Despite the fact that tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) and its receptors (TRAIL-Rs) are expressed in intestinal mucosa, little is known about the biological role of this system in intestinal cell physiology. The expression of surface TRAIL and TRAIL-R1, -R2, -R3, -R4 were examined by flow cytometry in the immortalized human cell line tsFHI under culture conditions promoting growth or growth arrest and expression of differentiated traits. A progressive increase of surface TRAIL expression paralleled tsFHI differentiation, consistently with immunohistochemistry analysis showing an increase of TRAIL immunostaining along the crypt-villus axis in normal jejuneal mucosa. In spite of the presence of TRAIL-R1 and TRAIL-R2 "death receptors," recombinant TRAIL was not cytotoxic for tsFHI cells. Exposure of tsFHI to recombinant TRAIL rather increased/anticipated the expression levels of the cyclin-dependent kinase inhibitors p21 and p27, which mediate the induction of growth arrest and the stabilization of differentiated traits, respectively, as well as of the canonical differentiation marker DPPIV. The differentiation inducing activity of TRAIL was abolished by pre-incubation with a Fc-TRAIL-R2 chimera. On the other hand, TRAIL did not significantly modulate the levels of osteoprotegerin (OPG), CXCL8/IL-8, CXCL9/MIG, and CXCL10/IP10 spontaneously released or induced by inflammatory cytokines. Taken together, these data suggest that TRAIL might act as a paracrine trophic cytokine on intestinal epithelium, promoting intestinal cell differentiation.

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.

The Herpes simplex virus-1 (HSV-1) is responsible for several clinical manifestations in humans, including encephalitis. To induce encephalitis, C57BL/6 mice were inoculated with 10(4) plaque-forming cells of HSV-1 by the intracranial route. Met-RANTES (regulated upon activation, normal T cell expressed and presumably secreted) (10 microg/mouse), a CC chemokine family receptor (CCR)1 and CCR5 antagonist, was given subcutaneously the day before, immediately after, and at days 1, 2, and 3 after infection. Treatment with Met-RANTES had no effect on the viral titers. In contrast, intravital microscopy revealed that treatment with Met-RANTES decreased the number of leukocytes adherent to the pial microvasculature at days 1 and 3 after infection. The levels of the chemokines CCL3, CCL5, CXCL1, and CXCL9 increased after infection and were enhanced further by the treatment with Met-RANTES. Treatment with a polyclonal anti-CCL5 antibody 2 h before the intravital microscopy decreased leukocyte adhesion in the microcirculation of infected mice. In conclusion, CCL5, a chemokine that binds to CCR1 and CCR5, is essential for leukocyte adhesion during HSV-1 encephalitis. However, blocking of CCR1 and CCR5 did not affect HSV-1 replication, suggesting that other immune mechanisms are involved in the process of infection control.

Monocyte chemoattractant protein-1 (CCL2/MCP-1) is a proinflammatory chemokine produced by several cell types, including pancreatic islets. High levels of donor-derived CCL2 have been associated with poor islet allograft outcome in patients with type 1 diabetes; however, the causal relationship is unknown. The constitutive and inducible expression of chemokines and their receptors by pancreatic islets in vitro were investigated, specifically the role of donor-derived CCL2 in marginal mass murine islet transplantation. The results showed that inflammatory cytokine stimulation of islets induced de novo expression of CCL2, CCL5/RANTES, CXCL9/MIG, and CXCL10/IP-10 and increased expression of CXCL2/macrophage-inflammatory protein-2. CCL2 mRNA and protein were highly expressed within 2 d in cultures. Transplantation of islets with high levels of CCL2 into syngeneic recipients led to a significantly greater influx of CCR2(+) cells and higher expression of monocyte/macrophage-associated inflammatory cytokines compared with low CCL2-donor islets. The level of pretransplantation CCL2 inversely correlated (P < 0.0001) with isograft function. In contrast, in CCR2-/- recipients, this correlation was not present. A direct toxic effect of CCL2 on islets was excluded by assessing cell viability and insulin release in vitro. In conclusion, CCL2 secreted by islets plays an important role in the immediate islet graft function. Strategies to decrease islet-derived CCL2 release may increase the success of islet transplantation.

Established tumors suppress antitumor immune responses and induce tolerance by incompletely characterized mechanisms, and this phenomenon is an important barrier to tumor immunotherapy. Single vaccination with tumor cells expressing gp96-Ig stimulates robust expansion of tumor-specific CTLs in tumor-naïve mice and this expansion is inhibited by established tumors. Interestingly, frequent vaccinations restore antitumor immune responses in the presence of established tumors. Syngeneic EG7 tumor-bearing mice have heterogeneous responses to frequent vaccination with EG7-gp96-Ig, with 32% complete responders and 68% partial responders. Comparison of responders to nonresponders revealed an inverse correlation between tumor-specific CTL expansion in the peripheral blood and tumor size. To identify immune cells and molecules associated with effective antitumor immune responses, reverse transcription-PCR arrays were performed using cells isolated from the vaccination site. ELISAs, cellular phenotyping, and tumor immunohistochemistry were also performed comparing vaccine responders to nonresponders. These data show that up-regulation of T-bet, RORgammat, IFNgamma, CCL8, CXCL9, and CXCL10 at the vaccination site are associated with vaccine-induced antitumor immunity. These data correlate with increased CTL expansion in the peripheral blood of responders, increased infiltration of responder tumors by CD8+ cells and interleukin-17+ cells, and decreased infiltration of responder tumors by CD11b+Gr-1+ cells and FoxP3+ cells. Furthermore, serum ELISAs revealed a significant elevation of transforming growth factor-beta in nonresponders as compared with responders. Interestingly, CD8+ T cells isolated from responders and nonresponders have equivalent cytotoxic activity in vitro. Taken together, our data suggest that established tumors may escape immunosurveillance by preventing clonal expansion of tumor-specific CTL without inducing anergy.

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.

Signal integration between IFNγ and TLRs in immune cells has been associated with the host defense against pathogens and injury, with a predominant role of STAT1. We hypothesize that STAT1-dependent transcriptional changes in vascular cells involved in cross-talk between IFNγ and TLR4, reflect pro-atherogenic responses in human atherosclerosis. Genome-wide investigation identified a set of STAT1-dependent genes that were synergistically affected by interactions between IFNγ and TLR4 in VSMCs. These included the chemokines Cxcl9, Ccl12, Ccl8, Ccrl2, Cxcl10 and Ccl5, adhesion molecules Cd40, Cd74, and antiviral and antibacterial genes Rsad2, Mx1, Oasl1, Gbp5, Nos2, Batf2 and Tnfrsf11a. Among the amplified genes was also Irf8, of which Ccl5 was subsequently identified as a new pro-inflammatory target in VSMCs and ECs. Promoter analysis predicted transcriptional cooperation between STAT1, IRF1, IRF8 and NFκB, with the novel role of IRF8 providing an additional layer to the overall complexity. The synergistic interactions between IFNγ and TLR4 also resulted in increased T-cell migration and impaired aortic contractility in a STAT1-dependent manner. Expression of the chemokines CXCL9 and CXCL10 correlated with STAT1 phosphorylation in vascular cells in plaques from human carotid arteries. Moreover, using data mining of human plaque transcriptomes, expression of a selection of these STAT1-dependent pro-atherogenic genes was found to be increased in coronary artery disease (CAD) and carotid atherosclerosis. Our study provides evidence to suggest that in ECs and VSMCs STAT1 orchestrates a platform for cross-talk between IFNγ and TLR4, and identifies a STAT1-dependent gene signature that reflects a pro-atherogenic state in human atherosclerosis.

Chronic lymphocytic leukemia (CLL) is an indolent lymphoproliferative disorder characterized by both circulating peripheral disease as well as involvement of the lymph nodes and bone marrow. Increasing evidence suggests that the stromal microenvironment provides anti-apoptotic and pro-survival signals to CLL cells, and may contribute significantly to resistance to a wide variety of treatments. Our understanding of the complex interactions involved in CLL cell trafficking continues to grow. Chemokines and corresponding chemokine receptors are key factors for organizing CLL cell trafficking and homing and the complex cellular interactions between CLL and accessory cells. Important chemokines include CCL3, CCL4, and CCL22, which are released by CLL cells, and CXCL12, CXCL13, CXCL9, 10, 11, CCL 19, and CCL21, which are constitutively secreted by various stromal cells. Integrins such as VLA-4 (CD49d) as well as selectins and CD44 also likely play a role in directing CLL cell migration within the tissue microenvironments. Data are also emerging that other molecules such as MMP-9 and cytoskeletal proteins also contribute to CLL cell trafficking. Though this interplay is complex, it is critical that we improve our understanding of CLL cell trafficking to facilitate the development of novel therapies that target these pathways. Several drugs in clinical development, such as CXCR4 antagonists and PI3K, Btk, and Syk inhibitors appear to modulate CLL cell trafficking and CLL-stroma interactions. Here, we review the current understanding of the molecular interactions that underlie CLL cell trafficking and we highlight some of the promising approaches underway to target these pathways therapeutically in CLL.

The IL-12Rbeta2 gene is expressed in human mature B cell subsets but not in transformed B cell lines. Silencing of this gene may be advantageous to neoplastic B cells. Our objective was to investigate the mechanism(s) and the functional consequence(s) of IL-12Rbeta2 gene silencing in primary B cell tumors and transformed B cell lines. Purified tumor cells from 41 patients with different chronic B cell lymphoproliferative disorders, representing the counterparts of the major mature human B cell subsets, tested negative for IL-12Rbeta2 gene expression. Hypermethylation of a CpG island in the noncoding exon 1 was associated with silencing of this gene in malignant B cells. Treatment with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine restored IL-12Rbeta2 mRNA expression in primary neoplastic B cells that underwent apoptosis following exposure to human recombinant IL-12 (hrIL-12). hrIL-12 inhibited proliferation and increased the apoptotic rate of IL-12Rbeta2-transfected B cell lines in vitro. Finally, hrIL-12 strongly reduced the tumorigenicity of IL-12Rbeta2-transfected Burkitt lymphoma RAJI cells in SCID-NOD mice through antiproliferative and proapoptotic effects, coupled with neoangiogenesis inhibition related to human IFN-gamma-independent induction of hMig/CXCL9. The IL-12Rbeta2 gene acts as tumor suppressor in chronic B cell malignancies, and IL-12 exerts direct antitumor effects on IL-12Rbeta2-expressing neoplastic B cells.

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.

BACKGROUND

Toll-like receptor (TLR)4 agonists are known potent immunostimulatory compounds. These compounds can be formulated as part of novel adjuvants to enhance vaccine medicated immune responses. However, the contribution of the formulation to the innate in vivo activity of TLR4 agonist compounds is not well understood.

RESULTS

We evaluated synthetic TLR4 agonist Glucopyranosyl Lipid A (GLA) for its effects on molecular and cellular innate immune responses in the murine model. Microarray techniques were used to compare the responses to GLA in an aqueous formulation or in an oil-in-water Stable Emulsion formulation (GLA-SE) versus either SE alone or the mineral salt aluminum hydroxide (alum) at the muscle injection site over multiple timepoints. In contrast to the minimal gene upregulation induced by SE and alum, both GLA and GLA-SE triggered MyD88- and TRIF-dependent gene expression. Genes for chemokines, cytokine receptors, signaling molecules, complement, and antigen presentation were also strongly upregulated by GLA and GLA-SE. These included chemokines for T(H)1-type T cells (CXCL9 and CXCL10) and mononuclear leukocytes (CCL2, CCL3) among others. GLA-SE induced stronger and more sustained gene upregulation than GLA in the muscle; GLA-SE induced genes were also detected in local draining lymph nodes and at lower levels in peripheral blood. Both GLA and GLA-SE resulted in increased cellular trafficking to the draining lymph nodes and upregulated MHC molecules and ICAM1 on local dendritic cells. GLA and GLA-SE transiently upregulated circulating MCP-1, TNFα, IFNγ and IP-10 in blood.

CONCLUSIONS

While GLA and GLA-SE activate a large number of shared innate genes and proteins, GLA-SE induces a quantitatively and qualitatively stronger response than GLA, SE or alum. The genes and proteins upregulated could be used to facilitate selection of appropriate adjuvant doses in vaccine formulations.

BACKGROUND

Vitiligo is a skin disorder characterized by loss of melanocytes from the epidermis. A recent study reported that CXCL10 is critical for the progression and maintenance of depigmentation in a mouse model of vitiligo, but there is very limited clinical data regarding this issue and little is known about the dynamic changes or correlations with disease severity of these chemokines throughout the disease course.

OBJECTIVE

To present clinical data that supports and identifies the pathway of CXCR3 and its ligands in T-lymphocytic cell recruitment in vitiligo.

METHODS

Cytometric bead array, flow cytometry, quantitative real-time polymerase chain reaction and immunohistology were used to examine their systemic and local expression in 80 patients with vitiligo and 40 controls.

RESULTS

We showed that serum CXCL9 and CXCL10 were significantly elevated in patients with vitiligo and were higher in patients in progressive stages than in stable stages. The relative expression of CXCR3 mRNA in peripheral blood mononuclear cells was higher in vitiligo. There were higher percentages of both circulating CXCR3(+) CD4(+) and CXCR3(+) CD8(+) T cells in patients with progressive vitiligo compared with controls, while only the expression of CXCR3(+) CD8(+) T cells increased in patients with stable vitiligo. Histological findings also demonstrated an abundance of CXCR3(+) cells within vitiligo lesions. Furthermore, serum CXCL10 levels were associated with Vitiligo Area Scoring Index scores of patients with progressive vitiligo and were reduced after successful treatment.

CONCLUSIONS

The CXCL10/CXCR3 axis mediates T-cell recruitment into the skin in progressive vitiligo. Blocking this chemotactic mechanism may present a new form of therapy. Serum CXCL10 may be a novel biomarker in monitoring disease activity and guiding treatment of progressive vitiligo.

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

Understanding the genital mucosal immunity and the factors involved in linking innate to adaptive immunity is crucial for the design of efficient preventive strategies against human immunodeficiency virus (HIV)-1.

METHODS

Levels of both genital mucosal and blood chemokines were compared between 58 HIV-1-uninfected and 50 HIV-1-infected female commercial sex workers (CSWs) as well as 53 HIV-1-uninfected non-CSW control women at low risk for exposure, recruited in Cotonou, Benin.

RESULTS

HIV-1-infected CSWs had significantly higher blood and genital levels of monocyte chemotactic protein (MCP-3/CCL7) and monokine induced by gamma interferon (MIG/CXCL9) compared with those in both the HIV-1-uninfected CSW and non-CSW groups. In the HIV-1-infected group, levels of MCP-3 and MIG were significantly higher in the genital mucosa than in the blood. However, the blood levels of macrophage inflammatory protein (MIP-1a/CCL3) and MIP-1b/CCL4 were higher in HIV-1-uninfected CSWs compared with those in the other groups.

CONCLUSIONS

Increased production of chemokines in the genital tract may favour the recruitment of HIV-1 target cells causing a mucosal environment that promotes viral replication and dissemination, whereas higher expression of beta-chemokines at the systemic level is associated with protection from HIV-1 infection.

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.

BACKGROUND

Chronic obstructive pulmonary disease (COPD) is a heterogeneous syndrome characterized by varying degrees of airflow limitation and diffusion impairment. There is increasing evidence to suggest that COPD is also characterized by systemic inflammation. The primary goal of this study was to identify soluble proteins in plasma that associate with the severity of airflow limitation in a COPD cohort with stable disease. A secondary goal was to assess whether unique markers associate with diffusion impairment, based on diffusion capacity of carbon monoxide (DLCO), independent of the forced expiratory volume in 1 second (FEV1).

METHODS

A cross sectional study of 73 COPD subjects was performed in order to examine the association of 25 different plasma proteins with the severity of lung function impairment, as defined by the baseline measurements of the % predicted FEV1 and the % predicted DLCO. Plasma protein concentrations were assayed using multiplexed immunobead-based cytokine profiling. Associations between lung function and protein concentrations were adjusted for age, gender, pack years smoking history, current smoking, inhaled corticosteroid use, systemic corticosteroid use and statin use.

RESULTS

Plasma concentrations of CCL2/monocyte chemoattractant protein-1 (CCL2/MCP-1), CCL4/macrophage inflammatory protein-1 beta (CCL4/MIP-1 beta), CCL11/eotaxin, and interleukin-13 (IL-13) were inversely associated with the % FEV1. Plasma concentrations of soluble Fas were associated with the % DLCO, whereas CXCL9/monokine induced by interferon-gamma (CXCL9/Mig), granulocyte- colony stimulating factor (G-CSF) and IL-13 showed inverse relationships with the % DLCO.

CONCLUSIONS

Systemic inflammation in a COPD cohort is characterized by cytokines implicated in inflammatory cell recruitment and airway remodeling. Plasma concentrations of IL-13 and chemoattractants for monocytes, T lymphocytes, and eosinophils show associations with increasing severity of disease. Soluble Fas, G-CSF and CXCL9/Mig may be unique markers that associate with disease characterized by disproportionate abnormalities in DLCO independent of the FEV1.

Acute graft-versus-host disease (aGVHD) is a major complication after allogeneic bone marrow transplantation (allo-BMT), and infiltration of donor leukocytes into aGVHD target organs is partially orchestrated by chemokines. Using a murine BMT model, the expression of 30 chemokines or chemokine receptors in the lung, liver, gut and tongue was analyzed using real-time PCR at 1, 2, 3 and 6 weeks after BMT during the development of clinical aGVHD and target organ histopathology. CXCL9-11 expression was linked to elevated expression of CXCR3 in the gut, lung and tongue. In contrast, hepatic CXCR3 expression was not changed, whereas a clear association was seen for CXCL16 and CXCR6 expression. An elevated intestinal CCL3 expression 1 week after allo-BMT was associated with an increased expression of CCR5 but not CCR1 or CCR3, and in the lung and liver CCL3-CCL5 expression was associated with increases in CCR1 and CCR5. Overexpression of CCL2, CCL8, CCL12 and their receptor CCR2 was found in the liver and lung, but not in the gut and tongue. On the basis of the differences in kinetics and organ distribution, more studies are required to better characterize specific targets within this network, as this will allow the development of novel preventive and therapeutic approaches by using single or multiple targeting reagents.

Mammalian pregnancy requires protection against immunological rejection of the developing fetus bearing discordant paternal antigens. Immune evasion in this developmental context entails silenced expression of chemoattractant proteins (chemokines), thereby preventing harmful immune cells from penetrating the maternal-fetal interface. Here, we demonstrate that fetal wastage triggered by prenatal Listeria monocytogenes infection is driven by placental recruitment of CXCL9-producing inflammatory neutrophils and macrophages that promote infiltration of fetal-specific T cells into the decidua. Maternal CD8+ T cells with fetal specificity upregulated expression of the chemokine receptor CXCR3 and, together with neutrophils and macrophages, were essential for L. monocytogenes-induced fetal resorption. Conversely, decidual accumulation of maternal T cells with fetal specificity and fetal wastage were extinguished by CXCR3 blockade or in CXCR3-deficient mice. Remarkably, protection against fetal wastage and in utero L. monocytogenes invasion was maintained even when CXCR3 neutralization was initiated after infection, and this protective effect extended to fetal resorption triggered by partial ablation of immune-suppressive maternal Tregs, which expand during pregnancy to sustain fetal tolerance. Together, our results indicate that functionally overriding chemokine silencing at the maternal-fetal interface promotes the pathogenesis of prenatal infection and suggest that therapeutically reinforcing this pathway represents a universal approach for mitigating immune-mediated pregnancy complications.

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.

The retinal pigment epithelial (RPE) cell is a potent regulatory cell that facilitates normal physiologic processes and plays a critical role in a variety of retinal diseases. We evaluated IFN-beta production in human RPE cells through TLR signaling and investigated the effects of IFN-beta on RPE cells. RPE cells treated with poly(I:C) or infected with an RNA virus produce IFN-beta. Kinetic studies revealed that IFN-beta levels continue to increase over a 48-h period and this was associated with the up-regulation of IRF-7 gene expression, a known positive feedback molecule for IFN-beta production. Microarray analysis revealed that in IFN-beta treated cells, 480 genes of 22,283 genes were up or down-regulated by >2-fold. We hypothesize that IFN-beta induction during TLR signaling in the retina is an immunosuppressive factor produced to limit immunopathologic damage. Cytokine activation of RPE cells results in the production of the chemokines, CXCL9 and CXCL10, and the adhesion molecule, ICAM-1. Pretreatment of RPE cells with IFN-beta resulted in inhibition of ICAM-1 production and elimination of CXCL9 production. This treatment did not alter CXCL10 production. Anti-IFN-beta Ab blocked the inhibitory action of IFN-beta. Real time PCR analysis revealed that IFN-beta treatment inhibited gene expression of sICAM-1 and CXCL9. The results indicate a critical role for RPE cell derived IFN-beta in the down-regulation of CXCL9 and ICAM-1 expression in the retina and suggest that the inhibition of CXCL9 is an immuno-suppressive mechanism that protects the retina from excessive inflammation.

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.

Early allograft dysfunction (EAD) occurring in the first week post-liver transplantation is associated with increased graft failure and mortality and is believed to be largely due to ischemia/reperfusion injury. We anticipated that the presence of EAD would be reflected by alterations in expression of serum proteins associated with an inflammatory response in the peri-operative period, and hypothesized that a specific pattern of expression might correlate with the development of EAD. The serum levels of 25 cytokines, chemokines, and immunoreceptors were measured by Luminex multiplex assays pre- and post-liver transplantation. Levels of each cytokine biomarker were compared in adult recipients with or without EAD at serial time points using samples collected pre-operatively and at 1, 7, 14, and 30 days post-transplant. EAD was defined according to standard criteria as maximum alanine transferase (ALT) or aspartate transferase (AST) levels on days 1-7 of >2000 U/ml, day 7 bilirubin level ≥10 mg/dl, or a day 7 international normalized ratio (INR) ≥1.7. Multivariable analyses showed that patients experiencing EAD had lower pre-operative IL-6 and higher IL-2R levels. Patients with EAD also showed higher MCP-1 (CCL2), IL-8 (CXCL8), and RANTES (CCL5) chemokine levels in the early post-operative period, suggesting up-regulation of the NF-kB pathway, in addition to higher levels of chemokines and cytokines associated with T cell immunity, including MIG (CXCL9), IP-10 (CXCL10) and IL-2R. These findings identify several possible biomarkers and pathways associated with EAD, that may guide future validation studies and investigation of specific cellular and molecular mechanisms of graft dysfunction. Furthermore, if validated, our findings may contribute to perioperative prediction of the occurrence of EAD and ultimately lead to identification of potential interventional therapies.

OBJECTIVE

The goal of this study was to investigate the chemokines CCL2, CXCL8, CXCL9 and CXCL10 as markers of the inflammatory responses in chronic subdural hematoma (CSDH).

METHODS

Samples of peripheral venous blood and CSDH fluid (obtained during surgery) in 76 adult patients were prospectively analyzed. Chemokine values were assessed by a Multiplex antibody bead kit.

RESULTS

We found significantly higher levels of chemokines CCL2, CXCL8, CXCL9 and CXCL10 in hematoma fluid compared with serum.

CONCLUSIONS

Chemokines are elevated in the hematoma cavity of patients with CSDH. It is likely that these signaling modulators play an important role in promoting local inflammation. Furthermore, biological activity of CCL2 and CXCL8 may promote neovascularization within the outer CSDH membrane, and a compensatory angiostatic activity of CXCL9 and CXCL10 may contribute to repairing this disorder. This phenomenon was restricted to the hematoma site, and the systemic chemokine levels might not reflect local immune responses.