Pulmonary macrophages are a target for inhaled therapies. Combinations of long-acting beta(2)-agonists (LABA) and glucocorticosteroids have been developed for asthma and chronic obstructive pulmonary disease (COPD). This study examined two LABA, salmeterol and formoterol, and the glucocorticosteroid, budesonide, on cytokine release from monocyte-derived macrophages (MDM) to determine whether anti-inflammatory effects observed in patients are due to inhibition of macrophages. MDM were incubated in the absence or presence of LABA or budesonide prior to stimulation with lipopolysaccharide (LPS). Tumour necrosis factor (TNF)-alpha, granulocyte macrophage-colony stimulating factor (GM-CSF) and CXC chemokine ligand (CXCL)8 were measured by ELISA. Formoterol and salmeterol inhibited LPS-stimulated release of TNF-alpha (mean effective concentration (EC(50)) 2.4+/-1.8 and 3.5+/-2.7 nM, respectively; n = 11-16), GM-CSF (EC(50) 24.6+/-2.1 and 52.4+/-40.8 nM, respectively, n = 11-12) but not CXCL8 from LPS-stimulated MDM. Budesonide inhibited release of all three cytokines (EC(50) TNF-alpha: 1.2+/-0.4 nM; GM-CSF: 0.4+/-0.2 nM; CXCL8: 0.4+/-0.1 nM; n = 3-4). Formoterol but not salmeterol elevated cAMP in these cells. These effects were attenuated by beta-adrenoceptor antagonists, propranolol and ICI118551. Salmeterol (10(-7) M) also inhibited formoterol-induced cAMP and formoterol-mediated attenuation of cytokine release. Combining budesonide (0.3 nM) with formoterol, inhibited TNF-alpha release additively. LABA may inhibit inflammatory cytokine release from macrophages in a cAMP-independent manner and act additively with budesonide.

In this second review on chemokines, we focus on the polymorphisms and alternative splicings and on their consequences in disease. Because chemokines are key mediators in the pathogenesis of inflammatory, autoimmune, vascular and neoplastic disorders, a large number of studies attempting to relate particular polymorphisms of chemokines to given diseases have already been conducted, sometimes with contradictory results. Reviewing the published data, it becomes evident that some chemokine genes that are polymorphic have alleles that are found repeatedly, associated with disease of different aetiologies but sharing some aspects of pathogenesis. Among CXC chemokines, single nucleotide polymorphisms (SNPs) in the CXCL8 and CXCL12 genes stand out, as they have alleles associated with many diseases such as asthma and human immunodeficiency virus (HIV), respectively. Of CC chemokines, the stronger associations occur among alleles from SNPs in CCL2 and CCL5 genes and a number of inflammatory conditions. To understand how chemokines contribute to disease it is also necessary to take into account all the isoforms resulting from differential splicing. The first part of this review deals with polymorphisms and the second with the diversity of molecular species derived from each chemokine gene due to alternative splicing phenomena. The number of molecular species and the level of expression of each of them for every chemokine and for each functionally related group of chemokines reaches a complexity that requires new modelling algorithms akin to those proposed in systems biology approaches.

The interaction between immune complexes (IC) and the receptors for the Fc portion of IgG (FcgammaRs) triggers regulatory and effector functions in the immune system. In this study, we investigated the effects of IC on differentiation, maturation, and functions of human monocyte-derived dendritic cells (DC). When IC were added on day 0, DC generated on day 6 (IC-DC) showed lower levels of CD1a and increased expression of CD14, MHC class II, and the macrophage marker CD68, as compared with normally differentiated DC. The use of specific blocking FcgammaR mAbs indicated that the effect of IC was exerted mainly through their interaction with FcgammaRI and to a lesser extend with FcgammaRII. Immature IC-DC also expressed higher levels of CD83, CD86, and CD40 and the expression of these maturation markers was not further regulated by LPS. The apparent lack of maturation following TLR stimulation was associated with a decreased production of IL-12, normal secretion of IL-10 and CCL22, and increased production of CXCL8 and CCL2. IC-DC displayed low endocytic activity and a reduced ability to induce allogeneic T cell proliferation both at basal and LPS-stimulated conditions. Altogether, these data reveal that IC strongly affect DC differentiation and maturation. Skewing of DC function from Ag presentation to a proinflammatory phenotype by IC resembles the state of activation observed in DC obtained from patients with chronic inflammatory autoimmune disorders, such as systemic lupus erythematosus disease and arthritis. Therefore, the altered maturation of DC induced by IC may be involved in the pathogenesis of autoimmune diseases.

Human immunodeficiency virus type 1 (HIV-1) entry into CD4(+) cells requires the chemokine receptors CCR5 or CXCR4 as co-fusion receptors. We have previously demonstrated that chemokine receptors are capable of cross-regulating the functions of each other and, thus, affecting cellular responsiveness at the site of infection. To investigate the effects of chemokine receptor cross-regulation in HIV-1 infection, monocytes and MAGIC5 and rat basophilic leukemia (RBL-2H3) cell lines co-expressing the interleukin-8 (IL-8 or CXCL8) receptor CXCR1 and either CCR5 (ACCR5) or CXCR4 (ACXCR4) were generated. IL-8 activation of CXCR1, but not the IL-8 receptor CXCR2, cross-phosphorylated CCR5 and CXCR4 and cross-desensitized their responsiveness to RANTES (regulated on activation normal T cell expressed and secreted) (CCL5) and stromal derived factor (SDF-1 or CXCL12), respectively. CXCR1 activation internalized CCR5 but not CXCR4 despite cross-phosphorylation of both. IL-8 pretreatment also inhibited CCR5- but not CXCR4-mediated virus entry into MAGIC5 cells. A tail-deleted mutant of CXCR1, DeltaCXCR1, produced greater signals upon activation (Ca(2+) mobilization and phosphoinositide hydrolysis) and cross-internalized CXCR4, inhibiting HIV-1 entry. The protein kinase C inhibitor staurosporine prevented phosphorylation and internalization of the receptors by CXCR1 activation. Taken together, these results indicate that chemokine receptor-mediated HIV-1 cell infection is blocked by receptor internalization but not desensitization alone. Thus, activation of chemokine receptors unrelated to CCR5 and CXCR4 may play a cross-regulatory role in the infection and propagation of HIV-1. Since DeltaCXCR1, but not CXCR1, cross-internalized and cross-inhibited HIV-1 infection to CXCR4, the data indicate the importance of the signal strength of a receptor and, as a consequence, protein kinase C activation in the suppression of HIV-1 infection by cross-receptor-mediated internalization.

The purpose of this study is to profile the changes in the serum levels of a range of chemokines, cytokines, and growth and angiogenic factors in MAPK inhibitor-treated metastatic melanoma patients and to correlate these changes with clinical outcome and changes in melanoma tissue biopsies taken from the same patients. Forty-two chemokine, cytokine, angiogenic, and growth factors were measured in the sera of 20 BRAF inhibitor-treated and four combination BRAF and MEK inhibitor-treated metastatic melanoma patients using a multiplex chemokine assay. The changes were correlated with Ki-67 and CD8(+) tumor-infiltrating lymphocytes in the tumor biopsies taken at the same time points, as well as clinical outcome, including response rate, progression-free survival, and overall survival. Serum levels of IFN-γ, CCL4, and TNF-α were significantly increased, whereas CXCL8 significantly decreased from pretreatment (PRE) to early during treatment (EDT) serum samples. The decrease in serum CXCL8 levels from PRE to EDT significantly correlated with decreases in markers of melanoma proliferation (Ki-67) and increases in cytotoxic tumor-infiltrating T cells in corresponding tumor biopsies. In addition, a greater fold reduction in CXCL8 serum levels from PRE to EDT serum samples was associated with decreased overall survival. These results suggest that BRAF inhibition causes decreased CXCL8 secretion from melanoma cells and induce an immune response against the tumor associated with increased IFN-γ, CCL4, and TNF-α. Further studies are needed to determine if CXCL8 is predictive of response and to confirm the functions of these chemokine and cytokine in BRAF-mutant melanoma under BRAF inhibition.

Cancer-associated fibroblasts (CAFs) have been shown to play an important role in angiogenesis, invasion, and metastasis. In the present study, we determined whether CAFs within the tumor microenvironment (TME) in head and neck squamous cell carcinoma (HNSCC) contributed to promoting immunosuppression and evasion from immune surveillance. Six pairs of CAFs and normal fibroblasts (NFs) were established from the resected tumor tissues of patients with HNSCC. The effects of CAFs and NFs on the functions of T cells were comparatively analyzed. CAFs expressed the co-regulatory molecules, B7H1 and B7DC, whereas NFs did not. The expression levels of cytokine genes, including those for IL6, CXCL8, TNF, TGFB1, and VEGFA, were higher in CAFs. T cell proliferation was suppressed more by CAFs or their supernatants than by NFs. Moreover, PBMCs co-cultured with the supernatants of CAFs preferentially induced T cell apoptosis and regulatory T cells over those co-cultured with the supernatants of NFs. A microarray analysis revealed that the level of genes related to the leukocyte extravasation and paxillin signaling pathways was higher in CAFs than in NFs. These results demonstrated that CAFs collaborated with tumor cells in the TME to establish an immunosuppressive network that facilitated tumor evasion from immunological destruction.

Toll-like receptors (TLRs) initiate innate immune responses and direct subsequent adaptive immunity. They play a major role in cutaneous host defense against micro-organisms and in the pathophysiology of several inflammatory skin diseases. To understand the role of TLRs in the acquisition of immunological competence, we conducted a comprehensive study to evaluate TLR expression and function in the developing human skin before and after birth and compared it with adults. We found that prenatal skin already expresses the same spectrum of TLRs as adult skin. Strikingly, many TLRs were significantly higher expressed in prenatal (TLRs 1-5) and infant and child (TLRs 1 and 3) skin than in adult skin. Surprisingly, neither dendritic cell precursors in prenatal skin nor epidermal Langerhans cells and dermal dendritic cells in adult skin expressed TLRs 3 and 6, whereas the staining pattern and intensity of both TLRs in fetal basal keratinocytes was almost comparable to those of adults. Stimulation of primary human keratinocytes from fetal, neonatal and adult donors with selected TLR agonists revealed that the synthetic TLR3 ligand poly (I:C) specifically, mimicking viral double-stranded RNA, induced a significantly enhanced secretion of CXCL8/IL8, CXCL10/IP-10 and TNFα in fetal and neonatal keratinocytes compared with adult keratinocytes. This study demonstrates quantitative age-specific modifications in TLR expression and innate skin immune reactivity in response to TLR activation. Thus, antiviral innate immunity already in prenatal skin may contribute to protect the developing human body from viral infections in utero in a scenario where the adaptive immune system is not yet fully functional.

BACKGROUND

Although inhaled glucocorticoids are the mainstays of asthma treatment, they are poorly effective at treating and preventing virus-induced asthma exacerbations. The major viruses precipitating asthma exacerbations are rhinoviruses.

OBJECTIVE

We sought to evaluate whether rhinovirus infection interferes with the mechanisms of action of glucocorticoids.

METHODS

Cultured primary human bronchial or transformed (A549) respiratory epithelial cells were infected with rhinovirus 16 (RV-16) before dexamethasone exposure. Glucocorticoid receptor (GR) α nuclear translocation, glucocorticoid response element (GRE) binding, and transactivation/transrepression functional readouts were evaluated by using immunocytochemistry, Western blotting, DNA binding assays, real-time quantitative PCR, coimmunoprecipitation, and ELISA techniques. Specific inhibitors of c-Jun N-terminal kinase (JNK) and of IκB kinase (IKK) were used to investigate the involvement of intracellular signaling pathways.

RESULTS

RV-16 infection impaired dexamethasone-dependent (1) inhibition of IL-1β-induced CXCL8 release, (2) induction of mitogen-activated protein kinase phosphatase 1 gene expression, and (3) binding of GR to GREs in airway epithelial cells. This was associated with impaired GRα nuclear translocation, as assessed by means of both immunochemistry (54.0% ± 6.8% vs 24.7% ± 3.8% GR-positive nuclei after 10 nmol/L dexamethasone treatment in sham- or RV-16-infected cells, respectively; P < .01) and Western blotting. RV-16 infection induced nuclear factor κB activation and GRα phosphorylation, which were prevented by inhibitors of IKK2 and JNK, respectively. In rhinovirus-infected cells the combination of JNK and IKK2 inhibitors totally restored dexamethasone suppression of CXCL8 release, induction of mitogen-activated protein kinase phosphatase 1 gene expression, and GRα nuclear translocation.

CONCLUSIONS

RV-16 infection of human airway epithelium induces glucocorticoid resistance. Inhibition of RV-16-induced JNK and nuclear factor κB activation fully reversed rhinovirus impairment of both GRα nuclear translocation and the transactivation/transrepression activities of glucocorticoids.

Regulatory molecules produced by stromal cells are often membrane bound until cleaved by matrix metalloproteinases (MMPs); cleavage can either activate or inactivate regulatory functions. We report here that marrow stromal cells induce the expression of MMP-9 in monocytes. Induction was contact independent and could be reproduced with recombinant MCP-1/CCL2, whereas IL-6, M-CSF, G-CSF, GM-CSF, IL-8/CXCL8, SDF-1/CXCL12, and MGSA/CXCL1 did not have this effect. Stroma-induced levels of MMP-9 in the monocyte population from healthy donors were relatively consistent, whereas induced levels varied significantly (P < .001) in the CD14+ population from 27 patients with myelodysplastic syndrome (MDS). In patients with a clonal chromosomal marker, the level of inducible MMP-9 expression in the monocyte population was inversely correlated with the percentage of marker-positive cells (n = 11, P = .01), suggesting that the ability to induce MMP-9 may be compromised in clonally derived monocytes. The inducible levels of MMP-9 were also inversely correlated with marrow cellularity observed in biopsies from MDS patients (P < .001). We conclude that monocytes can express MMP-9 in response to stromal factors and that this response may be significantly decreased in MDS-derived monocytes.

OBJECTIVE

The role of chemokines and their transporters in rheumatoid arthritis (RA) is poorly described. Evidence suggests that CXCL5 plays an important role, because it is abundant in RA tissue, and its neutralization moderates joint damage in animal models of arthritis. Expression of the chemokine transporter Duffy antigen receptor for chemokines (DARC) is also up-regulated in early RA. The aim of this study was to investigate the role of CXCL5 and DARC in regulating neutrophil recruitment, using an in vitro model of RA synovium.

METHODS

To model RA synovium, RA synovial fibroblasts (RASFs) were cocultured with endothelial cells (ECs) for 24 hours. Gene expression in cocultured cells was investigated using TaqMan gene arrays. The roles of CXCL5 and DARC were determined by incorporating cocultures into a flow-based adhesion assay, in which their function was demonstrated by blocking neutrophil recruitment with neutralizing reagents.

RESULTS

EC-RASF coculture induced chemokine expression in both cell types. Although the expression of CXC chemokines was modestly up-regulated in ECs, the expression of CXCL1, CXCL5, and CXCL8 was greatly increased in RASFs. RASFs also promoted the recruitment of flowing neutrophils to ECs. Anti-CXCL5 antibody abolished neutrophil recruitment by neutralizing CXCL5 expressed on ECs or when used to immunodeplete coculture-conditioned medium. DARC was also induced on ECs by coculture, and anti-Fy6 antibody or small interfering RNA targeting of DARC expression effectively abolished neutrophil recruitment.

CONCLUSIONS

This study is the first to demonstrate, in a model of human disease, that the function of DARC is essential for editing the chemokine signals presented by ECs and for promoting unwanted leukocyte recruitment.

BACKGROUND

Smokers who develop chronic obstructive pulmonary disease (COPD) have amplified inflammation within their lungs, involving selective tissue accumulation of neutrophils, macrophages and CD8+ T cells. CD11b (Mac-1, alphaMbeta(2)-integrin) is both a complement receptor (CR3) and a cell adhesion molecule present on the surface of peripheral blood leukocytes, and undergoes rapid surface upregulation from preformed cytoplasmic stores on activation. Cellular activation can also trigger chemotaxis and shape change, the activation itself being caused by the binding of chemokines to cell surface receptors.

METHODS

We developed a method of whole blood flow cytometry to measure neutrophil and monocyte CD11b upregulation on CD16+ and CD14+ cells, employing staining with the nuclear dye LDS-751 immediately before flow cytometry. In addition we assessed neutrophil shape change by modified gated autofluorescence with forward scatter (GAFS), this being correlated with chemotactic responses.

RESULTS

In smokers with COPD there was a lower maximal shape change for neutrophils in response to CXCL8 (IL-8) in comparison to healthy smokers (p=0.025), and a trend for lower expression of CD11b and shape change in response to CXCL1 (GRO-alpha). Neutrophils were found to predominantly express chemokine receptors CXCR1 and CXCR2 and respond to CXCL8 with CD11b upregulation, while monocytes express more CCR2 and upregulate CD11b preferentially to CCL2 (MCP-1). A CXCR2 antagonist (SB-656933) was found to inhibit neutrophil CD11b upregulation (IC50=260.7nM) and shape change (IC50=310.5nM) in COPD patients.

CONCLUSIONS

Neutrophils and monocytes participate in inflammatory processes in a range of diseases. These whole blood assays can be employed to monitor activity in disease and perform in vitro and ex vivo assessment of chemokine receptor (CXCR) antagonists.

Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a predominant cause of waterborne diarrheal disease that may lead to post-infectious functional gastrointestinal disorders. Although Giardia-infected individuals could carry as much as 106 trophozoites per centimetre of gut, their intestinal mucosa is devoid of overt signs of inflammation. Recent studies have shown that in endemic countries where bacterial infectious diseases are common, Giardia infections can protect against the development of diarrheal disease and fever. Conversely, separate observations have indicated Giardia infections may enhance the severity of diarrheal disease from a co-infecting pathogen. Polymorphonuclear leukocytes or neutrophils (PMNs) are granulocytic, innate immune cells characteristic of acute intestinal inflammatory responses against bacterial pathogens that contribute to the development of diarrheal disease following recruitment into intestinal tissues. Giardia cathepsin B cysteine proteases have been shown to attenuate PMN chemotaxis towards IL-8/CXCL8, suggesting Giardia targets PMN accumulation. However, the ability of Giardia infections to attenuate PMN accumulation in vivo and how in turn this effect may alter the host inflammatory response in the intestine has yet to be demonstrated. Herein, we report that Giardia infection attenuates granulocyte tissue infiltration induced by intra-rectal instillation of Clostridium difficile toxin A and B in an isolate-dependent manner. This attenuation of granulocyte infiltration into colonic tissues paralled decreased expression of several cytokines associated with the recruitment of PMNs. Giardia trophozoite isolates that attenuated granulocyte infiltration in vivo also decreased protein expression of cytokines released from inflamed mucosal biopsy tissues collected from patients with active Crohn's disease, including several cytokines associated with PMN recruitment. These results demonstrate for the first time that certain Giardia infections may attenuate PMN accumulation by decreasing the expression of the mediators responsible for their recruitment.

BACKGROUND

Rhinovirus (RV)-induced pulmonary exacerbations are common in cystic fibrosis (CF) and have been associated with impaired virus clearance by the CF airway epithelium in vitro. Here, we assess in vivo the association of RV prevalence and load with antiviral defense mechanisms, airway inflammation, and lung function parameters in children with CF compared with a control group and children with other chronic respiratory diseases.

METHODS

RV presence and load were measured by real-time reverse transcription-polymerase chain reaction in BAL samples and were related to antiviral and inflammatory mediators measured in BAL and to clinical parameters.

RESULTS

BAL samples were obtained from children with CF (n = 195), non-CF bronchiectasis (n = 40), or asthma (n = 29) and from a control group (n = 35) at a median (interquartile range [IQR]) age of 8.2 (4.0-11.7) years. RV was detected in 73 samples (24.4%). RV prevalence was similar among groups. RV load (median [IQR] x 10(3) copies/mL) was higher in children with CF (143.0 [13.1-1530.0]), especially during pulmonary exacerbations, compared with children with asthma (3.0 [1.3-25.8], P = .006) and the control group (0.5 [0.3-0.5], P < .001), but similar to patients with non-CF bronchiectasis (122.1 [2.7-4423.5], P = not significant). In children with CF, RV load was negatively associated with interferon (IFN)- b and IFN- l , IL-1ra levels, and FEV 1 , and positively with levels of the cytokines CXCL8 and CXCL10.

CONCLUSIONS

RV load in CF BAL is high, especially during exacerbated lung disease. Impaired production of antiviral mediators may lead to the high RV burden in the lower airways of children with CF. Whether high RV load is a cause or a consequence of inflammation needs further investigation in longitudinal studies.

Chronic inflammation is associated with epithelial to mesenchymal transition (EMT) and cancer progression however the relationship between inflammation and EMT remains unclear. Here, we have exploited zebrafish to visualize and quantify the earliest events during epithelial cell transformation induced by oncogenic HRas(V12). Live imaging revealed that expression of HRas(V12) in the epidermis results in EMT and chronic neutrophil and macrophage infiltration. We have developed an in vivo system to probe and quantify gene expression changes specifically in transformed cells from chimeric zebrafish expressing oncogenic HRas(V12) using translating ribosomal affinity purification (TRAP). We found that the expression of genes associated with EMT, including slug, vimentin and mmp9, are enriched in HRas(V12) transformed epithelial cells and that this enrichment requires the presence of neutrophils. An early signal induced by HRas(V12) in epithelial cells is the expression of il-8 (cxcl8) and we found that the chemokine receptor, Cxcr2, mediates neutrophil but not macrophage recruitment to the transformed cells. Surprisingly, we also found a cell autonomous role for Cxcr2 signaling in transformed cells for both neutrophil recruitment and EMT related gene expression associated with Ras transformation. Taken together, these findings implicate both autocrine and paracrine signaling through Cxcr2 in the regulation of inflammation and gene expression in transformed epithelial cells.

BACKGROUND

MicroRNAs (miRNAs) regulate gene expression by interfering translation or stability of target transcripts. This interplay between miRNA and their mRNA has been proposed as an important process in cancer development and progression. We have investigated molecular networks impacted by predicted mRNA targets of differentially expressed miRNAs in patients with clear cell renal cell carcinoma (ccRCC) diagnosed with or without metastasis.

METHODS

miRNA and mRNA microarray expression profiles derived from primary ccRCC from patients with (16 samples) or without diagnosed metastasis (22 samples) were used to identify anti-correlated miRNA-mRNA interaction in ccRCC. For this purpose, Ingenuity pathway analysis microRNA Target Filter, which enables prioritization of experimentally validated and predicted mRNA targets was used. By applying an expression pairing tool, the analysis was focused on targets exhibiting altered expression in our analysis, finding miRNAs and their target genes with opposite or same expression. The resulting identified interactions were revalidated by RT-qPCR in another cohort of ccRCC patients. A selection of the predicted miRNA-mRNA interactions was tested by functional analyses using miRNA knockdown and overexpression experiments in renal cancer cell lines.

RESULTS

Among the significantly differentially expressed miRNAs, we have identified three miRNAs (miR-146a-5p, miR-128a-3p, and miR-17-5p) that were upregulated in primary tumors from patients without metastasis and downregulated in primary tumors from patients with metastasis. We have further identified mRNA targets, which expression were inversely correlated to these 3 miRNAs, and have been previously experimentally demonstrated in cancer setting in humans. Specifically, we showed that CXCL8/IL8, UHRF1, MCM10, and CDKN3 were downregulated and targeted by miR-146a-5p. The interaction between miR-146a-5p and their targets CXCL8 and UHRF1 was validated in cell culture experiments.

CONCLUSIONS

We identified novel target genes of dysregulated miRNAs, which are involved in the transition from primary RCC without metastases into tumors generating distant metastasis.

Leishmania mexicana can cause both localized (LCL) and diffuse (DCL) cutaneous leishmaniasis, yet little is known about factors regulating disease severity in these patients. We analyzed if the disease was associated with single nucleotide polymorphisms (SNPs) in IL-1β (-511), CXCL8 (-251) and/or the inhibitor IL-1RA (+2018) in 58 Mexican mestizo patients with LCL, 6 with DCL and 123 control cases. Additionally, we analyzed the in vitro production of IL-1β by monocytes, the expression of this cytokine in sera of these patients, as well as the tissue distribution of IL-1β and the number of parasites in lesions of LCL and DCL patients. Our results show a significant difference in the distribution of IL-1β (-511 C/T) genotypes between patients and controls (heterozygous OR), with respect to the reference group CC, which was estimated with a value of 3.23, 95% CI = (1.2, 8.7) and p-value = 0.0167), indicating that IL-1β (-511 C/T) represents a variable influencing the risk to develop the disease in patients infected with Leishmania mexicana. Additionally, an increased in vitro production of IL-1β by monocytes and an increased serum expression of the cytokine correlated with the severity of the disease, since it was significantly higher in DCL patients heavily infected with Leishmania mexicana. The distribution of IL-1β in lesions also varied according to the number of parasites harbored in the tissues: in heavily infected LCL patients and in all DCL patients, the cytokine was scattered diffusely throughout the lesion. In contrast, in LCL patients with lower numbers of parasites in the lesions, IL-1β was confined to the cells. These data suggest that IL-1β possibly is a key player determining the severity of the disease in DCL patients. The analysis of polymorphisms in CXCL8 and IL-1RA showed no differences between patients with different disease severities or between patients and controls.

The aim of this study was to elucidate the characteristics, pathogenesis and treatment strategy of hypertrophic pachymeningitis that is associated with myeloperoxidase anti-neutrophil cytoplasmic antibody (ANCA). We retrospectively investigated clinical, radiological, immunological and pathological profiles of 36 patients with immune-mediated or idiopathic hypertrophic pachymeningitis, including 17 patients with myeloperoxidase-ANCA, four patients with proteinase 3-ANCA, six patients with other immune-mediated disorders, and nine patients with 'idiopathic' variety. Myeloperoxidase-ANCA-positive hypertrophic pachymeningitis was characterized by: (i) an elderly female predominance; (ii) 82% of patients diagnosed with granulomatosis with polyangiitis (previously known as Wegener's granulomatosis) according to Watts' algorithm; (iii) a high frequency of patients with lesions limited to the dura mater and upper airways, developing headaches, chronic sinusitis, otitis media or mastoiditis; (iv) a low frequency of patients with the 'classical or generalized form' of granulomatosis with polyangiitis involving the entire upper and lower airways and kidney, or progressing to generalized disease, in contrast to proteinase 3-ANCA-positive hypertrophic pachymeningitis; (v) less severe neurological damage according to the modified Rankin Scale and low disease activity according to the Birmingham Vasculitis Activity Score compared with proteinase 3-ANCA-positive hypertrophic pachymeningitis; (vi) increased levels of CXCL10, CXCL8 and interleukin 6 in cerebrospinal fluids, and increased numbers of T cells, neutrophils, eosinophils, plasma cells and monocytes/macrophages in autopsied or biopsied dura mater with pachymeningitis, suggesting TH1-predominant granulomatous lesions in hypertrophic pachymeningitis, as previously reported in pulmonary or renal lesions of granulomatosis with polyangiitis; and (vii) greater efficacy of combination therapy with prednisolone and cyclophosphamide compared with monotherapy with prednisolone. Proteinase 3-ANCA may be considered a marker for more severe neurological damage, higher disease activity and a higher frequency of the generalized form compared with myeloperoxidase-ANCA-positive hypertrophic pachymeningitis. However, categorization into 'granulomatosis with polyangiitis' according to Watts' algorithm and immunological or pathological features were common in both proteinase 3- and myeloperoxidase-ANCA-positive hypertrophic pachymeningitis. These data indicate that most patients with myeloperoxidase-ANCA-positive hypertrophic pachymeningitis should be categorized as having the central nervous system-limited form of ANCA-associated vasculitis, consistent with the concept of ophthalmic-, pulmonary- or renal-limited vasculitis.

OBJECTIVE

Pancreatic cancer is characterized by perineural invasion, early lymph node and liver metastases, and an extremely dismal prognosis. In the present study we aimed at investigating the expression profile of pro-inflammatory and angiogenic CXC chemokines as potential factors contributing to the aggressive biology of this gastrointestinal malignancy.

METHODS

Protein expression profiles of the CXC chemokines growth-related oncogene alpha (GRO-alpha/CXCL1), epithelial cell-derived neutrophil-activating peptide-78 (ENA-78/CXCL5), granulocyte chemoattractant protein-2 (GCP-2/CXCL6), neutrophil-activating protein-2 (NAP-2/CXCL7), and interleukin-8 (IL-8/CXCL8) were assessed by enzyme-linked immunosorbent assay in pancreatic carcinoma, cancer of the papilla of Vater, pancreatic cystadenoma, and chronic pancreatitis specimens.

RESULTS

IL-8 and ENA-78 protein expression was most pronounced in pancreatic carcinoma specimens, showing an 11-fold and 17-fold overexpression in comparison with non-affected neighbouring tissues, a 66-fold and 24-fold upregulation compared to pancreatic cystadenoma, and a 6-fold and 9-fold overexpression with respect to chronic pancreatitis, respectively (p < 0.05 between all groups). In addition, a close correlation between IL-8 and ENA-78 protein expression and advanced pancreatic carcinomas in relation to the T category was evident (p < 0.05).

CONCLUSIONS

Our results demonstrate that ELR+ CXC chemokines are differentially expressed in malignant and non-malignant human pancreatic specimens, suggesting a potential contribution of these chemokines to the pathogenesis of pancreatic carcinoma.

CXC chemokines bearing the glutamic acid-leucine-arginine (ELR) motif are crucial mediators in neutrophil-dependent acute inflammation. Interestingly, however, Interleukin (IL)-8/CXC ligand (CXCL) 8 is expressed in human milk in biologically significant concentrations, and may play a local maturational role in the developing human intestine. In this chemokine subfamily, there are six other known peptides beside IL-8/CXCL8, all sharing similar effects on neutrophil chemotaxis and angiogenesis. In this study, we measured the concentrations of these chemokines in human milk, sought their presence in human mammary tissue by immunohistochemistry, and confirmed chemokine expression in cultured human mammary epithelial cells (HMECs). Each of the seven ELR(+) CXC chemokines was measurable in milk, and except for NAP-2/CXCL7, these concentrations were higher than serum. The concentrations were higher in colostrum (except for GRO-beta/CXCL2 and NAP-2/CXCL7), and correlated negatively with time elapsed postpartum. IL-8/CXCL8, GRO-gamma/CXCL3, and ENA-78/CXCL5 concentrations were higher in preterm milk. There was intense immunoreactivity in mammary epithelial cells for all ELR(+) CXC chemokines, and the intensity of staining was higher in breast tissue with lactational changes. The supernatants from confluent HMEC cultures also contained measurable concentrations of all the seven ELR(+) CXC chemokines. These results confirm that all ELR(+) CXC chemokines are actively secreted by the mammary epithelial cells into human milk. Further studies are needed to determine if these chemokines share with IL-8/CXCL8 the protective effects on intestinal epithelial cells.

The house dust mite (Dermatophagoides pteronissinus) plays an important role in the pathogenesis of allergic diseases, including atopic dermatitis, and asthma. Monocyte chemotactic protein 1 (MCP-1/CCL2)/IL-6/IL-8 (CXCL8) plays a pivotal role in mediating the infiltration of various cells into the skin of atopic dermatitis and psoriasis. The aim of this study was to investigate the effect of D. pteronissinus extract (DpE) on expression of MCP-1/IL-6/IL-8 mRNA and protein and the signal transduction in the human monocytic cell line, THP-1. The mRNA and protein expression of MCP-1/CCL2, IL-6, and IL-8 were elevated by DpE in a time and dose-dependent manner in THP-1 cells. The increased expression of MCP-1, IL-6, and IL-8 was not affected by aprotinin (serine protease inhibitor) or E64 (cysteine protease inhibitor). We found that MCP-1 and IL-6 expression due to DpE was related to Src, protein kinase C delta (PKC delta), extracellular-signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) and IL-8 expression was involved in Src family tyrosine kinase, PKC delta, ERK. DpE increased the phosphorylation of ERK and p38 MAPK after 5min and peaked at 30min. The activation was significantly blocked by PP2, an inhibitor of Src family tyrosine kinase and rottlerin, an inhibitor of PKC delta (p<0.01). DpE increases MCP-1, IL-6, and IL-8 expression and transduces its signal via Src family tyrosine kinase, PKC, and ERK in a protease-independent manner. This finding may contribute to the elucidation of the pathogenic mechanism triggered by DpE .

Glioma cells release cytokines to stimulate inflammation that facilitates cell proliferation. Here, we show that Lipopolysaccharide (LPS) treatment could induce glioma cells to proliferate and this process was dependent on nucleotide receptor activation as well as interleukin-8 (IL-8/CXCL8) secretion. We observed that extracellular nucleotides controlled IL-8/CXCL8 and monocyte chemoattractant protein 1 (MCP-1/CCL2) release by U251MG and U87MG human glioma cell lines via P2X7 and P2Y6 receptor activation. The LPS-induced release of these cytokines was also modulated by purinergic receptor activation since IL-8 and MCP-1 release was decreased by the nucleotide scavenger apyrase as well as by the pharmacological P2Y6 receptor antagonists suramin and MRS2578. In agreement with these observations, the knockdown of P2Y6 expression decreased LPS-induced IL-8 release as well as the spontaneous release of IL-8 and MCP-1, suggesting an endogenous basal release of nucleotides. Moreover, high millimolar concentrations of ATP increased IL-8 and MCP-1 release by the glioma cells stimulated with suboptimal LPS concentration which were blocked by P2X7 and P2Y6 antagonists. Altogether, these data suggest that extracellular nucleotides control glioma growth via P2 receptor-dependent IL-8 and MCP-1 secretions.

Several dermatoses, including psoriasis, atopic dermatitis, and rosacea, alter the expression of the innate immune effector human cathelicidin antimicrobial peptide (CAMP). To elucidate the roles of aberrant CAMP in dermatoses, we performed cDNA array analysis in CAMP-stimulated human epidermal keratinocytes, the primary cells responding to innate immune stimuli and a major source of CAMP LL37 in skin. Among LL37-inducible genes, IL-1 cluster genes, particularly IL36G, are of interest because we observed coordinate increases in CAMP and IL-36γ in the lesional skin of psoriasis, whereas virtually no CAMP or IL-36γ was observed in nonlesional skin and normal skin. The production and release of IL-36γ were up to 20-30 ng/ml in differentiated keratinocytes cultured in high-calcium media. G-protein inhibitor pertussis toxin and p38 inhibitor suppressed IL-36γ induction by LL37. As an alarmin, LL37 induces chemokines, including CXCL1, CXCL8/IL8, CXCL10/IP-10, and CCL20/MIP3a, and IL-36 (10-100 ng/ml) augments the production of these chemokines by LL37. Pretreatment with small interfering RNA against IL36γ and IL-36R IL36R/IL1RL2 and IL1RAP suppressed LL37-dependent IL8, CXCL1, CXCL10/IP10, and CCL20 production in keratinocytes, suggesting that the alarmin function of LL37 was partially dependent on IL-36γ and its receptors. Counting on CAMP induction in innate stimuli, such as in infection and wounding, IL-36γ induction by cathelicidin would explain the mechanism of initiation of skin inflammation and occasional exacerbations of psoriasis and skin diseases by general infection.

We had reported elevated serum levels of the peptide neurotensin (NT) in children with autism spectrum disorders (ASD). Here, we show that NT stimulates primary human microglia, the resident immune cells of the brain, and the immortalized cell line of human microglia-SV40. NT (10 nM) increases the gene expression and release (P < 0.001) of the proinflammatory cytokine IL-1β and chemokine (C-X-C motif) ligand 8 (CXCL8), chemokine (C-C motif) ligand 2 (CCL2), and CCL5 from human microglia. NT also stimulates proliferation (P < 0.05) of microglia-SV40. Microglia express only the receptor 3 (NTR3)/sortilin and not the NTR1 or NTR2. The use of siRNA to target sortilin reduces (P < 0.001) the NT-stimulated cytokine and chemokine gene expression and release from human microglia. Stimulation with NT (10 nM) increases the gene expression of sortilin (P < 0.0001) and causes the receptor to be translocated from the cytoplasm to the cell surface, and to be secreted extracellularly. Our findings also show increased levels of sortilin (P < 0.0001) in the serum from children with ASD (n = 36), compared with healthy controls (n = 20). NT stimulation of microglia-SV40 causes activation of the mammalian target of rapamycin (mTOR) signaling kinase, as shown by phosphorylation of its substrates and inhibition of these responses by drugs that prevent mTOR activation. NT-stimulated responses are inhibited by the flavonoid methoxyluteolin (0.1-1 μM). The data provide a link between sortilin and the pathological findings of microglia and inflammation of the brain in ASD. Thus, inhibition of this pathway using methoxyluteolin could provide an effective treatment of ASD.

Secretion of proinflammatory cytokines by LPS activated endothelial cells contributes substantially to the pathogenesis of sepsis. However, the mechanism involved in this process is not well understood. In the present study, we determined the role of a nonreceptor proline-rich tyrosine kinase, Pyk2, in LPS-induced IL-8 (CXCL8) production in endothelial cells. First, we observed a marked activation of Pyk2 in response to LPS. Furthermore, inhibition of Pyk2 activity in these cells by transduction with the catalytically inactive Pyk2 mutant, transfection with Pyk2-specific small interfering RNA, or treatment with Tyrphostin A9 significantly blocked LPS-induced IL-8 production. The supernatants of LPS-stimulated cells exhibiting attenuated Pyk2 activity blocked transendothelial neutrophil migration in comparison to the supernatants of LPS-treated controls, thus confirming the inhibition of functional IL-8 production. Investigations into the molecular mechanism of this pathway revealed that LPS activates Pyk2 leading to IL-8 production through the TLR4. In addition, we identified the p38 MAPK pathway to be a critical step downstream of Pyk2 during LPS-induced IL-8 production. Taken together, these results demonstrate a novel role for Pyk2 in LPS-induced IL-8 production in endothelial cells.