines that are important for the recruitment of leukocytes and scavenger receptors that mediate foam cell formation. Several cytokines are involved in the regulation of chemokines and scavenger receptors in atherosclerosis. CXCL16 is a chemokine and scavenger receptor and found in macrophages in human atherosclerotic lesions. Using double-labelled immunohistochemistry, we identified that smooth muscle cells in human lesions express CXCL16. We then analysed the effects of IFN-gamma, TNF-alpha, IL-12, IL-15, IL-18, and LPS on CXCL16 expression in cultured aortic smooth muscle cells. IFN-gamma was the most potent CXCL16 inducer and increased mRNA, soluble form, membrane form, and total cellular levels of CXCL16. The IFN-gamma induction of CXCL16 was also associated with increased uptake of oxLDL into these cells. Taken together, smooth muscle cells express CXCL16 in atherosclerotic lesions, which may play a role in the attraction of T cells to atherosclerotic lesions and contribute to the cellular internalisation of modified LDL.     

Correction: Dynamic Changes of Post-Translationally Modified Forms of CXCL10 and Soluble DPP4 in HCV Subjects Receiving Interferon-Free Therapy

Serum levels of the interferon (IFN)-stimulated chemokine CXCL10 are increased during chronic HCV infection and associate with outcome of IFN-based therapy. Elevated levels of NH2-terminal truncated CXCL10 (3-77aa), produced by DPP4 cleavage, negatively associate with spontaneous clearance of acute HCV infection and sustained virological response (SVR) with IFN-based therapy for chronic infection. The association of different CXCL10 forms and DPP4 with outcome during IFN-free HCV therapy has not been examined. Using novel Simoa assays, plasma was analyzed from HCV genotype-1 (GT1) subjects who relapsed (n = 11) or achieved SVR (n = 10) after sofosbuvir and ribavirin (SOF/RBV) treatment, and from SOF/RBV relapsers who achieved SVR with a subsequent SOF/ledipasvir regimen (n = 9). While the NH2-truncated form of CXCL10 was elevated in HCV infection relative to healthy controls, pre-treatment plasma concentrations of CXCL10 forms failed to stratify subjects based on treatment outcome to IFN-free regimens. However, a trend (statistically non-significant) towards elevated higher levels of total and long CXCL10 was observed pre-treatment in subjects who relapsed. All forms of CXCL10 decreased rapidly following treatment initiation and were again elevated in subjects who experienced HCV relapse, indicating that CXCL10 production may be associated with active viral replication. While soluble DPP4 (sDPP4) and NH₂-truncated CXCL10 concentrations were highly correlated, on-treatment sDPP4 levels and activity declined more slowly than CXCL10, suggesting differential regulation. Conclusion: These data suggest post-translationally modified forms of CXCL10 will not support the prediction of treatment outcome in HCV GT1 subjects treated with SOF/RBV.     

Label-free impedance responses of endogenous and synthetic chemokine receptor CXCR3 agonists correlate with Gi-protein pathway activation

The chemokine receptor CXCR3 is a G-protein-coupled receptor that signals through the Gα(i) class of heterotrimeric G-proteins. CXCR3 is highly expressed on activated T cells and has been proposed to be a therapeutic target in autoimmune disease. CXCR3 is activated by the chemokines CXCL9, CXCL10 and CXCL11. CXCR3 signaling properties in response to CXCL10, CXCL11 and the synthetic agonist VUF10661 have previously been evaluated using conventional endpoint assays. In the present study, label-free impedance measurements were used to characterize holistic responses of CXCR3-expressing cells to stimulation with chemokines and VUF10661 in real time and to compare these responses with both G-protein and non-G-protein (β-arrestin2) mediated responses. Differences in response kinetics were apparent between the chemokines and VUF10661. Moreover, CXCR3-independent effects could be distinguished from CXCR3-specific responses with the use of the selective CXCR3 antagonist NBI-74330 and the Gα(i) inhibitor pertussis toxin. By comparing the various responses, we observed that CXCL9 is a biased CXCR3 agonist, stimulating solely G-protein-dependent pathways. Moreover, CXCR3-mediated changes in cellular impedance correlated with G-protein signaling, but not β-arrestin2 recruitment.     

Chemokines and chemokine receptors expression in the lesions of patients with American cutaneous leishmaniasis

American cutaneous leishmaniasis (ACL) presents distinct active clinical forms with different grades of severity, known as localised (LCL), intermediate (ICL) and diffuse (DCL) cutaneous leishmaniasis. LCL and DCL are associated with a polarised T-helper (Th)1 and Th2 immune response, respectively, whereas ICL, or chronic cutaneous leishmaniasis, is associated with an exacerbated immune response and a mixed cytokine expression profile. Chemokines and chemokine receptors are involved in cellular migration and are critical in the inflammatory response. Therefore, we evaluated the expression of the chemokines CXCL10, CCL4, CCL8, CCL11 and CXCL8 and the chemokine receptors CCR3, CXCR3, CCR5 and CCR7 in the lesions of patients with different clinical forms of ACL using immunohistochemistry. LCL patients exhibited a high density of CXCL10+, CCL4+ and CCL8+ cells, indicating an important role for these chemokines in the local Th1 immune response and the migration of CXCR3+ cells. LCL patients showed a higher density of CCR7+ cells than ICL or DCL patients, suggesting major dendritic cell (DC) migration to lymph nodes. Furthermore, DCL was associated with low expression levels of Th1-associated chemokines and CCL11+ epidermal DCs, which contribute to the recruitment of CCR3+ cells. Our findings also suggest an important role for epidermal cells in the induction of skin immune responses through the production of chemokines, such as CXCL10, by keratinocytes.