The developmentally regulated fetal enterocyte gene, ZP4, mediates anti-inflammation by the symbiotic bacterial surface factor polysaccharide A on Bacteroides fragilis.

Initial colonizing bacteria play a critical role in completing the development of the immune system in the gastrointestinal tract of infants. Yet, the interaction of colonizing bacterial organisms with the developing human intestine favors inflammation over immune homeostasis. This characteristic of bacterial-intestinal interaction partially contributes to the pathogenesis of necrotizing enterocolitis (NEC), a devastating premature infant intestinal inflammatory disease. However, paradoxically some unique pioneer bacteria (initial colonizing species) have been shown to have a beneficial effect on the homeostasis of the immature intestine and the prevention of inflammation. We have reported that one such pioneer bacterium, Bacteroides fragilis (B. fragilis), and its surface component polysaccharide A (PSA) inhibit IL-1ß-induced inflammation in a human primary fetal small intestinal cell line (H4 cells). In this study, using transcription profiling of H4 cellular RNA after pretreatment with or without PSA before an inflammatory stimulation of IL-1ß, we have begun to further determine the cellular mechanism for anti-inflammation. We show that a developmentally regulated gene, zona pellucida protein 4 (ZP4), is uniquely elevated after IL-1ß stimulation and reduced with PSA exposure. ZP4 was known as a sperm receptor-mediating species-specific binding protein in the initial life of mammals. However, its intestinal epithelial function is unclear. We found that ZP4 is a developmentally regulated gene involved with immune function and regulated by both Toll-like receptor 2 and 4. Knockdown of ZP4-affected PSA inhibited IL-8 mRNA expression in response to IL-1ß. This represents an initial study of ZP4 innate immune function in immature enterocytes. This study may lead to new opportunity for efficient treatment of NEC.NEW & NOTEWORTHY This study extends previous observations to define the cellular mechanisms of polysaccharide A-induced anti-inflammation in immature enterocytes using transcription profiling of enterocyte genes after preexposure to polysaccharide A before an inflammatory stimulus with IL-1ß.

Phospholipase Cε plays a crucial role in neutrophilic inflammation accompanying acute lung injury through augmentation of CXC chemokine production from alveolar epithelial cells.

BACKGROUND: We have shown that phospholipase Cε (PLCε), an effector of Ras and Rap1 small GTPases, plays pivotal roles in inflammation and inflammation-associated carcinogenesis by augmenting proinflammatory cytokine production from epithelial cells of various organs. The purpose of this study is to analyze its role in neutrophilic alveolar inflammation accompanying acute lung injury (ALI), focusing on that in alveolar epithelial cells (AECs), which are known to make a major contribution to the pathogenesis of ALI. METHODS: We examine the effect of the PLCε genotypes on the development of ALI induced by intratracheal administration of lipopolysaccharide (LPS) to PLCε wild-type (PLCε+/+) and knockout (PLCεΔX/ΔX) mice. Pathogenesis of ALI is analyzed by histological examination of lung inflammation and measurements of the levels of various cytokines, in particular neutrophil-attracting chemokines such as Cxcl5, by quantitative reverse transcription-polymerase chain reaction and immunostaining. Primary cultures of AECs, established from PLCε+/+ and PLCεΔX/ΔX mice, are used to analyze the roles of PLCε, protein kinase D (PKD) and nuclear factor-κB (NF-κB) in augmentation of LPS-induced Cxcl5 expression. RESULTS: Compared to PLCε+/+ mice, PLCεΔX/ΔX mice exhibit marked alleviation of lung inflammation as shown by great reduction in lung wet/dry weight ratios, accumulation of inflammatory cells in the alveolar space and thickening of alveolar walls as well as the number of neutrophils and the protein concentration in bronchoalveolar lavage fluid. Also, LPS-induced expression of the CXC family of chemokines, in particular Cxcl5, is substantially diminished in the total lung and AECs of PLCεΔX/ΔX mice. Moreover, LPS-induced Cxcl5 expression in primary cultured AECs is markedly suppressed on the PLCεΔX/ΔX background (p < 0.05 versus PLCε+/+ AECs), which is accompanied by the reduction in phosphorylation of inhibitor κB (IκB), PKD and nuclear translocation of NF-κB p65. Also, it is suppressed by the treatment with inhibitors of PKD and IκB kinase, suggesting the involvement of the PLCε-PKD-IκB-NF-κB pathway. CONCLUSIONS: PLCε-mediated augmentation of the production of the CXC family of chemokines, in particular Cxcl5, in AECs plays a crucial role in neutrophilic alveolar inflammation accompanying ALI, suggesting that PLCε may be a potential molecular target for the treatment of acute respiratory distress syndrome.

FTY720 attenuates intestinal injury and suppresses inflammation in experimental necrotizing enterocolitis via modulating CXCL5/CXCR2 axis.

Necrotizing enterocolitis (NEC) remains one of the leading causes of death in neonatal infants and new therapeutic strategies for NEC are urgently required. The immunomodulatory agent FTY720 has been shown to have protective effects in various inflammatory diseases. In this study, we hypothesized that treatment with FTY720 confers protection against experimental NEC. Experimental NEC was induced in five-day-old C57BL/6 neonatal mice by hyperosmolar formula feeding plus hypoxia and lipopolysaccharide (LPS) challenges. Induction of NEC resulted in substantial weight loss and high mortality compared to the control group, whereas FTY720 treatment significantly attenuated weight loss and improved survival in NEC-challenged neonatal mice. FTY720 treatment strongly ameliorated NEC-induced intestinal injury with reduced apoptosis and up-regulation of intestinal barrier proteins in the ileal tissues. Furthermore, FTY720 treatment abrogated NEC-initiated intestinal and systemic inflammation with markedly diminished inflammatory cytokines and chemokines. Moreover, FTY720 treatment suppressed NEC-activated CXCL5/CXCR2 axis with down-regulated expression of CXCL5 and CXCR2 at both mRNA and protein levels. Thus, we demonstrate that FTY720 protects neonatal mice against NEC-associated lethality by ameliorating intestinal injury and attenuating inflammation, possibly via its down-regulation of NEC-induced activation of intestinal CXCL5/CXCR2 axis.

Effects of extracellular orotic acid on acute contraction-induced adaptation patterns in C2C12 cells.

Dietary administration of orotic acid (OA), an intermediate in the pyrimidine biosynthetic pathway, is considered to provide a wide range of beneficial effects, including cardioprotection and exercise adaptation. Its mechanisms of action, when applied extracellularly, however, are barely understood. In this study, we evaluated potential effects of OA on skeletal muscle using an in vitro contraction model of electrically pulse-stimulated (EPS) C2C12 myotubes. By analyzing a subset of genes representing inflammatory, metabolic, and structural adaptation pathways, we could show that OA supplementation diminishes the EPS-provoked expression of inflammatory transcripts (interleukin 6, Il6; chemokine (C-X-C Motif) ligand 5, Cxcl5), and attenuated transcript levels of nuclear receptor subfamily 4 group A member 3 (Nr4A3), early growth response 1 (Egr1), activating transcription factor 3 (Atf3), and fast-oxidative MyHC-IIA isoform (Myh2). By contrast, OA had no suppressive effect on the pathogen-provoked inflammatory gene response in skeletal muscle cells, as demonstrated by stimulation of C2C12 myotubes with bacterial LPS. In addition, we observed a suppressive effect of OA on EPS-induced phosphorylation of AMP-activated protein kinase (AMPK), whereas EPS-triggered phosphorylation/activation of the mammalian target of rapamycin (mTOR) was not affected. Finally, we demonstrate that OA positively influences glycogen levels in EP-stimulated myotubes. Taken together, our results suggest that in skeletal muscle cells, OA modulates both the inflammatory and the metabolic reaction provoked by acute contraction. These results might have important clinical implications, specifically in cardiovascular and exercise medicine.

Lipopolysaccharide Upregulates the Expression of CINC-3 and LIX in Primary NG2 Cells.

Numerous NG2 cells, also called oligodendrocyte progenitor cells (OPCs), exist ubiquitously in the gray and white matter in the adult central nervous system (CNS). Although NG2 cells could become active by upregulation of NG2 expression and hypertrophy or extension of their processes under various neuropathological conditions, their actual role in the brain remains to be illustrated. In view of the fact that the synergy of cytokine and chemokine networks plays an important role in CNS inflammation and immunity, we have assumed that the NG2 cells might take part in brain inflammation and immunity by making a contribution to the pool of cytokines or chemokines. In the current study, NG2-expressing OPCs were prepared from cerebral hemispheres of postnatal day 0 or 1 Sprague-Dawley rats. Our results showed that NG2-expressing OPCs, verified by immunohistological staining of anti-NG2 antibody and anti-platelet-derived growth factor receptor alpha (PDGFRα) antibody, presented binding affinity to lipopolysaccharide (LPS), a commonly used stimulator in a neuroinflammatory model. Using cytokine antibody array, QPCR and ELISA, we have further shown that LPS could upregulate the expression of cytokine induced neutrophil chemoattractant-3 (CINC-3) and LPS induced CXC chemokine (LIX) in primary NG2-expressing OPCs, without the alteration in cell number of NG2-expressing OPCs. In addition, the cells bearing the receptor for these two cytokines included microglia and OPCs. Taken together, our results suggest that NG2-expressing OPCs could response to LPS and may take part in neuroinflammatory process, through secreting cytokines and chemokines to exert an effect on target cells (OPCs and microglia).

Cigarette smoke primes the pulmonary environment to IL-1α/CXCR-2-dependent nontypeable Haemophilus influenzae-exacerbated neutrophilia in mice.

Cigarette smoke has a broad impact on the mucosal environment with the ability to alter host defense mechanisms. Within the context of a bacterial infection, this altered host response is often accompanied by exacerbated cellular inflammation, characterized by increased neutrophilia. The current study investigated the mechanisms of neutrophil recruitment in a murine model of cigarette smoke exposure and, subsequently, a model of both cigarette smoke exposure and bacterial infection. We investigated the role of IL-1 signaling in neutrophil recruitment and found that cigarette smoke-induced neutrophilia was dependent on IL-1α produced by alveolar macrophages. In addition to being the crucial source of IL-1α, alveolar macrophages isolated from smoke-exposed mice were primed for excessive IL-1α production in response to bacterial ligands. To test the relevance of exaggerated IL-1α production in neutrophil recruitment, a model of cigarette smoke exposure and nontypeable Haemophilus influenzae infection was developed. Mice exposed to cigarette smoke elaborated an exacerbated CXCR2-dependent neutrophilia in response to nontypeable Haemophilus influenzae. Exacerbated neutrophilia was dependent on IL-1α priming of the pulmonary environment by cigarette smoke as exaggerated neutrophilia was dependent on IL-1 signaling. These data characterize a novel mechanism of cigarette smoke priming the lung mucosa toward greater IL-1-driven neutrophilic responses to bacteria, with a central role for the alveolar macrophage in this process.

Common and specific signatures of gene expression and protein-protein interactions in autoimmune diseases.

The aim of this study is to understand intracellular regulatory mechanisms in peripheral blood mononuclear cells (PBMCs), which are either common to many autoimmune diseases or specific to some of them. We incorporated large-scale data such as protein-protein interactions, gene expression and demographical information of hundreds of patients and healthy subjects, related to six autoimmune diseases with available large-scale gene expression measurements: multiple sclerosis (MS), systemic lupus erythematosus (SLE), juvenile rheumatoid arthritis (JRA), Crohn's disease (CD), ulcerative colitis (UC) and type 1 diabetes (T1D). These data were analyzed concurrently by statistical and systems biology approaches tailored for this purpose. We found that chemokines such as CXCL1-3, 5, 6 and the interleukin (IL) IL8 tend to be differentially expressed in PBMCs of patients with the analyzed autoimmune diseases. In addition, the anti-apoptotic gene BCL3, interferon-γ (IFNG), and the vitamin D receptor (VDR) gene physically interact with significantly many genes that tend to be differentially expressed in PBMCs of patients with the analyzed autoimmune diseases. In general, similar cellular processes tend to be differentially expressed in PBMC in the analyzed autoimmune diseases. Specifically, the cellular processes related to cell proliferation (for example, epidermal growth factor, platelet-derived growth factor, nuclear factor-κB, Wnt/ß-catenin signaling, stress-activated protein kinase c-Jun NH2-terminal kinase), inflammatory response (for example, interleukins IL2 and IL6, the cytokine granulocyte-macrophage colony-stimulating factor and the B-cell receptor), general signaling cascades (for example, mitogen-activated protein kinase, extracellular signal-regulated kinase, p38 and TRK) and apoptosis are activated in most of the analyzed autoimmune diseases. However, our results suggest that in each of the analyzed diseases, apoptosis and chemotaxis are activated via different subsignaling pathways. Analyses of the expression levels of dozens of genes and the protein-protein interactions among them demonstrated that CD and UC have relatively similar gene expression signatures, whereas the gene expression signatures of T1D and JRA relatively differ from the signatures of the other autoimmune diseases. These diseases are the only ones activated via the Fcɛ pathway. The relevant genes and pathways reported in this study are discussed at length, and may be helpful in the diagnoses and understanding of autoimmunity and/or specific autoimmune diseases.

IL-17A and TNF-α exert synergistic effects on expression of CXCL5 by alveolar type II cells in vivo and in vitro.

CXCL5, a member of the CXC family of chemokines, contributes to neutrophil recruitment during lung inflammation, but its regulation is poorly understood. Because the T cell-derived cytokine IL-17A enhances host defense by triggering production of chemokines, particularly in combination with TNF-α, we hypothesized that IL-17A would enhance TNF-α-induced expression of CXCL5. Intratracheal coadministration of IL-17A and TNF-α in mice induced production of CXCL1, CXCL2, and CXCL5, which was associated with increased neutrophil influx in the lung at 8 and 24 h. The synergistic effects of TNF-α and IL17A were greatly attenuated in Cxcl5(-/-) mice at 24 h, but not 8 h, after exposure, a time when CXCL5 expression was at its peak in wild-type mice. Bone marrow chimeras produced using Cxcl5(-/-) donors and recipients demonstrated that lung-resident cells were the source of CXCL5. Using differentiated alveolar epithelial type II (ATII) cells derived from human fetal lung, we found that IL-17A enhanced TNF-α-induced CXCL5 transcription and stabilized TNF-α-induced CXCL5 transcripts. Whereas expression of CXCL5 required activation of NF-κB, IL-17A did not increase TNF-α-induced NF-κB activation. Apical costimulation of IL-17A and TNF-α provoked apical secretion of CXCL5 by human ATII cells in a transwell system, whereas basolateral costimulation led to both apical and basolateral secretion of CXCL5. The observation that human ATII cells secrete CXCL5 in a polarized fashion may represent a mechanism to recruit neutrophils in host defense in a fashion that discriminates the site of initial injury.

CXCL1 regulates pulmonary host defense to Klebsiella Infection via CXCL2, CXCL5, NF-kappaB, and MAPKs.

Pulmonary bacterial infections are a leading cause of death. Since the introduction of antibiotics, multidrug-resistant Klebsiella pneumoniae became an escalating threat. Therefore, development of methods to augment antibacterial defense is warranted. Neutrophil recruitment is critical to clear bacteria, and neutrophil migration in the lung requires the production of ELR(+) CXC chemokines. Although lung-specific CXCL1/keratinocyte cell-derived chemokine (KC) transgene expression causes neutrophil-mediated clearance of K. pneumoniae, the mechanisms underlying KC-mediated host defense against K. pneumoniae have not been explored. In this study, we delineated the host defense functions of KC during pulmonary K. pneumoniae infection using KC(-/-) mice. Our findings demonstrate that KC is important for expression of CXCL2/MIP-2 and CXCL5/LPS-induced CXC chemokine, and activation of NF-κB and MAPKs in the lung. Furthermore, KC derived from both hematopoietic and resident cells contributes to host defense against K. pneumoniae. Neutrophil depletion in mice before K. pneumoniae infection reveals no differences in the production of MIP-2 and LPS-induced CXC chemokine or activation of NF-κB and MAPKs in the lung. Using murine bone marrow-derived and alveolar macrophages, we confirmed KC-mediated upregulation of MIP-2 and activation of NF-κB and MAPKs on K. pneumoniae infection. Moreover, neutralizing KC in bone marrow-derived macrophages before K. pneumoniae challenge decreases bacteria-induced production of KC and MIP-2, and activation of NF-κB and MAPKs. These findings reveal the importance of KC produced by hematopoietic and resident cells in regulating pulmonary host defense against a bacterial pathogen via the activation of transcription factors and MAPKs, as well as the expression of cell adhesion molecules and other neutrophil chemoattractants.

Modulatory effect of fenofibrate on endothelial production of neutrophil chemokines IL-8 and ENA-78.

PURPOSE: The PPAR-alpha agonists (fibrates) are commonly used in the treatment of dyslipidemia. It has been hypothesized that the cardio-protective effects of fibrates are partially due to immunomodulatory effects. However, there is a paucity of data regarding the effect of fibrates on neutrophilic chemokines such as epithelial neutrophil activating protein (ENA-78) and interleukin (IL)-8. We investigated the influence of fenofibrate on IL-1ß-stimulated production of ENA-78 and IL-8 from human endothelial cells (HUVECs). METHODS: HUVECs were cultured in the presence or absence of IL-1ß and fenofibrate ranging from 1-50 uM. ENA-78 and IL-8 were measured and normalized to total protein content in cell culture supernates by multiplex immunofluorescence detection. Experimental samples were measured in triplicate. Significance was set at P < 0.05 by ANOVA with correction for multiple comparisons. RESULTS: Endothelial production of both ENA-78 and IL-8 was induced by the proinflammatory cytokine IL-1ß. ENA-78 concentrations increased by more than 160-fold over constitutively produced ENA-78 upon IL-1ß stimulation (mean ± SEM: 10,129 ± 1591 pg/mg vs. 61 ± 9.5 mg/mg; P < 0.0001). IL-8 concentrations increased by slightly over 5-fold (6145 ± 860 pg/mg vs. 1160 ± 201 pg/mg; P = 0.0003). ENA-78 protein and mRNA were significantly reduced by fenofibrate while no drug effects were observed on IL-8 production. CONCLUSIONS: Fenofibrate blunts IL-1ß-mediated ENA-78 production with no effect on IL-8. This represents a novel mechanism by which fenofibrate exerts anti-inflammatory effects and should be further explored.

Reduced expression of lipopolysaccharide-induced CXC chemokine in Porphyromonas gingivalis-induced experimental periodontitis in matrix metalloproteinase-8 null mice.

BACKGROUND AND OBJECTIVE: Matrix metalloproteinase-8 (MMP-8) is a central mediator in chronic periodontitis. Recently developed MMP-8-deficient mice show an impaired polymorphonuclear neutrophil response and more severe alveolar bone loss in Porphyromonas gingivalis-induced experimental periodontitis. The main mediators involved in neutrophil and monocyte/macrophage recruitment and in bone loss include lipopolysaccharide-induced CXC chemokine (LIX/CXCL5), stromal-derived factor-1/CXC chemokine ligand 12 (SDF1/CXCL12) and RANKL. Therefore, the aim of this study was to characterize the expression of LIX/CXCL5, SDF1/CXCL12 and RANKL in Porphyromonas gingivalis-induced experimental periodontitis in MMP-8⁻/⁻ (knockout) and wild-type mice. MATERIAL AND METHODS: MMP-8 null and WT P. gingivalis-infected and uninfected mice were included. Histopathological changes were assessed and LIX/CXCL5, SDF1/CXCL12 and RANKL were immunodetected and quantified. RESULTS: Typical histopathological features of chronic periodontitis were seen in P. gingivalis-infected groups. LIX/CXCL5 expression was restricted to the gingival papilla in all four groups. Significantly lower expression of LIX/CXCL5 was seen in the knockout group compared with the wild-type infected group (p < 0.05). SDF1/CXCL12 and RANKL expression was mainly localized to the alveolar crest, including inflammatory leukocytes, vascular endothelium, osteoblasts and osteoclasts. Significant increases of SDF1/CXCL12 and RANKL were seen in both knockout and wild-type P. gingivalis-infected groups compared with uninfected groups (p < 0.05). CONCLUSION: RANKL and SDF1/CXCL12 are up-regulated in P. gingivalis-induced periodontitis and they appear to be associated with the pathogenesis of the disease. MMP-8 is associated with a reduced expression of LIX/CXCL5 in the P. gingivalis-induced experimental periodontitis model.

CXCL5/CXCR2 modulates inflammation-mediated neural repair after optic nerve injury.

BACKGROUND: Previous studies reported that mild inflammation promotes retinal ganglion cell (RGC) survival and axonal regeneration after optic nerve (ON) injury with involvement of infiltrating macrophages and neutrophils. Here we aimed to evaluate the involvement and regulation of the main inflammatory chemokine pathway CXCL5/CXCR2 in the inflammation-mediated RGC survival and axonal regeneration in mice after ON injury. METHODS: The expressions and cellular locations of CXCL5 and CXCR2 were confirmed in mouse retina. Treatment effects of recombinant CXCL5 and CXCR2 antagonist SB225002 were studied in the explant culture and the ON injury model with or without lens injury. The number of RGCs, regenerating axons, and inflammatory cells were determined, and the activation of Akt andSTAT3 signaling pathways were evaluated. RESULTS: Cxcr2 and Cxcl5 expressions were increased after ON and lens injury. Addition of recombinant CXCL5 promoted RGC survival and neurite outgrowth in retinal explant culture with increase in the number of activated microglia, which was inhibited by SB225002 or clodronate liposomes. Recombinant CXCL5 also alleviated RGC death and promoted axonal regeneration in mice after ON injury, and promoted the lens injury-induced RGC protection with increase in the number of activated CD68+ cells. SB225002 inhibited lens injury-induced cell infiltration and activation, and attenuated the promotion effect on RGC survival and axonal regeneration through reduction of lens injury-induced Akt activation. CONCLUSIONS: CXCL5 promotes RGC survival and axonal regeneration after ON injury and further enhances RGC protection induced by lens injury with CD68+ cell activation, which is attenuated by CXCR2 antagonist. CXCL5/CXCR2 could be a potential therapeutic target for RGC survival promotion after ON injury.

PAR1- and PAR2-induced innate immune markers are negatively regulated by PI3K/Akt signaling pathway in oral keratinocytes.

BACKGROUND: Protease-Activated Receptors (PARs), members of G-protein-coupled receptors, are activated by proteolytic activity of various proteases. Activation of PAR1 and PAR2 triggers innate immune responses in human oral keratinocytes (HOKs), but the signaling pathways downstream of PAR activation in HOKs have not been clearly defined. In this study, we aimed to determine if PAR1- and PAR2-mediated signaling differs in the induction of innate immune markers CXCL3, CXCL5 and CCL20 via ERK, p38 and PI3K/Akt. RESULTS: Our data show the induction of innate immunity by PAR1 requires both p38 and ERK MAP kinases, while PAR2 prominently signals via p38. However, inhibition of PI3K enhances expression of innate immune markers predominantly via suppressing p38 phosphorylation signaled by PAR activation. CONCLUSION: Our data indicate that proteases mediating PAR1 and PAR2 activation differentially signal via MAP kinase cascades. In addition, the production of chemokines induced by PAR1 and PAR2 is suppressed by PI3K/Akt, thus keeping the innate immune responses of HOK in balance. The results of our study provide a novel insight into signaling pathways involved in PAR activation.

Time-dependent mRNA expression of selected pro-inflammatory factors in the endometrium of primiparous cows postpartum.

BACKGROUND: Inflammatory processes and infections of the uterine wall must be accepted as a physiological event in dairy cows after calving. This might result in clinical or subclinical endometritis which is assumed to impair reproductive performance in the current lactation. Several cytokines and acute phase proteins have been discussed as local and systemic mediators of these inflammatory processes. The aim of the present study was to investigate the endometrial mRNA expression of the chemokine CXC ligand 5 (CXCL5), interleukin 1ß (IL1B), IL6, IL8, tumour necrosis factor alpha (TNF), prostaglandin-endoperoxide synthase 2 (PTGS2) and haptoglobin (HP) in the postpartum period. METHODS: Endometrial samples were obtained from primiparous cows (n = 5) on days 10, 17, 24, 31, 38 and 45 postpartum (pp) using the cytobrush technique. Cytological smears were prepared from cytobrush samples to determine the proportion of polymorphonuclear neutrophils (PMN). Total RNA was extracted from endometrial samples, and real-time RT-PCR was performed. RESULTS: A time-dependent mRNA expression of the investigated factors was found for the course of the postpartum period. In detail, a significantly higher expression of these factors was observed on day 17 pp compared to day 31 pp. Furthermore, the proportion of PMN peaked between days 10-24 pp and decreased thereafter to low percentages (< 5%) on day 31 pp and thereafter. In addition, CXCL5, IL1B, IL8 and HP mRNA expression correlated significantly with the proportion of PMN (P < 0.05). A significantly higher CXCL5, IL1B, IL6, IL8, PTGS2 and TNF mRNA content was observed in samples from cows with an inflamed endometrium compared with samples from cows with a healthy endometrium (P < 0.05). CONCLUSIONS: These results show that inflammatory cytokines and acute phase proteins are expressed in the bovine endometrium in a time-related manner during the postpartum period, with a significant expression peak on day 17 pp as a possible mucosal immune response in the uterus. The evaluation of the expression patterns of such candidate genes may reveal more information than only determining the percentage of PMN to judge the severity of an inflammation.

Inhibition of IL-1beta-mediated inflammatory responses by the IkappaB alpha super-repressor in human fibroblast-like synoviocytes.

The IL-1beta-NF-kappaB axis is a key pathway in the pathogenesis of rheumatoid arthritis (RA) and is central in the production of proinflammatory mediators in the inflamed synovium. Therefore, we examined whether fibroblast-like synoviocytes (FLS) could be spared from IL-1beta-induced toxicity by an overexpressing IkappaB super-repressor. Infection of FLS with Ad-IkappaB alpha (S32A, S36A), an adenovirus-containing mutant IkappaB alpha, inhibited IL-1beta-induced nuclear translocation and DNA binding of NF-kappaB. In addition, Ad-IkappaB alpha (S32A, S36A) prevented IL-1beta-induced inflammatory responses; namely, the production of chemokines, such as ENA-78 and RANTES, and activation of MMP-1 and MMP-3. Finally, increased cellular proliferation of FLS after IL-1beta treatment was significantly reduced by Ad-IkappaB alpha (S32A, S36A). However, Ad-IkappaB beta (S19A, S23A), the IkappaB beta mutant, was not effective in preventing IL-1beta toxicity. These results suggest that inhibition of IkappaB alpha degradation is a potential target for the prevention of joint destruction in patients with RA.

Interleukin-17 causes neutrophil mediated inflammation in ovalbumin-induced uveitis in DO11.10 mice.

T cell-mediated uveitis is strongly associated with many systemic inflammatory disorders. Th17 cells are a novel T cell subset characterized by production of interleukin (IL)-17. In this study, we used DO11.10 mice to investigate the role of IL-17 in the pathogenesis of uveitis. CD4(+) T cells in DO11.10 mice are genetically engineered to react with ovalbumin (OVA). IL-17 expression was determined by real-time PCR and ELISPOT. Uveitis was induced by intravitreal injection of OVA, and ocular inflammation was evaluated by intravital microscopy. OVA challenge significantly induced IL-17 production by DO11.10 splenocytes in vitro. Next, we examined whether OVA challenge could elicit local inflammation and induce IL-17 in vivo. OVA elicited marked neutrophil-predominant inflammatory cell infiltration in the eyes. This leukocyte influx was mediated by CD4(+) lymphocytes as evidenced by significant inhibition of the ocular inflammation by CD4+ depleting antibody. Compared to control mice, OVA treatment induced IL-17 expression. Moreover, anti-IL-17 antibody markedly reduced OVA-mediated ocular inflammation. Finally, the neutralization of IL-17 attenuated ocular expression of CXCL2 and CXCL5, two cytokines which are chemotactic for neutrophils. Our study suggests that IL-17 is implicated in the pathogenesis of this T cell-mediated model of uveitis in part through neutrophil chemotaxis as a downstream effect of IL-17.

Ethanol exposure impairs LPS-induced pulmonary LIX expression: alveolar epithelial cell dysfunction as a consequence of acute intoxication.

BACKGROUND: Alcohol intoxication impairs innate immune responses to bacterial pneumonia, including neutrophil influx. Lipopolysaccharide (LPS)-induced chemokine (LIX or CXCL5) is a recently described chemokine produced by type-II alveolar epithelial (AE2) cells which facilitates neutrophil recruitment. The effect of acute alcohol intoxication on AE2 cell expression of LIX is unknown. METHODS: C57BL/6 mice were given an intraperitoneal (i.p.) injection of ethanol (4 g/kg) or saline 30 minutes prior to intratracheal (i.t.) injection with 10 mug Escherichia coli LPS. In vitro stimulation of primary AE2 cells or murine AE2 cell line MLE-12 was performed with LPS and tumor necrosis factor-alpha (TNF-alpha). RESULTS: LIX protein is readily detectable in the lung but not in plasma following LPS administration, demonstrating "compartmentalization" of this chemokine during pulmonary challenge. In contrast to the CXC chemokines keratinocyte-derived chemokine and macrophage inflammatory protein-2, which are abundantly expressed in both lung tissue and alveolar macrophages, LIX expression is largely confined to the lung parenchyma. Compared to controls, intoxicated animals show a decrease in LIX and neutrophil number in bronchoalveolar lavage fluid following LPS challenge. Ethanol inhibits LIX at the transcriptional level. In vitro studies show that LPS and TNF-alpha are synergistic in inducing LIX by either primary AE2 or MLE-12 cells. Acute ethanol exposure potently and dose-dependently inhibits LIX expression by AE2 cells. Activation of nuclear factor-kappaB is critical to LIX expression in MLE-12 cells, and acute ethanol treatment interferes with early activation of this pathway as evidenced by impairing phosphorylation of p65 (RelA). Inhibition of p38 mitogen-activated protein kinase signaling, but not ERK1/2 activity, in MLE-12 cells by acute alcohol is likely an important cause of decreased LIX expression during challenge. CONCLUSIONS: These data demonstrate direct suppression of AE2 cell innate immune function by ethanol and add to our understanding of the mechanisms by which acute intoxication impairs the lung's response to microbial challenge.

CD44v6, MMP-7 and nuclear Cdx2 are significant biomarkers for prediction of lymph node metastasis in primary gastric cancer.

The aim of this study was to examine the expression of CD44v6, CD54, Cdx2, CXCL5, Cyclin B1, MMP-7, nm23, RCAS1 and Survivin in primary gastric cancer and to investigate whether these molecules were useful in predicting the lymph node status. They were selected as candidates for indicators of lymph node metastasis from various kinds of cancer-associated genes reported previously. In 135 cases of radically resected primary gastric adenocarcinoma, we investigated the association between the expression of these molecules and clinocopathologic factors by immunohistochemistry. The results revealed that the expression of CD44v6 and MMP-7 were significantly associated with lymph node status. By contrast, nuclear Cdx2 expression was found to be inversely correlated with lymph node metastasis. Moreover, multivariate analysis demonstrated that CD44v6, MMP-7 and nuclear Cdx2 were independent predictors for lymph node status. In conclusion, our results suggest that positive expression of both CD44v6 and MMP-7, and negative expression of nuclear Cdx2 may serve as powerful predictors of lymph node metastasis in gastric cancer. Combined evaluation of these markers could be further useful to predict lymph node status clinically.

CXCL5 as Regulator of Neutrophil Function in Cutaneous Melanoma.

Chemokines mold the tumor microenvironment by recruiting distinct immune cell populations, thereby strongly influencing disease progression. Previously, we showed that CXCL5 expression is upregulated in advanced stages of primary melanomas, which correlates with the presence of neutrophils in the tumor. The analysis of neutrophil populations in various tissues revealed a distinct phenotype of tumor-associated neutrophils. Tumor-associated neutrophils expressed PD-L1, CXCR4, CCR5, Adam17, and Nos2 and were immunosuppressive in a T-cell proliferation assay. To investigate the impact of CXCL5 and neutrophils in vivo, we established a syngeneic mouse tumor transplantation model using CXCL5-overexpressing and control melanoma cell lines. Growth behavior or vascularization of primary tumors was not affected by CXCL5 expression and neutrophils alone. However, in combination with Poly(I:C), tumor-associated neutrophils were able to attenuate induced antitumoral T-cell responses. CXCL5-overexpressing tumors had reduced lung metastasis compared with control tumors. Neutrophil depletion reversed this effect. In vitro, unstimulated lung-derived neutrophils had higher levels of reactive oxygen species compared with tumor-associated neutrophils, and CXCL5 stimulation further increased reactive oxygen species levels. In summary, in melanoma, neutrophils play a context-dependent role that is influenced by local or systemic factors, and interfere with therapies activating the acquired immune system. Actively switching neutrophils into antitumorigenic mode might be a new therapeutic strategy.

ELR+ CXC chemokine expression in benign and malignant colorectal conditions.

BACKGROUND: CXCR2 chemokine ligands CXCL1, CXCL5 and CXCL6 were shown to be involved in chemoattraction, inflammatory responses, tumor growth and angiogenesis. Here, we comparatively analyzed their expression profile in resection specimens from patients with colorectal adenoma (CRA) (n = 30) as well as colorectal carcinoma (CRC) (n = 48) and corresponding colorectal liver metastases (CRLM) (n = 16). METHODS: Chemokine expression was assessed by microdissection, quantitative real-time PCR (Q-RT-PCR), the enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC). RESULTS: In contrast to CXCL6, we demonstrated CXCL1 and CXCL5 mRNA and protein expression to be significantly up-regulated in CRC and CRLM tissue specimens in relation to their matched tumor neighbor tissues. Moreover, both chemokine ligands were demonstrated to be significantly higher expressed in CRC tissues than in CRA tissues thus indicating a progressive increase in the transition from the premalignant condition to the development of the malignant status. Although a comparative analysis of the CXCL1/CXCL5 protein expression profiles in CRC patients revealed that the absolute expression level of CXCL1 was significantly higher in comparison to CXCL5, mRNA- and protein overexpression of CXCL5 in CRC and CRLM tissues was much more pronounced (80- and 60- fold in CRC tissues, respectively) in comparison to CXCL1 (5- and 3.5- fold in CRC tissues, respectively). CONCLUSION: Our results demonstrate a significant association between CXCL1 and CXCL5 expression with CRC and CRLM suggesting for both chemokine ligands a potential role in the progression from CRA to CRC and thus, in the initiation of CRC.