Recent studies have demonstrated that human dental pulp cells sense pathogens and elicit innate and/or adaptive immunity. Particular attention has been paid to odontoblasts that are situated at the pulp-dentin interface and constitute the first line of defense to cariogenic bacteria entering dentin after enamel disruption. In this review, recent in vitro and in vivo data suggesting that odontoblasts initiate immune/inflammatory events within the dental pulp in response to cariogenic bacteria are discussed. These data include sensing of pathogens by Toll-like receptors (TLRs), production of chemokines upon cell stimulation with microbial by-products and induction of dendritic cell migration. Additional results presented here reveal that all TLR genes are expressed in the healthy human dental pulp that is thus well equipped to combat pathogens entering the tissue. Seventeen chemokine genes including CXCL12, CCL2, CXCL9, CX3CL1, CCL8, CXCL10, CCL16, CCL5, CXCL2, CCL4, CXCL11 and CCL3, and 9 chemokine receptor genes including CXCR4, CCR1, CCR5, CX3CR1, CCR10 and CXCR3, are also expressed in pulp. TLR2, CCL2 and CXCL1 are upregulated in odontoblasts both under caries lesions and upon stimulation with pathogen by-products. These molecules thus appear as preferential targets for the design of therapeutic agents able to reduce the immune/inflammatory response to cariogenic bacteria and favor pulp healing.

CD4(+) T(h)1 cells producing IFN-γ are of extreme importance in controlling infections by Mycobacterium tuberculosis both in mice and in men. In addition to IFN-γ-producing T cells, IL-17-producing T cells (T(h)17) have been observed during mycobacterial infections. Nevertheless, their contribution for the host immune response to mycobacteria as well as the signals triggering M. tuberculosis -specific T(h)17 cell differentiation and maintenance are not fully understood. We show that signaling via Toll-like receptor (TLR) 2 has a major impact on the regulation of p19 (IL-23) expression in response to M. tuberculosis and therefore on the establishment of T(h)17 cell responses to M. tuberculosis infection. Diminished T(h)17 responses in the lung of M. tuberculosis -infected TLR2-deficient animals were not caused by defective cell differentiation in the draining lymph node (LN) but rather by reduced maintenance at the site of infection. Consistent with the decreased numbers of T(h)17 cells in the lungs of infected TLR2-deficient animals, we observed reduced expression of CXCL9, CXCL10 and CXCL11, chemokines involved in recall responses to M. tuberculosis. Our data provides insights into the TLR2 role in infection with M. tuberculosis, with implications in pathophysiology of the disease and vaccine design.

Organ-specific lymphocyte homing is dependent on the expression of tissue-specific homing receptors and selected chemokine receptors. During the effector phase of an immune response, IgA and IgG antibody-secreting cells (ASC) are differently distributed in the body. Still, B cell expression of L-selectin and the mucosal homing receptor integrin alpha4beta7 is not related to the isotype produced, but only to the site of antigen encounter. In this study, we examined if differences in chemokine responsiveness between IgA+ and IgG+ B cells could explain their different tissue localization. Circulating CD19+ B cells were isolated and their expression of IgA, IgG, and selected chemokine receptors was determined by flow cytometry. Few Ig+ cells expressed CCR2, CCR3, or CCR9, and there was no difference in the expression of these receptors between IgA+ and IgG+ cells. In contrast, CCR4, CCR5, and CXCR3 was expressed on significantly more IgG+ than IgA+ cells. The function of chemokine receptors on memory B cells and ASC was then tested in the transwell system. IgG+ memory cells migrated to a higher extent than IgA+ cells towards the CXCR3 ligand CXCL11/I-TAC, while there was only a small migration towards the CCR4 ligand CCL17/TARC and the CCR9 ligand CCL25/TECK. ASC migrated poorly to all chemokines tested. In conclusion, this study shows that IgG+ and IgA+ memory B cells have a differential expression of the Th1 associated chemokine receptor CXCR3, as well as of CCR4 and CCR5. In contrast, none of the studied chemokine receptors was preferentially expressed by IgA+ cells.

Human IBD, including UC and Crohn's disease, is characterized by a chronic, relapsing, and remitting condition that exhibits various features of immunological inflammation and affects at least one/1000 people in Western countries. Polyphenol extracts from a variety of plants have been shown to have immunomodulatory and anti-inflammatory effects. In this study, treatment with APP was investigated to ameliorate chemically induced colitis. Oral but not peritoneal administration of APP during colitis induction significantly protected C57BL/6 mice against disease, as evidenced by the lack of weight loss, colonic inflammation, and shortening of the colon. APP administration dampened the mRNA expression of IL-1β, TNF-α, IL-6, IL-17, IL-22, CXCL9, CXCL10, CXCL11, and IFN-γ in the colons of mice with colitis. APP-mediated protection requires T cells, as protection was abated in Rag-1(-/-) or TCRα(-/-) mice but not in IL-10(-/-), IRF-1(-/-), μMT, or TCRδ(-/-) mice. Administration of APP during colitis to TCRα(-/-) mice actually enhanced proinflammatory cytokine expression, further demonstrating a requirement for TCRαβ cells in APP-mediated protection. APP treatment also inhibited CXCR3 expression by TCRαβ cells, but not B or NK cells, in the colons of mice with colitis; however, depletion of CD4(+) or CD8(+) T cells alone did not abolish APP-mediated protection. Collectively, these results show that oral administration of APP protects against experimental colitis and diminishes proinflammatory cytokine expression via T cells.

Respiratory epithelial cells and macrophages are the key innate immune cells that play an important role in the pathogenesis of influenza A virus infection. We found that these two cell types from both human and pig showed comparable susceptibilities to initial infection with a highly pathogenic avian influenza (HPAI) H5N1 virus (A/turkey/Turkey/1/05) and a moderately pathogenic human influenza H1N1 virus (A/USSR/77), but there were contrasting differences in host innate immune responses. Human cells mounted vigorous cytokine (tumor necrosis factor alpha [TNF-α] and interleukin-6 [IL-6]) and chemokine (CXCL9, CXCL10, and CXCL11) responses to H5N1 virus infection. However, pig epithelial cells and macrophages showed weak or no TNF-α and chemokine induction with the same infections. The apparent lack of a strong proinflammatory response, corroborated by the absence of TNF-α induction in H5N1 virus-challenged pigs, coincided with greater cell death and the reduced release of infectious virus from infected pig epithelial cells. Suppressor of cytokine signaling 3 (SOCS3), a protein suppressor of the JAK-STAT pathway, was constitutively highly expressed and transcriptionally upregulated in H5N1 virus-infected pig epithelial cells and macrophages, in contrast to the corresponding human cells. The overexpression of SOCS3 in infected human macrophages dampened TNF-α induction. In summary, we found that the reported low susceptibility of pigs to contemporary Eurasian HPAI H5N1 virus infections coincides at the level of innate immunity of respiratory epithelial cells and macrophages with a reduced output of viable virus and an attenuated proinflammatory response, possibly mediated in part by SOCS3, which could serve as a target in the treatment or prevention of virus-induced hypercytokinemia, as observed for humans.

Ectodysplasin (Eda), a member of the tumor necrosis factor (Tnf) family, regulates skin appendage morphogenesis via its receptor Edar and transcription factor NF-κB. In humans, inactivating mutations in the Eda pathway components lead to hypohidrotic ectodermal dysplasia (HED), a syndrome characterized by sparse hair, tooth abnormalities, and defects in several cutaneous glands. A corresponding phenotype is observed in Eda-null mice, where failure in the initiation of the first wave of hair follicle development is a hallmark of HED pathogenesis. In an attempt to discover immediate target genes of the Eda/NF-κB pathway, we performed microarray profiling of genes differentially expressed in embryonic skin explants after a short exposure to recombinant Fc-Eda protein. Upregulated genes included components of the Wnt, fibroblast growth factor, transforming growth factor-β, Tnf, and epidermal growth factor families, indicating that Eda modulates multiple signaling pathways implicated in skin appendage development. Surprisingly, we identified two ligands of the chemokine receptor cxcR3, cxcl10 and cxcl11, as new hair-specific transcriptional targets of Eda. Deficiency in cxcR3 resulted in decreased primary hair follicle density but otherwise normal hair development, indicating that chemokine signaling influences the patterning of primary hair placodes only.

Cytokines play important roles as regulators of cell functions, and over the last decades a number of cytokine assays have been developed. The aim of the present study was to investigate the effects of age and gender on a large number of cytokines. Plasma samples were collected from 33 healthy blood donors. The samples were analyzed using a multiplex proximity extension assay (PEA) allowing simultaneous measurement of 92 cytokines and four technical controls. Biomarkers with less than 80% quantitative results were excluded leaving 63 cytokines that were analyzed for the effects of gender and age. The plasma level of three of the investigated biomarkers (DNER, MCP-4 and MMP-10) were found to be significantly different for the two genders (adjusted p-value<0.05), and 15 of the biomarkers (CCL11, CCL25, CDCP1, CSF-1, CXCL11, CXCL9, FGF-23, Flt3L, HGF, IL-10RB, MCP-3, MCP-4, MMP-10, OPG, VEGF-A) were significantly associated with age. This study reveals the effects of age and gender on a large number of cytokine assays. CXCL5 and TNFB were significantly higher in females, while the other markers with significant gender-dependent differences were higher in males. For the markers that were significantly associated with age, only CXCL6 was found to decrease with age, while the other biomarkers increased with age.

The molecular mechanisms operating in human organ transplant rejection are best inferred from the mRNAs expressed in biopsies because the corresponding proteins often have low expression and short half-lives, while small non-coding RNAs lack specificity. Associations should be characterized in a population that rigorously identifies T cell-mediated (TCMR) and antibody-mediated rejection (ABMR). This is best achieved in kidney transplant biopsies, but the results are generalizable to heart, lung, or liver transplants. Associations can be universal (all rejection), TCMR-selective, or ABMR-selective, with universal being strongest and ABMR-selective weakest. Top universal transcripts are IFNG-inducible (eg, CXCL11 IDO1, WARS) or shared by effector T cells (ETCs) and NK cells (eg, KLRD1, CCL4). TCMR-selective transcripts are expressed in activated ETCs (eg, CTLA4, IFNG), activated (eg, ADAMDEC1), or IFNG-induced macrophages (eg, ANKRD22). ABMR-selective transcripts are expressed in NK cells (eg, FGFBP2, GNLY) and endothelial cells (eg, ROBO4, DARC). Transcript associations are highly reproducible between biopsy sets when the same rejection definitions, case mix, algorithm, and technology are applied, but exact ranks will vary. Previously published rejection-associated transcripts resemble universal and TCMR-selective transcripts due to incomplete representation of ABMR. Rejection-associated transcripts are never completely rejection-specific because they are shared with the stereotyped response-to-injury and innate immunity.

Dimethylfumarate (DMF) has been shown to reduce melanoma growth and metastasis in animal models. We addressed the question of whether DMF is as effective in its antitumor activity as the US Food and Drug Administration-approved alkylating agent dacarbazine (DTIC). We also tested the possibility of an improved antitumoral effect when both therapeutics were used together. Using our severe combined immunodeficiency (SCID) mouse model, in which xenografted human melanoma cells metastasize from primary skin sites to sentinel nodes, we show that these treatments, alone or in combination, reduce tumor growth at primary sites. Our main finding was that metastasis to sentinel nodes is significantly delayed only in mice treated with a combination of DTIC and DMF. Subsequent experiments were able to show that a combination of DTIC/DMF significantly reduced lymph vessel density in primary tumors as examined by real-time PCR and immunohistochemistry. In addition, DTIC/DMF treatment significantly impaired melanoma cell migration in vitro. In vivo, DTIC/DMF therapy significantly reduced mRNA expression and protein concentration of the promigratory chemokines CXCL2 and CXCL11. In addition, our data suggest that this xenotransplantation model is suitable for preclinical testing of various combinations of antimelanoma agents.

Interferon γ (IFNγ) is considered a seminal cytokine in the pathogenesis of giant cell arteritis (GCA), but its functional role has not been investigated. We explored changes in infiltrating cells and biomarkers elicited by blocking IFNγ with a neutralising monoclonal antibody, A6, in temporal arteries from patients with GCA.

Temporal arteries from 34 patients with GCA (positive histology) and 21 controls were cultured on 3D matrix (Matrigel) and exposed to A6 or recombinant IFNγ. Changes in gene/protein expression were measured by qRT-PCR/western blot or immunoassay. Changes in infiltrating cells were assessed by immunohistochemistry/immunofluorescence. Chemotaxis/adhesion assays were performed with temporal artery-derived vascular smooth muscle cells (VSMCs) and peripheral blood mononuclear cells (PBMCs).

Blocking endogenous IFNγ with A6 abrogated STAT-1 phosphorylation in cultured GCA arteries. Furthermore, selective reduction in CXCL9, CXCL10 and CXCL11 chemokine expression was observed along with reduction in infiltrating CD68 macrophages. Adding IFNγ elicited consistent opposite effects. IFNγ induced CXCL9, CXCL10, CXCL11, CCL2 and intracellular adhesion molecule-1 expression by cultured VSMC, resulting in increased PBMC chemotaxis/adhesion. Spontaneous expression of chemokines was higher in VSMC isolated from GCA-involved arteries than in those obtained from controls. Incubation of IFNγ-treated control arteries with PBMC resulted in adhesion/infiltration by CD68 macrophages, which did not occur in untreated arteries.

Our ex vivo system suggests that IFNγ may play an important role in the recruitment of macrophages in GCA by inducing production of specific chemokines and adhesion molecules. Vascular wall components (ie, VSMC) are mediators of these functions and may facilitate progression of inflammatory infiltrates through the vessel wall.

BACKGROUND

Although antibody mechanisms play a pathogenetic role in liver allograft rejection, no data exist on B lymphocytes, plasma cells, complement, and chemokines in rejected liver tissue.

METHODS

Liver biopsy specimens from 25 patients with acute allograft rejection (AR) (rejection activity index, RAI score: 1-9) were analyzed by immunohistochemistry (IH) and reverse transcriptase-polymerase chain reaction (RT-PCR) and compared with biopsy specimens taken prior to implantation (PI). The number of CD20 and CD138 cells was evaluated, and the presence and abundance of the chemokines macrophage inflammatory protein (MIP)-3alpha, CXCL9, CXCL10, CXCL11, CXCL12, and their receptors CCR-6, CXCR3, and CXCR4 were examined. Complement depositions were visualized by C4d IH.

RESULTS

The numbers of B lymphocytes (P=0.002) and plasma cells (P=0.022) were significantly higher in AR biopsy specimens compared with PI biopsy specimens. MIP-3alpha and CCR-6 cells were detected in the portal fields of all AR biopsy specimens. IH double staining revealed a colocalization of MIP-3alpha/CD20 cells; C4d deposits could be demonstrated along the portal capillaries. All examined chemokines and receptors could be detected in normal liver tissue and in AR biopsy specimens by RT-PCR and semiquantitative RT-PCR, demonstrating an overexpression of CXCL10 and -11.

CONCLUSIONS

The significant increase of B lymphocytes and plasma cells during acute rejection, together with the lack of a significant increase of proliferating cells, indicates that the migration of B lymphocytes and plasma cells-promoted by the expression of B-cell activating chemokines/receptors-plays a key role in acute liver rejection. The C4d deposits along the portal capillaries indicate a humorally mediated alloresponse caused by the accumulated B and plasma cells.

The chemokine receptor CXCR3 is predominantly expressed on activated T and natural killer (NK) cells. CXCR3 and its ligands, CXCL11, CXCL10, and CXCL9, play a major role in T-helper 1 (Th1)-dependent inflammatory responses. CXCL11 is the most dominant physiological inducer of adhesion, migration, and internalization of CXCR3. To study the role of CXCR3 carboxyl-terminus and the third intracellular (3i) loop in chemokine-mediated migration, adhesion, and CXCR3 internalization, we generated CXCR3 receptors mutated in their distal (Ser-Thr domain) or proximal (trileucine domain) membrane carboxyl terminus, and/or the third intracellular loop. We found that migration of CXCR3-expressing HEK 293 cells toward CXCL11 was pertussis toxin-dependent and required the membrane proximal carboxyl terminus of CXCR3. Internalization induced by CXCL11 and protein kinase C (PKC) activation was also regulated by the membrane proximal carboxyl terminus; however, only CXCL11-induced internalization required the LLL motif of this region. Internalization and Ca(2+) flux induced by CXCL11 were independent of the 3i loop S245, whereas migration at high CXCL11 concentrations, integrin-dependent adhesion, and actin polymerization were S245 dependent. Our findings indicate that CXCL11-dependent CXCR3 internalization and cell migration are regulated by the CXCR3 membrane proximal carboxyl terminus, whereas adhesion is regulated by the 3i loop S245. Thus, distinct conformational changes induced by a given CXCR3 ligand trigger different downstream effectors of adhesion, motility, and CXCR3 desensitization.

The selective CXC chemokine receptor 3 (CXCR3) agonists, monokine induced by interferon-gamma (IFN- gamma)/CXC chemokine ligand 9 (CXCL9), IFN-inducible protein 10/CXCL10, and IFN-inducible T cell alpha chemoattractant (I-TAC)/CXCL11, attract CXCR3+ cells such as CD45RO+ T lymphocytes, B cells, and natural killer cells. Further, all three chemokines are potent, natural antagonists for chemokine receptor 3 (CCR3) and feature defensin-like, antimicrobial activities. In this study, we show that I-TAC, in addition to these effects, acts as an antagonist for CCR5. I-TAC inhibited the binding of macrophage-inflammatory protein-1alpha (MIP-1alpha)/CC chemokine ligand 3 (CCL3) to cells transfected with CCR5 and to monocytes. Furthermore, cell migration evoked by regulated on activation, normal T expressed and secreted (RANTES)/CCL5 and MIP-1beta/CCL4, the selective agonist of CCR5, was inhibited in transfected cells and monocytes, respectively. In two other functional assays, namely the release of free intracellular calcium and actin polymerization, I-TAC reduced CCR5 activities to minimal levels. Sequence and structure analyses indicate a potential role for K17, K49, and Q51 of I-TAC in CCR5 binding. Our results expand on the potential role of I-TAC as a negative modulator in leukocyte migration and activation, as I-TAC would specifically counteract the responses mediated by many "classical," inflammatory chemokines that act not only via CCR3 but via CCR5 as well.

OBJECTIVE

To investigate the effects of T-cell-derived cytokines on gene and protein expression of chemokines in a human RPE cell line (ARPE-19).

METHODS

We used an in vitro coculture system in which the RPE and CD3/CD28-activated T-cells were separated by a membrane. RPE cell expression of chemokine genes was quantified using three different types of microarrays. Protein expression was determined by single and multiplex ELISA and immunoblotting.

RESULTS

Coculture with activated T-cells increased RPE mRNA and protein expression of chemokines CCL2 (MCP-1); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (MCP-2); CXCL1 (GRO-α); IL8 (CXCL8); CXCL9 (MIG); CXCL10 (IP10); CXCL11 (ITAC); and CX3CL1 (fractalkine). CCL7, CXCL9, CXCL10, and CXCL11 were secreted significantly more in the apical direction. Using recombinant human cytokines and neutralizing antibodies, we identified IFNγ and TNFα as the two major T-cell-derived cytokines responsible for the RPE response. For CCL5, CXCL9, CXCL10, CXCL11, CXCL16, and CX3CL1, we observed a synergistic effect of IFNγ and TNFα in combination. CCL20, CXCL1, CXCL6, and IL8 were negatively regulated by IFNγ.

CONCLUSIONS

RPE cells responded to exposure to T-cell-derived cytokines by upregulating expression of multiple chemokines related to microglial, T-cell, and monocyte chemotaxis and activation. This inflammatory stress response may have implications for immune homeostasis in the retina, and for the further understanding of inflammatory ocular diseases such as uveitis and AMD.

Graft versus host disease (GVHD) is a major complication of haematopoietic SCT (HSCT). A number of inflammatory cytokines/chemokines are implicated in GVHD and have been identified in numerous single centre studies as potential biomarkers for acute and/or chronic GVHD. In this study, we analysed candidate inflammatory biomarkers (B-cell activating factor (BAFF), interleukin 33 (IL-33), CXCL10 and CXCL11) in a two-centre study. Biomarkers were evaluated pre-transplant and at serial time points post transplant in acute and chronic GVHD patient sera with time-matched control samples from patients without GVHD. Further validation was performed using the human skin explant assay, clinical GVHD biopsies and mRNA expression analysis. BAFF was significantly increased pre-transplant. BAFF, IL-33, CXCL10 and CXCL11 showed increased levels in acute GVHD patient sera and high protein expression in grades II-III of the in vitro skin explant graft versus host reaction (GVHR) group. BAFF, CXCL10 and CXCL11 also showed increased mRNA expression levels in clinical biopsies compared with the no/low-grade GVHD group. BAFF, CXCL10 and CXCL11 levels were increased in chronic GVHD patient sera. The results identify BAFF and CXCL10 as predictive biomarkers for acute GVHD and BAFF, CXCL10 and CXCL11 as useful diagnostic biomarkers for acute GVHD and chronic GVHD.

OBJECTIVE

Chemokines are involved in the migration of inflammatory cells to the central nervous system in multiple sclerosis (MS). The aim of our study was to estimate the concentrations of CCL5, CXCL10 and CXCL11 in serum and cerebrospinal fluid (CSF) samples of relapsing-remitting MS (RRMS) patients during both relapse and stable disease, and to compare the results with those of controls. We also decided to evaluate the effect of methylprednisolone (MP) therapy on CCL5, CXCL10 and CXCL11 serum concentrations in MS patients with relapse.

METHODS

The study groups consisted of 17 RRMS patients during relapse, 30 RRMS patients in remission and 25 patients with tension headache with no symptoms of inflammatory disease as controls. In the group of relapsing MS patients, blood samples were obtained before steroid therapy and after a 5-day treatment with MP at a dose of 1 g i.v. once daily. Chemokine levels were measured by ELISA.

RESULTS

CXCL10 levels were significantly higher in the CSF of MS patients both during relapse (mean ± SD, 298.2 ± 143.8 pg/ml) and stable disease (323.7 ± 183 pg/ml) in comparison with the control group (152.4 ± 97.7 pg/ml; p < 0.001). CSF levels of CCL5 were significantly higher in relapsing MS patients (8.74 ± 6.18 pg/ml) in comparison with stable MS patients (4.4 ± 3.9 pg/ml, p = 0.005). CXCL11 levels of MS patients did not significantly differ from control values. There was no effect of MP therapy on serum levels of CCL5, CXCL10 and CXCL11.

CONCLUSIONS

These observations suggest involvement of CXCL10 and CCL5 but not CXCL11 in the pathogenesis of MS. CCL5 may induce the recruitment of inflammatory cells in acute-stage MS.

BACKGROUND

The role of Mycobacteria in the etiology of Crohn's disease (CD) has been a contentious subject for many years. Recently, our laboratory showed that spontaneous colitis in IL-10-/- mice is driven in part by antigens (Ags) conserved in Mycobacteria. The present study dissects some of the common cellular and molecular mechanism that drive Mycobacteria-mediated and spontaneous colitis in IL-10-/- mice.

RESULTS

We show that serum from inflammatory bowel disease (IBD) patients contain significantly higher levels of Mycobacterium avium paratuberculosis-specific IgG1 and IgG2 antibodies (Abs), serum amyloid A (SAA) as well as CXCR3 ligands than serum from healthy donors. To study the cellular mechanisms of Mycobacteria-associated colitis, pathogen-free IL-10-/- mice were given heat-killed or live M. avium paratuberculosis. The numbers of mucosal T cells, neutrophils, NK/NKT cells that expressed TNFalpha, IFN-gamma, and/or CXCL10 were significantly higher in mice that received live Mycobacteria than other groups. The numbers of mucosal CXCR3+, CXCL9+, CXCL11+ and/or IFN-gamma+ dendritic cells (DCs) were also significantly higher in M. avium paratuberculosis-challenged mice, than compared to control mice.

CONCLUSIONS

The present study shows that CD and UC patients mount significant Mycobacteria-specific IgG1>> IgG2 and CXCR3 ligand responses. Several cellular mechanisms that drive spontaneous colitis also mediate Mycobacteria-enhanced colitis in IL-10-/- mice. Similar to IL-10-/- mice under conventional housing, we show that Mycobacteria-challenge IL-10-/- mice housed under otherwise pathogen-free conditions develop colitis that is driven by CXCR3- and CXCR3 ligand-expressing leukocytes, which underscores another important hallmark and molecular mechanism of colitis. Together, the data show that Mycobacteria-dependent host responses, namely CXCL10+ T cells and NK cells, assist in the recruitment and activation of CXCR3+ and CXCL11+ leukocytes to enhance colitis of susceptible hosts.

BACKGROUND

In recent years, many immunoregulatory functions have been ascribed to soluble HLA-G (sHLA-G). Since chemotaxis is crucial for an efficient immune response, we have investigated for the first time the effects of sHLA-G on chemokine receptor expression and function in different human T cell populations.

RESULTS

T cell populations isolated from peripheral blood were stimulated in the presence or absence of sHLA-G. Chemokine receptors expression was evaluated by flow cytometry. sHLA-G downregulated expression of i) CCR2, CXCR3 and CXCR5 in CD4(+) T cells, ii) CXCR3 in CD8(+) T cells, iii) CXCR3 in Th1 clones iv) CXCR3 in TCR Vdelta2gamma9 T cells, and upregulated CXCR4 expression in TCR Vdelta2gamma9 T cells. sHLA-G inhibited in vitro chemotaxis of i) CD4(+) T cells towards CCL2, CCL8, CXCL10 and CXCL11, ii) CD8(+) T cells towards CXCL10 and CXCL11, iii) Th1 clones towards CXCL10, and iv) TCR Vdelta2gamma9 T cells towards CXCL10 and CXCL11. Downregulation of CXCR3 expression on CD4+ T cells by sHLA-G was partially reverted by adding a blocking antibody against ILT2/CD85j, a receptor for sHLA-G, suggesting that sHLA-G downregulated chemokine receptor expression mainly through the interaction with ILT2/CD85j. Follicular helper T cells (T(FH)) were isolated from human tonsils and stimulated as described above. sHLA-G impaired CXCR5 expression in T(FH) and chemotaxis of the latter cells towards CXCL13. Moreover, sHLA-G expression was detected in tonsils by immunohistochemistry, suggesting a role of sHLA-G in local control of T(FH) cell chemotaxis. Intracellular pathways were investigated by Western Blot analysis on total extracts from CD4+ T cells. Phosphorylation of Stat5, p70 s6k, beta-arrestin and SHP2 was modulated by sHLA-G treatment.

CONCLUSIONS

Our data demonstrated that sHLA-G impairs expression and functionality of different chemokine receptors in T cells. These findings delineate a novel mechanism whereby sHLA-G modulates T cell recruitment in physiological and pathological conditions.

Inflammatory bowel disease (IBD) is a group of disorders that are characterized by chronic, uncontrolled inflammation in the intestinal mucosa. Although the aetiopathogenesis is poorly understood, it is widely believed that IBD stems from a dysregulated immune response towards otherwise harmless commensal bacteria. Chemokines induce and enhance inflammation through their involvement in cellular trafficking. Reducing or limiting the influx of these proinflammatory cells has previously been demonstrated to attenuate inflammation. CXCR3, a chemokine receptor in the CXC family that binds to CXCL9, CXCL10 and CXCL11, is strongly overexpressed in the intestinal mucosa of IBD patients. We hypothesised that CXCR3 KO mice would have impaired cellular trafficking, thereby reducing the inflammatory insult by proinflammatory cell and attenuating the course of colitis. To investigate the role of CXCR3 in the progression of colitis, the development of dextran sulfate sodium (DSS)-induced colitis was investigated in CXCR3-/- mice over 9 days. This study demonstrated attenuated DSS-induced colitis in CXCR3-/- mice at both the macroscopic and microscopic level. Reduced colitis correlated with lower recruitment of neutrophils (p = 0.0018), as well as decreased production of IL-6 (p<0.0001), TNF (p = 0.0038), and IFN-γ (p = 0.0478). Overall, our results suggest that CXCR3 plays an important role in recruiting proinflammatory cells to the colon during colitis and that CXCR3 may be a therapeutic target to reduce the influx of proinflammatory cells in the inflamed colon.

Structurally related chemotactic cytokines (chemokines) regulate cell trafficking through interactions with 7-transmembrane domain, G protein-coupled receptors. Biased signaling or functional selectivity is a concept that describes a situation where a 7-transmembrane domain receptor preferentially activates one of several available cellular signaling pathways. It can be divided into 3 distinct cases: ligand bias, receptor bias, and tissue or cell bias. Many studies, including those coming from our lab, have shown that only a limited number of chemokines are key drivers of inflammation. We have referred to them as "driver chemokines." They include the CXCR3 ligands CXCL9 and CXCL10, the CCR2 ligand CCL2, all 3 CCR5 ligands, and the CCR9 ligand CCL25. As for CXCR3, despite the proinflammatory nature of CXCL10 and CXCL9, transgenic mice lacking CXCR3 display an aggravated manifestation of different autoimmune disease, including Type I diabetes and experimental autoimmune encephalomyelitis. Recently, we showed that whereas CXCL9 and CXCL10 induce effector Th1/Th17 cells to promote inflammation, CXCL11, with a relatively higher binding affinity to CXCR3, drives the development of the forkhead box P3-negative IL-10(high) T regulatory 1 cell subset and hence, dampens inflammation. We also showed that CXCL9/CXCL10 activates a different signaling cascade than CXCL11, despite binding to the same receptor, CXCR3, which results in these diverse biologic activities. This provides new evidence for the role of biased signaling in regulating biologic activities, in which CXCL11 induces ligand bias at CXCR3 and receptor-biased signaling via atypical chemokine receptor 3.

In areas where polyparasitism is highly prevalent, the impact of multiple parasites on the host response is underestimated. In particular, the presence of helminth infection coincident with malaria profoundly alters the production of malaria-specific IFN-γ, IL-12p70, CXCL9, CXCL10 and CXCL11, cytokines/chemokines known to be critical in mediating malaria-specific immunity. In order to elucidate the mechanisms underlying the suppression of malaria-specific cytokines/chemokines, we assessed the expression of malaria-specific IL-12Rβ1, IL-12Rβ2 and interferon regulatory factor (IRF)-1 in blood obtained from 18 filaria-infected (Fil(+)) and 17 filaria-uninfected (Fil(-)) individuals in a filaria-malaria co-endemic region of Mali. We found that Fil(+) individuals had significantly lower RNA expression of IRF-1 but not IL-12Rβ1 or IL-12Rβ2 in response to malaria antigen stimulation. We also measured the frequency of IL-12-producing DCs from these subjects and found that Fil(+) subjects had lower frequencies of IL-12(+) mDCs after malaria antigen stimulation than did the Fil(-) subjects. Modeling these data in vitro, we found that mDCs pre-exposed to live microfilariae not only produced significantly lower levels of CXCL-9, CXCL-10, IL-12p35, IL-12p40, IL-12p19 and CXCL-11 following stimulation with malaria antigen but also markedly downregulated the expression of IRF-1, IRF-2 and IRF-3 compared with microfilaria-unexposed mDCs. siRNA-inhibition of irf-1 in mDCs downregulated the production of IL-12p70 through repression of IL-12p35. Our data demonstrate that the modulation of IRFs seen in filarial (and presumably other tissue-invasive helminths) infection underlies the suppression of malaria-specific cytokines/chemokines that play a crucial role in immunity to malaria.

GM-CSF plays an important role in inflammation by promoting the production, activation, and survival of granulocytes and macrophages. In this study, GM-CSF knockout (GM-CSF(-/-)) mice were used to investigate the role of GM-CSF in a model of allergic airway inflammation. In allergic GM-CSF(-/-) mice, eosinophil recruitment to the airways showed a striking pattern, with eosinophils present in perivascular areas, but almost completely absent in peribronchial areas, whereas in wild-type mice, eosinophil infiltration appeared in both areas. In the GM-CSF(-/-) mice, mucus production in the airways was also reduced, and eosinophil numbers were markedly reduced in the bronchoalveolar lavage (BAL)(3) fluid. IL-5 production was reduced in the lung tissue and BAL fluid of GM-CSF(-/-) mice, but IL-4 and IL-13 production, airway hyperresponsiveness, and serum IgE levels were not affected. The presence of eosinophils in perivascular but not peribronchial regions was suggestive of a cell migration defect in the airways of GM-CSF(-/-) mice. The CCR3 agonists CCL5 (RANTES) and CCL11 (eotaxin-1) were expressed at similar levels in GM-CSF(-/-) and wild-type mice. However, IFN-gamma mRNA and protein were increased in the lung tissue and BAL fluid in GM-CSF(-/-) mice, as were mRNA levels of the IFN-gamma-inducible chemokines CXCL9 (Mig), CXCL10 (IP-10), and CXCL11 (I-Tac). Interestingly, these IFN-gamma-inducible chemokines are natural antagonists of CCR3, suggesting that their overproduction in GM-CSF(-/-) mice contributes to the lack of airway eosinophils. These findings demonstrate distinctive abnormalities to a model of allergic asthma in the absence of GM-CSF.

OBJECTIVE

Pancreatic ductal adenocarcinoma is a deadly disease because of late diagnosis and chemoresistance. We aimed to find a panel of serum cytokines representing diagnostic and predictive biomarkers for pancreatic cancer.

METHODS

A cytokine antibody array was performed to simultaneously identify 507 cytokines in sera of patients with pancreatic cancer and healthy controls. The nonparametric Mann-Whitney U test was used to pairwise compare the controls, the pretreated patients, and the posttreated patients. Fold changes greater than or equal to 1.5 or less than or equal to 1/1.5 were considered significant. Receiver operating characteristic curves were used to assess the performance of the model. A leave-one-out cross-validation was used for estimating prediction error.

RESULTS

Comparing the sera of pretreated patients against the control samples, the cytokines fibroblast growth factor 10 (FGF-10/keratinocyte growth factor-2 (KGF-2), chemokine (C-X-C motif) ligand 11 interferon inducible T cell alpha chemokine (I-TAC)/chemokine [C-X-C motif] ligand 11 (CXCL11), oncostatin M (OSM), osteoactivin/glycoprotein nonmetastatic melanoma protein B, and stem cell factor (SCF) were found significantly overexpressed. Besides, the cytokines CD30 ligand/tumor necrosis factor superfamily, member 8 (TNFSF8), chordin-like 2, FGF-10/KGF-2, growth/differentiation factor 15, I-TAC/CXCL11, OSM, and SCF were differentially expressed in response to treatment.

CONCLUSIONS

We propose a role for FGF-10/KGF-2, I-TAC/CXCL11, OSM, osteoactivin/glycoprotein nonmetastatic melanoma protein B, and SCF as novel diagnostic biomarkers. CD30 ligand/TNFSF8, chordin-like 2, FGF-10/KGF-2, growth/differentiation factor 15, I-TAC/CXCL11, OSM, and SCF might represent as predictive biomarkers for gemcitabine and erlotinib response of patients with pancreatic cancer.