C-X-C Motif Chemokine Receptor 3 Research Articles

CXCR3 mediates chondrocyte injury through regulating nitric oxide.

As a common joint disease, osteoarthritis exhibits increasing trend in recent years. C-X-C motif chemokine receptor 3 (CXCR3) is a kind of chemokine with the characteristic of recruiting inflammatory cells. Its function in osteoarthritis has not been clarified. This study aims to explore the role of CXCR3 in cartilage injury by affecting unfolded protein response (UPR) pathway. The sample was obtained from osteoarthritis patients to test CXCR3 expression by Real-time polymerase chain reaction (PCR). Chondrocyte apoptosis model was established in vitro induced by interleukin 1β (IL-1β) and sodium nitroprusside (SNP). CXCR3 level was downregulated by using siRNA. Cell apoptosis was determined by using transferase-mediated deoxyuridine triphosphate-biotin nick end labeling(TUNEL) assay. UPR pathway related factors C/EBP homology protein (CHOP) and glucose regulated protein78 (GRP78) protein expressions were tested by using Western blot. CXCR3 protein level significantly increased in osteoarthritis patients (2.66 ± 0.25 vs. 1.00 ± 0.05, p<0.05). CXCR3 siRNA significantly reduced nitrate level in chondrocytes induced by IL-β (35.22 ± 1.76 vs. 17.82 ± 0.89, p<0.05) without affecting cell apoptosis (1.13 ± 0.05 vs. 0.859 ± 0.04, p>0.05). CXCR3 siRNA markedly downregulated nitrate level in chondrocytes (50.63 ± 2.53 vs. 30.63 ± 1.63, p<0.05) and alleviated cell apoptosis induced by SNP (1.98 ± 0.10 vs. 1.25 ± 0.06, p<0.05). UPR pathway C/EBP homology protein (CHOP) and glucose regulated protein78 (GRP78) participated in the process of chondrocyte apoptosis. Endoplasmic reticulum (ER) stress signaling pathway CHOP and GRP78 are involved in CXCR3 receptor attenuating chondrocyte apoptosis induced by SNP.

Autoantibodies in Serum of Systemic Scleroderma Patients: Peptide-Based Epitope Mapping Indicates Increased Binding to Cytoplasmic Domains of CXCR3.

Systemic sclerosis (SSc) is a severe chronic autoimmune disease with high morbidity and mortality. Sera of patients with SSc contain a large variety of autoantibody (aab) reactivities. Among these are functionally active aab that bind to G protein-coupled receptors (GPCR) such as C-X-C motif chemokine receptor 3 (CXCR3) and 4 (CXCR4). Aab binding to the N-terminal portion of these two GPCRs have been shown to be associated with slower disease progression in SSc, especially deterioration of lung function. Aabs binding to GPCRs exhibit functional activities by stimulating or inhibiting GPCR signaling. The specific functional activity of aabs crucially depends on the epitopes they bind to. To identify the location of important epitopes on CXCR3 recognized by aabs from SSc patients, we applied an array of 36 overlapping 18-20mer peptides covering the entire CXCR3 sequence, comparing epitope specificity of SSc patient sera (N = 32, with positive reactivity with CXCR3) to healthy controls (N = 30). Binding of SSc patient and control sera to these peptides was determined by ELISA. Using a Bayesian model approach, we found increased binding of SSc patient sera to peptides corresponding to intracellular epitopes within CXCR3, while the binding signal to extracellular portions of CXCR3 was found to be reduced. Experimentally determined epitopes showed a good correspondence to those predicted by the ABCpred tool. To verify these results and to translate them into a novel diagnostic ELISA, we combined the peptides that represent SSc-associated epitopes into a single ELISA and evaluated its potential to discriminate SSc patients (N = 31) from normal healthy controls (N = 47). This ELISA had a sensitivity of 0.61 and a specificity of 0.85. Our data reveals that SSc sera preferentially bind intracellular epitopes of CXCR3, while an extracellular epitope in the N-terminal domain that appears to be target of aabs in healthy individuals is not bound by SSc sera. Based upon our results, we could devise a novel ELISA concept that may be helpful for monitoring of SSc patients.

JN-2, a C-X-C motif chemokine receptor 3 antagonist, ameliorates arthritis progression in an animal model

Rheumatoid arthritis (RA) is a chronic autoimmune disease that is characterized by uncontrolled joint inflammation and destruction of bone and cartilage. Previous studies have shown that C-X-C motif chemokine 10 (CXCL10) has important roles in RA development and that blocking CXCL10 expression effectively inhibits arthritis progression in animal models. However, clinical study using anti-CXCL10 monoclonal antibody (MDX-1100) to block CXCL10 expression in patients with RA did not show significant effectiveness. Therefore, we turned our attention to C-X-C motif chemokine receptor 3 (CXCR3), which is a receptor for CXCL9, CXCL10, and CXCL11, to treat RA. In the present study, administration of JN-2, our newly developed CXCR3 antagonist, ameliorated the progression of arthritis in a collagen-induced arthritis animal model. JN-2 also inhibited CXCR3-induced cell migration and pro-inflammatory cytokine expression of bone marrow–derived macrophages and CD4+ T cells in vitro. In addition, we found that CXCL10 formed an auto-amplification loop through activation of NFκB. Furthermore, Phosphorylation of p65 at serine 536 played an important role in the auto-amplification of CXCL10. Overall, the present results demonstrated that JN-2 decreased inflammation by inhibiting CXCR3-enhanced cell migration and pro-inflammatory cytokine expression, which then ameliorated arthritis progression.

T helper 1/T helper 2-associated chemokine and chemokine receptor expression in immune thrombocytopenia

Background Immune thrombocytopenia (ITP) is an acquired immune disorder. Chemokines have complicated role in different autoimmune disorders including ITP. C-C motif chemokine ligand 2 (CCL2) chemokine represents a T helper (Th)2 polarizing chemokine, whereas C-X-C motif chemokine receptor 3 (CXCR3) and C-C motif chemokine receptor 2 (CCR2) represent chemokine members with Th1 polarization effect on the immune system. ITP is associated with an imbalance in Th1/Th2 ratio and favors Th1 polarization. This study aimed to explore the role of CCL2 and its receptor CCR2 in addition to CXCR3 receptor gene expression in the pathogenesis and severity of ITP.Participants and methods Expression of CCL2, CCR2, and CXCR3 was assayed using real-time quantitative polymerase chain reaction in peripheral blood mononuclear cells of 21 normal healthy participants and 68 patients with ITP: 24 acute cases, 25 chronic responder cases, and 19 chronic non-responder cases.Results Acute ITP group showed a 1.85 median fold change in CCL2 gene expression from the control group. CCR2 and CXCR3 showed a higher median fold change from the control group in acute ITP (7.36 and 5.42, respectively) and in chronic non-responder patients (3.38 and 4.32, respectively), whereas the chronic responder patients showed the least changes (1.63 and 2.35, respectively). There was no significant difference in chemokine or chemokine receptors gene expression between different ITP groups (P>0.05). Statistically significant positive correlations were detected between CCL2 and CCR2 (r=0.453, P<0.001) and between CXCR3 and CCR2 (r=0.583, P<0.001) among patients with ITP.Conclusion CCR2 and CXCR3 but not CCL2 may have a role in ITP pathogenesis. Further studies investigating the role of the complicated chemokine network may help better understanding of ITP pathogenesis.

CXCR3 ligands in pleural fluid as markers for the diagnosis of tuberculous pleural effusion.

A tertiary care academic medical centre. To evaluate the clinical usefulness of C-X-C motif chemokine receptor 3 (CXCR3) ligands in tuberculous pleural effusion (TPE). We recruited 336 patients with pleural effusion due to various causes. Concentrations of interferon-gamma (IFN-γ) and the CXCR3 ligands CXCL9 and CXCL11 were determined using enzyme immunoassays; adenosine deaminase (ADA) activity was measured in pleural fluid and serum. TPE was diagnosed in 106 patients. Non-TB conditions included lung cancer (n = 95), para-pneumonic effusion (n = 52), non-lung malignancy (n = 30), other exudate (n = 24) and transudate (n = 29) disorders. All marker levels in serum samples and pleural fluid were significantly higher in the TPE group. Analyses of receiver operating characteristic curves for differentiating TPE from non-TB effusions produced the following results for the area under the curve (AUC) for CXCL9, CXCL11, IFN-γ and ADA, respectively: 0.982, 0.952, 0.982, and 0.952. Marker AUCs in lymphocytic exudates were also high. Combining the levels of CXCL9, IFN-γ and ADA in pleural fluid improved the diagnostic performance. Serum levels of CXCL9 had the highest AUC (0.848) for diagnosing TPE. Levels of CXR3 ligands in pleural effusion are useful for diagnosing TPE.

Advanced glycation end products induce the apoptosis of and inflammation in mouse podocytes through CXCL9-mediated JAK2/STAT3 pathway activation.

Diabetic nephropathy (DN) is a serious and one of the most common microvascular complications of diabetes. There is accumulating evidence to indicate that advanced glycation end products (AGEs), senescent macroprotein derivatives formed at an accelerated rate under conditions of diabetes, play a role in DN. In this study, we found that the serum and urine levels of C-X-C motif chemokine ligand 9 (CXCL9) were significantly elevated in patients with DN compared with healthy controls. Based on an in vitro model of mouse podocyte injury, AGEs decreased the proliferation of podocytes and increased the expression of CXCL9 and C-X-C motif chemokine receptor 3 (CXCR3), and promoted the activation of signal transducer and activator of transcription 3 (STAT3). The knockdown of CXCL9 by the transfection of mouse podoyctes with specific siRNA significantly increased the proliferation and decreased the apoptosis of the podoyctes. Moreover, the levels of inflammatory factors, such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 were also decreased in the podoyctes transfected with siRNA-CXCL9, accompanied by the increased expression of nephrin and podocin, and decreased levels of Bax/Bcl-2 and activated caspase-3. The knockdown of CXCL9 also led to the inactivation of the Janus kinase 2 (JAK2)/STAT3 pathway. Importantly, the use of the JAK2 inhibitor, AG490, and valsartan (angiotensin II receptor antagonist) attenuated the injury induced to mouse podoyctes by AGEs. On the whole, and to the best of our knowledge, this study demonstrates for the first time that AGEs exert pro-apoptotic and pro-inflammatory effects in mouse podoyctes through the CXCL9-mediated activation of the JAK2/STAT3 pathway. Thus, our data provide a potential therapeutic target for DN.

T helper type 1-related molecules as well as interleukin-15 are hyperexpressed in the skin lesions of patients with pyoderma gangrenosum.

Pyoderma gangrenosum (PG) is a rare, immune-mediated skin disease classified into the group of neutrophilic dermatoses. Although a number of studies confirmed the central role of innate immunity, only few studies have investigated the possible contributing role of acquired immunity. In particular, no reports concerning T helper type 1 (Th1) and Th2 cells are available as yet. Therefore, 15 patients with PG, five with Sweet's syndrome (SS) and nine skin specimens from healthy controls (HC) were investigated, evaluating the expression of Th1-related markers interleukin (IL)-12, interferon (IFN)-γ, C-X-C motif chemokine receptor 3 (CXCR3) and C-C motif chemokine receptor 5 (CCR5), of the Th2-related molecules IL-4, IL-5, IL-13 and CCR3, of the co-stimulatory axis CD40/CD40 ligand, of IL-15 and the natural killer (NK) cell marker CD56 in skin lesions by immunohistochemistry. Patients with PG and SS showed a higher expression of Th1 markers than HC. Conversely, IL-5- and CCR3-expressing cells were less numerous in PG skin lesions compared to SS (P = 0·0157 and <0·0001, respectively). Both CD40 and CD40L were expressed more in PG than in SS and HC (P < 0·0001 for both). Finally, the number of IL-15+ and CD56+ cells was higher in the skin of patients with PG than in those of SS and HC (P < 0·0001 for both). Our results suggest that Th2 cells are down-regulated in PG. At the same time, over-expression of the co-stimulatory axis CD40/CD40L amplifies the impairment of the Th1/Th2 balance. Both these findings might explain the most aggressive behaviour of PG in comparison to SS. Moreover, over-expression of IL-15+ and CD56+ cells may suggest a possible role of NK cells in the pathogenesis of the disease.

C-X-C Motif Chemokine Receptor 3 Splice Variants Differentially Activate Beta-Arrestins to Regulate Downstream Signaling Pathways.

Biased agonism, the ability of different ligands for the same receptor to selectively activate some signaling pathways while blocking others, is now an established paradigm for G protein-coupled receptor signaling. One group of receptors in which endogenous bias is critical is the chemokine system, consisting of over 50 ligands and 20 receptors that bind one another with significant promiscuity. We have previously demonstrated that ligands for the same receptor can cause biased signaling responses. The goal of this study was to identify mechanisms that could underlie biased signaling between different receptor splice variants. The C-X-C motif chemokine receptor 3 (CXCR3) has two splice variants, CXCR3A and CXCR3B, which differ by 51 amino acids at its N-terminus. Consistent with an earlier study, we found that C-X-C motif chemokine ligands 4, 9, 10, and 11 all activated G αi at CXCR3A, while at CXCR3B these ligands demonstrated no measurable G αi or G αs activity. β-arrestin (βarr) was recruited at a reduced level to CXCR3B relative to CXCR3A, which was also associated with differences in βarr2 conformation. βarr2 recruitment to CXCR3A was attenuated by both G protein receptor kinase (GRK) 2/3 and GRK5/6 knockdown, while only GRK2/3 knockdown blunted recruitment to CXCR3B. Extracellular regulated kinase 1/2 phosphorylation downstream from CXCR3A and CXCR3B was increased and decreased, respectively, by βarr1/2 knockout. The splice variants also differentially activated transcriptional reporters. These findings demonstrate that differential splicing of CXCR3 results in biased responses associated with distinct patterns of βarr conformation and recruitment. Differential splicing may serve as a common mechanism for generating biased signaling and provides insights into how chemokine receptor signaling can be modulated post-transcriptionally.

Induction of cathepsin B by the CXCR3 chemokines CXCL9 and CXCL10 in human breast cancer cells.

Cathepsin B (CTSB) is a lysosomal cysteine protease that has been linked to the progression of breast cancer, for example by activation of other proteases and tumor-promoting cytokines, thereby supporting tumor invasion and metastasis. Previously, it was shown that CTSB cleaves and inactivates C-X-C motif chemokine receptor 3 (CXCR3) chemokines. As CXCR3 ligands have been demonstrated to induce proteases in cancer cells, the present study hypothesized that they may also affect CTSB in breast cancer cells. The results demonstrated that the human breast cancer tumor cell lines MCF-7 and MDA-MB-231 express the CXCR3 splice variants A and B and CTSB. Upon binding to CXCR3, the two chemokine ligands C-X-C motif chemokine ligand (CXCL) 9 and CXCL10 trigger upregulation of CTSB in these breast cancer cells, whereas the CXCR3-B-specific ligand CXCL4 has no such effect, suggesting the involvement of CXCR3-A in the regulation of CTSB. In early-stage human breast cancer specimens (n=81), overexpression of CXCR3 is associated with statistically significant poorer overall survival, independent of lymph node status, tumor size and nuclear grading (hazard ratio=1.99; 95% confidence interval=1.00–3.97; P=0.050). In conclusion, the data from the current study propose a so far unknown mechanism by which breast cancer cells may exploit tumor-suppressive chemokines to enhance their invasiveness and reduce immune cell infiltration by the degradation of these chemokines. This mechanism may support the established unfavorable prognostic feature of CXCR3 expression in breast cancer.

Pro-tumoural CXCL10/CXCR3-A autocrine loop in invasive mucinous lung adenocarcinoma.

Invasive mucinous adenocarcinoma (IMA) is a mucinous variant of lepidic predominant lung adenocarcinoma (LPA) and associated with a worse prognosis. We postulated that cytokine expression would enable us to differentiate IMA from LPA in terms of prognosis and acquisition of pro-tumoural capacities.A 30-cytokine panel was assessed in bronchoalveolar lavage fluids (BALF) from IMA (n=38), LPA (n=25) and control samples (n=7). We investigated the expression of differentially expressed cytokines and splice variants of their receptors in surgical samples. The presence of EGFR and KRAS mutations were determined. We also examined the expression of cytokines and splice variants of their receptors in different cell lines, exploring their functional impact on signalling pathways, proliferation and migration.Only C-X-C motif chemokine 10 (CXCL10) was differentially expressed, namely overexpressed in IMA BALF compared with LPA. CXCL10 overexpression in BALF was linked to a worse prognosis. In surgical samples, CXCL10 and its receptor C-X-C motif chemokine receptor 3 (CXCR3) were overexpressed in IMA compared to LPA. A pro-tumoural CXCR3-A splice variant was overexpressed in IMA, suggesting a CXCL10/CXCR3-A autocrine loop in IMA. CXCL10 and CXCR3 expression were not correlated with EGFR or KRAS status. CXCL10 up-regulated CXCR3-A expression, Erk1/2 phosphorylation and enhanced migration in the mucinous H2228 cell line.CXCL10/CXCR3-A may play a pro-tumoural role in IMA via an autocrine mechanism.

Critical Role of IP-10 on Reducing Experimental Corneal Neovascularization

Aim and scope: To address the role of interferon-induced protein of 10 kDa (IP-10) in the course of corneal neovascularization (CrNV) in a mouse model of experimental corneal neovascularization. Material and method: BALB/c mice that were 7- to 8-week-old male were included in the study. Corneal injury was induced by NaOH. Mice were randomly divided into 2 groups of IP-10 and vehicle. The alkali-treated eyes received 5 μl of 5 μg/ml IP-10 dissolved in 0.2% sodium hyaluronate for IP-10-treated group, or 5 μl of 0.2% sodium hyaluronate for vehicle-treated group twice a day for 7 days immediately after the alkali injury. 2 weeks after alkali injury, corneas were removed and used for whole mount CD31 staining. The percentages of neovascularization on corneal photographs were examined with digital image analysis. In other experiments, at indicated time intervals, the corneas were removed. Angiogenic factor expression in the early phase after injury was quantified by real-time PCR and western blot. The VEGF expression in macrophages infiltrating into burned corneas was examined by Flow cytometry (FCM) and immunofluorescence. Tube formation and cell proliferation of human retinal endothelial cells (HRECs) were detected after being stimulated with IP-10 in vitro. Results: The mRNA and protein expression of IP-10 and C-X-C motif chemokine receptor 3 (CXCR3) was augmented after the alkali injury (p < 0.05). Compared with vehicle-treated mice, IP-10-treated mice exhibited reduced CrNV 2 weeks after injury, as evidenced by diminished CD31-positive areas (p < 0.05). Concomitantly, the intracorneal mRNA and protein expression enhancement of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) was lower in IP-10-treated mice than in vehicle-treated mice after injury (p < 0.05). Moreover, IP-10 inhibited HREC tube formation and proliferation in vitro. Conclusion: IP-10-treated mice exhibited reduced alkali-induced CrNV through decreasing intracorneal VEGF and bFGF expression, and inhibiting endothelial cell proliferation and tube formation.

CXCR3-Mediated Skin Homing of Autoreactive CD8 T Cells Is a Key Determinant in Murine Graft-Versus-Host Disease

The pathomechanisms underlying the development of cutaneous graft-versus-host disease (GVHD) are incompletely defined. We previously reported that K14-mOVA mice expressing membrane ovalbumin (mOVA), driven by the keratin 14 (K14) promoter, developed GVHD-like mucocutaneous disease and weight loss following transfer of OVA-specific, CD8(+) OT-I T cells. In this study, we demonstrate that early in the course of disease, the kinetics of epidermal expression of C-X-C motif chemokine ligand 9 (CXCL9) and CXCL10, interferon-γ-inducible chemokines that bind the C-X-C motif chemokine receptor 3 (CXCR3) receptor, coincides with CXCR3 expression by OT-I cells in secondary lymphoid organs. Recruitment of OT-I cells into the skin began by day 5 with progressive accumulation through day 13 post transfer. Transfer of CXCR3-knockout (CXCR3KO) OT-I cells into K14-mOVA mice resulted in strikingly attenuated skin disease. CXCR3KO OT-I cells retained full activation and effector function, but preferentially accumulated in the spleen, in contrast to wild-type (WT) OT-I cells that accumulated in skin-draining lymph nodes. Moreover, OT-I cells accounted for a significantly reduced percentage of skin-infiltrating lymphocytes in mice receiving CXCR3KO OT-I cells compared with WT OT-I cells. These results identify CXCR3 as being critical to the skin-selective effector T-cell recruitment underlying autoreactive GVHD, suggesting CXCR3 as a potential target in the treatment of GVHD and related skin diseases.

Therapeutic effect of anti-C-X-C motif chemokine 10 (CXCL10) antibody on C protein-induced myositis mouse.

IntroductionC-X-C motif chemokine 10 (CXCL10) is a chemokine that plays a critical role in the infiltration of T cells in autoimmune diseases and is reported to be expressed in muscle tissue of polymyositis. To determine the therapeutic efficacy of CXCL10 blockade, we investigated the role of CXCL10 and the effect of anti-CXCL10 antibody treatment in C protein-induced myositis (CIM), an animal model of polymyositis.MethodsCIM was induced with human skeletal muscle C protein fragment in female C57BL/6 mice. Immunohistochemistry of CXCL10 and C-X-C motif chemokine receptor 3 (CXCR3) and measurement of serum CXCL10 were performed. Cell surface markers and interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) in CIM lymph node cells was investigated by flow cytometry. Mice with CIM were treated with anti-CXCL10 antibody or control antibody (anti-RVG1) and the inflammation in muscle tissue was assessed.ResultsImmunohistochemistry showed increased expression of CXCL10 and CXCR3 in the inflammatory lesions of muscle in CIM. Especially, CD8+ T cells invading myofiber expressed CXCR3. Serum level of CXCL10 was increased in CIM compared to the level in normal mice (normal mouse, 14.3 ± 5.3 pg/ml vs. CIM, 368.5 ± 135.6 pg/ml, P < 0.001). CXCR3 positivity in CD8+ T cells was increased compared to that of CD4+ T cells in the lymph node cells of CIM (CXCR3+ among CD8+ T cell, 65.9 ± 2.1% vs. CXCR3+ among CD4+ T cell, 23.5 ± 4.7%, P <0.001). Moreover, IFN-γ+ cells were increased among CXCR3+CD8+ T cells compared to CXCR3–CD8+ T cells (CXCR3+CD8+ T cell, 28.0 ± 4.2% vs. CXCR3-CD8+ T cell, 9.5 ± 1.5%, P = 0.016). Migration of lymph node cells was increased in response to CXCL10 (chemotactic index was 1.91 ± 0.45). CIM mice treated with anti-CXCL10 antibody showed a lower inflammation score in muscles than those with anti-RVG1 (median, anti-CXCL10 treatment group, 0.625 vs. anti-RVG1 treatment group, 1.25, P = 0.007).ConclusionsCXCL10/CXCR3 expression was increased in the inflammation of CIM model and its blockade suppressed inflammation in muscle.