Substance P (SP) is a neuropeptide with neuroimmunoregulatory activity that may play a role in susceptibility to infection. Human mast cells, which are important in innate immune responses, were analysed for their responses to pathogen-associated molecules via Toll-like receptors (TLRs) in the presence of SP. Human cultured mast cells (LAD2) were activated by SP and TLR ligands including lipopolysaccharide (LPS), Pam3CysSerLys4 (Pam3CSK4) and lipoteichoic acid (LTA), and mast cell leukotriene and chemokine production was assessed by enzyme-linked immunosorbent assay (ELISA) and gene expression by quantitative PCR (qPCR). Mast cell degranulation was determined using a β-hexosaminidase (β-hex) assay. SP treatment of LAD2 up-regulated mRNA for TLR2, TLR4, TLR8 and TLR9 while anti-immunoglobulin E (IgE) stimulation up-regulated expression of TLR4 only. Flow cytometry and western blot confirmed up-regulation of TLR2 and TLR8. Pretreatment of LAD2 with SP followed by stimulation with Pam3CSK4 or LTA increased production of leukotriene C4 (LTC(4) ) and interleukin (IL)-8 compared with treatment with Pam3CSK4 or LTA alone (>2-fold; P<0·01). SP alone activated 5-lipoxygenase (5-LO) nuclear translocation but also augmented Pam3CSK4 and LTA-mediated 5-LO translocation. Pam3CSK4, LPS and LTA did not induce LAD2 degranulation. SP primed LTA and Pam3CSK4-mediated activation of JNK, p38 and extracellular-signal-regulated kinase (ERK) and activated the nuclear translocation of c-Jun, nuclear factor (NF)-κB, activating transcription factor 2 (ATF-2) and cyclic-AMP-responsive element binding protein (CREB) transcription factors. Pretreatment with SP followed by LTA stimulation synergistically induced production of chemokine (C-X-C motif) ligand 8 (CXCL8)/IL-8, chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemotactic protein 1 (MCP-1), tumour necrosis factor (TNF) and IL-6 protein. SP primes TLR2-mediated activation of human mast cells by up-regulating TLR expression and potentiating signalling pathways associated with TLR. These results suggest that neuronal responses may influence innate host defence responses.

BACKGROUND

Systemic adiponectin is reduced in patients with cardiovascular disease (CVD) and low adiponectin may contribute to the pathogenesis of atherosclerosis. However, circulating adiponectin is elevated in type 1 diabetes (T1D) patients, who have also a higher incidence to develop CVD. Because monocytes play an important role in atherosclerosis, we analysed the influence of adiponectin on cytokine and chemokine release in monocytes from T1D patients and controls.

METHODS

Systemic adiponectin was determined in the plasma and the high-molecular weight (HMW) form of adiponectin was analysed by immunoblot. Monocytes were isolated from T1D patients and controls and the adiponectin-stimulated release of interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1, CCL2) and interleukin-8 (IL-8, CXCL8) was analysed.

RESULTS

Systemic adiponectin was higher in T1D patients. Immunoblot analysis of the plasma indicate abundance of HMW adiponectin in T1D patients and controls. IL-6, CCL2 and CXCL8 secretion in response to adiponectin were found induced in monocytes from controls whereas only IL-6 was upregulated in T1D cells. The induction of IL-6 by adiponectin was abrogated by an inhibitor of the NFkappaB pathway.

CONCLUSIONS

These data indicate that adiponectin-mediated induction of IL-6, CCL2 and CXCL8 is disturbed in monocytes from T1D patients and therefore elevated systemic adiponectin in T1D patients may be less protective when compared to controls.

CXCL8 is a CXC chemokine that recruits leukocytes to sites of inflammation. Expression of CXCL8 in the CNS has been demonstrated in neuroinflammatory diseases, including human immunodeficiency virus (HIV-1) encephalitis, but the mechanism of secretion of this chemokine is not fully understood. CD40 is a 50-kDa protein on the surface of microglia, and we have previously shown that it is increased in expression in HIV-1-infected brain tissue as well as by interferon-gamma (IFNgamma) in tissue culture. We examined the expression and regulation of CXCL8 in cultured human fetal microglia after ligation of CD40 with soluble trimeric CD40 ligand (sCD40L) as well as the expression of CXCL8 on microglia in HIV encephalitic brain tissue sections. Treatment of cultured microglia with IFNgamma + sCD40L resulted in significant induction of CXCL8. This expression was mediated by activation of the ERK1/2 MAPK pathway, as demonstrated by ELISA and Western blot using a specific inhibitor (U0126). Gel shift analyses demonstrated that NFkappaB and AP-1, but not C/EBPbeta, mediate microglial CXCL8 production. We also found increased colocalization of CXCL8 with CD68/CD40-positive cells in HIV encephalitic brain tissue compared with HIV-infected nonencephalitic and normal tissue. Thus, CD40-CD40L interactions facilitate chemokine expression, leading to the influx of inflammatory cells into the CNS. These events can lead to the pathology that is associated with neuroinflammatory diseases.

Although homo- and heterodimerization are reported for some chemokine receptors, it remains unclear whether these functional states are in dynamic equilibrium and how receptor/ligand levels influence oligomerization. In human neutrophils and in cell lines that coexpress the chemokine receptors CXCR1 and CXCR2, we used fluorescence resonance energy transfer techniques to show that these two receptors form homo- and heterodimers. Receptor expression and ligand activation were found to regulate the balance between these complexes, adapting the response to changes in the milieu. CXCL8, a ligand for both receptors, alters heterodimeric complexes, whereas it stabilizes homodimers and promotes receptor internalization. Oligomerization of receptors, together with the regulation of their expression and desensitization, could thus contribute to the fine control of chemokine functions.

BACKGROUND

Neutrophilic airway inflammation is considered to be a major factor in the pathogenesis of chronic obstructive pulmonary disease (COPD), with sputum and bronchoalveolar lavage neutrophil counts broadly correlating with disease severity. The mechanisms responsible for neutrophil accumulation are poorly understood, but they could involve increased influx and/or survival of these cells.

OBJECTIVE

To investigate whether neutrophil chemotactic responsiveness and/or chemotactic activity in airway secretions are increased in subjects with COPD.

METHODS

Chemotaxis experiments were performed using induced sputum supernatants from subjects with and without COPD as a source of chemotactic activity, and neutrophils from healthy donors as responder cells. In addition, chemotactic responses to N-formyl-Met-Leu-Phe (fMLP) and interleukin-8 (IL-8/CXCL8) were studied using neutrophils from healthy subjects and subjects with COPD.

RESULTS

As reported in the literature, sputum neutrophil counts were significantly increased in subjects with COPD compared with healthy subjects. However, this was associated with reduced chemotactic activity in sputum in COPD, as judged by reduced chemotaxis to the fluid phase of sputum from subjects with COPD compared with healthy subjects. Furthermore, whereas neutrophils from subjects with stage I COPD had normal responses to fMLP and IL-8, subjects with more severe stage II-IV COPD showed reduced levels of spontaneous migration and chemotaxis to fMLP and IL-8.

CONCLUSIONS

Neither increased chemotactic activity in the airways nor increased chemotactic responsiveness of neutrophils explains the increased number of these cells in subjects with stable COPD. The implications of the observed reduction in neutrophil chemotactic activity remain to be established.

Chemokines critically regulate basal and inflammatory leukocyte trafficking and may play a role in angiogenesis. This review summarizes our current understanding of the regulation and potential role of the chemokines in myocardial ischemia and reperfusion. Reperfused myocardial infarction is associated with an inflammatory response leading to leukocyte recruitment, healing and scar formation. Neutrophil chemoattractants, such as the CXC chemokine CXCL8/Interleukin (IL)-8, are upregulated in the infarcted area inducing polymorphonuclear leukocyte infiltration. In addition, mononuclear cell chemoattractants, such as the CC chemokine CCL2/Monocyte Chemoattractant Protein (MCP)-1, are expressed, leading to monocyte and lymphocyte recruitment in the ischemic area. However, chemokines may have additional effects in healing infarcts beyond their leukotactic properties. We have recently described a marked transient induction of the angiostatic CXC chemokine CXCL10/Interferon-gamma inducible Protein (IP)-10 in the infarct. Upregulation of angiostatic factors, such as IP-10, in the first few hours following injury may inhibit premature angiogenesis, until the infarct is debrided and appropriate supportive matrix is formed. Suppression of IP-10 synthesis during the healing phase may allow formation of the wound neovessels, a critical process for infarct healing. Chemokine expression is also noted after a single brief ischemic insult in the absence of myocardial infarction, suggesting a potential role for a chemokine-induced inflammatory response in noninfarctive ischemic cardiomyopathy. Unlike cytokines, which have pleiotropic effects, chemokines have more specific cellular targets. Understanding of their role in myocardial infarction may allow us to design specific therapeutic strategies aiming at optimizing cardiac repair and preventing ventricular remodeling.

Chemokines are mediators of innate and acquired immunity. CCL18, also designated pulmonary and activation-regulated chemokine (PARC), dendritic cell-derived CC chemokine-1 (DC-CK1), alternative macrophage activation-associated CC chemokine-1 (AMAC-1) and macrophage inflammatory protein-4 (MIP-4), was for the first time isolated from peripheral blood mononuclear cells (PBMC) and biochemically characterized. We found that CCL18/PARC protein is spontaneously secreted by PBMC and is selectively induced in PBMC by staphylococcal enterotoxins (SEA, SEB) and IL-4, but not by IFN-gamma and the CXCL8/IL-8 inducers lipopolysaccharide (LPS) or Concanavalin A. Human fibroblasts, chondrocytes and endothelial cells did not produce CCL18/PARC in response to inflammatory mediators such as measles virus, double-stranded RNA, LPS or IL-1beta, whereas up to 150 ng/ml of CCL2/MCP-1 was induced under these conditions. In synovial fluids from septic and rheumatoid arthritis patients, fourfold-enhanced CCL18/PARC levels (150 ng/ml) were detected compared to those in crystal-induced arthritis and osteoarthritis. In septic arthritis, the synovial levels of CCL18/PARC were fivefold higher than those of CXCL8/IL-8. Immunochemistry revealed CD68(+) monocytes/macrophages as the main CCL18/PARC-producing cell type in both PBMC and arthritic synovial tissue. In addition, CD1a(+) blood dendritic cells expressed CCL18/PARC. These findings suggest that monocytic cells respond to Gram-positive bacterial infection by the production of CCL18/PARC in the synovial cavity.

Periodontitis is a chronic inflammatory disease characterized by destruction of periodontal tissue ultimately leading to bone destruction and has been associated with other inflammatory diseases, such as atherosclerosis. Attachment loss of periodontal tissue is primarily caused by host cell-derived immune responses against subgingival biofilm. The aim of the present study was to determine the cytokine profile in serum, saliva and gingival crevicular fluid (GCF) patients with periodontitis and healthy controls. We show that periodontitis patients exhibit higher numbers of periodontal pathogens and their immune responses are significantly altered. The levels of IL-6 in saliva and GCF were significantly suppressed, and while CXCL8 was not altered in serum, its expression levels were significantly suppressed in saliva and elevated in GCF. The T-cell-derived cytokine IL-2 did not differ between patients and controls in serum and saliva, but there was a significant suppression in GCF of patients. Interestingly, TGF-β1 levels were significantly elevated in serum, saliva and GCF in patients compared to controls. Furthermore, by using cultured gingival fibroblasts stimulated with wild type and proteinase mutant strains of Porphyromonas gingivalis, we show that the suppression of CXCL8 and IL-6, and the induction of TGF-β1 is primarily mediated by the proteolytic activity of lysine-specific proteinases. These results indicate that P. gingivalis is a major contributor to the altered immune responses and the pathology of periodontitis. Furthermore, the ease of sampling and analyzing cytokine expression profiles, including TGF-β1, in saliva and GCF may serve to predict the progression of periodontitis and associated systemic inflammatory diseases.

Given the role played by chemokines in the selective homing of immune cells, we sought to characterize the profile of chemokines produced by human dendritic cells (DC) following in vitro Aspergillus fumigatus infection and their ability to recruit cells involved in the antifungal defense. At the onset of A. fumigatus infection, DC released elevated amounts of CXCL8 that promote the migration of polymorphonuclear cells (PMN). Moreover, soluble factors released from A. fumigatus-infected DC increased also the surface expression of two activation markers, CD11b and CD18, on PMN. A. fumigatus infection resulted also in CCL3, CCL4, CXCL10 and CCL20 productions that induce the migration of effector memory Th1 cells. Moreover, the late expression of CCL19 suggests that A. fumigatus-infected DC could be implicated in the migration of CCR7+ naïve T cells and mature DC in lymph nodes. Together these results suggested the involvement of human DC in the regulation of innate and adaptive immunity against A. fumigatus through the recruitment of cells active in the fungal destruction.

The incidence of cancers that metastasize to the peritoneum increases with age. Intraperitoneal cancer dissemination depends largely on angiogenesis and interactions with the peritoneal mesothelium. We assessed the proangiogenic potential of human peritoneal mesothelial cells. Conditioned media collected from these cells at senescence stimulated proliferation of endothelial cells to a significantly greater extent compared to media from early-passage cells. The effect was accompanied by a significantly increased release of proangiogenic mediators -- VEGF, CXCL1/GROalpha, CXCL8/IL-8, and CCL2/MCP-1. These results indicate that the senescent mesothelium exhibits increased angiogenic activity, which may contribute to accelerated intraperitoneal cancer progression in the aged.

BACKGROUND

During the inflammatory process, chemokine CXCL8 plays a pivotal role in recruitment of human neutrophilic granulocytes. A diversity of sequences similar to CXCL8 was reported in fish, but their evolutionary relationships and functional homology with their human homolog remain unclear.

RESULTS

We screened fish genomes to seek for sequences related to CXCL8. A first lineage was retrieved in all teleosts, while a second CXCL8 lineage was found in zebrafish and carp only. An early inflammatory function for both lineages was indicated by several lines of evidence. The induction of carp CXCL8s, CXCb, and CXC receptor-1 and -2 was analyzed after in vitro stimulation of leukocyte subpopulations and in two in vivo inflammation models. Recombinant proteins of carp CXCL8 proteins were produced and showed significant chemotactic activity for carp leukocytes.

CONCLUSIONS

While both carp CXCL8s appear to be functional homologs of mammalian CXCL8, their different induction requirements and kinetics evoke a gene-specific sub-functionalization.

Macrophages produce nitric oxide (NO) via the inducible nitric oxide synthase as part of a successful response to infection. The gene norB of Neisseria meningitidis encodes a NO reductase which enables utilization and consumption of NO during microaerobic respiration and confers resistance to nitrosative stress-related killing by human monocyte-derived macrophages (MDM). In this study we confirmed that NO regulates cytokine and chemokine release by resting MDM: accumulation of TNF-alpha, IL-12, IL-10, CCL5 (RANTES) and CXCL8 (IL-8) in MDM supernatants was significantly modified by the NO-donor S-nitroso-N-penicillamine (SNAP). Using a protein array, infection of MDM with N. meningitidis was shown to be associated with secretion of a wide range of cytokines and chemokines. To test whether NO metabolism by N. meningitidis modifies release of NO-regulated cytokines, we infected MDM with wild-type organisms and an isogenic norB strain. Resulting expression of the cytokines TNF-alpha and IL-12, and the chemokine CXCL8 was increased and production of the cytokine IL-10 and the chemokine CCL5 was decreased in norB-infected MDM, in comparison to wild-type. Addition of SNAP to cultures infected with wild-type mimicked the effect observed in cultures infected with the norB mutant. In conclusion, NorB-catalysed removal of NO modifies cellular release of NO-regulated cytokines and chemokines.

Human gammadelta T cells play a vital role in the innate and adaptive immune response to microbial antigens by acting as antigen-presenting cells while at the same time being capable of directly activating CD4(+) T cells. Pathogenic microbes or loss of tolerance toward the host's own microflora trigger many diseases including inflammatory bowel diseases. We previously demonstrated that Escherichia coli Nissle 1917 directly interacts with the adaptive immune system by regulating central T cell functions. Here we aimed to investigate whether E. coli Nissle regulates gammadelta T cell function, thereby linking the innate and adaptive immune system. In our study, we demonstrate that, in contrast to the other probiotic strains tested, E. coli Nissle increased activation, cell cycling and expansion of gammadelta, but not alphabeta T cells. In gammadelta T cells, E. coli Nissle reduced tumor necrosis factor-alpha secretion but increased IL-6 and CXCL8 release. However, after activation, only E. coli Nissle induced gammadelta T cell apoptosis, mediated via Toll-like receptor-2 by caspase- and FasLigand-dependent pathways. gammadelta T cells play an important role in the recognition of microbial antigens and the perpetuation of inflammatory processes. The demonstration that E. coli Nissle, but not the other bacteria tested, profoundly regulate gammadelta T cell function contributes to explaining the biological function of this probiotic strain in inflammatory diseases and provides us with a better understanding of the role of gammadelta T cells.

As the current staging system is imprecise for estimating prognosis of early stage non-small cell lung cancer (NSCLC), it is important to identify other methods for selecting high-risk patients after failed surgical treatment. The aim of the study was to evaluate the expression of 23 genes as putative prognostic markers in early stage NSCLC. The study was performed on 109 pairs of tumor and matched unaffected lung tissue surgical specimens taken from stage I and II NSCLC patients. We evaluated the mRNA level of 23 genes using the real-time PCR method. The difference in the expression between the tumor and normal tissue for each gene was analyzed using a general linear model. The influence of gene expression on survival was analyzed by using the proportional hazards model. Eighteen out of the 23 genes showed statistically significant differences in expression between the tumor and non-tumor tissue. For 12 genes (ITGB1, ITGB3, CXCL1, CXCL8, CXCL9, CXCL10, CXCL11, CXCR3, CXCR4, TNF, CHKA, AGFG1, and CTC1), the expression was lower, and for six genes (ITGA5, IL8, IL6, CXCL2, CXCL3, and CXCL12), it was higher in the tumor tissue as compared to the matched normal tissue. Expression changes were more pronounced in squamous cell carcinomas than in adenocarcinomas or large cell carcinomas. Of all the analyzed genes, only CXCL5 was found to statistically significantly (p = 0.04) influence both overall and disease-free survival. Among the 23 genes previously suggested to be relevant for early staged NSCLC patients' postoperative outcome, only CXCL5 showed a statistically significant prognostic effect.

The objective of this study was to examine the effects of sex hormones on IL-1beta-mediated responses by uterine epithelial cells. The mRNA expression and secretion of human beta-defensin-2 and CXCL8 by uterine epithelial cells was examined following stimulation with IL-1beta in the presence of estradiol or progesterone. Estradiol inhibited the IL-1beta-mediated mRNA expression and secretion of human beta-defensin-2 and CXCL8 by uterine epithelial cells while progesterone had no effect. Inhibition of the IL-1beta-mediated response by estradiol was dose dependent, with maximal inhibition observed using 10(-7) to 10(-10) M, and was shown to be mediated through the estrogen receptor because addition of a pure estrogen receptor antagonist abrogated this effect. The mechanism by which estradiol inhibits IL-1beta-mediated responses by uterine epithelial cells appears to be the down-modulation of the IL-1R type I, thereby reducing the uterine epithelial cell's ability to respond to IL-1beta. These results suggest that the inhibitory effect of estradiol on IL-1beta-mediated inflammatory responses by uterine epithelial cells indicates a link between the endocrine and immune systems and may be crucial for dampening proinflammatory responses during the time of ovulation or pregnancy.

The inability of epithelial cells from the cornea and other tissues to respond to LPS is reportedly due to low expression of the TLR4 co-receptor MD-2. We generated MD-2(-/-) bone marrow chimeras, and showed that MD-2 expression on non-myeloid cells was sufficient to mediate LPS-induced corneal inflammation. As IFN-γ is produced during Pseudomonas aeruginosa corneal infection, we examined the role of this cytokine on MD-2 expression by primary human corneal epithelial (HCE) cells and HCE cell lines. Exogenous IFN-γ was found to induce MD-2 mRNA, MD-2 cell surface expression, and LPS responsiveness as determined by p65 translocation to the nucleus and production of IL-6, CXCL1, and CXCL8/IL-8. Incubation with either the AG490 JAK2 inhibitor or with STAT1 siRNA blocked STAT1 phosphorylation and MD-2 transcription. Furthermore, EMSA analysis demonstrated that STAT1 binds to the MD-2 promoter, indicating that STAT1 is an MD-2 transcription factor. Together, these findings demonstrate that IFN-γ induces MD-2 expression and LPS responsiveness in HCE cells by JAK-2-dependent STAT1 activation and direct binding to the MD-2 promoter. Furthermore, given our findings on LPS-induced corneal inflammation, it is likely that IFN-γ-induced MD-2 expression by corneal epithelial cells contributes to the host response in vivo, determining the extent of tissue damage and bacterial clearance.

Airway epithelial cells release proinflammatory mediators that may contribute to airway remodeling and leukocyte recruitment. We explored the hypothesis that leukotriene D₄ (LTD₄) may trigger the release of proremodeling factors through activation of the EGF receptor (EGFR). We particularly focused on the effects of LTD₄ on release of heparin-binding EGF-like factor (HB-EGF) and IL-8 (CXCL8), a potent neutrophil chemoattractant that may be released downstream of EGFR activation. To address this hypothesis, both primary (NHBE) and transformed bronchial human epithelial cells (BEAS-2B) were grown on an air-liquid interface and stimulated with LTD₄. HB-EGF and CXCL8 were evaluated by ELISA in cell culture supernatants. To explore the EGFR signaling pathway, we used a broad-spectrum matrix metalloproteinase (MMP) inhibitor, GM-6001, two selective EGFR tyrosine kinase inhibitors, AG-1478 and PD-153035, an HB-EGF neutralizing antibody, and a specific small interfering RNA (siRNA) against the EGFR. Expression of the CysLT₁ cysteinyl leukotriene receptor was demonstrated by RT-PCR and immunocytochemistry in both BEAS-2B and NHBE cells. Four hours after stimulation with LTD₄, HB-EGF and CXCL8 were significantly increased in cell culture supernatant. GM-6001 and montelukast, a specific CysLT₁ receptor antagonist, blocked the LTD₄-induced increase in HB-EGF. All inhibitors/antagonists decreased LTD₄-induced CXCL8 release. siRNA against EGFR abrogated CXCL8 release following stimulation with LTD₄ and exogenous HB-EGF. These findings suggest LTD₄ induced EGFR transactivation through the release of HB-EGF in human bronchial epithelial cells with downstream release of CXCL8. These effects may contribute to epithelial-mediated airway remodeling in asthma and other conditions associated with cysteinyl leukotriene release.

BACKGROUND

Infection with human immunodeficiency virus type-1 (HIV)-1 leads to some form of HIV-1-associated neurocognitive disorders (HAND) in approximately half of the cases. The mechanisms by which astrocytes contribute to HIV-1-associated dementia (HAD), the most severe form of HAND, still remain unresolved. HIV-1-encephalitis (HIVE), a pathological correlate of HAD, affects an estimated 9-11% of the HIV-1-infected population. Our laboratory has previously demonstrated that HIVE brain tissues show significant upregulation of CD38, an enzyme involved in calcium signaling, in astrocytes. We also reported an increase in CD38 expression in interleukin (IL)-1β-activated astrocytes. In the present investigation, we studied regulatory mechanisms of CD38 gene expression in astrocytes activated with HIV-1-relevant stimuli. We also investigated the role of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB in astrocyte CD38 regulation.

METHODS

Cultured human astrocytes were transfected with HIV-1(YU-2) proviral clone and levels of CD38 mRNA and protein were measured by real-time PCR gene expression assay, western blot analysis and immunostaining. Astrocyte activation by viral transfection was determined by analyzing proinflammatory chemokine levels using ELISA. To evaluate the roles of MAPKs and NF-κB in CD38 regulation, astrocytes were treated with MAPK inhibitors (SB203580, SP600125, U0126), NF-κB interfering peptide (SN50) or transfected with dominant negative IκBα mutant (IκBαM) prior to IL-1β activation. CD38 gene expression and CD38 ADP-ribosyl cyclase activity assays were performed to analyze alterations in CD38 levels and function, respectively.

RESULTS

HIV-1(YU-2)-transfection significantly increased CD38 mRNA and protein expression in astrocytes (p < 0.01) in a dose-dependent manner and induced astrocyte activation. IL-β-activation of HIV-1(YU-2)-transfected astrocytes significantly increased HIV-1 gene expression (p < 0.001). Treatment with MAPK inhibitors or NF-κB inhibitor SN50 abrogated IL-1β-induced CD38 expression and activity in astrocytes without altering basal CD38 levels (p < 0.001). IκBαM transfection also significantly inhibited IL-1β-mediated increases in CD38 expression and activity in astrocytes (p < 0.001).

CONCLUSIONS

The present findings demonstrate a direct involvement of HIV-1 and virus-induced proinflammatory stimuli in regulating astrocyte-CD38 levels. HIV-1(YU-2)-transfection effectively induced HIV-1p24 protein expression and activated astrocytes to upregulate CCL2, CXCL8 and CD38. In astrocytes, IL-1β-induced increases in CD38 levels were regulated through the MAPK signaling pathway and by the transcription factor NF-κB. Future studies may be directed towards understanding the role of CD38 in response to infection and thus its role in HAND.

Recruitment of mesenchymal stem cells (MSC) to tissue damages is a promising approach for in situ tissue regeneration. The physiological mechanisms and regulatory processes of MSC trafficking to injured tissue remain poorly understood. However, the pivotal role of chemokines in MSC recruitment has already been shown. The aim of this study was to determine the migratory potential and the gene expression profile of MSC stimulated with the CC chemokine CCL25 (TECK). Bone marrow derived human MSC were exposed to different doses of CCL25 in a standardized chemotaxis assay. Microarray gene expression profiling and pathway analysis were performed for CCL25 stimulated MSC. Maximum migration of MSC towards CCL25 was observed at 10(3) nM. Microarray analysis revealed an induction of molecules directly involved in chemotaxis and homing of bone marrow cells (CXCL1-3, CXCL8, PDE4B), cytoskeletal and membrane reorganisation (CXCL8, PLD1, IGFBP1), cellular polarity (PLD1), and cell movement (CXCL1-3, CXCL6, CXCL8, PTGS2, PDE4B, TGM2). Respective chemokine secretion was confirmed by protein membrane-array analysis. The activation of CXCR2 ligands (CXCL1-3, CXCL5-6, CXCL8) and a LIF-receptor/gp130 ligand (LIF) indicated an involvement of the respective signaling pathways during initiation of chemotaxis and migration. These results suggest CCL25 as a new potential candidate for further in situ regeneration approaches.

In situ tissue engineering is a promising approach in regenerative medicine, with the possibility that adult stem or progenitor cells will be guided chemotactically to a tissue defect and subsequently differentiate into the surrounding tissue type. Mesenchymal stem cells (MSC) represent attractive candidate cells. Chemokines such as CXCL12 (SDF-1alpha) chemoattract MSC, but little is known about the molecular processes involved in the chemotaxis and migration of MSC. In this study, MSC recruitment by CXCL12 was investigated by genome-wide microarray analysis. The dose-dependent migration potential of bone-marrow-derived MSC toward CXCL12 was measured in an in vitro assay, with a maximum being recorded at a concentration of 1,000 nM CXCL12. Microarray analysis of MSC stimulated with CXCL12 and non-stimulated controls showed 30 differentially expressed genes (24 induced and six repressed). Pathway analysis revealed 11 differentially expressed genes involved in cellular movement and cytokine-cytokine receptor interaction, including those for migratory inducers such as the chemokines CXCL8 and CCL26, the leukocyte inhibitory factor, secretogranin II, and prostaglandin endoperoxide synthase 2. These results were confirmed by real-time polymerase chain reaction for selected genes. The obtained data provide further insights into the molecular mechanisms involved in chemotactic processes in cell migration and designate CXCL12 as a promising candidate for in situ recruitment in regenerative therapies.

Aqueous humor (AH) samples from patients with Behçet's disease (BD) (n=29), Vogt-Koyanagi-Harada (VKH) disease (n=21), and HLA-B27-associated uveitis (n=8), and 42 control patients were assayed for the neutrophil chemoattractants CXCL1/GRO-α and CXCL8/IL-8 and the lymphocyte chemoattractants CXCL9/MIG, CXCL10/IP-10 and CXCL12/SDF-1 with the use of a multiplex chemokine assay. Chemokine levels except SDF-1 were significantly higher in the 3 disease groups than in normal controls. Considering all patients, mean GRO-α levels were 15-fold higher than IL-8 levels and mean IP-10 levels were 22-fold higher than MIG levels. In patients with the same disease activity, AH levels of GRO-α and IP-10 were significantly higher in patients with BD than in patients with VKH disease and HLA-B27-associated uveitis (p=0.0474; p<0.001, respectively). These data suggest that GRO-α and IP-10 are the predominant CXC chemokines involved in neutrophil and activated T lymphocyte chemoattraction in endogenous uveitis, particularly in BD.

BACKGROUND

Microarray studies on the epidermal transcriptome in psoriasis and atopic dermatitis (AD) have revealed genes with disease-specific expression in keratinocytes of lesional epidermis. These genes are possible candidates for disease-specific pathogenetic changes, but could also provide a tool for molecular diagnostics of inflammatory skin conditions in general.

OBJECTIVE

To analyse if gene expression signatures as found in purified epidermal cells from AD are also present in other eczematous conditions such as allergic and irritant contact dermatitis.

METHODS

We used real-time quantitative polymerase chain reaction, immunohistochemistry and bioinformatics to investigate gene expression in different forms of eczema. Normal epidermis and psoriatic epidermis were analysed for comparison.

RESULTS

Carbonic anhydrase II was highly induced in epidermis from all forms of eczema but not in psoriasis. Remarkably, the presumed neuron-specific Nel-like protein 2 showed a strong induction only in AD epidermis. Interleukin-1F9, elafin, beta-defensin-2 and vanin-3 were strongly induced in psoriasis, but not in any type of eczema. High levels of the chemokines CCL17 and CXCL10 were predominantly found in epidermis of allergic contact dermatitis. The chemokine CXCL8 was highly expressed in psoriasis, AD and allergic contact dermatitis, but not in irritant contact dermatitis. Cluster analysis or multinomial logistic regression indicated that expression levels of a set of seven genes are a strong predictor of the type of inflammatory response.

CONCLUSIONS

These observations contribute to molecular diagnostic criteria for inflammatory skin conditions.

Proper healing of cutaneous wounds progresses through a series of overlapping phases. Nonhealing wounds are defective in one or more of these processes and represent a major clinical problem. A critical issue in developing treatments for chronic wounds is the paucity of animal models to study the mechanisms underlying the defects in healing. Here we show that deletion of tumor necrosis factor superfamily member 14 (TNFSF14/LIGHT) leads to impaired wounds in mice that have the characteristics of nonchronic and chronic ulcers. These wounds show: (1) excessive production of cytokines, in particular three chemokines (KC/CXCL8, MCP-1/CCL2, IP-10/CXCL10), that may be key to the abnormal initiation and resolution of inflammation; (2) defective basement membranes, explaining blood vessel leakage and disruption of dermal/epidermal interactions; and (3) granulation tissue that contains high levels of Coll III, whereas Coll I is virtually absent and does not form fibrils. We also see major differences between nonchronic and chronic wounds, with the latter populated by bacterial films and producing eotaxin, a chemokine that attracts leukocytes that combat multicellular organisms (which biofilms can be considered to be). This new mouse model captures many defects observed in impaired and chronic human wounds and provides a vehicle to address their underlying cell and molecular mechanisms.

The aim of this study is to investigate if macrophage-colony stimulating factor (M-CSF) or interleukin-34 (IL-34) induces cytokines or chemokines using human whole blood (HWB) and if an M-CSF- or IL-34-induced cytokine or chemokine production from HWB is inhibited by soluble M-CSF receptor or c-FMS kinase inhibitors. Among eight cytokines or growth factors tested, only IL-6 level was increased by up to 6-fold by M-CSF or IL-34 in HWB. In contrast, chemokine levels (IP-10/CXCL10, IL-8/CXCL8, and MCP-1/CCL2) were dramatically increased by M-CSF or IL-34 in HWB while exhibiting a large variation among donors. Variability of the MCP-1 signal induced by M-CSF or IL-34 was relatively less among donors compared to the IP-10 and IL-8 signals. The elevation of these chemokine levels was significantly decreased by soluble M-CSF receptor, indicating the elevation of these chemokines was mediated by M-CSF or IL-34. Furthermore, GW2580, a c-FMS kinase inhibitor, inhibited the induction of MCP-1 by M-CSF or IL-34 in a concentration dependent manner. These indicate MCP-1 is the most appropriate chemokine target for a chemokine release assay to evaluate the potency of c-FMS kinase inhibitors and MCP-1 release assay using HWB would be useful, relevant tool for translational pharmacology of c-FMS kinase inhibitors.