OBJECTIVE

Beta cell failure is a crucial component in the pathogenesis of type 2 diabetes. One of the proposed mechanisms of beta cell failure is local inflammation, but the presence of pancreatic islet inflammation in type 2 diabetes and the mechanisms involved remain under debate.

METHODS

Chemokine and cytokine expression was studied by microarray analysis of laser-capture microdissected islets from pancreases obtained from ten non-diabetic and ten type 2 diabetic donors, and by real-time PCR of human islets exposed to oleate or palmitate at 6 or 28 mmol/l glucose. The cellular source of the chemokines was analysed by immunofluorescence of pancreatic sections from individuals without diabetes and with type 2 diabetes.

RESULTS

Microarray analysis of laser-capture microdissected beta cells showed increased chemokine and cytokine expression in type 2 diabetes compared with non-diabetic controls. The inflammatory response in type 2 diabetes was mimicked by exposure of non-diabetic human islets to palmitate, but not to oleate or high glucose, leading to the induction of IL-1beta, TNF-alpha, IL-6, IL-8, chemokine (C-X-C motif) ligand 1 (CXCL1) and chemokine (C-C motif) ligand 2 (CCL2). Interference with IL-1beta signalling abolished palmitate-induced cytokine and chemokine expression but failed to prevent lipotoxic human islet cell death. Palmitate activated nuclear factor kappaB (NF-kappaB) in human pancreatic beta and non-beta cells, and chemically induced endoplasmic reticulum stress caused cytokine expression and NF-kappaB activation similar to that occurring with palmitate.

CONCLUSIONS

Saturated-fatty-acid-induced NF-kappaB activation and endoplasmic reticulum stress may contribute to IL-1beta production and mild islet inflammation in type 2 diabetes. This inflammatory process does not contribute to lipotoxicity ex vivo, but may lead to local chemokine release.

Keratinocytes play a crucial role in the regulation of skin inflammation, responding to environmental and immune cells stimuli. They produce soluble factors that can act in an autocrine or paracrine manner on immune cells or directly on aggressors. A screening of the activities of 36 cytokines on keratinocyte gene expression identified IL-17A, IL-22, oncostatin M, TNF-alpha, and IL-1alpha as potent cytokines in inducing cutaneous inflammation. These five proinflammatory cytokines synergistically increased production of CXCL8 and beta-defensin 2 (BD2). In addition, ex vivo studies on human skin explants demonstrated upregulation of BD2, S100A7, and CXCL8 expression in response to the same combination of cytokines. In vivo intradermal injection of these five cytokines in mouse increased CXCL1, CXCL2, CXCL3, S100A9, and BD3 expression, associated with neutrophil infiltration. We confirmed and extended this synergistic effect using quantitative real-time PCR analysis and observed increased expression of nine chemokines and 12 antimicrobial peptides. Production of CXCL, CXCL5, and CXCL8 by keratinocytes stimulated in the presence of this cytokine combination was associated with increased neutrophil chemotactic activity. Similarly, high production of BD2, BD3, and S100A7 was associated with an increased antimicrobial activity. Finally, the transcriptional profile observed in this in vitro model of inflammatory keratinocytes correlated with the one of lesional psoriatic skin. Our results demonstrate the important potentiating activities of IL-17A, IL-22, oncostatin M, TNF-alpha, and IL-1alpha on keratinocytes. This is particularly interesting in the context of psoriasis where these cytokines are overexpressed and could synergize to play an important role in upregulation of chemokines and antimicrobial peptides production.

TNF-like weak inducer of apoptosis, or TWEAK, is a relatively new member of the TNF-ligand superfamily. Ligation of the TWEAK receptor Fn14 by TWEAK has proinflammatory effects on fibroblasts, synoviocytes, and endothelial cells. Several of the TWEAK-inducible cytokines are important in the pathogenesis of kidney diseases; however, whether TWEAK can induce a proinflammatory effect on kidney cells is not known. We found that murine mesangial cells express cell surface TWEAK receptor. TWEAK stimulation of mesangial cells led to a dose-dependent increase in CCL2/MCP-1, CCL5/RANTES, <em>CXCL1</em>0/IFN-gamma-induced protein 10 kDa, and <em>CXCL1</em>/KC. The induced levels of chemokines were comparable to those found following mesangial cell exposure to potent proinflammatory stimuli such as TNF-alpha + IL-1beta. <em>CXCL1</em>1/interferon-inducible T cell alpha chemoattractant, CXCR5, mucosal addressin cell adhesion molecule-1, and VCAM-1 were up-regulated by TWEAK as well. TWEAK stimulation of mesangial cells resulted in an increase in phosphorylated Ikappa-B, while pretreatment with an Ikappa-B phosphorylation inhibitor significantly blocked chemokine induction, implicating activation of the NF-kappaB signaling pathway in TWEAK-induced chemokine secretion. Importantly, the Fn14-mediated proinflammatory effects of TWEAK on kidney cells were confirmed using mesangial cells derived from Fn14-deficient mice and by injection in vivo of TWEAK into wild-type vs Fn14-deficient mice. Finally, TWEAK-induced chemokine secretion was prevented by treatment with novel murine anti-TWEAK Abs. We conclude that TWEAK induces mesangial cells to secrete proinflammatory chemokines, suggesting a prominent role for TWEAK in the pathogenesis of renal injury. Our results support Ab inhibition of TWEAK as a potential new approach for the treatment of chemokine-dependent inflammatory kidney diseases.

Although mast cells (MCs) often are abundant in the synovial tissues of patients with rheumatoid arthritis, the contribution of MCs to joint inflammation and cartilage loss remains poorly understood. MC-restricted tryptase/heparin complexes have proinflammatory activity, and significant amounts of human tryptase beta (hTryptase-beta) are present in rheumatoid arthritis synovial fluid. Mouse MC protease-6 (mMCP-6) is the ortholog of hTryptase-beta, and this serine protease is abundant in the synovium of arthritic mice. We now report that C57BL/6 (B6) mice lacking their tryptase/heparin complexes have attenuated arthritic responses, with mMCP-6 as the dominant tryptase responsible for augmenting neutrophil infiltration in the K/BxN mouse serum-transfer arthritis model. While inflammation in this experimental arthritis model was not dependent on protease-activated receptor-2, it was dependent on the chemokine receptor CXCR2. In support of the latter data, exposure of synovial fibroblasts to hTryptase-beta/heparin or mMCP-6/heparin complexes resulted in expression of the neutrophil chemotactic factors CXCL1/KC, CXCL5/LIX, and CXCL8/IL-8. Our proteomics, histochemistry, and immunohistochemistry data also revealed substantial loss of cartilage-derived aggrecan proteoglycans in the arthritic joints of wild-type B6 mice but not mMCP-6-null B6 mice. These observations demonstrate the functional contribution of MC-restricted tryptase/heparin complexes in the K/BxN mouse arthritis model and connect our mouse findings with rheumatoid arthritis pathophysiology.

Transforming growth factor (TGF)-beta signaling has been associated with early tumor suppression and late tumor progression; however, many of the mechanisms that mediate these processes are not known. Using Cre/LoxP technology, with the whey acidic protein promoter driving transgenic expression of Cre recombinase (WAP-Cre), we have now ablated the type II TGF-beta receptor (T beta RII) expression specifically within mouse mammary alveolar progenitors. Transgenic expression of the polyoma virus middle T antigen, under control of the mouse mammary tumor virus enhancer/promoter, was used to produce mammary tumors in the absence or presence of Cre (T beta RII((fl/fl);PY) and T beta RII((fl/fl);PY;WC), respectively). The loss of TGF-beta signaling significantly decreased tumor latency and increased the rate of pulmonary metastasis. The loss of TGF-beta signaling was significantly correlated with increased tumor size and enhanced carcinoma cell survival. In addition, we observed significant differences in stromal fibrovascular abundance and composition accompanied by increased recruitment of F4/80(+) cell populations in T beta RII((fl/fl);PY;WC) mice when compared with T beta RII((fl/fl);PY) controls. The recruitment of F4/80(+) cells correlated with increased expression of known inflammatory genes including Cxcl1, Cxcl5, and Ptgs2 (cyclooxygenase-2). Notably, we also identified an enriched K5(+) dNp63(+) cell population in primary T beta RII((fl/fl);PY;WC) tumors and corresponding pulmonary metastases, suggesting that loss of TGF-beta signaling in this subset of carcinoma cells can contribute to metastasis. Together, our current results indicate that loss of TGF-beta signaling in mammary alveolar progenitors may affect tumor initiation, progression, and metastasis through regulation of both intrinsic cell signaling and adjacent stromal-epithelial interactions in vivo.

The severity of disease caused in humans by H5N1 influenza viruses remains unexplained. The NS gene of Hong Kong H5N1/97 viruses was shown to contribute to high pathogenicity of reassortants in a pig model. However, the molecular pathogenesis and host immune response underlying this phenomenon remain unclear. Here, in a mouse model, H1N1 A/Puerto Rico/8/34 (PR/8) reassortants that contained the H5N1/97 NS gene, the H5N1/01 NS gene, or an altered H5N1/97 NS gene encoding a Glu92->>Asp substitution in NS1 was studied. The pathogenicity of reassortant viruses, the induction of cytokines and chemokine CXCL1 (KC) in the lungs and specific B- and T-cell responses was characterized. In mice infected with reassortant virus containing the H5N1/97 NS gene, the mouse lethal dose (50%) and lung virus titres were similar to those of PR/8, which is highly pathogenic to mice. This reassortant virus required two more days than PR/8 to be cleared from the lungs of infected mice. Reassortants containing the altered H5N1/97 NS gene or the H5N1/01 NS gene demonstrated attenuated pathogenicity and lower lung titres in mice. Specific B- and T-cell responses were consistent with viral pathogenicity and did not explain the delayed clearance of the H5N1/97 NS reassortant. The reassortant induced elevated pulmonary concentrations of the inflammatory cytokines IL1alpha, IL1beta, IL6, IFN-gamma and chemokine KC, and decreased concentrations of the anti-inflammatory cytokine IL10. This cytokine imbalance is reminiscent of the clinical findings in two humans who died of H5N1/97 infection and may explain the unusual severity of the disease.

Pseudomonas aeruginosa is a major cause of blindness and visual impairment in the United States and worldwide. Using a murine model of keratitis in which abraded corneas are infected with P. aeruginosa parent and ΔfliC (aflagellar) strains 19660 and PAO1, we found that F4/80(+) macrophages were the predominant cell type in the cornea expressing TLR2, TLR4, and TLR5. Depletion of macrophages and dendritic cells using transgenic Mafia mice, in which Fas ligand is selectively activated in these cells, resulted in diminished cytokine production and cellular infiltration to the corneal stroma and unimpaired bacterial growth. TLR4(-/-) mice showed a similar phenotype postinfection with ΔfliC strains, whereas TLR4/5(-/-) mice were susceptible to corneal infection with parent strains. Bone marrow-derived macrophages stimulated with ΔfliC bacteria induced Toll/IL-1R intracellular domain (TIR)-containing adaptor inducing IFN-β (TRIF)-dependent phosphorylation of IFN regulatory factor 3 in addition to TIR-containing adaptor protein/MyD88-dependent phosphorylation of IκB and nuclear translocation of the p65 subunit of NFκB. Furthermore, TRIF(-/-) mice showed a similar phenotype as TLR4(-/-) mice in regulating only ΔfliC bacteria, whereas MyD88(-/-) mice were unable to clear parent or ΔfliC bacteria. Finally, IL-1R1(-/-) and IL-1α/β(-/-) mice were highly susceptible to infection. Taken together, these findings indicate that P. aeruginosa activates TLR4/5 on resident corneal macrophages, which signal through TRIF and TIR-containing adaptor protein/MyD88 pathways, leading to NF-κB translocation to the nucleus, transcription of CXCL1 and other CXC chemokines, recruitment of neutrophils to the corneal stroma, and subsequent bacterial killing and tissue damage. IL-1α and IL-1β are also produced, which activate an IL-1R1/MyD88-positive feedback loop in macrophages and IL-1R on other resident cells in the cornea.

Functional interleuin-8 (IL-8) receptors (IL-8RA and IL-8RB: CXCR1 and CXCR2, respectively) have been described in human, monkey, dog, rabbit, and guinea pig. Although three IL-8R homologues have been found in rat, only one of these, rat CXCR2, appears to be functional based on responsiveness to ligands. Similarly, CXC chemokines induce biological responses through the murine homolog of CXCR2, but the identification of functional rodent CXCR1 homologues has remained elusive. We have identified and characterized the mouse CXCR1 homologue (mCXCR1). Murine CXCR1 shares 68 and 88% amino acid identity with its human and rat counterparts, respectively. Similar to the tissue distribution pattern of rat CXCR1, we found murine CXCR1 mRNA expression predominantly in lung, stomach, bone marrow, and leukocyte-rich tissues. In contrast to previous reports, we determined that mCXCR1 is a functional receptor. We show predominant engagement of this receptor by mouse GCP-2/CXCL6, human GCP-2, and IL-8/CXCL8 by binding, stimulation of GTPgammaS exchange, and chemotaxis of mCXCR1-transfected cells. Furthermore, murine CXCR1 is not responsive to the human CXCR2 ligands ENA-78/CXCL5, NAP-2/CXCL7, GRO-alpha, -beta, -gamma/CXCL1-3, or rat CINC-1-3. In addition, we show concomitant elevation of mCXCR1 and its proposed major ligand, GCP-2, positively correlated with paw swelling in murine collagen-induced arthritis. This report represents the first description of a functional CXCR1-like receptor in rodents.

Polymorphonuclear leukocytes (PMNs) are key in innate immunity, but their role in viral pathogenesis is incompletely understood. In infection due to West Nile virus (WNV), we found that expression of 2 PMN-attracting chemokines, Cxcl1 and Cxcl2, was rapidly and dramatically elevated in macrophages. PMNs are rapidly recruited to the site of WNV infection in mice and support efficient replication of WNV. Mice depleted of PMNs after WNV inoculation developed higher viremia and experienced earlier death, compared with the control group, which suggest a protective role for PMNs. In contrast, when PMNs were depleted prior to infection with WNV, and in mice deficient in Cxcr2 (a chemokine receptor gene), viremia was reduced and survival was enhanced. Collectively, these data suggest that PMNs have a biphasic response to WNV infection, serving as a reservoir for replication and dissemination in early infection and later contributing to viral clearance.

Pericytes and mesenchymal stem cells (MSCs) are ontogenically related, and in fact, no significant phenotypic differences could be observed by flow cytometry. Transcriptome analysis of human pericytes and MSCs revealed that 43 genes were up-regulated more than 10-fold in pericytes compared with MSCs. Identification of Toll-like receptor 4 (TLR4) as one of the most abundant RNA species in pericytes with respect to MSCs and confirmation of TLR4 expression on the cell surface led us to obtain a comprehensive overview of the expression program of lipopolysaccharide (LPS)-stimulated pericytes. Transcriptional profiling of LPS-treated cells revealed that 22 genes were up-regulated more than 5-fold. Of them, 10 genes encoded chemokines and cytokines (CXCL1CXCL1, IL6, CCL2, IL1B, CXCL2, IL1A, and CXCL6), and three genes encoded adhesion molecules (ICAM1, VCAM1, and SELE). LPS induced nuclear translocation of the transcription factor NF-κB in stimulated pericytes. Moreover, inhibition of NF-κB activation by SC-514 blocked LPS-induced up-regulation of a subset of chemokine genes, confirming the key role of NF-κB in LPS signaling in pericytes. At the protein level, we assessed the secretion of the proinflammatory cytokines and chemokines IL-6, IL-8, CXCL1, CXCL2, CXCL3, and CCL2 not only after LPS treatment but also in HMGB1-stimulated pericytes. Up-regulation of the adhesion molecules ICAM-1 and VCAM-1 resulted in an increased adhesion of peripheral blood leukocytes to an LPS-treated pericyte monolayer. The role of pericytes in the inflammatory context has been scarcely addressed; according to these results, pericytes should be considered as active players in the inflammatory cascade with potential physiopathological implications.

Apoptosis is commonly thought to represent an immunologically silent or even anti-inflammatory mode of cell death, resulting in cell clearance in the absence of explicit activation of the immune system. However, here we show that Fas/CD95-induced apoptosis is associated with the production of an array of cytokines and chemokines, including IL-6, IL-8, CXCL1, MCP-1, and GMCSF. Fas-induced production of MCP-1 and IL-8 promoted chemotaxis of phagocytes toward apoptotic cells, suggesting that these factors serve as "find-me" signals in this context. We also show that RIPK1 and IAPs are required for optimal production of cytokines and chemokines in response to Fas receptor stimulation. Consequently, a synthetic IAP antagonist potently suppressed Fas-dependent expression of multiple proinflammatory mediators and inhibited Fas-induced chemotaxis. Thus, in addition to provoking apoptosis, Fas receptor stimulation can trigger the secretion of chemotactic factors and other immunologically active proteins that can influence immune responsiveness toward dying cells.

Constitutive IKK activity associated with increased IkappaBalpha phosphorylation and degradation contribute to the high level of endogenous nuclear factor-kappaB (NF-kappaB) activation in Hs294T melanoma cells as compared with RPE cells (R. L. Shattuck-Brandt and A. Richmond, Cancer Res., 57: 3032-3039, 1997; M. N. Devalaraja et al., Cancer Res., 59: 1372-1377, 1999). To determine whether this endogenous NF-kappaB activation was characteristic of melanoma, we examined the level of constitutive activation of NF-kappaB in a number of melanoma cell lines. We demonstrate here that eight melanoma cell lines exhibit increased IkappaB kinase (IKK) activity, enhanced phosphorylation of IkappaBalpha and p65, and enhanced nuclear localization of p65/p50 in comparison to normal human epidermal melanocytes. The chemokines, CXC ligand 1 (CXCL1) and CXCL8, but not CXCL5, are highly expressed in most of the melanoma cell lines, suggesting that the constitutive production of chemokines is highly correlated to endogenous NF-kappaB activity. Our failure to observe a direct relationship between the fold activation of IKK, CXCL1, or CXCL8 mRNA levels and secretion of these chemokines into the culture medium suggest that regulation of chemokine expression also occurs at the posttranscription level of mRNA stability and/or translational control. Moreover, recombinant CXCL1 can directly induce IKK activity in normal human epidermal melanocytes in a concentration-dependent manner after up-modulation of CXCL1 protein expression, whereas inhibition of IKKbeta activity results in down-modulation of CXCL1 protein expression. Finally, CXCL1 antibody blocks IKK activity and inhibits the proliferation of melanoma cells to further support the concept that the constitutive activation of NF-kappaB and autocrine effects of CXCL1 play an important role in the pathogenesis of melanoma.

Cisplatin is an important chemotherapeutic agent; however, its nephrotoxicity limits its clinical use. Enhanced inflammatory response and oxidative/nitrosative stress seem to play a key role in the development of cisplatin-induced nephropathy. Activation of cannabinoid-2 (CB(2)) receptors with selective agonists exerts anti-inflammatory and tissue-protective effects in various disease models. We have investigated the role of CB(2) receptors in cisplatin-induced nephrotoxicity using the selective CB(2) receptor agonist HU-308 and CB(2) knockout mice. Cisplatin significantly increased inflammation (leukocyte infiltration, CXCL1/2, MCP-1, TNFalpha, and IL-1beta levels) and expression of adhesion molecule ICAM-1 and superoxide-generating enzymes NOX2, NOX4, and NOX1 and enhanced ROS generation, iNOS expression, nitrotyrosine formation, and apoptotic and poly(ADP-ribose) polymerase-dependent cell death in the kidneys of mice, associated with marked histopathological damage and impaired renal function (elevated serum BUN and creatinine levels) 3 days after the administration of the drug. CB(2) agonist attenuated the cisplatin-induced inflammatory response, oxidative/nitrosative stress, and cell death in the kidney and improved renal function, whereas CB(2) knockouts developed enhanced inflammation and tissue injury. Thus, the endocannabinoid system, through CB(2) receptors, protects against cisplatin-induced kidney damage by attenuating inflammation and oxidative/nitrosative stress, and selective CB(2) agonists may represent a promising novel approach to preventing this devastating complication of chemotherapy.

Subsequent to demyelination in multiple sclerosis, myelin repair occurs but, as lesions age, the ability to remyelinate diminishes. Molecular pathways underlying oligodendrocyte behaviour during CNS remyelination remain to be elucidated. In this study, we report for the first time constitutive expression of the CXC/alpha chemokine receptors, CXCR1, CXCR2 and CXCR3, on oligodendrocytes in normal adult human CNS tissue, the levels of which were upregulated in multiple sclerosis and other neurological diseases (OND). In addition, both immature (A2B5+/O4+) and more mature (CNPase+) human oligodendrocytes in vitro expressed the same three receptors. The respective ligands to CXCR1, CXCR2 and CXCR3 [i.e. CXCL8/IL-8, <em>CXCL1</em>/GRO-alpha and <em>CXCL1</em>0/IP-10), were absent in CNS tissue from normals and subjects with OND, but were present at high levels on hypertrophic (reactive) astrocytes at the edge of active (but not silent) multiple sclerosis lesions. Astrocytes in vitro could be induced to express chemokines following stimulation with pro-inflammatory cytokines. CXCL8 and <em>CXCL1</em> production by human astrocytes at both the RNA and protein levels could be induced by interleukin (IL)-1beta, while <em>CXCL1</em>0 was induced by both IL-1beta and interferon-gamma. Since these cytokines are integral to inflammatory events occurring at the margins of active multiple sclerosis lesions, their upregulation in these regions may underlie the dynamics of chemokine expression observed herein. The simultaneous expression of different CXC chemokine receptors on oligodendrocytes, and their ligands on astrocytes around multiple sclerosis lesions, may bespeak novel functional roles for these immune system molecules in the recruitment of oligodendrocytes and remyelination.

Recent studies have indicated an important role of chemokines such as CCL2 in the development of chronic pain. However, the distinct roles of different chemokines in the development and maintenance of neuropathic pain and in their interactions with neurons have not been clearly elucidated. We found that spinal nerve ligation (SNL) not only induced persistent neuropathic pain symptoms, including mechanical allodynia and heat hyperalgesia, but also produced sustained CXCL1 upregulation in the spinal cord. Double staining of immunofluorescence and in situ hybridization revealed that CXCL1 was primarily induced in spinal astrocytes. In cultured astrocytes, tumor necrosis factor-α induced robust CXCL1 expression via the activation of the c-jun N-terminal kinase. Intrathecal administration of CXCL1 neutralizing antibody transiently reduced SNL-induced pain hypersensitivity, suggesting an essential role of CXCL1 in neuropathic pain sensitization. In particular, intraspinal delivery of CXCL1 shRNA lentiviral vectors, either before or after SNL, persistently attenuated SNL-induced pain hypersensitivity. Spinal application of CXCL1 not only elicited pain hypersensitivity but also induced rapid neuronal activation, as indicated by the expression of phosphorylated extracellular signal-regulated kinase and cAMP response element binding protein, and c-Fos in spinal cord neurons. Interestingly, CXCR2, the primary receptor of CXCL1, was upregulated in dorsal horn neurons after SNL, and the CXCR2 antagonist SB225002 completely blocked the CXCL1-induced heat hyperalgesia. SB225002 also attenuated SNL-induced pain hypersensitivity. Collectively, our results have demonstrated a novel form of chemokine-mediated glial-neuronal interaction in the spinal cord that can drive neuropathic pain. Inhibition of the CXCL1-CXCR2 signaling may offer a new therapy for neuropathic pain management.

The chemokine sink hypothesis pertaining to erythrocyte Duffy Antigen Receptor for Chemokines (DARC) during inflammation has received considerable attention, but lacks direct in vivo evidence. Here we demonstrate, using mice with a targeted deletion in CXCL5, that CXCL5 bound erythrocyte DARC and impaired its chemokine scavenging in blood. CXCL5 increased the plasma concentrations of CXCL1 and CXCL2 in part through inhibiting chemokine scavenging, impairing chemokine gradients and desensitizing CXCR2, which led to decreased neutrophil influx to the lung, increased lung bacterial burden and mortality in an Escherichia coli pneumonia model. In contrast, CXCL5 exerted a predominant role in mediating neutrophil influx to the lung during inflammation after LPS inhalation. Platelets and lung resident cells were the sources of homeostatic CXCL5 in blood and inflammatory CXCL5 in the lung respectively. This study presents a paradigm whereby platelets and red cells alter chemokine scavenging and neutrophil-chemokine interaction during inflammation.

Mesothelial cells that line the serous cavities and outer surface of internal organs are involved in inflammatory responses induced by microbial stimuli and bacterial infection. Upon exposure to bacterial products, mesothelial cells secrete chemokines, but the signaling pathways by which these cells recognize bacteria to mediate innate immune responses remain largely unknown. We report that stimulation of primary peritoneal mesothelial cells via nucleotide-binding oligomerization domain (Nod)1, a member of the intracytoplasmic Nod-like receptor family, induced potent secretion of the chemokines CXCL1 and CCL2 as well as expression of inducible NO synthase and such responses required the kinase RICK. Mesothelial cells also produced chemokines in response to TLR2, TLR3, TLR4, and TLR5 agonists, but unlike that induced by Nod1 stimulation, the TLR-mediated responses were independent of RICK. Yet, Nod1 stimulation of mesothelial cells via RICK enhanced chemokine secretion induced by LPS or IFN-gamma and cooperated with IFN-gamma in the production of NO. The i.p. administration of KF1B, a synthetic Nod1 agonist, elicited chemokine production in the serum and peritoneal fluid as well as the recruitment of neutrophils into the peritoneal cavity of wild-type mice, but not RICK-deficient mice. Finally, infection of mesothelial cells with Listeria monocytogenes induced production of CXCL1 and this response was significantly reduced in Nod1- or RICK-deficient cells. These results define mesothelial cells as microbial sensors through TLRs and Nod-like receptors and identify Nod1 and RICK as important mediators of chemokine and antimicrobial responses in mesothelial cells.

Streptococcus suis, an important swine and human pathogen, causes septic shock and meningitis. The pathogenesis of both systemic and CNS infections caused by S. suis is poorly understood. A hematogenous model of infection in CD1 mice was developed to study the systemic release of cytokines during the septic shock phase and the proinflammatory events in the CNS associated with this pathogen. Using a liquid array system, high levels of systemic TNF-alpha, IL-6, IL-12, IFN-gamma, CCL2, CXCL1, and CCL5 were observed 24 h after infection and might be responsible for the sudden death of 20% of animals. Infected mice that survived the early sepsis later developed clinical signs of meningitis and exhibited lesions in the meninges and in numerous regions of the brain, such as the cortex, hippocampus, thalamus, hypothalamus, and corpus callosum. Bacterial Ags were found in association with microglia residing only in the affected zones. In situ hybridization combined with immunocytochemistry showed transcriptional activation of TLR2 and TLR3 as well as CD14, NF-kappaB, IL-1beta, CCL2, and TNF-alpha, mainly in myeloid cells located in affected cerebral structures. Early transcriptional activation of TLR2, CD14, and inflammatory cytokines in the choroid plexus and cells lining the brain endothelium suggests that these structures are potential entry sites for the bacteria into the CNS. Our data indicate an important role of the inflammatory response in the pathogenesis of S. suis infection in mice. This experimental model may be useful for studying the mechanisms underlying sepsis and meningitis during bacterial infection.

The recruitment of selected dendritic cell (DC) subtypes conditions the class of the immune response. Here we show that the migration of human plasmacytoid DCs (pDCs), the blood natural interferon alpha-producing cells, is induced upon the collective action of inducible and constitutive chemokines. Despite expression of very high levels of CXCR3, pDCs do not respond efficiently to CXCR3 ligands. However, they migrate in response to the constitutive chemokine stromal cell-derived factor 1 (SDF-1)/<em>CXCL1</em>2 and CXCR3 ligands synergize with SDF-1/<em>CXCL1</em>2 to induce pDC migration. This synergy reflects a sensitizing effect of CXCR3 ligands, which, independently of a gradient and chemoattraction, decrease by 20-50-fold the threshold of sensitivity to SDF-1/<em>CXCL1</em>2. Thus, the ability of the constitutive chemokine SDF-1/<em>CXCL1</em>2 to induce pDC recruitment might be controlled by CXCR3 ligands released during inflammation such as in virus infection. SDF-1/<em>CXCL1</em>2 and the CXCR3 ligands Mig/CXCL9 and ITAC/<em>CXCL1</em> display adjacent expression both in secondary lymphoid organs and in inflamed epithelium from virus-induced pathologic lesions. Because pDCs express both the lymph node homing molecule l-selectin and the cutaneous homing molecule cutaneous lymphocyte antigen, the cooperation between inducible CXCR3 ligands and constitutive SDF-1/<em>CXCL1</em>2 may regulate recruitment of pDCs either in lymph nodes or at peripheral sites of inflammation.

Recent data suggest that chemokines could be essential players in breast carcinogenesis. We previously showed that the CXC chemokine CXCL8 (interleukin-8) was overexpressed in estrogen receptor alpha (ERalpha)-negative breast cell lines. Analysis of CXCL8 chromosomal location showed that several CXC chemokines (CXCL1, CXCL2, CXCL3, CXCL4, CXCL4V1, CXCL5, CXCL6, CXCL7, and CXCL8) were localized in the same narrow region (360 kb in size) of chromosome 4. We thus hypothesized that they could belong to the same cluster. Quantification of these chemokines in breast tumors showed that samples expressing high CXCL8 also produced elevated levels of CXCL1, CXCL3, and CXCL5, and displayed low content of ERalpha. CXCL1, CXCL2, CXCL3, CXCL5, and CXCL8 were co-regulated both in tumors and in breast cancer cell lines. CXCL5 and CXCL8 were mainly produced by epithelial cells, whereas CXCL1, CXCL2, and CXCL3 had a high expression in blood cells. The overexpression of these chemokines in tumor cells was not the result of gene amplification, but rather of an enhanced gene transcription. Our data suggest that high CXCL8 expression in tumors is mainly correlated to activating protein-1 (AP-1) pathway and to a minor extent to NF-kappaB pathway. Interestingly, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, and CXCL8 chemokines were present at higher levels in metastases when compared with grade I and III biopsies. High levels of CXCL8, CXCL1, and CXCL3 accounted for a shorter relapse-free survival of ERalpha-positive patients treated with tamoxifen. In summary, we present evidences that multiple CXC chemokines are co-expressed in CXCL8-positive breast tumors. In addition, these chemokines could account for the higher aggressiveness of these types of tumors.

Dietary fiber protects against chronic inflammatory diseases by dampening immune responses through short-chain fatty acids (SCFAs). Here we examined the effect of dietary fiber in viral infection, where the anti-inflammatory properties of SCFAs in principle could prevent protective immunity. Instead, we found that fermentable dietary fiber increased survival of influenza-infected mice through two complementary mechanisms. High-fiber diet (HFD)-fed mice exhibited altered bone marrow hematopoiesis, characterized by enhanced generation of Ly6c- patrolling monocytes, which led to increased numbers of alternatively activated macrophages with a limited capacity to produce the chemokine CXCL1 in the airways. Blunted CXCL1 production reduced neutrophil recruitment to the airways, thus limiting tissue immunopathology during infection. In parallel, diet-derived SCFAs boosted CD8+ T cell effector function by enhancing cellular metabolism. Hence, dietary fermentable fiber and SCFAs set an immune equilibrium, balancing innate and adaptive immunity so as to promote the resolution of influenza infection while preventing immune-associated pathology.

OBJECTIVE

To provide a more complete picture of the effect of interleukin-1 beta (IL-1beta) on adult human articular chondrocyte gene expression, in contrast to the candidate gene approach.

METHODS

Chondrocytes from human knee cartilage were cultured in medium containing IL-1beta. Changes in gene expression were analyzed by microarray and reverse transcriptase-polymerase chain reaction analysis. The ability of transforming growth factor beta-1 (TGF-beta1), fibroblast growth factor (FGF)-18, and bone morphogenetic protein 2 (BMP-2) to alter the effects of IL-1beta was analyzed. Computational analysis of the promoter regions of differentially expressed genes for transcription factor binding motifs was performed.

RESULTS

IL-1beta-treated human chondrocytes showed significant increases in the expression of granulocyte colony stimulating factor-3, endothelial leukocyte adhesion molecule 1 and leukemia inhibitory factor as well as for a large group of chemokines that include CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, CCL2, CCL3, CCL4, CCL5, CCL8, CCL20, CCL3L1, CX3CL1 and the cytokine IL-6. As expected, the mRNA for matrix metalloproteinase (MMP)-13 and BMP-2 also increased while mRNA for the matrix genes COL2A1 and aggrecan was down-regulated. A subset of chemokines increased rapidly at very low levels of IL-1beta. The phenotype induced by IL-1beta was partially reversed by TGF-beta1, but not by BMP-2. In the presence of IL-1beta, FGF-18 increased expression of ADAMTS-4, aggrecan, BMP-2, COL2A1, CCL3, CCL4, CCL20, CXCL1, CXCL3, CXCL6, IL-1beta, IL-6, and IL-8 and decreased ADAMTS-5, MMP-13, CCL2, and CCL8. Computational analysis revealed a high likelihood that the most up-regulated chemokines are regulated by the transcription factors myocyte enhancer binding factor-3 (MEF-3), CCAAT/enhancer binding protein (C/EBP) and nuclear factor-kappa B (NF-kappaB).

CONCLUSIONS

IL-1beta has a diverse effect on gene expression profile in human chondrocytes affecting matrix genes as well as chemokines and cytokines. TGF-beta1 has the ability to antagonize some of the phenotype induced by IL-1beta.

BACKGROUND

Airway inflammation is a central feature of chronic obstructive pulmonary disease (COPD). COPD exacerbations are often triggered by rhinovirus (RV) infection.

OBJECTIVE

We hypothesized that airway epithelial cells from patients with COPD maintain a proinflammatory phenotype compared with control subjects, leading to greater RV responses.

METHODS

Cells were isolated from tracheobronchial tissues of 12 patients with COPD and 10 transplant donors. Eight patients with COPD had severe emphysema, three had mild to moderate emphysema, and one had no emphysema. All had moderate to severe airflow obstruction, and six met criteria for chronic bronchitis or had at least one exacerbation the previous year. Cells were grown at air-liquid interface and infected with RV serotype 39. Cytokine and IFN expression was measured by ELISA. Selected genes involved in inflammation, oxidative stress, and proteolysis were assessed by focused gene array and real-time polymerase chain reaction.

RESULTS

Compared with control subjects, cells from patients with COPD demonstrated increased mRNA expression of genes involved in oxidative stress and the response to viral infection, including NOX1, DUOXA2, MMP12, ICAM1, DDX58/RIG-I, STAT1, and STAT2. COPD cells showed elevated baseline and RV-stimulated protein levels of IL-6, IL-8/CXCL8, and growth-related oncogene-alpha/<em>CXCL1</em>. COPD cells demonstrated increased viral titer and copy number after RV infection, despite increased IL-29/IFN-lambda1, IL-28A/IFN-lambda2, and IFN-inducible protein-10/<em>CXCL1</em>0 protein levels. Finally, RV-infected COPD cultures showed increased mRNA expression of IL28A/IFNlambda2, IL29/IFNlambda1, IFIH1/MDA5, DDX58/RIG-I, DUOX1, DUOX2, IRF7, STAT1, and STAT2.

CONCLUSIONS

Airway epithelial cells from patients with COPD show higher baseline levels of cytokine expression and increased susceptibility to RV infection, despite an increased IFN response.

Th1 effector CD4+ cells contribute to the pathogenesis of proliferative and crescentic glomerulonephritis, but whether effector Th17 cells also contribute is unknown. We compared the involvement of Th1 and Th17 cells in a mouse model of antigen-specific glomerulonephritis in which effector CD4+ cells are the only components of adaptive immunity that induce injury. We planted the antigen ovalbumin on the glomerular basement membrane of Rag1(-/-) mice using an ovalbumin-conjugated non-nephritogenic IgG1 monoclonal antibody against alpha3(IV) collagen. Subsequent injection of either Th1- or Th17-polarized ovalbumin-specific CD4+ effector cells induced proliferative glomerulonephritis. Mice injected with Th1 cells developed progressive albuminuria over 21 d, histologic injury including 5.5 +/- 0.9% crescent formation/segmental necrosis, elevated urinary nitrate, and increased renal NOS2, CCL2, and CCL5 mRNA. Mice injected with Th17 cells developed albuminuria by 3 d; compared with Th1-injected mice, their glomeruli contained more neutrophils and greater expression of renal CXCL1 mRNA. In conclusion, Th1 and Th17 effector cells can induce glomerular injury. Understanding how these two subsets mediate proliferative forms of glomerulonephritis may lead to targeted therapies.