BACKGROUND

Neutrophil functions have long been regarded as limited to acute inflammation and the defense against microbes. The role(s) of neutrophils in cancer remain poorly understood. Neutrophils infiltrate tumors and are key effector cells in the orchestration of inflammatory responses. Thyroid cancer (TC) is the most recurrent endocrine malignant tumor and is responsible for 70% of deaths due to endocrine cancers. No studies are so far available on the role of neutrophils in TC.

OBJECTIVE

Our purpose was to study the involvement of tumor-associated neutrophils in TC.

METHODS

Highly purified human neutrophils (>99%) from healthy donors were stimulated in vitro with conditioned media derived from TC cell lines TPC1 and 8505c (TC-CMs). Neutrophil functions (e.g., chemotaxis, activation, plasticity, survival, gene expression, and protein release) were evaluated.

RESULTS

TC-derived soluble factors promoted neutrophil chemotaxis and survival. Neutrophil chemotaxis toward a TC-CM was mediated, at least in part, by CXCL8/IL-8, and survival was mediated by granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition, each TC-CM induced morphological changes and activation of neutrophils (e.g., CD11b and CD66b upregulation and CD62L shedding) and modified neutrophils' kinetic properties. Furthermore, each TC-CM induced production of reactive oxygen species, expression of proinflammatory and angiogenic mediators (CXCL8/IL-8, VEGF-A, and TNF-α), and a release of matrix metalloproteinase 9 (MMP-9). Moreover, in TC patients, tumor-associated neutrophils correlated with larger tumor size.

CONCLUSIONS

TC cell lines produce soluble factors able to "educate" neutrophils toward an activated functional state. These data will advance the understanding of the molecular and cellular mechanisms of innate immunity in TC.

Esophageal squamous cell carcinoma (ESCC) is a highly aggressive tumor with frequent recurrence even after curative resection. The tumor microenvironment, which consists of non-cancer cells, such as cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs), was recently reported to promote several cancers, including ESCC. However, the role of CAF as a coordinator for tumor progression in ESCC remains to be elucidated. In our immunohistochemical investigation of ESCC tissues, we observed that the intensity of expression of two CAF markers-alpha smooth muscle actin (αSMA) and fibroblast activation protein (FAP)-in the tumor stroma was significantly correlated with the depth of tumor invasion, lymph node metastasis, advanced pathological stage, and poor prognosis. We co-cultured human bone marrow-derived mesenchymal stem cells (MSCs) with ESCC cells and confirmed the induction of FAP expression in the co-cultured MSCs. These FAP-positive MSCs (which we defined as CAF-like cells) promoted the cell growth and migration of ESCC cells and peripheral blood mononuclear cell-derived macrophage-like cells. CAF-like cells induced the M2 polarization of macrophage-like cells. A cytokine array and ELISA revealed that CAF-like cells secreted significantly more CCL2, Interleukin-6, and CXCL8 than MSCs. These cytokines promoted the migration of tumor cells and macrophage-like cells. The silencing of FAP in CAF-like cells attenuated cytokine secretion. We compared cell signaling of MSCs, CAF-like cells, and FAP-silenced CAF-like cells; PTEN/Akt and MEK/Erk signaling were upregulated and their downstream targets, NF-κB and β-catenin, were also activated with FAP expression. Silencing of FAP attenuated these effects. Cytokine secretion from CAF-like cells were attenuated by inhibitors against these signaling pathways. These findings indicate that the collaboration of CAFs with tumor cells and macrophages plays a pivotal role in tumor progression, and that FAP expression is responsible for the tumor promotive and immunosuppressive phenotypes of CAFs.

Chemokines are chemotactic cytokines that mediate immune cell chemotaxis and lymphoid tissue development. Recent advances have indicated that chemokines and their cognate receptors play critical roles in cancer-related inflammation and cancer progression. On the basis of these findings, the chemokine system has become a new potential drug target for cancer immunotherapy. In this review, we summarize the essential roles of the complex network of chemokines and their receptors in cancer progression. Furthermore, we discuss the potential value of the chemokine system as a cancer prognostic marker. The chemokine system regulates the infiltration of immune cells into the tumor microenvironment, which induces both pro- and anti-immunity and promotes or suppresses tumor growth and proliferation, angiogenesis, and metastasis. Increasing evidence indicates the promising prognostic value of the chemokine system in cancer patients. While CCL2, CXCL10, and CX3CL1/CX3CR1 can serve as favorable or unfavorable prognostic factors depending on the cancer types, CCL14 and XCL1 possess good prognostic value. Other chemokines such as CXCL1, CXCL8, and CXCL12 are poor prognostic markers. Despite vast advances in our understanding of the complex nature of the chemokine system in tumor biology, knowledge about the multifaceted roles of the chemokine system in different types of cancers is still limited. Further studies are necessary to decipher distinct roles within the chemokine system in terms of cancer progression and to validate their potential value in cancer prognosis.

In established tumors, tumor-associated macrophages (TAMs) orchestrate non-resolving cancer-related inflammation and produce mediators favoring tumor growth, metastasis and angiogenesis. However, the factors conferring inflammatory and pro-tumor properties on human macrophages remain largely unknown. Most solid tumors have high lactate content. We therefore analyzed the impact of lactate on human monocyte differentiation. We report that prolonged lactic acidosis induces the differentiation of monocytes into macrophages with a phenotype including pro-tumor and inflammatory characteristics. These cells produce tumor growth factors, inflammatory cytokines and chemokines as well as low amounts of IL10. These effects of lactate require its metabolism and are associated with HIF-1α stabilization. The expression of some lactate-induced genes is dependent on autocrine M-CSF consumption. Finally, TAMs with pro-tumor and inflammatory characteristics (VEGFhigh CXCL8+ IL1β+) are found in solid ovarian tumors. These results show that tumor-derived lactate links the pro-tumor features of TAMs with their inflammatory properties. Treatments that reduce tumor glycolysis or tumor-associated acidosis may help combat cancer.

BACKGROUND

In osteoarthritis (OA), an inflammatory environment is responsible for the imbalance between the anabolic and catabolic activity of chondrocytes and, thus, for articular cartilage derangement. This study was aimed at providing further insight into the impairment of the anabolic cytokine IL-4 and its receptors in human OA cartilage, as well as the potential ability of IL-4 to antagonize the catabolic phenotype induced by IL-1β.

RESULTS

The in vivo expression of IL-4 and IL-4 receptor subunits (IL-4R, IL-2Rγ, IL-13Rα1) was investigated on full thickness OA or normal knee cartilage. IL-4 expression was found to be significantly lower in OA, both in terms of the percentage of positive cells and the amount of signal per cell. IL-4 receptor type I and II were mostly expressed in mid-deep cartilage layers. No significant difference for each IL-4 receptor subunit was noted. IL-4 anti-inflammatory and anti-catabolic activity was assessed in vitro in the presence of IL-1β and/or IL-4 for 24 hours using differentiated high density primary OA chondrocyte also exhibiting the three IL-4 R subunits found in vivo. Chemokines, extracellular matrix degrading enzymes and their inhibitors were evaluated at mRNA (real time PCR) and protein (ELISA or western blot) levels. IL-4 did not affect IL-1β-induced mRNA expression of GRO-α/CXCL1, IL-8/CXCL8, ADAMTS-5, TIMP-1 or TIMP-3. Conversely, IL-4 significantly inhibited RANTES/CCL5, MIP-1α/CCL3, MIP-1β/CCL4, MMP-13 and ADAMTS-4. These results were confirmed at protein level for RANTES/CCL5 and MMP-13.

CONCLUSIONS

Our results indicate for the first time that OA cartilage has a significantly lower expression of IL-4. Furthermore, we found differences in the spectrum of biological effects of IL-4. The findings that IL-4 has the ability to hamper the IL-1β-induced release of both MMP-13 and CCL5/RANTES, both markers of OA chondrocytes, strongly indicates IL-4 as a pivotal anabolic cytokine in cartilage whose impairment impacts on OA pathogenesis.

Chemokine CXCL8 and its receptor CXCR1 are key mediators in combating infection and have also been implicated in the pathophysiology of various diseases including chronic obstructive pulmonary disease (COPD) and cancer. CXCL8 exists as monomers and dimers but monomer alone binds CXCR1 with high affinity. CXCL8 function involves binding two distinct CXCR1 sites - the N-terminal domain (Site-I) and the extracellular/transmembrane domain (Site-II). Therefore, higher monomer affinity could be due to stronger binding at Site-I or Site-II or both. We have now characterized the binding of a human CXCR1 N-terminal domain peptide (hCXCR1Ndp) to WT CXCL8 under conditions where it exists as both monomers and dimers. We show that the WT monomer binds the CXCR1 N-domain with much higher affinity and that binding is coupled to dimer dissociation. We also characterized the binding of two CXCL8 monomer variants and a trapped dimer to two different hCXCR1Ndp constructs, and observe that the monomer binds with ∼10- to 100-fold higher affinity than the dimer. Our studies also show that the binding constants of monomer and dimer to the receptor peptides, and the dimer dissociation constant, can vary significantly as a function of pH and buffer, and so the ability to observe WT monomer peaks is critically dependent on NMR experimental conditions. We conclude that the monomer is the high affinity CXCR1 agonist, that Site-I interactions play a dominant role in determining monomer vs. dimer affinity, and that the dimer plays an indirect role in regulating monomer function.

The natural niche of Streptococcus pneumoniae is the nasopharyngeal mucosa and nasopharyngeal carriage of pneumococci is widely prevalent. Pneumolysin (Ply), a pore-forming protein produced by S. pneumonia, may be important in driving the innate immune response of the nasopharynx. We studied the Ply-induced production of CXCL8 by nasopharyngeal cells and further analysed the mechanism of this induction. Detroit nasopharyngeal cells were stimulated with supernatants derived from bacterial cultures of Ply-deficient, wild-type pneumococci and recombinant Ply, and CXCL8 measured by ELISA. The role of MAP kinase family members in Ply-induced CXCL8 production was analysed using specific inhibitors, NF-κB activity was measured by immunoblot and Ply-mediated TLR4 activation analysed by a CXCL8 promotor luciferase assay. Ply significantly increased production of CXCL8 in Detroit and primary nasal cells. Flow cytometric analysis showed that Detroit cells express cell surface TLR4. CXCL8 production was dependent on changes in the intracellular Ca(2+) levels and also by NF-κB via activation of TLR4, and MAP kinase signalling. Ply induces production of CXCL8 by nasopharyngeal cells using signalling mechanisms involving Ca(2+) mobilisation and activation of MAPK and NF-κB via TLR4. This may be important in regulating nasopharyngeal immunity against pneumococcal colonization.

Outer membrane protein A (OmpA) is a class of bacterial cell wall protein that is immunogenic without adjuvant. As specific immune responses are initiated in the lymph nodes (LN, we analyzed the effect of the OmpA from Klebsiella pneumoniae (KpOmpA) onchemokine/ chemokine receptor expression by APC and on cell migration to the LN. Upon contact with KpOmpA, human immature DC and macrophages acquire CCR7 expression and responsiveness to CCL21. In parallel, CCR1 and CCR5 expression is down-regulated and CXCL8, CCL2, CCL3 and CCL5 production is up-regulated. Mice injected subcutaneously with KpOmpA present a transient inflammatory reaction at the site of injection accompanied by an enlargement of the draining LN with a higher proportion of DC and macrophages. Lastly, when exposed to KpOmpA prior injection, DC but not macrophages migrate to the draining LN. In conclusion, KpOmpA confers a migratory phenotype to DC and triggers their migration to the regional LN. This property contributes to explain how innate cells initiate adaptive immune response upon recognition of conserved bacterial components and also why OmpA is immunogenic in the absence of adjuvant.

OBJECTIVE

Hypoxia is a potent stimulus for inflammation and remodeling. Hypoxia develops in chronic sinusitis as shown via tissue oxygen concentrations and colonization with obligate anaerobes. This hypoxia reflects occlusion of the sinus ostia and thereby failure of transepithelial oxygenation, nonvascularized exudates, and the tendency of inflammatory hyperplasia to exceed neovascularization.

RESULTS

Hypoxia-induced transcription factors are responsible for transcription of numerous inflammatory cytokines and growth factors, including vascular endothelial growth factor, CXCL8, CCL11, transforming growth factor-beta, inducible nitric oxide synthase, as well as matrix remodeling proteins such as procollagen and matrix metalloproteinases.

CONCLUSIONS

Many diseases, such as asthma, share the tendency to afflict respiratory epithelium of the lower (bronchi) and upper (sinus) airway. Although the histopathology and inflammation of asthma and its associated sinusitis share many features, aggressive fibrosis, polyp formation and intense hyperplasia are not features of asthma, a disease seldom associated with significant chronic hypoxia. In contrast, fibrosis is a cardinal feature of hypoxic diseases of the lungs such as interstitial lung diseases and primary pulmonary hypertension. Arguably, chronic sinusitis can be viewed as reflecting both 'asthma' and 'primary pulmonary hypertension' of the upper airway.

BACKGROUND

The aim of this study was to characterize the presence and roles of CXCL12, CXCL8, CXCL1, and HGF in five human uveal melanoma cell lines, using different methods, in order to ascertain their significance in this disease.

METHODS

Five human uveal melanoma cell lines (92.1, SP6.5, MKT-BR, OCM-1, and UW-1) of known proliferative, invasive, and metastatic potential were used in this experiment. A migration assay was used in order to assess the responsiveness of each cell line towards the four chosen chemotactic factors. Immunohistochemistry was then performed for all five cell lines (cytospins) using antibodies directed toward CXCL1, CXCL8 and their receptors CXCR2 and CXCR1 respectively. Quantitative real-time PCR was then performed on all five cell lines in order to establish the presence of these four chemotactic factors.

RESULTS

All five human uveal melanoma cell lines migrated towards the four chosen chemotactic factors at a level greater than that of the negative control. Chemokines CXCL1 and CXCL8 resulted in the greatest number of migrating cells in all five of our cell lines. Immunohistochemistry confirmed the expression of CXCL1, CXCL8, and their receptors CXCR2 and CXCR1 in all five of the cell lines. Quantitative real-time PCR results established expression of CXCL8, CXCL1, and HGF in all 5 cell lines tested. CXCL1 and CXCL8 are highly expressed in SP6.5 and UW-1. None of the five cell lines expressed any detectable levels of CXCL12.

CONCLUSIONS

The migratory ability of the 5 human uveal melanoma cell lines was positively influenced by the four chemotactic factors tested, namely CXCL12, CXCL8, CXCL1, and HGF. Self-expression of chemotactic factors CXCL8, CXCL1, and HGF may indicate an autocrine system, which perhaps contributes to the cells' metastatic ability in vivo.

BACKGROUND

The role of CXCL1 in prostate cancer (PCa) progression has been poorly investigated. A limitation of previous studies is linked to the use of human PCa cell lines PC3 and DU145, producing CXCL8 at levels strongly exceeding CXCL1 levels. Moreover, in mouse models the sharing of CXCR2 receptor by both ligands makes the phenotype induced by CXCL8 and CXCL1 almost indistinguishable. To overcome this problem we used the murine TRAMP-C2 cell line, not expressing CXCL8 and expressing CXCL1 at low levels.

METHODS

The effect of CXCL1 overexpression was examined by in vivo subcutaneous tumor studies and in vitro functional assays of invasion and adhesion. Biochemical modifications were evaluated by Western blotting and antibody arrays.

RESULTS

Our data show that the overexpression of CXCL1 in TRAMP-C2 cells represses tumor establishment and in situ invasion. In vitro, the main action of CXCL1 expression in TRAMP cells is associated with the perturbation of molecules linked to cell adhesion and migration thus explaining in vivo data. Other in vitro findings also suggest that signaling by CXCL1 might activate a secretory network limiting in vivo tumor growth by reinforcing senescence. Immunohistochemical staining of human PCa, BPH, and normal prostate biopsies strengthen our observations on the mouse model: when expressed, CXCL1 is limited to small areas with faint staining and PCa progression does not rely on CXCL1 expression.

CONCLUSIONS

We could speculate that CXCL1 overexpression acts as a suppressor of malignancy limiting the escape of tumor cells from the primary tumor and reinforcing growth arrest.

OBJECTIVE

The aetiology of idiopathic intracranial hypertension (IIH) is not known, but its association with obesity is well-recognized. Recent studies have linked obesity with abnormalities in circulating inflammatory and adiposity related cytokines. The aim of this study was to characterize adipokine and inflammatory cytokine profiles in IIH.

METHODS

Paired serum and cerebrospinal fluid (CSF) specimens were collected from 26 patients with IIH and compared to 62 control subjects. Samples were analysed for leptin, resistin, adiponectin, insulin, IL-1beta, IL-6, IL-8 (CXCL8), TNFalpha, MCP-1 (CCL2), hepatocyte growth factor, nerve growth factor and PAI-1 using multiplex bead immunoassays.

RESULTS

CSF leptin was significantly higher in patients with IIH (P = 0.001) compared to controls after correction for age, gender and body mass index (BMI). In the control population, BMI correlated with serum leptin (r = 0.34; P = 0.007) and CSF leptin (r = 0.51; P < 0.0001), but this was not the case for the IIH population. Profiles of other inflammatory cytokines and adipokines did not differ between IIH patients and controls once anthropometric factors had been accounted for.

CONCLUSIONS

IIH was characterized by significantly elevated CSF leptin levels which did not correlate with BMI. We suggest that CSF leptin may be important in the pathophysiology of IIH and that obesity in IIH may occur as a result of hypothalamic leptin resistance.

Chemokines and chemokine receptors are key modulators in inflammatory diseases and malignancies. Here, we describe the identification and pharmacologic characterization of nanobodies selectively blocking CXCR2, the most promiscuous of all chemokine receptors. Two classes of selective monovalent nanobodies were identified, and detailed epitope mapping showed that these bind to distinct, nonoverlapping epitopes on the CXCR2 receptor. The N-terminal-binding or class 1 monovalent nanobodies possessed potencies in the single-digit nanomolar range but lacked complete efficacy at high agonist concentrations. In contrast, the extracellular loop-binding or class 2 monovalent nanobodies were of lower potency but were more efficacious and competitively inhibited the CXCR2-mediated functional response in both recombinant and neutrophil in vitro assays. In addition to blocking CXCR2 signaling mediated by CXCL1 (growth-related oncogene α) and CXCL8 (interleukin-8), both classes of nanobodies displayed inverse agonist behavior. Bivalent and biparatopic nanobodies were generated, respectively combining nanobodies from the same or different classes via glycine/serine linkers. Interestingly, receptor mutation and competition studies demonstrated that the biparatopic nanobodies were able to avidly bind epitopes within one or across two CXCR2 receptor molecules. Most importantly, the biparatopic nanobodies were superior over their monovalent and bivalent counterparts in terms of potency and efficacy.

Clearance of apoptotic neutrophils is a central feature of the resolution of inflammation. Findings indicate that immuno-modulation and induction of neutrophil apoptosis by macrolide antibiotics generate anti-inflammatory benefits via mechanisms that remain obscure. Tulathromycin (TUL), a new antimicrobial agent for bovine respiratory disease, offers superior clinical efficacy for reasons not fully understood. The aim of this study was to identify the immuno-modulating effects of tulathromycin and, in this process, to establish tulathromycin as a new model for characterizing the novel anti-inflammatory properties of antibiotics. Bronchoalveolar lavage specimens were collected from Holstein calves 3 and 24 h postinfection, challenged intratracheally with live Mannheimia haemolytica (2 × 10(7) CFU), and treated with vehicle or tulathromycin (2.5 mg/kg body weight). Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining and enzyme-linked immunosorbent assay (ELISA) revealed that tulathromycin treatment significantly increased leukocyte apoptosis and reduced levels of proinflammatory leukotriene B(4) in M. haemolytica-challenged calves. In vitro, tulathromycin concentration dependently induced apoptosis in freshly isolated bovine neutrophils from healthy steers in a capase-3-dependent manner but failed to induce apoptosis in bovine fibroblasts, epithelial cells, and endothelial cells, as well as freshly isolated bovine blood monocytes and monocyte-derived macrophages. The proapoptotic effects of TUL were also, in part, drug specific; equimolar concentrations of penicillin G, oxytetracycline, and ceftiofur failed to cause apoptosis in bovine neutrophils. In addition, tulathromycin significantly reduced levels of phosphorylated IκBα, nuclear translocation of NF-κB p65, and mRNA levels of proinflammatory interleukin-8 in lipopolysaccharide (LPS)-stimulated bovine neutrophils. The findings illustrate novel mechanisms through which tulathromycin confers anti-inflammatory benefits.

Interleukin-8/CXCL8 (IL-8) is a prominent factor that modulates endothelial cell proliferation, migration, and angiogenesis. Therefore, the present study focused on the regulatory mechanisms of IL-8 expression induced by environmental pollutants such as polychlorinated biphenyls (PCBs). Treatment of human microvascular endothelial cells (HMECs) with specific PCB congener, 2,2',4,6,6'-pentachlorobiphenyl (PCB 104), dose dependently increased levels of IL-8 mRNA and secreted protein. IL-8-neutralizing antibody inhibited migration of endothelial cells stimulated by conditioned media derived from PCB 104-treated HMECs. Site-directed mutagenesis of the IL-8 promoter- and DNA-binding assays revealed that activator protein 1 (AP-1) and nuclear factor kappaB (NF-kappaB) sites are required for PCB 104-induced IL-8 transcription. Most importantly, pharmacological inhibition of Src kinase activity or overexpression of dominant-negative c-src in HMECs resulted in a significant decrease in IL-8 expression and promoter activity. In contrast, ectopic expression of activated c-Src markedly increased promoter activity of IL-8. These stimulatory effects of dominant-positive c-src were abrogated by mutagenesis of AP-1- and NF-kappaB-binding sites in the IL-8 promoter.

BACKGROUND

Most patients with cystic fibrosis (CF) die of respiratory failure due to chronic infection and destructive neutrophilic inflammation.

OBJECTIVE

To identify potential therapeutic targets by characterising the neutrophil stimulating mediators in the CF airway.

METHODS

Spontaneously expectorated CF sputum was extracted in phosphate buffered saline for assays of neutrophil chemotaxis, intracellular calcium mobilisation and cell shape change. Mediators were purified by ion exchange, C(18) reversed phase and size exclusion chromatography.

RESULTS

A pool of CF sputum contained considerable neutrophil stimulating activity but neutralisation of interleukin (IL)8/CXCL8 had little inhibitory effect on neutrophil chemotactic (10149 (2023) migrating cells vs 8661 (2597) at 62 mg sputum/ml; NS) or shape change (% forward scatter increase 46 (8) vs 38 (5) at 19 mg sputum/ml; p<0.05) responses. Furthermore, the CF sputum pool induced an elevation in intracellular calcium ions even after desensitisation of the neutrophils to IL8. Chromatography identified contributions to the neutrophil shape change inducing activity from IL8, other CXC chemokines, leukotriene (LT) B(4) and two formyl peptides. There was also suggestive evidence for contributions from platelet activating factor (PAF) and C5a. Using non-chromatographed individual sputum samples, anti-IL8 alone did have an inhibitory effect on neutrophil chemotaxis (median inhibition 41%; p = 0.0002). However, even in this experiment, there were clearly significantly important, non-IL8 mediated, effects of CF sputum on neutrophils, and an inhibitor cocktail of anti-IL8 plus CXCR2, LTB(4), formyl peptide, PAF and C5a receptor antagonists inhibited chemotaxis by a median of 97% (p = 0.0002).

CONCLUSIONS

Many chemoattractants contribute to the neutrophil stimulating activity in CF sputum although the relative contribution of these mediators differs in different patients. Selective blockade of single mediators may not be sufficient to control neutrophil recruitment and activation in the CF airway.

BACKGROUND

Platelet-rich plasma therapies for tendinopathy appear to provide moderate pain reduction. However, the biological mechanisms behind the observed clinical effects remain poorly characterized.

OBJECTIVE

The purpose of this study was to explore whether platelet-rich plasma modifies the inflammatory/angiogenic status of already inflamed tenocytes by examining (1) gene expression; (2) modulation of chemokine and interleukin secretion; and (3) differences between healthy and tendinopathic tenocytes.

METHODS

Cells from both healthy and tendinopathic tendons were exposed to interleukin (IL)-1ß and after treated with platelet-rich plasma. Modifications in the expression of selected genes were assessed by real-time reverse transcription-polymerase chain reaction and changes in secretion of angiogenic/inflammatory molecules by enzyme-linked immunosorbent assay. Platelet-rich plasma-induced changes in tendinopathic cells were compared with normal after normalizing platelet-rich plasma data against IL-1ß status in each specific sample.

RESULTS

In IL-1ß-exposed cells, platelet-rich plasma downregulates expression of IL-6/CXCL-6 (mean, 0.015; 95% confidence interval [CI], 0.005-0.025; p = 0.026), IL-6R (mean, 0.61; 95% CI, 0.27-0.95; p = 0.029), and IL-8/CXCL-8 (mean, 0.02; 95% CI, 0.007-0.023; p = 0.026). Secretion of IL-6/CXCL6, 0.35 (95% CI, 0.3-0.4; p = 0.002), IL-8/CXCL8, 0.55 (95% CI, 0.5-0.7; p = 0.01), and monocyte chemoattractant protein-1/CCL2, 0.40 (95% CI, 0.2-0.6; p = 0.001) was reduced by platelet-rich plasma, whereas vascular endothelial growth factor increased by twofold, (95% CI, 1.7-2.3; p < 0.001). RANTES/CCL5 increased by10-fold (95% CI, 4-17) and hepatocyte growth factor by 21-fold (95% CI, 0.2-42) in tendinopathic and by 2.3-fold (95% CI, 2-3) and threefold (95% CI, 1-5) in normal cells (p = 0.005 for both).

CONCLUSIONS

Platelet-rich plasma induces an immunomodulatory and proangiogenic phenotype consistent with healing mechanisms with few differences between tendinopathic and normal cells.

CONCLUSIONS

Platelet-rich plasma injections in pathological and nearby tissue might help to recover tendon homeostasis.

Hypoxia-inducible factor (HIF-1alpha) and cyclooxygenase-2 (COX-2) have been implicated in the regulation of inflammatory-like processes that lead to angiogenesis and fibrotic disorders. Here we demonstrate that in human lung fibroblasts (HLFs) treated with mixed exposures to chemical and microbial stimuli, HIF-1alpha stabilization plays a pivotal role in the induction of COX-2 mRNA and protein, driving the release of vascular endothelial growth factor (VEGF) and proangiogenic and profibrotic chemokines. Upon costimulation with Ni and the mycoplasma-derived lipopeptide macrophage-activating lipopeptide-2 (MALP-2), there was a synergistic induction of CXCL1 and CXCL5 mRNA and protein release from HLF, as well as an enhanced response in VEGF compared to either stimulus alone. Consistent with our previous findings that Ni and MALP-2 stimulates the induction of CXCL8 via a COX-2-mediated pathway, CXCL1, CXCL5, and VEGF release were also regulated by COX-2. Ni induced the stabilization of HIF-1alpha protein in HLF, which was further enhanced in the presence of MALP-2. Depletion of HIF-1alpha using siRNA blocked COX-2 induction by Ni and MALP-2 along with the release of VEGF, CXCL1, CXCL5, and CXCL8. Our results indicate that Ni and MALP-2 interact to promote an angiogenic profibrotic phenotype in HLF. Moreover, these findings reveal a potential role for HIF-1alpha in mediating chemical-induced alterations in cellular response to microbial stimuli, modulating pulmonary inflammation and its consequences such as fibrosis and angiogenesis.

The casual relationship between inflammation and tumour progression has been widely accepted and the etiology of breast cancer has been associated with inflammatory processes. Interleukin (IL)-1β, besides its central role in inflammation, has also been recognised as a powerful player in tumour progression, angiogenesis and invasiveness. Recently, there has been considerable interest in understanding the non-hypoxic upregulation of the hypoxia-inducible factor (HIF)-1α by IL-1 in neoplastic cells since aberrant expression of HIF-1α correlates with tumour progression. Here, using the highly invasive human breast cancer cell line MDAMB231, we studied the effect of IL-1β on tumour cell migration along with HIF-1α accumulation. We observed that non-hypoxic induction of HIF-1α by IL-1β in MDAMB231 was associated with increased cell migration, paralleled by upregulation of p38 MAPK phosphorylation and CXCL8/CXCR1 expression. Inhibition of HIF-1α by siRNA resulted in a significant reduction of CXCR1 expression and IL-1β-induced cell migration in MDAMB231 cells, thus confirming a role of HIF-1α in the non-hypoxic-IL-1β-dependent induction of migratory potentials. Our observation that IL-1 induces HIF-1α accumulation in MDAMB231 cells was confirmed in tumour cells growing in vivo using an experimental approach, mimicking the endogenous release of IL-1 in mice bearing MDAMB231 xenografts. Our in vivo data, along with the fact that inhibition of HIF-1α resulted in the decrease of IL-1β-promoted cell migration, further support the link between inflammation and cancer. The overall results may have important implications in those therapeutic approaches aimed to inhibit IL-1-mediated activities in tumour cells, specifically in breast cancer.

BACKGROUND

Activated mast cell densities are increased on the airway smooth muscle in asthma where they may modulate muscle functions and thus contribute to airway inflammation, remodelling and airflow obstruction.

OBJECTIVE

To determine the effects of human lung mast cells on the secretory and proliferative functions of airway smooth muscle cells from donors with and without asthma.

METHODS

Freshly isolated human lung mast cells were stimulated with IgE/anti-IgE. Culture supernatants were collected after 2 and 24 h and the mast cells lysed. The supernatants/lysates were added to serum-deprived, subconfluent airway smooth muscle cells for up to 48 h. Released chemokines and extracellular matrix were measured by ELISA, proliferation was quantified by [(3) H]-thymidine incorporation and cell counting, and intracellular signalling by phospho-arrays.

RESULTS

Mast cell 2-h supernatants reduced CCL11 and increased CXCL8 and fibronectin production from both asthmatic and nonasthmatic muscle cells. Leupeptin reversed these effects. Mast cell 24-h supernatants and lysates reduced CCL11 release from both muscle cell types but increased CXCL8 release by nonasthmatic cells. The 24-h supernatants also reduced asthmatic, but not nonasthmatic, muscle cell DNA synthesis and asthmatic cell numbers over 5 days through inhibiting extracellular signal-regulated kinase (ERK) and phosphatidylinositol (PI3)-kinase pathways. However, prostaglandins, thromboxanes, IL-4 and IL-13 were not involved in reducing the proliferation.

CONCLUSIONS

Mast cell proteases and newly synthesized products differentially modulated the secretory and proliferative functions of airway smooth muscle cells from donors with and without asthma. Thus, mast cells may modulate their own recruitment and airway smooth muscle functions locally in asthma.

CXC chemokines are particularly significant for leukocyte infiltration in inflammatory diseases. Recent reports have shown that inflammation is one of potential pathogenic mechanisms for diabetic nephropathy. However, information on inflammation related with CXC chemokines in human Type 2 diabetic nephropathy still remains scarce. We measured urinary and serum levels of three CXC chemokines, CXCL5, CXCL8 and CXCL9, in 45 Type 2 diabetic patients (DM), 42 primary renal disease (PRD) patients and 22 healthy controls by enzyme-linked immunosorbent assay. Urinary levels of CXCL5, CXCL8 and CXCL9 in DM were significantly elevated compared to those in controls (P<.0001, P<.01, P<.001; respectively). They increased consistent with urinary albumin excretion rate (UAER) and correlated with UAER in partial correlation analyses (r=0.41, P<.01; r=0.40, P<.01; r=0.45, P<.01; respectively). Urinary levels of CXCL5 in DM were significantly interrelated to HbA(1c) (r=0.42, P<.01). On the other hand, PRD showed significant increased levels of urinary CXCL8 and CXCL9 compared to controls (P<.001, P<.01; respectively), and so did PRD as UAER increased. However, there were no significant elevations of urinary levels of CXCL5 in PRD in spite of the increased UAER. We found significant associations of UAER in DM with diabetes duration, 1/serum creatinine, urinary CXCL5 (adjusted R(2)=0.67, P<.0001) or CXCL9 (adjusted R(2)=0.69, P<.0001) in a stepwise multiple regression analysis. These results suggest that these three CXC chemokines may be involved in the progression of human Type 2 diabetic nephropathy and that CXCL5 may be of use for telling diabetic nephropathy from primary renal diseases.

Inflammatory cytokines, particularly the neutrophil chemoattractant IL-8, are elevated in the cystic fibrosis (CF) airway, even in the absence of detectable infection. The transcriptional regulation of many inflammatory genes, including IL8 (CXCL8), involves chromatin remodeling through histone acetylation. NF-kappaB is known to facilitate histone acetylation of IL8 and other proinflammatory gene promoters, but we find that increased NF-kappaB activation cannot explain the elevated IL8 expression and promoter acetylation seen in CFTR-deficient cells. Recognized components of the NF-kappaB-coactivator complex, acetyltransferase CBP, p300, and the histone deacetylase HDAC1, are unchanged by CFTR activity. However, we find that the histone acetyltransferase (HAT)/HDAC balance is sensitive to CFTR function, as cells with reduced or absent CFTR function have decreased HDAC2 protein, resulting in hyperacetylation of the IL8 promoter and increased IL8 transcription. Reduced HDAC2 and HDAC2 activity, but not HDAC2 mRNA, is observed in cells deficient in CFTR. Suppressing HDAC2 expression with HDAC2 short hairpin RNA (shRNA) results in increased IL8 expression and promoter acetylation comparable with CFTR-deficient cells. Treating CFTR-deficient cells with N-acetyl-cysteine (NAC) increases HDAC2 expression to near control levels. Our data suggest that there is an intrinsic alteration in the HAT/HDAC balance in cells lacking CFTR function in vitro and in native CF tissue and that oxidative stress is likely contributing to this alteration. This mechanism, found in other inflammatory airway diseases, provides an explanation for the apparent dysregulation of inflammatory mediators seen in the CF airway, as reduced histone deacetylation would potentially influence many genes.

OBJECTIVE

Adenosine is a major endogenous regulator of macrophage function, and activates four specific adenosine receptors (A(1), A(2A), A(2B) and A(3)). Here, we have assessed in human lung macrophages the modulation of the expression of adenosine receptor mRNA by lipopolysaccharide (LPS), and the relative contributions of the different adenosine receptors to LPS-induced production of tumour necrosis factor (TNF)-alpha and chemokines.

METHODS

Lung macrophages isolated from resected lungs were stimulated with LPS and treated with adenosine receptor agonists or/and antagonists. Adenosine receptor expression was assessed with qRT-PCR. Cytokines were measured in lung macrophage supernatants with elisa.

RESULTS

LPS increased (about 400-fold) mRNA for A(2A) adenosine receptors, decreased mRNA for A(1) and A(2B), but had no effect on A(3) adenosine receptor mRNA. The adenosine receptor agonist NECA inhibited TNF-alpha production concentration dependently, whereas the A(1) receptor agonist, CCPA, and the A(3) receptor agonist, AB-MECA, inhibited TNF-alpha production only at concentrations affecting A(2A) receptors. NECA also inhibited the production of CCL chemokines (CCL2, CCL3, CCL4, CCL5) and CXCL chemokines (CXCL9 and CXCL10), but not that of CXCL1, CXCL8 and CXCL5. Reversal of NECA-induced inhibition of TNF-alpha and chemokine production by the selective A(2A) adenosine receptor antagonist ZM 241385, but not the A(2B) receptor antagonist, MRS 1754, or the A(3) receptor antagonist, MRS 1220, indicated involvement of A(2A) receptors.

CONCLUSIONS

LPS up-regulated A(2A) adenosine receptor gene transcription, and this receptor subtype mediated inhibition of the LPS-induced production of TNF-alpha and of a subset of chemokines in human lung macrophages.

OBJECTIVE

Sarcoidosis is a chronic multisystem granulomatous disorder characterized by an accumulation of activated CD4+ T cells and monocytes/macrophages in involved organs. Chemokines are required for the extravasation of leukocytes to the inflammation site. This study was undertaken to determine which chemokines are augmented in the serum of patients with active ocular sarcoidosis.

METHODS

Seventeen patients with diagnosed ocular sarcoidosis, 28 with suspected ocular sarcoidosis, 16 with Behçet's disease, 17 with Vogt-Koyanagi-Harada disease, and 18 healthy subjects were studied. Serum levels of CCL2, CCL5, CXCL8, CXCL9, and CXCL10 were simultaneously measured by cytometric bead array using flow cytometer. In addition, serum CXCL9 and CXCL10 levels in the patients with diagnosed or suspected ocular sarcoidosis were compared with respect to ocular disease activity, the presence of bilateral hilar lymphadenopathy (BHL), and laboratory data.

RESULTS

Serum levels of both CXCL9 and CXCL10 were markedly elevated in the patients with diagnosed or suspected ocular sarcoidosis compared with patients with other types of uveitis and healthy subjects. Although CCL2 and CXCL8 were detected in the serum of all subjects, the levels were extremely low with no significant differences between groups. Elevation of serum CXCL9 and CXCL10 in ocular sarcoidosis correlated significantly with ocular disease activity and ACE (angiotensin converting enzyme) levels and was unrelated to the presence of BHL, erythrocyte sedimentation rate, white blood cell count, serum IgG, or serum lysozyme.

CONCLUSIONS

The results demonstrated that serum levels of CXCL9 and CXCL10 were elevated markedly in the patients with ocular sarcoidosis and correlated with ocular disease activity and ACE level.