BACKGROUND

Chemokines are chemotactic mediators that are implicated in liver diseases. In viral hepatitis and primary biliary cirrhosis, a predominant chemokine receptor expressed in the liver is CXCR3, suggesting that its specific ligands are important in the progression of chronic liver diseases across different aetiologies.

RESULTS

We analysed the serum concentrations of the CXCR3 ligands, CXCL9 (monokine induced by interferon-γ), CXCL10 (interferon-γ-inducible protein 10) and CXCL11 (interferon-inducible T cell α chemo-attractant) in healthy controls (n=53), subjects with histologically determined liver fibrosis (n=109) and patients with different stages of cirrhosis (n=153) of various disease aetiologies. Chemokine concentrations were determined by cytometric bead assay or ELISA respectively. Serum concentrations of all three chemokines were significantly increased in patients with chronic liver diseases compared with healthy controls (P<0.001). In the biopsied fibrosis cohort, CXCL9 and CXCL10 were positively associated with the severity of liver fibrosis (histology and serum markers), while CXCL11 was not. In cirrhotic patients, CXCL9 was increased in early Child-Pugh stages, while CXCL11 was elevated only in Child B and C patients and CXCL10 across all stages. Notably, CXCR3 chemokines were also associated with the development of clinical complications of cirrhosis, especially portal hypertension. All chemokines significantly correlated with serum levels of the hepatoprotective cytokines interleukin (IL)-6 and IL-10, suggesting their involvement in a counter-regulatory response during the progression of liver disease, shedding new light on their involvement in the pathophysiology of chronic liver diseases.

CONCLUSIONS

CXCR3 chemokines are differentially expressed during chronic liver diseases across different disease stages and aetiologies. Their association with portal hypertension and hepatoprotective cytokines implies biological functions beyond immune cell recruitment, thereby provoking new diagnostic and therapeutic concepts.

Because IFN-gamma-inducible chemokines, Mig (CXCL9), IP-10 (CXCL10), I-TAC (CXCL11) and their receptor, CXCR3, are critical molecules in T cell trafficking and generation of effector T cells, we examined their expression in the muscle biopsies of patients with sporadic inclusion body myositis (s-IBM) and disease controls. The functional role of these molecules was also studied by examining the effect and time kinetics of IFN-gamma in inducing Mig and IP-10 expression in human myotubes in vitro. We found significantly high levels of Mig and IP-10 mRNA expression in s-IBM muscles compared to controls. IFN-gamma upregulated the mRNA expression of Mig and IP-10 by human myotubes in a dose-dependent manner. By double-label immunohistochemistry, Mig was expressed on a subset of CD8(+) cells and the areas of the muscle fiber in contact or contiguous to the T cells; CXCR3 was expressed only on a subset of the autoinvasive CD8(+) T cells but not the myofibers. IP-10 and I-TAC were not detected by immunocytochemistry. The findings indicate that in s-IBM, IFN-gamma is involved in the upregulation and in situ production of proinflammatory chemokines, which, in turn, participate in the recruitment of activated T cells and contribute to the self-sustaining nature of endomysial inflammation.

The inflamed synovial tissue of rheumatoid arthritis (RA) is characterized by an infiltration with Th1 cells that predominantly express the chemokine receptors CXCR3 and CCR5. In this study, we investigated the production of the CXCR3-agonistic chemokines CXCL9, CXCL10, and CXCL11 by synovial tissue cells and synovial fibroblast-cell lines (fourth or fifth passage) from RA patients. Concentrations of all CXCR3 ligands in synovial fluids were markedly higher in RA patients than in osteoarthritis (OA) patients. Synovial tissue cells from RA patients more strongly expressed mRNAs for CXCR3 ligands and spontaneously secreted larger amounts of these chemokine proteins than the cells from OA patients. The mRNA expression of all CXCR3 ligands was induced in synovial fibroblasts from RA patients after stimulation with interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), or interleukin-1 beta (IL-1beta). However, synovial fibroblasts significantly secreted CXCL9 and CXCL10 proteins, but not CXCL11 protein, after IFN-gamma stimulation and secreted only CXCL10 protein after TNF-alpha or IL-1beta stimulation. When stimulated with a combination of IFN-gamma and TNF-alpha, these cells were able to secrete large amounts of all three chemokines. These results indicate that synovial fibroblasts may be involved in perpetuating the Th1 immune response by producing the Th1-associated CXCR3 ligands, and the synergistic effect of IFN-gamma and TNF-alpha may be important for their chemokine production in RA joints.

BACKGROUND

STAT1 has been attributed a function as tumor suppressor. However, in breast cancer data from microarray analysis indicated a predictive value of high mRNA expression levels of STAT1 and STAT1 target genes belonging to the interferon-related signature for a poor response to therapy. To clarify this issue we have determined STAT1 expression levels and activation by different methods, and investigated their association with tumor infiltration by immune cells. Additionally, we evaluated the interrelationship of these parameters and their significance for predicting disease outcome.

METHODS

Expression of STAT1, its target genes SOCS1, IRF1, CXCL9, CXCL10, CXCL11, IFIT1, IFITM1, MX1 and genes characteristic for immune cell infiltration (CD68, CD163, PD-L1, PD-L2, PD-1, CD45, IFN-γ, FOXP3) was determined by RT-PCR in two independent cohorts comprising 132 breast cancer patients. For a subset of patients, protein levels of total as well as serine and tyrosine-phosphorylated STAT1 were ascertained by immunohistochemistry or immunoblotting and protein levels of CXCL10 by ELISA.

RESULTS

mRNA expression levels of STAT1 and STAT1 target genes, as well as protein levels of total and serine-phosphorylated STAT1 correlated with each other in neoplastic tissue. However, there was no association between tumor levels of STAT1 mRNA and tyrosine-phosphorylated STAT1 and between CXCL10 serum levels and CXCL10 expression in the tumor. Tumors with increased STAT1 mRNA amounts exhibited elevated expression of genes characteristic for tumor-associated macrophages and immunosuppressive T lymphocytes. Survival analysis revealed an association of high STAT1 mRNA levels and bad prognosis in both cohorts. A similar prognostically relevant correlation with unfavorable outcome was evident for CXCL10, MX1, CD68, CD163, IFN-γ, and PD-L2 expression in at least one collective. By contrast, activation of STAT1 as assessed by the level of STAT1-Y701 phosphorylation was linked to positive outcome. In multivariate Cox regression, the predictive power of STAT1 mRNA expression was lost when including expression of CXCL10, MX1 and CD68 as confounders.

CONCLUSIONS

Our study confirms distinct prognostic relevance of STAT1 expression levels and STAT1 tyrosine phosphorylation in breast cancer patients and identifies an association of high STAT1 levels with elevated expression of STAT1 target genes and markers for infiltrating immune cells.

BACKGROUND

After lung transplantation, insults to the allograft generally result in one of four histopathologic patterns of injury: (1) acute rejection, (2) lymphocytic bronchiolitis, (3) organizing pneumonia, and (4) diffuse alveolar damage (DAD). We hypothesized that DAD, the most severe form of acute lung injury, would lead to the highest risk of chronic lung allograft dysfunction (CLAD) and that a type I immune response would mediate this process.

OBJECTIVE

Determine whether DAD is associated with CLAD and explore the potential role of CXCR3/ligand biology.

METHODS

Transbronchial biopsies from all lung transplant recipients were reviewed. The association between the four injury patterns and subsequent outcomes were evaluated using proportional hazards models with time-dependent covariates. Bronchoalveolar lavage (BAL) concentrations of the CXCR3 ligands (CXCL9/MIG, CXCL10/IP10, and CXCL11/ITAC) were compared between allograft injury patterns and "healthy" biopsies using linear mixed-effects models. The effect of these chemokine alterations on CLAD risk was assessed using Cox models with serial BAL measurements as time-dependent covariates.

RESULTS

There were 1,585 biopsies from 441 recipients with 62 episodes of DAD. An episode of DAD was associated with increased risk of CLAD (hazard ratio, 3.0; 95% confidence interval, 1.9-4.7) and death (hazard ratio, 2.3; 95% confidence interval, 1.7-3.0). There were marked elevations in BAL CXCR3 ligand concentrations during DAD. Furthermore, prolonged elevation of these chemokines in serial BAL fluid measurements predicted the development of CLAD.

CONCLUSIONS

DAD is associated with marked increases in the risk of CLAD and death after lung transplantation. This association may be mediated in part by an aberrant type I immune response involving CXCR3/ligands.

Lipopolysaccharide (LPS) in the outer membrane of Neisseria meningitidis plays a dominant role as an inflammation-inducing molecule in meningococcal disease. We have used microarray analysis to study the global gene expression after exposure of human monocytes for 3 h to wild-type N. meningitidis (10(6)), LPS-deficient N. meningitidis (10(6) and 10(8)), and purified N. meningitidis LPS (1 ng [33 endotoxin units]/ml) to identify LPS-inducible genes. Wild-type N. meningitidis (10(6)) induced 4,689 differentially expressed genes, compared with 72 differentially expressed genes induced by 10(6) LPS-deficient N. meningitidis organisms. However, 10(8) LPS-deficient N. meningitidis organisms induced 3,905 genes, indicating a dose-response behavior of non-LPS cell wall molecules. A comparison of the gene expression patterns from 10(6) wild-type N. meningitidis and 10(8) LPS-deficient N. meningitidis organisms showed that 2,401 genes in human monocytes were not strictly LPS dependent. A list of "particularly LPS-sensitive" genes (2,288), differentially induced by 10(6) wild-type N. meningitidis but not by 10(8) LPS-deficient N. meningitidis organisms, showed an early expression of beta interferon (IFN-beta), most likely through the Toll-like receptor-MyD88-independent pathway. Subsequently, IFN-beta may activate the type I IFN signaling pathway, and an unknown number of IFN-beta-inducible genes, such as those for CXCL9, CXCL10, CXCL11, IFIT1, IFIT2, IFIT3, and IFIT5, are transcribed. Supporting this, human monocytes secreted significantly higher levels of CXCL10 and CXCL11 when stimulated by 10(6) wild-type N. meningitidis organisms than when stimulated by 10(8) LPS-deficient N. meningitidis organisms. Plasma CXCL10, but not CXCL11, was positively correlated (r = 0.67; P < 0.01) to LPS in patients (n = 24) with systemic meningococcal disease. Thus, new circulating biomarkers in meningococcal disease may be suggested through LPS-induced gene expression changes in human monocytes.

The Th1-associated chemokines CXCL9, CXCL10, and CXCL11 coordinate migration of CXCR3(+) Th1 cells. The objective of this study was to evaluate the role of the innate immune system in stimulating chemokine expression in an experimental model of dry eye and bridge the gap between innate and adaptive immunity. Desiccating stress (DS) induced very early (6 h) expression and production of Th1-associated chemokines in cornea and conjunctiva of C57BL/6 and RAG1 knockout (KO) mice, demonstrating that chemokine expression does not require innate T cells. We then demonstrated that activating the innate immune system prior to adoptive transfer of T cells to RAG1KO mice increased disease severity. Interestingly, lack of induction of chemokines CXCL9, CXCL10, and CXCL11 in IFN-γKO mice provided evidence that their expression requires IFN-γ for induction. Treatment of RAG1KO mice with anti-NK1.1 prevented the increase of CXCL9, CXCL10, and CXCL11 in response to DS, compared with isotype controls. Additionally, DS increased the expression of NKG2D in the conjunctiva. The expression of the NKG2D ligand, retinoic acid early inducible gene 1, also increased at the ocular surface at both the protein and gene levels. Neutralization of NKG2D at the ocular surface decreased the expression of CXCL9, CXCL10, CXCL11, and IFN-γ. In summary, upregulation of CXCL9, CXCL10, and CXCL11 expression in experimental dry eye is T cell-independent, requiring IFN-γ-producing NKG2D(+) NK cells that are activated in response to DS-induced stress signals. This study provides insight into the events that trigger the initial immune response in dry eye pathology.

BACKGROUND

Alterations in serum CXCR3 ligand levels were examined in interstitial cystitis (IC) patients; similar expression patterns in serum as well as CXCR3, CXCR3 ligands, and cytokines expressed by peripheral and local leukocyte subpopulations were characterized during cyclophosphamide (CYP)-induced acute cystitis in mice.

RESULTS

Serum levels of monokine-induced by interferon-gamma (IFN-gamma) (MIG/CXCL9), IFN-gamma-inducible protein-10 (IP-10/CXCL10), and IFN-gamma-inducible T cell alpha chemoattractant (I-TAC/CXCL11) were elevated in patients with IC. These clinical features closely correlated with CYP-induced cystitis in mice. Serum levels of these CXCR3 ligands and local T helper type 1 (Th1) cytokines were also increased. We demonstrate that CXCR3 as well as CXCL9, CXCL10 and CXCL11 mRNA were significantly expressed by urinary bladder lymphocytes, while CXCR3 and CXCL9 transcripts were significantly expressed by iliac lymph node leukocytes following CYP treatment. We also show that the number of CD4+ T cells, mast cells, natural killer (NK) cells, and NKT cells were increased at systemic (spleen) and mucosal (urinary bladder and iliac lymph nodes) sites, following CYP-induced cystitis in mice. Importantly, CXCL10 blockade attenuated these increases caused by CYP.

CONCLUSIONS

Antibody (Ab)-mediated inhibition of the most abundant serum CXCR3 ligand, CXCL10, in mice decreased the local production of CXCR3 ligands as well as Th1 cytokines expressed by local leukocytes, and lowered corresponding serum levels to reduce the severity of CYP-induced cystitis. The present study is among the first to demonstrate some of the cellular and molecular mechanisms of chemokines in cystitis and may represent new drug target for this disease.

IL-27, a member of the IL-12 cytokine family, primes Th1 cell differentiation, whereas it suppresses Th17 cell development. We have previously reported that serum IL-27 levels are elevated in psoriatic patients and that IL-27 greatly induces in vitro production of Th1-type chemokines through STAT1 activation. In this study, to further investigate the in vivo role of IL-27 in the pathogenesis of psoriasis, we induced psoriasis-like inflammation on mouse back skin with topical application of imiquimod (IMQ), and continuously injected IL-27 or PBS subcutaneously. IMQ-treated skin showed an increase of IL-27 mRNA levels and the infiltration of IL-27-producing cells in the papillary dermis. The injection of IL-27 to the IMQ-treated skin exacerbated the disease compared with PBS injection. The IL-27 injection further augmented mRNA levels of IFN-γ, CXCL9, CXCL10, CXCL11, and TNF-α, without altering those of IL-17A, IL-17F, IL-22, and CCL20. Finally, IL-27 antagonism attenuated the upregulation of IFN-γ, CXCL9, CXCL10, CXCL11, and TNF-α mRNA levels, and induced clinical and histological improvement in the IMQ-treated skin. These results indicate that IL-27 would act in a proinflammatory manner, and thereby exacerbate the psoriasis-like skin inflammation induced by IMQ.

BACKGROUND

CXC chemokines are induced by inflammatory stimuli in epithelial cells and some, like MIG/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11, are antibacterial for Streptococcus pyogenes.

RESULTS

SpeB from S. pyogenes degrades a wide range of chemokines (i.e. IP10/CXCL10, I-TAC/CXCL11, PF4/CXCL4, GROalpha/CXCL1, GRObeta/CXCL2, GROgamma/CXCL3, ENA78/CXCL5, GCP-2/CXCL6, NAP-2/CXCL7, SDF-1/CXCL12, BCA-1/CXCL13, BRAK/CXCL14, SRPSOX/CXCL16, MIP-3alpha/CCL20, Lymphotactin/XCL1, and Fractalkine/CX3CL1), has no activity on IL-8/CXCL8 and RANTES/CCL5, partly degrades SRPSOX/CXCL16 and MIP-3alpha/CCL20, and releases a 6 kDa CXCL9 fragment. CXCL10 and CXCL11 loose receptor activating and antibacterial activities, while the CXCL9 fragment does not activate the receptor CXCR3 but retains its antibacterial activity.

CONCLUSIONS

SpeB destroys most of the signaling and antibacterial properties of chemokines expressed by an inflamed epithelium. The exception is CXCL9 that preserves its antibacterial activity after hydrolysis, emphasizing its role as a major antimicrobial on inflamed epithelium.

TNF-stimulated gene-6 (TSG-6) is a multifunctional protein secreted in response to pro-inflammatory stimuli by a wide range of cells, including neutrophils, monocytes, and endothelial cells. It has been shown to mediate anti-inflammatory and protective effects when administered in disease models, in part, by reducing neutrophil infiltration. Human TSG-6 inhibits neutrophil migration by binding CXCL8 through its Link module (Link_TSG6) and interfering with the presentation of CXCL8 on cell-surface glycosaminoglycans (GAGs), an interaction that is vital for the function of many chemokines. TSG-6 was also found to interact with chemokines CXCL11 and CCL5, suggesting the possibility that it may function as a broad specificity chemokine-binding protein, functionally similar to those encoded by viruses. This study was therefore undertaken to explore the ability of TSG-6 to regulate the function of other chemokines. Herein, we demonstrate that Link_TSG6 binds chemokines from both the CXC and CC families, including CXCL4, CXCL12, CCL2, CCL5, CCL7, CCL19, CCL21, and CCL27. We also show that the Link_TSG6-binding sites on chemokines overlap with chemokine GAG-binding sites, and that the affinities of Link_TSG6 for these chemokines (KD values 1-85 nm) broadly correlate with chemokine-GAG affinities. Link_TSG6 also inhibits chemokine presentation on endothelial cells not only through a direct interaction with chemokines but also by binding and therefore masking the availability of GAGs. Along with previous work, these findings suggest that TSG-6 functions as a pluripotent regulator of chemokines by modulating chemokine/GAG interactions, which may be a major mechanism by which TSG-6 produces its anti-inflammatory effects in vivo.

Ig-like transcript 3 (ILT3), an inhibitory receptor expressed by APC is involved in functional shaping of T cell responses toward a tolerant state. We have previously demonstrated that membrane (m) and soluble (s) ILT3 induce allogeneic tolerance to human islet cells in humanized NOD/SCID mice. Recombinant sILT3 induces the differentiation of CD8(+) T suppressor cells both in vivo and in vitro. To better understand the molecular mechanisms by which ILT3 suppresses immune responses, we have generated ILT3 knockdown (ILT3KD) dendritic cells (DC) and analyzed the phenotypic and functional characteristics of these cells. In this study, we report that silencing of ILT3 expression in DC (ILT3KD DC) increases TLR responsiveness to their specific ligands as reflected in increased synthesis and secretion of proinflammatory cytokines such as IL-1alpha, IL-1beta, and IL-6 and type I IFN. ILT3KD-DC also secretes more CXCL10 and CXCL11 chemokines in response to TLR ligation, thus accelerating T cell migration in diffusion chamber experiments. ILT3KD-DC elicit increased T cell proliferation and synthesis of proinflammatory cytokines IFN-gamma and IL-17A both in MLC and in culture with autologous DC pulsed with CMV protein. ILT3 signaling results in inhibition of NF-kappaB and, to a lesser extent, MAPK p38 pathways in DC. Our results suggest that ILT3 plays a critical role in the control of inflammation.

High levels of the intermediate filament keratin 17 (K17) correlate with a poor prognosis for several types of epithelial tumors. However, the causal relationship and underlying mechanisms remain undefined. A recent study suggested that K17 promotes skin tumorigenesis by fostering a specific type of inflammation. We report here that K17 interacts with the RNA-binding protein hnRNP K, which has also been implicated in cancer. K17 is required for the cytoplasmic localization of hnRNP K and for its role in regulating the expression of multiple pro-inflammatory mRNAs. Among these are the CXCR3 ligands CXCL9, CXCL10, and CXCL11, which together form a signaling axis with an established role in tumorigenesis. The K17-hnRNP K partnership is regulated by the ser/thr kinase RSK and required for CXCR3-dependent tumor cell growth and invasion. These findings functionally integrate K17, hnRNP K, and gene expression along with RSK and CXCR3 signaling in a keratinocyte-autonomous axis and provide a potential basis for their implication in tumorigenesis.

BACKGROUND

Exacerbations of asthma and chronic obstructive pulmonary disease (COPD) are heterogeneous.

OBJECTIVE

We sought to investigate the sputum cellular, mediator, and microbiome profiles of both asthma and COPD exacerbations.

METHODS

Patients with severe asthma or moderate-to-severe COPD were recruited prospectively to a single center. Sputum mediators were available in 32 asthmatic patients and 73 patients with COPD assessed at exacerbation. Biologic clusters were determined by using factor and cluster analyses on a panel of sputum mediators. Patterns of clinical parameters, sputum mediators, and microbiome communities were assessed across the identified clusters.

RESULTS

The asthmatic patients and patients with COPD had different clinical characteristics and inflammatory profiles but similar microbial ecology. Three exacerbation biologic clusters were identified. Cluster 1 was COPD predominant, with 27 patients with COPD and 7 asthmatic patients exhibiting increased blood and sputum neutrophil counts, proinflammatory mediators (IL-1β, IL-6, IL-6 receptor, TNF-α, TNF receptors 1 and 2, and vascular endothelial growth factor), and proportions of the bacterial phylum Proteobacteria. Cluster 2 had 10 asthmatic patients and 17 patients with COPD with increased blood and sputum eosinophil counts, type 2 mediators (IL-5, IL-13, CCL13, CCL17, and CCL26), and proportions of the bacterial phylum Bacteroidetes. Cluster 3 had 15 asthmatic patients and 29 patients with COPD with increased type 1 mediators (CXCL10, CXCL11, and IFN-γ) and proportions of the phyla Actinobacteria and Firmicutes.

CONCLUSIONS

A biologic clustering approach revealed 3 subgroups of asthma and COPD exacerbations, each with different percentages of patients with overlapping asthma and COPD. The sputum mediator and microbiome profiles were distinct between clusters.

The CXC chemokine receptor 3 (CXCR3) is predominantly expressed on T helper type 1 (Th1) cells that are involved in inflammatory diseases. The three CXCR3 ligands CXCL9, CXCL10, and CXCL11 are produced at sites of inflammation and elicit migration of pathological Th1 cells. Here, we are the first to characterize the pharmacological potencies and specificity of a CXCR3 antagonist, N-1R-[3-(4-ethoxy-phenyl)-4-oxo-3,4-dihydro-pyrido[2,3-d]pyrimidin-2-yl]-ethyl-N-pyridin-3-ylmethyl-2-(4-fluoro-3-trifluoromethyl-phenyl)-acetamide (NBI-74330), from the T487 small molecule series. NBI-74330 demonstrated potent inhibition of [(125)I]CXCL10 and [(125)I]CXCL11 specific binding (K(i) of 1.5 and 3.2 nM, respectively) and of functional responses mediated by CXCR3, such as ligand-induced guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding, calcium mobilization, and cellular chemotaxis (IC(50) of 7 to 18 nM). NBI-74330 was selective for CXCR3 because it showed no significant inhibition of chemotactic responses to other chemokines and did not inhibit radioligand binding to a panel of nonchemokine G-protein coupled receptors. There was a striking difference in potencies among the three CXCR3 ligands, with CXCL11>>) CXCL10>> CXCL9. A comparison of the rank order of K(i) values with the rank order of monocyte production levels of these three ligands revealed a precise inverse correlation, suggesting that the weaker receptor affinities of CXCL9 and CXCL10 were physiologically compensated for by an elevated expression, perhaps to maintain effectiveness of each ligand under physiological conditions.

OBJECTIVE

To investigate the involvement of the chemokines CXCL10/IP-10, CXCL11/I-TAC, CXCL8/1L-8, CXCL6/GCP-2, CCL3/MIP-1alpha, and CCL18/PARC, and gelatinases A and B in uveitis.

METHODS

Prospective, experimental, case-control study.

METHODS

Aqueous humor samples from 30 patients with active uveitis, and 14 control patients and paired serum samples were assayed for chemokines with specific enzyme-linked immunosorbent assays (ELISAs) and for gelatinase levels by quantitative zymography.

RESULTS

In control AH, none of the chemokines was detected. Gelatinase A was detected in all samples, and gelatinase B was detected in only one sample. In patients with uveitis, IP-10 was detected in all AH samples, whereas I-TAC, IL-8, GCP-2, MIP-1alpha, and PARC were detected in three, 16, six, two, and 12 samples, respectively. IP-10 levels were significantly higher in AH samples than those of serum (P =.006). Gelatinase A was detected in 29 AH samples and gelatinase B was detected in 26 samples. Gelatinase A levels were significantly higher in AH samples from patients than those of controls (P <.0001). In 11 AH samples, gelatinase B was detected in complex with lipocalin (NGAL). Disease activity correlated significantly with the levels of IP-10 (r =.627; P <.0001), gelatinase A (r =.508; P =.002), gelatinase B (r =.685; P <.0001), and NGAL-gelatinase B complex (r =.595; P <.0001).

CONCLUSIONS

These data suggest a pathogenic role of the T lymphocyte chemoattractant IP-10 and gelatinases in the recruitment and activity of T cells into the eye in patients with uveitis and in the pathogenesis of uveitis.

Community-acquired respiratory viruses (CARV) can accelerate the development of lung allograft dysfunction, but the immunologic mechanisms are poorly understood. The chemokine receptor CXCR3 and its chemokine ligands, CXCL9, CXCL10 and CXCL11 have roles in the immune response to viruses and in the pathogenesis of bronchiolitis obliterans syndrome, the predominant manifestation of chronic lung allograft rejection. We explored the impact of CARV infection on CXCR3/ligand biology and explored the use of CXCR3 chemokines as biomarkers for subsequent lung allograft dysfunction. Seventeen lung transplant recipients with CARV infection had bronchoalveolar lavage fluid (BALF) available for analysis. For comparison, we included 34 BALF specimens (2 for each CARV case) that were negative for infection and collected at a duration posttransplant similar to a CARV case. The concentration of each CXCR3 chemokine was increased during CARV infection. Among CARV infected patients, a high BALF concentration of either CXCL10 or CXCL11 was predictive of a greater decline in forced expiratory volume in 1 s, 6 months later. CXCR3 chemokine concentrations provide prognostic information and this may have important implications for the development of novel treatment strategies to modify outcomes after CARV infection.

Chemokines play an important role in regulating tumor-mediated immunity, angiogenesis, and tumor cell metastasis. The chemokine receptor, CXCR3, is expressed in various human tumors, including renal cell carcinoma (RCC). CXCR3 is also associated with antiangiogenic effects in multiple tumors, and we hypothesized that interleukin-2 (IL-2) treatment of patients with metastatic clear cell RCC could augment CXCR3 levels on circulating mononuclear cells and correlate to outcome. The kinetics of CXCR3 expression on circulating mononuclear cells and its ligands (CXCL9, CXCL10, and CXCL11) in plasma were evaluated in 20 patients with metastatic clear cell RCC during cycles 1 and 2 of high dose IL-2 therapy. Subpopulations of peripheral blood mononuclear cells (PBMCs) were studied by dual color flow cytometry. Angiogenic ligands were measured and an "angiogenic ratio" was calculated prehigh and posthigh dose IL-2. CXCR3 expression on PBMC at baseline was similar in patients with metastatic RCC and normal controls. PBMC CXCR3 expression increased during treatment, and peaked during cycle 2. Plasma from RCC patients displayed similar baseline levels of CXCR3 ligands to normal controls. However, the angiogenic ratio was significantly increased in patients with metastatic RCC at baseline. Plasma levels of CXCR3 ligands increased during treatment, resulting in a reversal in the angiogenic ratio to favor angiostatic chemokines. The CXCR3/CXCR3 ligand biologic axis and angiogenic ratio may be important biomarkers in clear cell RCC patients who are undergoing high dose IL-2 therapy.

Trafficking of leukocytes to sites of inflammation is an important step in the establishment of an immune response. Chemokines are critical regulators of leukocyte trafficking and are widely studied molecules for their important role in disease and for their potential as new therapeutic targets. The ability of chemokines to induce leukocyte recruitment has been mainly measured by in vitro chemotaxis assays, which lack many components of the complex biological process of leukocyte migration and therefore provide incomplete information about chemokine function in vivo. In vivo assays to study the activity of chemokines to induce leukocyte recruitment have been difficult to establish. We describe here the development of a robust in vivo recruitment assay for CD8(+) and CD4(+) T lymphocytes induced by the CXCR3 ligands IP-10 (CXCL10) and I-TAC (CXCL11). For this assay, in vitro activated T lymphocytes were adoptively transferred into the peritoneum of naïve mice. Homing of these transferred T lymphocytes into the airways was measured following intratracheal instillation of chemokines. High recruitment indices were achieved that were dependent on chemokine concentration and CXCR3 expression on the transferred lymphocytes. Recruitment was also inhibited by antibodies to the chemokine. The assay models the natural condition of chemokine-mediated lymphocyte migration into the airways as chemokines are expressed in the airways during inflammation. The nature of this model allows flexibility to study wildtype and mutant chemokines and chemokine receptors and the ability to evaluate chemokine antagonists and antibodies in vivo. This assay will therefore help elucidate a deeper understanding of the chemokine system in vivo.

OBJECTIVE

The CXC chemokine CXCL10 is up-regulated in the infarcted myocardium and limits cardiac fibrosis by inhibiting growth factor-mediated fibroblast migration. CXCL10 signals by binding to its receptor CXCR3; however, recently CXCR3-independent CXCL10 actions have been suggested. Our study explores the role of CXCR3 signalling in myocardial infarction and investigates its involvement in mediating the anti-fibrotic effects of CXCL10.

RESULTS

Wild-type and CXCR3 null mice underwent reperfused infarction protocols. CXCL10 was markedly induced in the infarct; in contrast, expression of the other two CXCR3 ligands, CXCL9 and CXCL11 was extremely low. CXCR3 loss did not affect scar size, geometric ventricular remodelling, collagen deposition, and systolic dysfunction of the infarcted heart. CXCR3 null mice had increased peak neutrophil recruitment and delayed myofibroblast infiltration in the infarcted heart, but exhibited comparable myocardial expression of pro-inflammatory cytokines and chemokines. In vitro, CXCL10 did not modulate Transforming Growth Factor (TGF)-β signalling, but inhibited basic fibroblast growth factor (bFGF)-induced cardiac fibroblast migration in both wild-type and CXCR3 null cells. Treatment of fibroblasts with heparinase and chondroitinase to cleave glycosaminoglycan chains abrogated the inhibitory effects of CXCL10 on cell migration.

CONCLUSIONS

CXCR3 signalling does not critically regulate cardiac remodelling and dysfunction following myocardial infarction. The anti-fibrotic effects of CXCL10 in the healing infarct and in isolated cardiac fibroblasts are CXCR3-independent and may be mediated through proteoglycan signalling. Thus, administration of CXCR3-defective forms of CXCL10 may be an effective anti-fibrotic strategy in the remodelling myocardium without activating a potentially injurious, CXCR3-driven T cell response.

The chemokine CXCL12 has a decisive role in tumor progression by mediating pro-angiogenic and pro-metastatic effects through its receptor CXCR4. The CXCL12 pathway is connected with another chemokine, CXCL11, through its second receptor CXCR7. CXCL11 also binds to the CXCR3 receptor. CXCL11 function in tumor angiogenesis is likely receptor dependent because CXCR3 predominantly mediates angiostatic signals whereas CXCR7 mediated signaling is rather angiogenic. We therefore studied the interaction of CXCL12 and CXCL11 in an in vivo model of colorectal cancer metastasis. GFP-transfected CT26.WT colorectal cancer cells were implanted into the dorsal skinfold chamber of syngeneic BALB/c mice. The animals received either peritumoral application of CXCL11 or intraperitoneal injections with neutralizing antibodies against CXCL11, CXCL12 or both. Tumor growth characteristics, angiogenesis, cell migration, invasive tumor growth, tumor cell proliferation and apoptosis were studied by intravital fluorescence microscopy and immunohistochemistry during an observation period of 14 days. Local exposure to CXCL11 significantly stimulated tumor growth compared to controls and enhanced invasive growth characteristics without affecting tumor angiogenesis and tumor cell migration. Neither CXCL11 nor CXCL12-blockade had a significant impact on tumor growth and angiogenesis, whereas the combined neutralization of CXCL11 and CXCL12 almost completely abrogated tumor vessel formation. As a consequence, tumor growth and invasive growth characteristics were reduced compared to the other groups. The results of the present study underline the interaction of CXCL12 and CXCL11 during tumor angiogenesis. The combined blockade of both signaling pathways may provide an interesting anti-angiogenic approach for anti-tumor therapy.

BACKGROUND

Ligands for CXCR3 chemokines [IFN-gamma-inducible protein of 10 kD (IP-10/CXCL10), monokine induced by IFN-gamma (Mig/CXCL9), IFN-inducible T cell alpha chemoattractant (I-TAC/CXCL11)] and those for CCR4 [macrophage-derived chemokine (MDC/CCL22), thymus- and activation-regulated chemokine (TARC/CCL17)] have been shown to play the central roles for T helper-cell recruitment into the tissues. To examine the role of these chemokines in tumor progression of lung cancer, we investigated their expression in human lung cancer tissues to determine the possible relationship between their expression and the prognosis of patients.

METHODS

Total RNA was prepared from lung cancer tissues of 40 patients (24 adenocarcinoma and 16 squamous cell carcinoma). We measured gene expression levels of chemokines (IP-10, Mig, I-TAC, MDC and TARC) by real-time quantitative RT-PCR.

RESULTS

Higher gene expression of MDC in lung cancer was significantly correlated with longer disease-free survival time and lower risk of recurrence after tumor resection. We could not find any significant relationship of IP-10, Mig, I-TAC and TARC gene expression with disease-free survival or lower risk of recurrence after surgery.

CONCLUSIONS

These results suggest that increased gene expression of MDC in tumor tissues may be a predictive marker for improving the prognosis of lung cancer.

Stimulator of IFN genes (STING) spontaneously contributes to anti-tumor immunity by inducing type I interferons (IFNs) following sensing of tumor-derived genomic DNAs in the tumor-bearing host. Although direct injection of STING ligands such as cyclic diguanylate monophosphate (c-di-GMP) and cyclic [G(2',5')pA(3',5')p] (cGAMP) into the tumor microenvironment exerts anti-tumor effects through strong induction of type I IFNs and activation of innate and adaptive immunity, the precise events caused by STING in the tumor microenvironment remain to be elucidated. We describe here our finding that a CD45+ CD11bmid Ly6C+ cell subset transiently accumulated in mouse tumor microenvironment of 4T1 breast cancer, squamous cell carcinomas, CT26 colon cancer, or B16F10 melanoma tissue after intratumoral injection of cGAMP. The accumulated cells displayed a macrophage (M ) phenotype since the cells were positive for F4/80 and MHC class II and negative for Ly6G. Intratumoral cGAMP treatment did not induce Mφ accumulation in STING-deficient mice. Depletion of CD8+ T cell using anti-CD8 mAb impaired the anti-tumor effects of cGAMP treatment. Depletion of the Mφ using clodronate liposomes impaired the anti-tumor effects of cGAMP treatment. Functional analysis indicated that the STING-triggered tumor-migrating Mφ exhibited phagocytic activity, production of tumor necrosis factor alpha TNFα), and high expression levels of T cell-recruiting chemokines, Cxcl10 and Cxcl11, IFN-induced molecules, MX dynamin-like GTPase 1 (Mx1) and 2'-5' oligoadenylate synthetase-like 1 (Oasl1), nitric oxide synthase 2 (Nos2), and interferon beta 1 (Ifnb1). These results indicate that the STING-triggered tumor-migrating Mφ participate in the anti-tumor effects of STING-activating compounds.

BACKGROUND

Perivascular infiltrating mononuclear cells have been described in the vasculopathy found in multiple types of pulmonary arterial hypertension (PAH). We determined the expression of a specific type 1 immune response cytokine-chemokine cascade-interleukin (IL)-18 → (monokine induced by γ-interferon [MIG]/chemokine [C-X-C motif] ligand [CXCL] 9, interferon γ-induced protein [IP]-10/CXCL10 and interferon-inducible T-cell α chemoattractant [ITAC]/CXCL11)-in plasma samples from individuals with World Health Organization (WHO) Group 1 PAH.

METHODS

We analyzed cytokine and chemokine protein levels in plasma from 43 individuals with WHO Group 1 PAH by enzyme-linked immunosorbent assay compared with 35 healthy individuals. Immunohistochemical studies on tissue specimens from WHO Group 1 PAH patients were performed for cytokines and chemokines and their respective receptors.

RESULTS

Plasma IL-18 levels from WHO Group 1 PAH patients were significantly increased compared with healthy controls. Downstream chemokine CXCL10, but not CXCL9 or CXCL11, was markedly elevated compared with controls. Cellular sources of IL-18 were medial but not intimal smooth muscle cells. IL-18Rα was expressed from medial smooth muscle cells, endothelial cells, and mononuclear cells. CXCL10 and its main receptor, CXCR3, were expressed from infiltrating vascular wall mononuclear cells.

CONCLUSIONS

These data suggest that augmented expression of IL-18 and CXCL10 may perpetuate an inflammatory milieu that eventually contributes to the vascular obstruction characteristic of PAH.