Airborne particulate matter (PM) has a complex composition, and the relative contribution of different compounds to PM-induced effects is only partly understood. The present study compared the capability of selected components commonly found in PM, to induce pro-inflammatory responses in lung epithelial cells. Ultrafine carbon black (ufCB), ZnCl(2), FeSO(4), 1-nitropyrene (1-NP), lipopolysaccharide (LPS), and crystalline silica (positive control) were screened for effects on the expression of 84 inflammation-related genes in the bronchial epithelial cell line, BEAS-2B. A total of 22 genes were up-regulated by one or more of the tested compounds, and 5 cytokine and 11 chemokine genes were selected for further studies. After 10h exposure, silica induced significantly increased expression of CCL20, CXCL1/-3/-8/-10/-11, lymphotoxin (LT)beta and interleukin (IL)-6; ufCB induced CXCL8/-10 and -11; ZnCl(2) induced CCL11/-20/-26, CXCL1/-5/-8/-14 and tumor necrosis factor (TNF)-alpha; FeSO(4) induced a weak up-regulation of CXCL8 and TNF-alpha; LPS induced CCL20, CXCL1/-5/-8/-10/-11, LTbeta and IL-6; and 1-NP induced expression of CCL20, CXCL1/-3/-8, TNF-alpha and IL-6. Despite obvious differences, all compounds induced response-patterns that correlated relatively well with that of silica, the positive control. The predominant response appeared to be increased gene expression of neutrophil-recruiting CXC-chemokines. CXCL8 was the only gene induced by all tested PM-components, the most up-regulated on average, and also dominating the gene-expression patterns induced by coarse PM. The data show quantitative, and to a certain extent qualitative differences in cytokine/chemokine gene-expression profiles of the compounds tested. However, there were also striking similarities in the response-patterns induced by these physically/chemically widely different compounds.

BACKGROUND

We previously showed that tumor-free peritoneum of patients with epithelial ovarian cancer (EOC) exhibited enhanced expression of several inflammatory response genes compared to peritoneum of benign disease. Here, we examined peritoneal inflammatory cell patterns to determine their concordance with selected enhanced genes.

METHODS

Expression patterns of selected inflammatory genes were mined from our previously published data base. Bilateral pelvic peritoneal and subjacent stromal specimens were obtained from 20 women with EOC and 7 women with benign pelvic conditions. Sections were first stained by indirect immunoperoxidase and numbers of monocytes/macrophages (MO/MA), T cells, B cells, and NK cells counted. Proportions of CD68+ cells and CD3+ cells that coexpressed MO/MA differentiation factors (CD163, CCR1, CXCR8, VCAM1, and phosphorylated cytosolic phospholipase A2 [pcPLA2]), which had demonstrated expression in EOC peritoneal samples, were determined by multicolor immunofluorescence.

RESULTS

MO/MA were present on both sides of the pelvic peritoneum in EOC patients, with infiltration of the subjacent stroma and mesothelium. CD68+ MO/MA, the most commonly represented population, and CD3+ T cells were present more often in EOC than in benign pelvic tumors. NK cells, B cells, and granulocytes were rare. CXCL8 (IL-8) and the chemokine receptor CCR1 were coexpressed more frequently on MO/MA than on CD3+ cells contrasting with CD68+/CD163+ cells that coexpressed CXCL8 less often. An important activated enzyme in the eicosanoid pathway, pcPLA2, was highly expressed on both CD68+ and CD163+ cells. The adherence molecule Vascular Cell Adhesion Molecule-1 (VCAM1) was expressed on CD31+ endothelial cells and on a proportion of CD68+ MO/MA but rarely on CD3+ cells.

CONCLUSIONS

The pelvic peritoneum in EOC exhibits a general pattern of chronic inflammation, represented primarily by differentiated MO/MA, and distinct from that in benign conditions concordant with previous profiling results.

Giardia duodenalis (syn. G. intestinalis, G. lamblia) infections are a leading cause of waterborne diarrheal disease that can also result in the development of postinfectious functional gastrointestinal disorders via mechanisms that remain unclear. Parasite numbers exceed 10(6) trophozoites per centimeter of gut at the height of an infection. Yet the intestinal mucosa of G. duodenalis-infected individuals is devoid of signs of overt inflammation. G. duodenalis infections can also occur concurrently with infections with other proinflammatory gastrointestinal pathogens. Little is known of whether and how this parasite can attenuate host inflammatory responses induced by other proinflammatory stimuli, such as a gastrointestinal pathogen. Identifying hitherto-unrecognized parasitic immunomodulatory pathways, the present studies demonstrated that G. duodenalis trophozoites attenuate secretion of the potent neutrophil chemoattractant interleukin-8 (CXCL8); these effects were observed in human small intestinal mucosal tissues and from intestinal epithelial monolayers, activated through administration of proinflammatory interleukin-1β or Salmonella enterica serovar Typhimurium. This attenuation is caused by the secretion of G. duodenalis cathepsin B cysteine proteases that degrade CXCL8 posttranscriptionally. Furthermore, the degradation of CXCL8 via G. duodenalis cathepsin B cysteine proteases attenuates CXCL8-induced chemotaxis of human neutrophils. Taken together, these data demonstrate for the first time that G. duodenalis trophozoite cathepsins are capable of attenuating a component of their host's proinflammatory response induced by a separate proinflammatory stimulus.

BACKGROUND

Preeclampsia is associated with elevated levels of proinflammatory cytokines, excess decidual macrophages, and dendritic cells. IL-1β- or TNF-α-stimulated leukocyte-free first trimester decidual cells produced abundant macrophage- and dendritic cell-recruiting chemokines identified in preeclamptic decidua.

OBJECTIVE

The relative potency of IL-1β- or TNF-α-induced first trimester decidual cell-secreted chemokines in chemoattracting macrophages or dendritic cells and the signaling pathways involved in the expression of these chemokines were evaluated.

METHODS

First trimester decidual cells were treated with estradiol + medroxyprogesterone acetate ± IL-1β or TNF-α. The chemotaxis assay was performed by incubating conditioned medium from first trimester decidual cells with neutralizing antibody for six chemokines. The activation of each signaling pathway was examined by Western blotting, flow cytometry, confocal microscopy, and ELISA with or without kinase and nuclear factor κB (NFκB) inhibitors.

RESULTS

Neutralization of CCL2 and CCL5 significantly reduced chemotaxis of monocyte and dendritic cells up to 50 and 36%, respectively. NFκB and MAPK (MAPK kinase, JUN NH₂-terminal kinase, p38 kinase) pathways were activated by IL-1β or TNF-α in first trimester decidual cells. In IL-1β- or TNF-α-stimulated first trimester decidual cells, NFκB inhibitor suppressed production of all six chemokines; JUN NH₂-terminal kinase inhibitor inhibited secretion of CCL2, CCL4, and CCL5; and MAPK kinase and p38 inhibitor decreased production of CXCL8.

CONCLUSIONS

Up-regulation of CCL2 and CCL5 by first trimester decidual cells in response to proinflammatory stimuli may account for the accumulation of macrophages and dendritic cells in preeclamptic decidua. These chemokines and underlying IL-1β- or TNF-α-induced signaling molecules are potential diagnostic and therapeutic targets for preeclampsia.

Previous studies of acute generalized exanthematous pustulosis, a peculiar drug hypersensitivity reaction, suggested that CXCL8-producing T cells regulate sterile, polymorphonuclear neutrophil-rich skin inflammations. In this study, we test the hypothesis of whether CXCL8-producing T cells are present in autoinflammatory diseases like pustular psoriasis and Behçet's disease. Immunohistochemistry of normal skin revealed few CD4+ and CD8+ T cells, few CXCL8+ cells, and no neutrophilic infiltration, whereas in acute exacerbations of atopic dermatitis, numerous CD4+ T cells but few CD8+ T cells, neutrophils, or CXCL8+ cells were detected. In contrast, a pronounced infiltration of neutrophils and of predominantly CD4+ T cells was observed in skin biopsies from pustular psoriasis, Behçet's disease, and acute generalized exanthematous pustulosis, with infiltrating T cells strongly positive for CXCL8 and the chemokine receptor CCR6. Skin-derived T cell clones from pustular skin reactions were positive for CCR6 but negative for CCR8 and secreted high amounts of CXCL8 and GM-CSF, often together with IFN-gamma and TNF-alpha after in vitro stimulation. Moreover, some skin-derived T cell clones from Behçet's disease and from pustular psoriasis predominantly produced CXCL8 and GM-CSF, but failed to secrete IL-5 and IFN-gamma. These cells might represent a particular subset as they differ from both Th1 as well as Th2 T cells and are associated with a unique, neutrophil-rich sterile inflammation. Our findings suggest that CXCL8/GM-CSF-producing T cells may orchestrate neutrophil-rich pathologies of chronic autoinflammatory diseases like pustular psoriasis and Behçet's disease.

Cells undergo senescence in response to various conditions, including telomere erosion, oncogene activation and multiple cytokines. One of these cytokines, interleukin-6 (IL‑6), not only functions in the immune system, but also promotes cellular senescence and cancer. Here we demonstrate that IL‑6 and the soluble IL‑6 receptor (sIL‑6R) induce premature senescence in normal human fibroblasts by establishing a senescence-inducing circuit involving the signal transducer and activator of transcription 3 (STAT3) and insulin-like growth factor-binding protein 5 (IGFBP5). Stimulating TIG3 fibroblast cells with IL‑6/sIL‑6R sequentially caused an increase in reactive oxygen species (ROS) as early as day 1, followed by the DNA damage response, p53 accumulation and, finally, senescence on days 8-10. We found that STAT3 was required for the events leading to senescence, including the initial early-phase ROS increase and the induction of IL‑1α/β, IL‑6 and CXCL8 mRNAs 4-5 d after IL‑6/sIL‑6R stimulation, suggesting that STAT3's role is indirect. We searched for STAT3-downstream molecule(s) responsible for the senescence-inducing activity in the supernatants of stimulated TIG3 and identified IGFBP5 as a major STAT3 mediator, because IGFBP5 was expressed from the early phase through the entire senescence process and was responsible for IL‑6/STAT3-induced ROS increase and premature senescence. Thus, IL‑6/sIL‑6R forms a senescence-inducing circuit involving the STAT3-IGFBP5 axis as a key triggering and reinforcing component.

BACKGROUND

Virus-induced inflammation contributes to respiratory syncytial virus (RSV) pathogenesis. We sought to determine the specific mediators that are associated with more severe illness in young children.

METHODS

Children ≤ 5 years of age seen in our emergency department for respiratory symptoms from September 1998 to May 2008 were eligible for enrollment. Nasopharyngeal wash samples were collected from all eligible patients, and clinical data were recorded. Individuals were included in this study if nasopharyngeal wash samples were positive for RSV only. Patients enrolled in the study were stratified by disease severity, defined as mild (not hospitalized), moderate (hospitalized) or severe (requiring intensive care unit stay). Concentrations of individual inflammatory biomarkers in nasopharyngeal wash fluids were determined using the Luminex human 30-plex assay.

RESULTS

Eight hundred fifty-one patients met study criteria: 268 (31.5%) with mild, 503 (59.1%) with moderate and 80 (9.4%) with severe illness. As expected, illness severity was directly associated with young age, prematurity, heart or lung disease, infection with RSV group A and elevated concentrations of interleukin (IL)-2R, IL-6, CXCL8, tumor necrosis factor-α, interferon-α, CCL3, CCL4 and CCL2. In addition, we report several novel and mechanistically important inflammatory biomarkers of severe RSV disease, including IL-1β, IL1-RA, IL-7, epidermal growth factor and hepatocyte growth factor.

CONCLUSIONS

In a large, longitudinal study (10 years, 851 enrolled patients) limited to RSV infection only, in which well-known risk factors are confirmed, we identified 5 novel biomarkers specifically of severe disease. These markers may ultimately serve to elucidate disease mechanisms.

OBJECTIVE

Circumstantial evidence exists that certain neutrophilic inflammatory processes are regulated by T cells, but how this occurs is not well understood. The present review presents data on how T cells may directly orchestrate a neutrophilic inflammation by specific release of the neutrophil-attracting chemokine CXCL8 (formerly known as interleukin-8).

RESULTS

Acute generalized exanthematous pustulosis (AGEP) is an uncommon cutaneous eruption that is most often provoked by drugs, by acute infections with enteroviruses, or by mercury. It is characterized by acute, extensive formation of nonfollicular sterile pustules on an erythematous background, fever and elevated numbers of blood neutrophils. Involvement of T cells in drug-induced AGEP was suggested by positive patch tests and lymphocyte transformation tests. Moreover, drug-specific CD4+ and CD8+ T cells could be isolated and propagated in vitro from patch test sites and blood from AGEP patients. Their main characteristic is a high level of CXCL8 production.

CONCLUSIONS

T cells are involved even in some neutrophil-rich inflammatory responses, and they may orchestrate the immune reaction directly by high CXCL8 production or indirectly via interleukin-17 production, which induces CXCL8 production in various cell types. AGEP serves as a valuable model for characterizing T cells with a particular function--namely production of CXCL8--leading to neutrophilic inflammation. It is tempting to speculate that elucidation of this pathomechanism will help to improve our understanding of similar neutrophilic eruptions (e.g. pustular psoriasis) and may reveal new targets for pharmacotherapeutic interventions in such diseases.

Inhalation is an important route of cadmium (Cd) exposure, and the lung is considered to be one of the main target organs of Cd toxicity. Pulmonary inflammation seems to be involved in development of many lung diseases. In the present study we show that Cd(2+) at fairly low concentrations affects gene expression of several different cytokines/chemokines in human M1 fibroblasts. The chemokines CXCL2, CXCL3, IL-8/CXCL8 and CCL26, the pro-inflammatory cytokine IL-6 and the receptor IL-1RL1 were expressed at high levels after exposure to 7 microM Cd(2+) for 7h. The expression of some important cytokines was further studied in two different primary cell cultures from rat lungs. Cd(2+) induced cytokine responses at low concentrations (3-6 microM) and early time-points both in type 2 epithelial cell-enriched cultures and alveolar macrophages. However, the two primary lung cells displayed different patterns of cytokine release. Cd(2+) induced an increased release of IL-6 and MIP-2/CXCL2 from the epithelial cells and MIP-2, IL-1beta and TNF-alpha from alveolar macrophages. In conclusion, the marked up-regulation of different cytokines in these cell types, that are important in development of lung injury and disease, suggests that inflammation may contribute in Cd-induced lung damage.

Infectious mastitis cuts down milk production profitability and is a major animal welfare problem. Bacteria-induced inflammation in the mammary gland (MG) is driven by innate immunity, but adaptive immunity can modulate the innate response. Several studies have shown that it is possible to elicit inflammation in the MG by sensitization to an antigen subsequently infused into the lumen of the gland. The objective of our study was to characterize the inflammation triggered in the MG of cows sensitized to ovalbumin, by identifying the cytokines and chemokines likely to play a part in the reaction. Among immunized cows, responders mobilized locally high numbers of leukocytes. An overexpression of the genes encoding IL-17a, IL-17F, IL-21, IL-22 and INF-γ was found in milk cell RNA extracts in the early phase of the inflammatory response. At the protein level, IL-17A was detected in milk as soon as the first sampling time (8 h post-challenge), and both IL-17A and IFN-γ concentrations peaked at 12 to 24 h post-challenge. In mammary tissue from challenged quarters, overexpression of the genes encoding IL-17A, IL-17F, IL-21, IL-22, IL-26 and IFN-γ was observed. Neutrophil-attracting chemokines (CXCL3 and CXCL8) were found in milk, and overexpressed transcripts of chemokines attracting lymphocytes and other mononuclear leukocytes (CXCL10, CCL2, CCL5, CCL20) were detected in mammary tissue. Expression of IL-17A, as revealed by immunohistochemistry, was located in epithelial cells, in leukocytes in the connective tissue and in association with the epithelium, and in migrated alveolar leukocytes of challenged quarters. Altogether, these results show that antigen-specific inflammation in the MG was characterized by the production of IL-17 and IFN-γ. The orientation of the inflammatory response induced by the antigen-specific response has the potential to strongly impact the outcome of bacterial infections of the MG.

BACKGROUND

Choline-binding protein A (CbpA) and pneumolysin (Ply) can induce the expression and release of chemokines by human cells, which might modulate specific immune responses. In dendritic cells (DCs), such effects could be important for the size and character of the immunity induced if administered as vaccines. We studied the induction of CCL and CXCL chemokines by CbpA and Ply in DCs and related signaling pathways.

METHODS

Proteins derived from bacterial cultures and cloning were used as stimulants. DCs were generated from CD14+ human monocytes by negative selection, followed by coculture with recombinant human granulocyte-macrophage colony-stimulating factor and recombinant interleukin-4. The role played by Toll-like receptors (TLRs) was assessed using anti-TLR antibodies. Likewise, specific inhibitors (given in parentheses) of signaling molecules were used: NF-kappaB (SN50), extracellular signal-regulated kinase (PD98059), p38 (SB203580), and Jun N-terminal kinase (SP600125).

RESULTS

Both CbpA and Ply significantly up-regulated DC mRNA of several CCL (2, 4, 5, and 8) and CXCL (8 and 10) chemokines studied as well as the expression of 3 proteins studied: CCL2, CCL5, and CXCL8. Ply stimulation was blocked by anti-TLR4. Inhibition of NF-kappaB and several mitogen-activated protein kinase signaling pathways also reduced chemokine release.

CONCLUSIONS

Chemokine induction in DCs by CbpA and Ply may be important for their potential use in future pneumococcal vaccines.

BACKGROUND

In Crohn's disease high tissue expression and serum levels of chemokines and their receptors are known to correlate with disease activity. Because statins can reduce chemokine expression in patients with coronary diseases, we wanted to test whether this can be achieved in patients with Crohn's disease.

RESULTS

We investigated plasma levels of chemokines (CCL2, CCL4, CCL11, CCL13, CCL17, CCL22, CCL26, CXCL8, CXCL10) and endothelial cytokines (sP-selectin, sE-selectin, sICAM-3, thrombomodulin) in ten Crohn's disease patients before and after thirteen weeks' daily treatment with 80 mg atorvastatin. Of the 13 substances investigated, only CXCL10 was found to be significantly reduced (by 34%, p = 0.026) in all of the treated patients. Levels of CXCL10 correlated with C-reactive protein (r = 0.82, p<0.01).

CONCLUSIONS

CXCL10 is a ligand for the CXCR3 receptor, the activation of which results in the recruitment of T lymphocytes and the perpetuation of mucosal inflammation. Hence the reduction of plasma CXCL10 levels by atorvastatin may represent a candidate for an approach to the treatment of Crohns disease in the future.

BACKGROUND

(ClinicalTrials.gov) NCT00454545.

Chronic obstructive pulmonary disease (COPD) is characterized by an abnormal innate immune response. We have investigated the changes in the innate immune response of COPD alveolar macrophages exposed to both cigarette smoke and Toll-like receptor (TLR) stimulation. COPD and control alveolar macrophages were exposed to cigarette smoke extract (CSE) followed by TLR-2, -4 and -5 ligands [Pam3CSK4, lipopolysaccharide (LPS) and phase I flagellin (FliC), respectively] or non-typeable Haemophilus influenzae (NTHi). CSE exposure suppressed TLR-induced tumour necrosis factor (TNF)-α, interleukin (IL)-6, IL-10 and regulated on activation, normal T cell expressed and secreted (RANTES) production in both COPD and control alveolar macrophages, but had no effect on interleukin 8 (CXCL8) production. Similarly, CSE suppressed NTHi-induced TNF-α but not NTHi-induced CXCL8 production in COPD alveolar macrophages. Gene expression analysis showed that CSE suppressed LPS-induced TNF-α transcription but not CXCL8 transcription in COPD alveolar macrophages. The dampening effect of CSE on LPS-induced cytokine production was associated with a reduction in p38, extracellular signal regulated kinase (ERK) and p65 activation. In conclusion, CSE caused a reduced innate immune response in COPD alveolar macrophages, with the exception of persistent CXCL8 production. This could be a mechanism by which alveolar macrophages promote neutrophil chemotaxis under conditions of oxidative stress and bacterial exposure.

We have reported recently that the chemokine interleukin 8 (IL-8)/CXCL8 was overexpressed in invasive estrogen receptor (ERalpha)-negative breast cancer cells compared with ERalpha-positive breast cancer cells. We now demonstrate that histone deacetylases (HDACs) play an essential role in the regulation of IL-8 gene expression in ERalpha-positive MCF-7 breast cancer cells. Treatment of MCF-7 cells with the HDAC inhibitor trichostatin A (TSA) led to a strong up-regulation of IL-8 protein and RNA levels in MCF-7 cells. The up-regulation of IL-8 in MCF-7 cells was time- and concentration-dependent. Moreover, run-on and transfection experiments demonstrated that IL-8 induction by HDAC inhibitors was transcriptional and involved mainly the nuclear factor-kappaB (NF-kappaB) site of the IL-8 promoter. These observations are corroborated by an up-regulation of NF-kappaB activity in MCF-7 cells in the presence of TSA. In addition, blocking NF-kappaB pathway by adenoviral delivery of a dominant-negative IkappaBorIkappaB kinase complex 2 (IKK2) mutant abolished IL-8 gene induction by histone deacetylase inhibitors. HDAC inhibitors triggered IKK phosphorylation and up-regulated p65 nuclear translocation, although they decreased the protein levels of IkappaBalpha, which accounts for NF-kappaB activation. TSA increased binding of acetylated histone 3 to the IL-8 gene promoter. In summary, our results demonstrate that NF-kappaB pathway repression by HDAC is responsible for the low expression of IL-8 in ERalpha-positive breast cancer cells.

The anti-VEGF targeted antibody bevacizumab (BVZ) has been approved for treating renal cell carcinomas (RCCs). Although BVZ increases the progression-free survival of patients with metastatic RCC, the effect on overall survival is poor. To gain insight into the limited efficacy of BVZ on overall survival, we analyzed patient samples of RCC for angiogenic factors that may participate in escape from anti-VEGF therapy. Our study shows that the level of vascular endothelial growth factor (VEGF) in tumors was increased compared with normal tissue. The level of interleukin-8/CXCL8, a pro-angiogenic member of the CXCL family of cytokines, was also increased in tumors. These observations gave us a good reason to analyze the combined effects of BVZ and anti-CXCL8 antibodies on tumor growth. Surprisingly, we report that BVZ accelerates the growth of RCC in nude mice with in vivo selection of tumor cells with an increased growth capacity. Downregulation of receptor tyrosine phosphatase-κ, a phosphatase implicated in EGF receptor regulation, may partly explain this phenomenon. Modification of the vascular network and development of lymphatic vessels through VEGF-C production and compensatory production of pro-angiogenic CXCL cytokines were also observed. The apparent normalization of the vascular network prompted us to associate BVZ with the chemotherapeutic agent paclitaxel. While efficient in vitro, paclitaxel did not reverse the anti-VEGF effects in vivo. Anti-CXCL8-targeting antibodies were promising as they decreased intra-tumor VEGF production; decreased the pro-angiogenic CXCL/anti-angiogenic CXCL ratio and did not induce lymphangiogenesis. These observations hold clinical implication as they highlight putative markers implicated in escape from BVZ treatment. They also recommend proceeding with caution in the use of anti-VEGF therapy alone for treatment of RCC.

High levels of donor-derived CCL2 have been associated with poor islet allograft outcome in patients with type 1 diabetes. The aim of our work was to determine whether CCL2 secreted by the islet has independent proinflammatory effects that influence engraftment and graft acceptance. Both in mice and humans CCL2 is significantly positively associated with other cytokines/chemokines, in particular with the highly released "proinflammatory" IL-6 and CXCL8 or CXCL1. Transplantation of CCL2-/- islets into syngenic recipients did not improve the transplant function. Transplantation of islets into CCL2-/- syngenic recipients led to a significant improvement of transplant function and partial abrogation of local hepatic inflammation. When evaluated in human islets CCL2 release was strongly related to the immediate local inflammatory response in the liver and impacted short-term human islet function dependently by the induced inflammatory response and independently by the immunosuppressive therapy. The data showed that islet CCL2 release is a sign of "inflamed" islets without having a direct role in graft failure. On the other hand, a causal effect for developing detrimental proinflammatory conditions after transplant was proved for recipient CCL2. Strategies to selectively decrease recipient, but not donor, CCL2 release may increase the success of islet transplantation.

The innate immune response to inhaled bacteria, such as the opportunist Pseudomonas aeruginosa, is initiated by TLR2 displayed on the apical surface of airway epithelial cells. Activation of TLR2 is accompanied by an immediate Ca(2+) flux that is both necessary and sufficient to stimulate NF-kappaB and MAPK proinflammatory signaling to recruit and activate polymorphonuclear leukocytes in the airway. In human airway cells, gap junction channels were found to provide a regulated conduit for the movement of Ca(2+) from cell to cell. In response to TLR2 stimulation, by either lipid agonists or P. aeruginosa, gap junctions functioned to transiently amplify proinflammatory signaling by communicating Ca(2+) fluxes from stimulated to adjacent, nonstimulated cells thus increasing epithelial CXCL8 production. P. aeruginosa stimulation also induced tyrosine phosphorylation of connexin 43 and association with c-Src, events linked to the closure of these channels. By 4 h postbacterial stimulation, gap junction communication was decreased indicating an autoregulatory control of the connexins. Thus, gap junction channels comprised of connexin 43 and other connexins in airway cells provide a mechanism to coordinate and regulate the epithelial immune response even in the absence of signals from the immune system.

BACKGROUND

Rhinoviruses (RVs) are responsible for the majority of acute asthma and chronic obstructive pulmonary disease (COPD) exacerbations. RVs infect the lower airways and induce the production of pro-inflammatory and remodelling-associated mediators. Budesonide (BUD) and formoterol (FORM) synergize in controlling asthma and COPD exacerbations; however, their effects on virus-induced inflammation and remodelling are less known.

OBJECTIVE

We investigated whether BUD and FORM synergize in suppressing RV-induced inflammation and remodelling in the airways.

METHODS

In vitro models of RV infection of BEAS-2B and primary normal human bronchial epithelial (NHBE) cells were used. We assessed the effects of individual and combined drugs administered post-infection, at a clinically relevant concentration range (10(-6)-10(-10) m), on the production of CCL5, CXCL10, CXCL8, IL-6 and the remodelling-associated VEGF and bFGF, using ELISA and RT-PCR.

RESULTS

BUD effectively suppressed RV-mediated induction of all mediators studied, in a concentration-dependent manner. FORM alone suppressed the production of CXCL8 and bFGF. The combination of BUD and FORM had concentration-dependent, additive or synergistic effects in the suppression of RV-induced CCL5, CXCL8 and CXCL10 in both cell types as well as VEGF in NHBE only. Combination treatment also resulted in an enhanced suppression of RV-induced IL-6, and CCL5 at the mRNA level as compared with BUD or FORM alone.

CONCLUSIONS

BUD and FORM suppress RV-induced chemokines and growth factors in bronchial epithelial cells in a concentration-dependent, synergistic or additive manner. These data further support the combined use of BUD and FORM in asthma and COPD and intensification of this therapy during exacerbations.

OBJECTIVE

To identify and characterize which endothelial heparan sulfate proteoglycans (HSPGs) bind the chemokine CXCL8 (interleukin-8) in human rheumatoid arthritis (RA) and nonrheumatoid synovia.

METHODS

CXCL8 binding to endothelial HSPGs in RA and nonrheumatoid synovia was determined by heparinase treatment followed by an in situ binding assay and autoradiography. Endothelial HSPGs were characterized by immunohistochemical analysis and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Phosphatidyinositol-specific phospholipase C (PI-PLC) and antibodies to HSPGs were used in in situ binding experiments to identify which HSPGs bound CXCL8.

RESULTS

The expression of heparan sulfate on microvascular endothelial cells was demonstrated in RA and nonrheumatoid synovia. Using antibodies to syndecan-1-4 and glypican-1, -3, and -4, the selective expression of syndecan-3 by endothelial cells was detected in RA and nonrheumatoid synovia. In addition, RT-PCR showed the presence of syndecan-3 messenger RNA in endothelial cells extracted from RA and nonrheumatoid synovia. (125)I-CXCL8 bound to venular endothelial cells; treatment with heparinases I and III significantly reduced this binding in RA but not nonrheumatoid synovia. (125)I-CXCL8 binding was not reduced after treatment with PI-PLC, which cleaves glycosyl phosphatidylinositol linkages, suggesting that CXCL8 did not bind to glypicans. Treatment of synovia with a syndecan-3 antibody reduced CXCL8 binding to RA but not nonrheumatoid endothelial cells; however, no reduction in binding was observed with syndecan-2 or glypican-4 antibodies.

CONCLUSIONS

Our results show the selective induction of a CXCL8 binding site on endothelial syndecan-3 in RA synovium. This site may be involved in leukocyte trafficking into RA synovial tissue.

The pathogenesis of idiopathic nephrotic syndrome (INS) remains unknown. Several findings suggest a role for the immune system. This study aimed to evaluate immune mediators in INS by measuring plasma and urinary levels of transforming growth factor beta1 (TGF-beta1), monocyte chemoattractant protein-1 (MCP-1/CCL2), regulated on activation normal T-cell expressed and secreted (RANTES/CCL5) and IL-8 (IL-8/CXCL8) in pediatric patients with INS and in age-matched healthy controls. Patients were divided according to their response to corticosteroids: steroid-sensitive (SS, n = 8), or steroid-resistant (SR, n = 24). Immune mediators were also compared in regard with disease activity (relapse and remission). Immune mediators were measured by ELISA. Plasma TGF-beta1 levels in SR patients were approximately 2.8-fold higher than control values (p < 0.05). Urinary IL-8/CXCL8 was 2.9-fold higher in INS patients in relapse (proteinuria >100 mg/m2/24 h) when compared with patients in remission (p < 0.05), and levels had a positive correlation with individual proteinuria values (p < 0.05). Urinary IL-8/CXCL8 was significantly higher in relapsed SR than in SS patients in remission. No changes in MCP-1/CCL2 and RANTES/CCL5 levels were detected. Our findings suggest that IL-8/CXCL8 and TGF-beta1 are involved in the pathogenesis of INS: IL-8/CXCL8 associated with local changes in glomerular permeability and TGF-beta1 could be related to worse response to corticosteroids.

BACKGROUND

Much evidence exists regarding the fact that blood DCs, both myeloid DCs (MDCs) and plasmacytoid DCs (PDCs), are negatively affected in different types of cancer, with both reduced numbers and impaired functionality. Functional impairment of DCs in patients with pancreatic ductal adenocarcinoma (PDAC), may contribute to the poor clinical outcome. The aim of this study was to examine the effects PDAC had on blood DCs and elucidate the underlying mechanism responsible for the DC impairment.

RESULTS

We examined the systemic influence PDAC exerted on blood DCs by ex vivo measuring numerous activation and maturation markers expressed on these cells. Furthermore, the effect patient plasma and the inflammatory factors CXCL8 and PGE(2) had on purified MDCs and PDCs from healthy donors was assessed and compared to the DCs existing in PDAC patients. We found a partial maturation of the blood MDCs and PDCs in PDAC patients with significantly enhanced expression of CD83, CD40, B7H3, PDL-1, CCR6, and CCR7 and decreased expression of ICOSL, and DCIR. These changes lead to impairment in their immunostimulatory function. Furthermore, chronic pancreatitis gave rise to DCs with similar semi-mature phenotype as seen in PDAC. Low expression of ICOSL was associated with poor prognosis. We found that the mechanism underlying this semi-maturation of DCs was inflammatory factors existing in the PDAC patients' plasma. Of note, PGE(2), which is elevated PDAC patient plasma, was one contributing factor to the changes seen in MDCs and PDCs phenotype.

CONCLUSIONS

Our findings point to a role for the systemic inflammation in transforming blood MDCs and PDCs into semi-mature cells in PDAC patients and we show a correlation between maturation status and clinical outcome. Thus, means to preserve a functional blood DC compartment in PDAC patients by diminishing the inflammation could facilitate their ability to control the disease and improve survival.

The CXCL1/CXCR2 axis plays a crucial role in recruiting neutrophils in response to microbial infection and tissue injury, and dysfunction in this process has been implicated in various inflammatory diseases. Chemokines exist as monomers and dimers, and compelling evidence now exists that both forms regulate in vivo function. Therefore, knowledge of the receptor activities of both CXCL1 monomer and dimer is essential to describe the molecular mechanisms by which they orchestrate neutrophil function. The monomer-dimer equilibrium constant (~20 μm) and the CXCR2 binding constant (1 nm) indicate that WT CXCL1 is active as a monomer. To characterize dimer activity, we generated a trapped dimer by introducing a disulfide across the dimer interface. This disulfide-linked CXCL1 dimer binds CXCR2 with nanomolar affinity and shows potent agonist activity in various cellular assays. We also compared the receptor binding mechanism of this dimer with that of a CXCL1 monomer, generated by deleting the C-terminal residues that stabilize the dimer interface. We observe that the binding interactions of the dimer and monomer to the CXCR2 N-terminal domain, which plays an important role in determining affinity and activity, are essentially conserved. The potent activity of the CXCL1 dimer is novel: dimers of the CC chemokines CCL2 and CCL4 are inactive, and the dimer of the CXC chemokine CXCL8 (which is closely related to CXCL1) is marginally active for CXCR1 but shows variable activity for CXCR2. We conclude that large differences in dimer activity among different chemokine-receptor pairs have evolved for fine-tuned leukocyte function.

Syndecans are important cell surface proteoglycans with many functions; yet, they have not been studied to a very large extent in primary human endothelial cells. The purpose of this study was to investigate syndecan-4 expression in cultured human umbilical vein endothelial cells (HUVECs) and assess its role in inflammatory reactions and experimental wound healing. qRT-PCR analysis revealed that syndecan-3 and syndecan-4 were highly expressed in HUVECs, whereas the expression of syndecan-1 and -2 was low. HUVECs were cultured with the inflammatory mediators lipopolysaccharide (LPS) and interleukin 1β (IL-1β). As a result, syndecan-4 expression showed a rapid and strong increase. Syndecan-1 and -2 expressions decreased, whereas syndecan-3 was unaffected. Knockdown of syndecan-4 using siRNA resulted in changes in cellular morphology and focal adhesion sites, delayed wound healing and tube formation, and increased secretion of the pro-inflammatory and angiogenic chemokine, CXCL8. These data suggest functions for syndecan-4 in inflammatory reactions, wound healing and angiogenesis in primary human endothelial cells.