BACKGROUND

The expression of pro-angiogenic cytokines, such as vascular endothelial growth factor (VEGF) and interleukin-8/CXCL8 (IL-8), plays an important role in tumor growth and metastasis. Low oxygen tension within poorly-vascularized tumors is thought to be the prime stimulus causing the secretion of VEGF. The expression of IL-8 by solid tumors is thought to be primarily due to intrinsic influences, such as constitutive activation of nuclear factor kappa B (NF-kappaB). However, VEGF expression is responsive to glucose deprivation, suggesting that low concentrations of nutrients other than oxygen may play a role in triggering the pro-angiogenic phenotype. Glucose deprivation causes endoplasmic reticulum (ER) stress and alters gene expression through the unfolded protein response (UPR) signaling pathway. A branch of the UPR, known as the ER overload response (EOR), can cause NF-kappaB activation. Thus, we hypothesized that treatments that cause ER stress and deprivation of other nutrients, such as amino acids, would trigger the expression of angiogenic cytokines by breast cancer cell lines.

RESULTS

We found that glutamine deprivation and treatment with a chemical inducer of ER stress (tunicamycin) caused a marked induction of the secretion of both VEGF and IL-8 protein by a human breast adenocarcinoma cell line (TSE cells). Glutamine deprivation, glucose deprivation and several chemical inducers of ER stress increased VEGF and IL-8 mRNA expression in TSE and other breast cancer cell lines cultured under both normoxic and hypoxic conditions, though hypoxia generally diminished the effects of glucose deprivation. Of all amino acids tested, ambient glutamine availability had the largest effect on VEGF and IL-8 mRNA expression. The induction of VEGF mRNA expression, but not IL-8, was sustained and closely corresponded with the upregulated expression of the ER stress-responsive genes glucose-regulated protein 78 (GRP78) and growth arrest and DNA damage inducible gene 153 (GADD153).

CONCLUSIONS

These results suggest that nutrient deprivation within the solid tumor microenvironment might contribute to the activation of a pro-angiogenic phenotype. The angiogenic switch may act to increase blood supply in response to nutrient deprivation as well as hypoxia.

Viruses are associated with the majority of exacerbations of asthma and chronic obstructive pulmonary disease. Virus induction of neutrophil and lymphocyte chemokines in bronchial epithelium is important in exacerbation pathogenesis. Combined corticosteroid/beta2 agonists synergistically suppress airway smooth muscle chemokine production. Because bronchial epithelium expresses glucocorticoid and beta2 receptors, we investigated whether combination therapy can synergistically suppress rhinovirus-induced bronchial epithelial cell neutrophil (CXCL5, CXCL8) and lymphocyte (CCL5, CXCL10) chemokine production. We investigated modulation of rhinovirus- and IL-1beta-induced bronchial epithelial cell chemokine production by salmeterol and fluticasone propionate, used at therapeutic concentrations, alone and in combination. After 1 h pretreatment, combined treatment significantly inhibited rhinovirus 16, 1B, and IL-1beta-induced CCL5 and CXCL8 protein and mRNA production in BEAS-2B cells compared with fluticasone alone. When used 4 h after treatment, the combination significantly reduced virus-induced CCL5 but not CXCL8. Salmeterol alone had no effect; therefore, this inhibition was synergistic. Kinetic analysis demonstrated that combination therapy reduced by 5-fold the concentration of corticosteroid required to inhibit CXCL8 mRNA expression. In primary cells, salmeterol alone reduced rhinovirus-induced CCL5 and CXCL10 and increased CXCL5 production in a dose-dependent manner but had no effect on CXCL8. Fluticasone alone reduced CCL5, CXCL8, and CXCL10 but had no effect on CXCL5. Combination therapy augmented inhibition of CXCL8, CCL5, and CXCL10 but had no effect on CXCL5. Corticosteroids and beta2 agonists suppress rhinovirus-induced chemokines in bronchial epithelial cells through synergistic and additive mechanisms. This effect was greater for lymphocyte- than for neutrophil-related chemokines.

OBJECTIVE

Vasoactive intestinal peptide (VIP) has demonstrated therapeutic effects in arthritis by inhibiting both innate and acquired immune responses. We investigated the potential effects of VIP in the regulation of Toll-like receptor (TLR) expression and function in synovial fibroblasts from patients with rheumatoid arthritis (RA) and osteoarthritis (OA).

METHODS

Cultured fibroblast-like synoviocytes (FLS) were obtained from patients with RA and OA. The effects of VIP on basal or TNF-alpha or lipopolysaccharide (LPS)-induced TLR2, TLR4 and MyD88 expression and its effects on TLR4-mediated CCL2 and CXCL8 chemokine production were studied by reverse transcription-polymerase chain reaction, western blotting and enzyme-linked immunosorbent assay.

RESULTS

TLR2, TLR4 and MyD88 mRNA expression was increased in RA FLS compared with OA FLS. The largest increase was observed for TLR4 and there was also overexpression at the protein level in RA FLS. TLR4 and MyD88 mRNA and proteins were induced by LPS and TNF-alpha in RA FLS. VIP down-regulated the induced but not the constitutive expression of TLR4 and MyD88 in RA FLS. VIP treatment decreased CCL2 and CXCL8 chemokine production in response to TLR4 activation with LPS in RA FLS.

CONCLUSIONS

We demonstrate that VIP down-regulates LPS and TNF-alpha activation of TLR4 expression and the TLR4 functional response in terms of proinflammatory chemokine production. These studies suggest that the pleiotropic anti-inflammatory actions of VIP involve inhibitory effects on TLR4 expression and signalling.

Interleukin-8 (IL-8 or CXCL8) plays a critical role in orchestrating the immune response by binding and activating the receptor CXCR1 that belongs to the GPCR class. IL-8 exists as both monomers and dimers, and both bind CXCR1 but with differential affinities. It is well established that the monomer is the high-affinity ligand and that the interactions between the ligand N-loop and receptor N-domain play a critical role in determining binding affinity. In order to characterize the structural basis of differential binding of the IL-8 monomer and dimer to the CXCR1 N-domain, we analyzed binding-induced NMR chemical shift and peak intensity changes and show that they are exquisitely sensitive and can provide detailed insights into the binding process. We used three IL-8 variants, a designed monomer, a trapped disulfide-linked dimer, and WT at dimeric concentrations. NMR data for the monomer show that nonsequential residues that span the entire N-loop are involved in the binding process and that the binding is mediated by a network of extensive direct and indirect coupled interactions. Interestingly, in the case of WT, binding induces dissociation of the dimer-receptor complex to the monomer-receptor complex, and in the case of the trapped dimer, binding results in increased global conformational flexibility. Increased dynamics is evidence of unfavorable interactions, indicating that binding of the WT dimer triggers conformational changes that disrupt dimer-interface interactions, resulting in its dissociation. These results together provide evidence that binding is not a localized event but results in extensive coupled interactions within the monomer and across the dimer interface and that these interactions play a fundamental role in determining binding affinity.

More than 40% of colon cancers have a mutation in K-RAS or N-RAS, GTPases that operate as central hubs for multiple key signaling pathways within the cell. Utilizing an isogenic panel of colon carcinoma cells with K-RAS or N-RAS variations, we observed differences in tumor necrosis factor-alpha (TNFalpha)-induced apoptosis. When the dynamics of phosphorylated ERK response to TNFalpha were examined, K-RAS mutant cells showed lower activation whereas N-RAS mutant cells exhibited prolonged duration. These divergent trends were partially explained by differential induction of two ERK-modulatory circuits: negative feedback mediated by dual-specificity phosphatase 5 and positive feedback by autocrine transforming growth factor-alpha. Moreover, in the various RAS mutant colon carcinoma lines, the transforming growth factor-alpha autocrine loop differentially elicited a further downstream chemokine (CXCL1/CXCL8) autocrine loop, with the two loops having opposite effects on apoptosis. Although the apoptotic responses of the RAS mutant panel to TNFalpha treatment showed significant dependence on the respective phosphorylated ERK dynamics, successful prediction across the various cell lines required contextual information concerning additional pathways including IKK and p38. A quantitative computational model based on weighted linear combinations of these pathway activities successfully predicted not only the spectrum of cell death responses but also the corresponding chemokine production responses. Our findings indicate that diverse RAS mutations yield differential cell behavioral responses to inflammatory cytokine exposure by means of (a) differential effects on ERK activity via multiple feedback circuit mechanisms, and (b) differential effects on other key signaling pathways contextually modulating ERK-related dependence.

BACKGROUND

Initiation of antiretroviral therapy (ART) in human immunodeficiency virus patients with treated or unrecognized Mycobacterium tuberculosis infection may result in tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) or ART-associated tuberculosis (ART-TB), respectively. Both conditions appear to be immune restoration disease but their immunopathogenesis is not completely understood.

METHODS

Chemokines and cytokines produced by the innate immune system (CCL2, CXCL8, CXCL9, CXCL10, and interleukin 18 [IL-18]) were assayed in plasma from unstimulated whole blood cultures obtained from 15 TB-IRIS case patients, 11 ART-TB case patients, and matched control participants over 24 weeks of ART.

RESULTS

When compared with control participants, levels of IL-18 and CXCL10 were higher in TB-IRIS case patients (P = .002 and .006, respectively), whereas CCL2 was lower (P = .006). IL-18 level was higher in ART-TB case patients (P = .002), but CXCL10 was only marginally higher (P = .06). When TB-IRIS case patients were compared with ART-TB case patients, IL-18 was higher in ART-TB (P = .03), whereas CXCL10 was higher in TB-IRIS (P = .001). Using receiver operating characteristic curves, pre-ART levels of CCL2, CXCL10, and IL-18 were predictive of TB-IRIS and additive to IFN-γ responses.

CONCLUSIONS

Perturbations of the innate immune response to M. tuberculosis before and during ART may contribute to the immunopathology of TB-IRIS, whereas elevated IL-18 alone suggests adaptive immune responses predominate in ART-TB. These findings may have implications for therapy in TB-IRIS.

Cytokines and chemokines in cerebrospinal fluid (CSF) can have implications on the pathogenesis of neuro-Behçet's disease (NB). CSF and serum samples from 33 patients with NB, 25 with multiple sclerosis (MS), 20 patients with infectious and/or inflammatory neurological diseases (IN) and 14 with other noninflammatory neurological diseases (NIN) were investigated by ELISA. In the CSF, CXCL10 levels were significantly higher in NB and IN than NIN and MS, whereas CXCL8 was increased in NB compared to NIN. CCL2 levels in MS CSF and sera were lower, whereas CXCL8 in MS sera was higher than the other groups. IL-12 was elevated in CSF of IN compared to NB and NIN and also in the CSF of MS compared to NIN. No difference was detected for IL-10 and IL-17. These results reflect that NB has a mediator pattern in resemblance with non-specific inflammations such as neuro-infections compared to autoimmune disorders such as multiple sclerosis, suggesting that a currently unknown infection might be the trigger of a vasculitic process in the central nervous system (CNS).

CXCL8 (interleukin-8) interacts with two receptors, CXCR1 and CXCR2, to activate leukocytes. Upon activation, CXCR2 internalizes very rapidly relative to CXCR1 ( approximately 90% versus approximately 10% after 5 min). The C termini of the receptors have been shown to be necessary for internalization but are not sufficient to explain the distinct kinetics of down-regulation. To determine the structural determinant(s) that modulate receptor internalization, various chimeric and point mutant receptors were generated by progressively exchanging specific domains or amino acids between CXCR1 and CXCR2. The receptors were stably expressed in rat basophilic leukemia 2H3 cells and characterized for receptor binding, intracellular Ca(2+) mobilization, phosphoinositide hydrolysis, phosphorylation, internalization, and MAPK activation. The data herein indicate that the second extracellular loop (2ECL) of the receptors is critical for the distinct rate of internalization. Replacing the 2ECL of CXCR2 with that of CXCR1 (B(2ECL)A) or Asp(199) with its CXCR1 valine counterpart (B(D199V)A) delayed CXCR2 internalization similarly to CXCR1. Replacing Asp(199) with Asn (B(D199N)) restored CXCR2 rapid internalization. Structure modeling of the 2ECL of the receptors also suggested that Asp(199) plays a critical role in stabilizing and modulating CXCR2 rapid internalization relative to CXCR1. B(D199N) internalized rapidly but migrated as a single phosphorylated form like CXCR1 ( approximately 75 kDa), whereas B(2ECL)A and B(D199V)A showed slow and fast migrating forms like CXCR2 ( approximately 45 and approximately 65 kDa, respectively) but internalized like CXCR1. These data further undermine the role of receptor oligomerization in CXCL8 receptor internalization. Like CXCR1, B(D199V)A also induced sustained ERK activation and cross-desensitized Ca(2+) mobilization to CCR5 relative to B(D199N) and CXCR2. Altogether, the data suggest that the 2ECL of the CXCL8 receptors is important in modulating their distinct rate of down-regulation and thereby signal length and post-internalization activities.

MERS-CoV, a highly pathogenic virus in humans, is associated with high morbidity and case fatality. Inflammatory responses have a significant impact on MERS-CoV pathogenesis and disease outcome. However, CD4⁺ T-cell induced immune responses during acute MERS-CoV infection are barely detectable, with potent inhibition of effector T cells and downregulation of antigen presentation. The local pulmonary immune response, particularly the Th1 and Th2-related immune response during acute severe MERS-CoV infection is not fully understood. In this study, we offer the first insights into the pulmonary gene expression profile of Th1 and Th2-related cytokines/chemokines (Th1 & Th2 responses) during acute MERS-CoV infection using RT² Profiler PCR Arrays. We also quantified the expression level of primary inflammatory cytokines/chemokines. Our results showed a downregulation of Th2, inadequate (partial) Th1 immune response and high expression levels of inflammatory cytokines IL-1α and IL-1β and the neutrophil chemoattractant chemokine IL-8 (CXCL8) in the lower respiratory tract of MERS-CoV infected patients. Moreover, we identified a high viral load in all included patients. We also observed a correlation between inflammatory cytokines, Th1, and Th2 downregulation and the case fatality rate. Th1 and Th2 response downregulation, high expression of inflammatory cytokines, and high viral load may contribute to lung inflammation, severe infection, the evolution of pneumonia and ARDS, and a higher case fatality rate. Further study of the molecular mechanisms underlying the Th1 and Th2 regulatory pathways will be vital for active vaccine development and the identification of novel therapeutic strategies.

CXCR1 and CXCR2 are receptors for angiogenic ELR+CXC chemokines and are differentially expressed on endothelial cells; however, their functional significance in angiogenesis remains unclear. In this study, we determined the functional significance of these receptors in modulating endothelial cell phenotype by knocking-down the expression of CXCR1 and/or CXCR2 in human microvascular endothelial cells (HMEC-1) using short-hairpin RNA (shRNA). Cell proliferation, migration, invasion and capillary-like structure (CLS) formation were analyzed. Our data demonstrate that knock-down of CXCR1 and/or CXCR2 expression inhibited endothelial cell proliferation, survival, migration, invasion and CLS formation. Additionally, we examined the mechanism of CXCL8-dependent CXCR1 and/or CXCR2 mediated phenotypic changes by evaluating ERK phosphorylation and cytoskeletal rearrangement and observed inhibition of ERK phosphorylation and cytoskeletal rearrangement in HMEC-1-shCXCR1, HMEC-1-shCXCR2 and HMEC-1-shCXCR1/2 cells. Together, these data demonstrate that CXCR1 and CXCR2 expression plays a critical role in regulating multiple biological activities in human microvascular endothelial cells.

There is considerable interest in the use of azithromycin for the treatment of lung disease in patients with cystic fibrosis (CF). Although its mechanism of action as an inhibitor of bacterial protein synthesis has been well-established, it is less clear how azithromycin ameliorates the lung disease associated with Pseudomonas aeruginosa, which is considered to be resistant to the drug. We tested the effects of azithromycin on clinical isolates (CIs) from CF patients and compared them with laboratory reference strains to establish how this drug might interfere with the production of bacterial virulence factors that are relevant to the pathogenesis of airway disease in CF patients. Azithromycin inhibited P aeruginosa PAO1 protein synthesis by 80%, inhibiting bacterial growth and the expression of immunostimulatory exoproducts such as pyocyanin, as well as the gene products necessary for biofilm formation. In contrast, the effects of azithromycin on CIs of P aeruginosa were much more variable, due in large part to their slow growth and limited exoproduct expression. Culture supernatants for two of three clinical strains induced appreciable CXCL8 expression from cultured epithelial cells. Azithromycin treatment of the organisms inhibited 65 to 70% of this induction; azithromycin had no direct effect on the ability of either normal cells or CF epithelial cells to produce CXCL8. Azithromycin does decrease the P aeruginosa synthesis of immunostimulatory exoproducts and is likely to be most effective against planktonic, actively growing bacteria. This effect is less predictable against CIs than the prototypic strain PAO1.

OBJECTIVE

The aim of this study was to analyze both the constitutive and induced expression and function of double-stranded RNA (dsRNA; Toll-like receptor 3 [TLR-3], retinoic acid-inducible gene I [RIG-I], and melanoma differentiation-associated gene 5 [MDA5]) and single-stranded RNA (ssRNA; TLR-7) receptors in osteoarthritis (OA) and rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS), by studying the transcription factors involved and the subsequent effects on antiviral interferon-β (IFNβ), the proinflammatory CXCL8 chemokine, and matrix metalloproteinase 3 (MMP-3). An additional goal was to study the effect of vasoactive intestinal peptide (VIP).

METHODS

The expression of TLR-3, TLR-7, RIG-I, and MDA5 in cultured FLS was studied by reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and Western blotting. Transcription factors were studied using the ELISA-based TransAM transcription factor kit. The expression of IFNβ, CXCL8 (interleukin-8), and MMP-3 was analyzed by RT-PCR and ELISA.

RESULTS

FLS expressed TLR-3, TLR-7, RIG-I, and MDA5. The expression of TLR-3 and RIG-I was higher in RA FLS, while the expression of TLR-7 and MDA5 was higher in OA FLS. Stimulation with poly(I-C) induced the activation of IFN regulatory factor 3 (IRF-3), NF-κB, and activator protein 1 (AP-1) c-Jun as well as the subsequent production of IFNβ, CXCL8, and MMP-3. VIP reduced the activation of IRF-3 and the production of IFNβ in both OA and RA FLS. Imiquimod induced the activation of NF-κB, AP-1 c-Fos, and AP-1 c-Jun and the synthesis of CXCL8 and MMP-3. VIP significantly diminished MMP-3 production only in imiquimod-treated RA FLS.

CONCLUSIONS

The results of this study revealed a prominent function of FLS in the recognition of both dsRNA and ssRNA, which may be present in the joint microenvironment. This study also advances the healing function of the endogenous neuroimmune peptide VIP, which inhibited TLR-3-, RIG-I-, MDA5-, and TLR-7-mediated stimulation of antiviral, proinflammatory, and joint destruction mediators.

Large DNA viruses, such as herpesvirus and poxvirus, encode proteins that target and exploit the chemokine system of their host. UL146 and UL147 in the cytomegalovirus (CMV) genome encode the two CXC chemokines vCXCL1 and vCXCL2. In this study, vCXCL1 was probed against a panel of the 18 classified human chemokine receptors. In calcium mobilization assays vCXCL1 acted as an agonist on both CXCR1 and CXCR2 but did not activate or block any of the other 16 chemokine receptors. vCXCL1 was characterized and compared with CXCL1/GROalpha, CXCL2/GRObeta, CXCL3/GROgamma, CXCL5/ENA-78, CXCL6/GCP-2, CXCL7/NAP-2 and CXCL8/IL-8 in competition binding, calcium mobilization, inositol triphosphate turnover, and chemotaxis assays using CXCR1- and CXCR2-expressing Chinese hamster ovary, 300.19, COS7, and L1.2 cells. The affinities of vCXCL1 for the CXCR1 and CXCR2 receptors were 44 and 5.6 nm, respectively, as determined in competition binding against radioactively labeled CXCL8. In calcium mobilization, phosphatidylinositol turnover, and chemotaxis assays, vCXCL1 acted as a highly efficacious activator of both receptors, with a rather low potency for the CXCR1 receptor but comparable with CXCL5 and CXCL7. It is suggested that CMV uses the UL146 gene product expressed in infected endothelial cells to attract neutrophils by activating their CXCR1 and CXCR2 receptors, whereby neutrophils can act as carriers of the virus to uninfected endothelial cells. In that way a lasting pool of CMV-infected endothelial cells could be maintained.

OBJECTIVE

To address the hypothesis that physiologic interactions between astrocytes and endothelial cells (EC) at the blood-brain barrier (BBB) are afflicted by pathogenic inflammatory signaling when astrocytes are exposed to aquaporin-4 (AQP4) antibodies present in the immunoglobulin G (IgG) fraction of serum from patients with neuromyelitis optica (NMO), referred to as NMO-IgG.

METHODS

We established static and flow-based in vitro BBB models incorporating co-cultures of conditionally immortalized human brain microvascular endothelial cells and human astrocyte cell lines with or without AQP4 expression.

RESULTS

In astrocyte-EC co-cultures, exposure of astrocytes to NMO-IgG decreased barrier function, induced CCL2 and CXCL8 expression by EC, and promoted leukocyte migration under flow, contingent on astrocyte expression of AQP4. NMO-IgG selectively induced interleukin (IL)-6 production by AQP4-positive astrocytes. When EC were exposed to IL-6, we observed decreased barrier function, increased CCL2 and CXCL8 expression, and enhanced leukocyte transmigration under flow. These effects were reversed after application of IL-6 neutralizing antibody.

CONCLUSIONS

Our results indicate that NMO-IgG induces IL-6 production by AQP4-positive astrocytes and that IL-6 signaling to EC decreases barrier function, increases chemokine production, and enhances leukocyte transmigration under flow.

BACKGROUND

Eosinophils are seen at sites of inflammation in diseases such as helminthic infestation, asthma, ulcerative colitis and some neoplastic diseases. They are also associated with connective tissue remodelling, for example in longstanding asthma. In the present study, we investigated whether eosinophils express the CXC chemokine epithelial cell-derived neutrophil activating peptide (ENA-78/CXCL5), a chemokine that can activate neutrophils and in addition possesses angiogenic properties. Immunocytochemistry detected CXCL5 in eosinophils and the peptide was localized in the specific granules by immunoelectron microscopy.

RESULTS

In eosinophil lysates, 12 +/- 2 pg (mean +/- SEM) of CXCL5 was detected per 106 cells by enzyme-linked immunosorbent assay (ELISA). Weak constitutive expression of CXCL5, as well as the related CXC chemokine IL-8/CXCL8, could be detected in freshly isolated eosinophils by RT-PCR. However, during prolonged incubation of eosinophils, a strong increase in both CXCL5 and IL-8/CXCL8 expression was seen, as detected by RT-PCR, and increasing amounts of CXCL5 peptide with time were detected in the incubation medium by ELISA. Addition of TNF-alpha neutralizing antibodies during prolonged incubation significantly inhibited CXCL5 production, demonstrating involvement of auto- and paracrine effects from TNF-alpha produced by eosinophils themselves. Addition of IFN-gamma showed a strong inhibitory effect on CXCL5 synthesis.

CONCLUSIONS

These findings suggest that, through expression of CXCL5, eosinophils can recruit and activate CXC receptor 2 (CXCR2)-bearing cells such as neutrophils at sites of inflammation. Eosinophils may also promote connective tissue remodelling through release of this peptide.

CCL5 (or RANTES (regulated upon activation, normal T cell expressed and secreted)) recruits T lymphocytes and monocytes. The source and regulation of CCL5 in pulmonary tuberculosis are unclear. Infection of the human alveolar epithelial cell line (A549) by Mycobacterium tuberculosis caused no CCL5 secretion and little monocyte secretion. Conditioned medium from tuberculosis-infected human monocytes (CoMTB) stimulated significant CCL5 secretion from A549 cells and from primary alveolar, but not upper airway, epithelial cells. Differential responsiveness of small airway and normal human bronchial epithelial cells to CoMTB but not to conditioned medium from unstimulated human monocytes was specific to CCL5 and not to CXCL8. CoMTB induced CCL5 mRNA accumulation in A549 cells and induced nuclear translocation of nuclear factor kappaB (NFkappaB) subunits p50, p65, and c-rel at 1 h; nuclear binding of activator protein (AP)-1 (c-Fos, FosB, and c-Jun) at 4-8 h; and binding of NF-interleukin (IL)-6 at 24 h. CCL5 promoter-reporter analysis using deletion and site-specific mutagenesis constructs demonstrated a key role for AP-1, NF-IL-6, and NFkappaB in driving CoMTB-induced promoter activity. The IL-1 receptor antagonist inhibited A549 and small airway epithelial cell CCL5 secretion, gene expression, and promoter activity. CoMTB contained IL-1beta, and recombinant IL-1beta reproduced CoMTB effects. Monocyte alveolar, but not upper airway, epithelial cell networks in pulmonary tuberculosis cause AP-1-, NF-IL-6-, and NFkappaB-dependent CCL5 secretion. IL-1beta is the critical regulator of tuberculosis-stimulated CCL5 secretion in the lung.

BACKGROUND

WNT signalling is activated during lung tissue damage and inflammation. We investigated whether lung epithelial expression of WNT ligands, receptors (frizzled; FZD) or target genes is dysregulated on cigarette smoking and/or in chronic obstructive pulmonary disease (COPD).

METHODS

We studied this in human lung epithelial cell lines and primary bronchial epithelial cells (PBEC) from COPD patients and control (non-)smokers, at baseline and on cigarette smoke extract (CSE) exposure.

RESULTS

CSE significantly decreased WNT-4, WNT-10B and FZD2 and increased WNT-5B mRNA expression in 16HBE, but did not affect WNT-4 protein. The mRNA expression of WNT-4, but not other WNT ligands, was lower in PBEC from smokers than non-smokers and downregulated by CSE in PBEC from all groups, yet higher in PBEC from COPD patients than control smokers. Moreover, PBEC from COPD patients displayed higher WNT-4 protein expression than both smokers and non-smokers. Exogenously added WNT-4 significantly increased CXCL8/IL-8, IL-6, CCL5/RANTES, CCL2/MCP-1 and vascular endothelial growth factor (VEGF) secretion in 16HBE, but did not affect the canonical WNT target genes MMP-2, MMP-9, fibronectin, β-catenin, Dickkopf and axin-2, and induced activation of the non-canonical signalling molecule p38. Moreover, WNT-4 potentiated the CSE-induced upregulation of IL-8 and VEGF.

CONCLUSIONS

WNT-4 mRNA and protein levels are higher in PBEC from COPD patients than control (non-)smokers, while cigarette smoke downregulates airway epithelial WNT-4 mRNA, but not protein expression. As WNT-4 further increases CSE-induced pro-inflammatory cytokine release in bronchial epithelium, we propose that higher epithelial WNT-4 levels in combination with cigarette smoking may have important implications for the development of airway inflammation in COPD.

Anoikis is a form of apoptosis which occurs when anchorage-dependent cells either show loss of adhesion or inappropriate adhesion. Only a few cancer cells that detach from the primary site of the tumor acquire the ability to resist anoikis and form metastasis. The mechanism underlying the resistance of colorectal cancer (CRC) cells to anoikis remains unclear. Interleukin-8 (alternatively known as CXCL8) is associated with CRC angiogenesis and progression. Here, we found that a high abundance of CXCL8 or TOPK strongly correlated with poor overall and disease-free survival of 186 patients with CRC. A combination of high CXCL8 and high TOPK expressions had the worst prognosis. We showed that CXCL8 expression was negatively correlated with anoikis in CRC cells. CXCL8 treatment enhanced the resistance of CRC cells to apoptosis, which was accompanied by the increase of TOPK, and the activation of AKT and ERK. Moreover, we demonstrated that the inhibition of either ERK or AKT by specific chemical inhibitors attenuated the CXCL8-mediated resistance to anoikis. Treatment with AKT inhibitor abolished the effects of CXCL8 on TOPK expression, suggesting that TOPK was downstream of AKT in the process of anoikis. Taken together, we demonstrated that CXCL8 is strongly implicated in the resistance of CRC cells to anoikis, and that the AKT, TOPK and ERK pathway may be a potential therapeutic target for CRC.

p38α mitogen-activated protein kinase (MAPK) is activated in cancer cells in response to environmental factors, oncogenic stress, radiation, and chemotherapy. p38α MAPK phosphorylates a number of substrates, including MAPKAP-K2 (MK2), and regulates the production of cytokines in the tumor microenvironment, such as TNF-α, interleukin-1β (IL-1β), IL-6, and CXCL8 (IL-8). p38α MAPK is highly expressed in human cancers and may play a role in tumor growth, invasion, metastasis, and drug resistance. LY2228820 dimesylate (hereafter LY2228820), a trisubstituted imidazole derivative, is a potent and selective, ATP-competitive inhibitor of the α- and β-isoforms of p38 MAPK in vitro (IC(50) = 5.3 and 3.2 nmol/L, respectively). In cell-based assays, LY2228820 potently and selectively inhibited phosphorylation of MK2 (Thr334) in anisomycin-stimulated HeLa cells (at 9.8 nmol/L by Western blot analysis) and anisomycin-induced mouse RAW264.7 macrophages (IC(50) = 35.3 nmol/L) with no changes in phosphorylation of p38α MAPK, JNK, ERK1/2, c-Jun, ATF2, or c-Myc ≤ 10 μmol/L. LY2228820 also reduced TNF-α secretion by lipopolysaccharide/IFN-γ-stimulated macrophages (IC(50) = 6.3 nmol/L). In mice transplanted with B16-F10 melanoma, tumor phospho-MK2 (p-MK2) was inhibited by LY2228820 in a dose-dependent manner [threshold effective dose (TED)(70) = 11.2 mg/kg]. Significant target inhibition (>40% reduction in p-MK2) was maintained for 4 to 8 hours following a single 10 mg/kg oral dose. LY2228820 produced significant tumor growth delay in multiple in vivo cancer models (melanoma, non-small cell lung cancer, ovarian, glioma, myeloma, breast). In summary, LY2228820 is a p38 MAPK inhibitor, which has been optimized for potency, selectivity, drug-like properties (such as oral bioavailability), and efficacy in animal models of human cancer.

Interleukin-17A (IL-17A) and IL-17F have been shown to mediate a crucial crosstalk between the immune system and various epithelial tissues, stimulating various defensive mechanisms to bacterial infections. A number of studies have characterized the response to IL-17A and IL-17F of epithelial cells from airways, intestine, and skin, but not from the mammary gland. To evaluate the potential contribution of IL-17 to the immune defense of the mammary gland, we analyzed the effects of recombinant bovine IL-17A and IL-17F on primary bovine mammary epithelial cells (MEC) by quantitative PCR and ELISA. We found expression (mRNA) of the two components of the IL-17 receptor complex, IL-17RA and IL-17RC, in mammary tissue and MEC in vitro. The expression of a number of genes encoding cytokines, chemokines and proteins endowed with antibacterial activities was increased by IL-17A, and to a lesser extent by IL-17F, but the magnitude of responses was modest. As expected, responses were augmented by the combination of IL-17A or IL-17F with TNF-α. Interestingly, responses of a few of the tested genes, such as IL8, CCL20, iNOS, and CfB, were augmented by the combination of IL-17A with staphylococcal lipoteichoic acid or muramyl dipeptide, bacterial agonists of the innate immune system. This can be interpreted as indicating that IL-17A and IL-17F are tailored to exert their full potential in a septic environment. MEC responses were characterized by the expression of chemokines targeting not only neutrophils (CXCL3 and CXCL8) but also mononuclear leucocytes (CCL2, CCL20). Production of IL-6 was low and the inflammatory cytokines TNF-α and IL-1β were expressed (mRNA) but proteins were not secreted. Altogether, our results suggest that IL-17A and IL-17F have a potential to modulate the mammary gland immune response to mastitis-causing pathogens.

Chemokines are important mediators of chemotaxis, cell adherence, and proliferation and exert specific functions in bone remodeling. Despite the potential intriguing role of chemokines in the regulation of osteoclast (OC) functions, little is known about the expression of chemokines and their receptors in human OCs at different stages of differentiation. Therefore, we analyzed the expression of CXC chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4 and CXCR5) and ligands (CXCL8, CXCL10, CXCL12 and CXCL13) both at molecular and protein levels, in human OCs grown on plastic or calcium phosphate-coated slides at different stages of differentiation. Real-time PCR showed that CXCR1, CXCR2, CXCR3, CXCR4, CXCR5 and CXCL8 were expressed in undifferentiated cells and significantly decreased during OC differentiation. By contrast, CXCL10 and CXCL12 were strongly upregulated from day 0 to day 8 in cells grown on calcium phosphate-coated slides. Immunocytochemistry showed that OCs grown on plastic expressed CXCR3, CXCR4, CXCR5, CXCL8 and CXCL12, while they were negative for CXCR1, CXCR2 and CXCL10. Interestingly, both at molecular and protein levels CXCL10 and CXCL12 significantly increased only when cells were differentiated on calcium phosphate-coated slides. These data suggest that the selection of a substrate that better mimics the tridimensional structure of bone tissue, thus favoring OC maturation and differentiation, may be necessary when studying osteoclastogenesis in vitro.

Immature dendritic cells (DCs), unlike mature DCs, require the viral determinant nef to drive immunodeficiency virus (SIV and HIV) replication in coculture with CD4(+) T cells. Since immature DCs may capture and get infected by virus during mucosal transmission, we hypothesized that Nef associated with the virus or produced during early replication might modulate DCs to augment virus dissemination. Adenovirus vectors expressing nef were used to introduce nef into DCs in the absence of other immunodeficiency virus determinants to examine Nef-induced changes that might activate immature DCs to acquire properties of mature DCs and drive virus replication. Nef expression by immature human and macaque DCs triggered IL-6, IL-12, TNF-alpha, CXCL8, CCL3, and CCL4 release, but without up-regulating costimulatory and other molecules characteristic of mature DCs. Coincident with this, nef-expressing immature DCs stimulated stronger autologous CD4(+) T cell responses. Both SIV and HIV nef-expressing DCs complemented defective SIVmac239 delta nef, driving replication in autologous immature DC-T cell cultures. In contrast, if DCs were activated after capturing delta nef, virus growth was not exacerbated. This highlights one way in which nef-defective virus-bearing immature DCs that mature while migrating to draining lymph nodes could induce stronger immune responses in the absence of overwhelming productive infection (unlike nef-containing wild-type virus). Therefore, Nef expressed in immature DCs signals a distinct activation program that promotes virus replication and T cell recruitment but without complete DC maturation, thereby lessening the likelihood that wild-type virus-infected immature DCs would activate virus-specific immunity, but facilitating virus dissemination.

During acute inflammation, neutrophil recruitment into extravascular tissue requires neutrophil tethering and rolling on cytokine-activated endothelial cells (ECs), tight adhesion, crawling towards EC junctions and transendothelial migration (TEM). Following TEM, neutrophils must still traverse the subendothelial basement membrane and network of pericytes (PCs). Until recently, the contribution of the PC layer to neutrophil recruitment was largely ignored. Here we analyze human neutrophil interactions with interleukin (IL)-1β-activated human EC monolayers, PC monolayers and EC/PC bilayers in vitro. Compared to EC, PC support much lower levels of neutrophil binding (54.6% vs. 7.1%, respectively) and transmigration (63.7 vs. 8.8%, respectively) despite comparable levels of IL-8 (CXCL8) synthesis and display. Remarkably, EC/PC bilayers support intermediate levels of transmigration (37.7%). Neutrophil adhesion to both cell types is Mac-1-dependent and while ICAM-1 transduction of PCs increases neutrophil adhesion to (41.4%), it does not increase transmigration through PC monolayers. TEM, which increases neutrophil Mac-1 surface expression, concomitantly increases the ability of neutrophils to traverse PCs (19.2%). These data indicate that contributions from both PCs and ECs must be considered in evaluation of microvasculature function in acute inflammation.

BACKGROUND

Human colostrum and milk contain components that influence development. Our aim was to use a protein array to determine the cytokine profile of human lacteal secretions and changes that occur during the early postpartum period.

METHODS

We collected 17 samples of colostrum during the first 2 days postpartum and a 2nd group of 5 sets of 2 to 3 sequential colostrum or milk samples (at 20- to 30-h intervals). We analyzed the samples with array membranes consisting of 42 or 79 antibodies directed against cytokines.

RESULTS

In most samples, we detected the previously described cytokines interleukin-8 (IL-8)/CXCL8, epidermal growth factor (EGF), growth-related oncoprotein (GRO)/CXCL1-3, angiogenin, transforming growth factor beta-2, and monocyte chemotactic protein 1 (MCP-1/CCL2). In addition, we found 32 cytokines that have not been described before in colostrum. Cytokine concentrations differed among mothers, and the spectrum of cytokines changed with time after delivery. A significant decrease occurred in IL-12 and macrophage inflammatory protein-1delta/CCL15 and a significant increase in MCP-1/CCL2. The production of angiogenin, vascular endothelial growth factor, GRO/CXCL1-3, EGF, and IL-8/CXCL8 remained high throughout. The concentrations of 2 selected cytokines measured with the array technique and ELISA showed moderate to strong correlation (r = 0.63 for EGF and r = 0.84 for IL-8/CXCL8).

CONCLUSIONS

Despite the lack of precise quantification, the protein array might be suitable for cytokine screening. It allows simultaneous detection of a broad spectrum of cytokines (including those not described before) in lacteal secretions.