BACKGROUND

The mechanisms by which viruses induce asthma exacerbations are not well understood.

OBJECTIVE

We characterized fluctuations in nasal aspirate cytokines during naturally occurring respiratory viral infections in children with asthma.

METHODS

Sixteen children underwent home collections of nasal aspirates when they were without cold symptoms and again during self-reported respiratory illnesses. The presence of viral infection was ascertained by multiplex PCR. Cytokines were measured using multiplex immune assay. mRNA expression for selected markers of viral infection was measured using RT-PCR. A cumulative respiratory symptom score was calculated for each day of measurement. Generalized estimated equations were used to evaluate associations between viral infection and marker elevation, and between marker elevation and symptom score.

RESULTS

The 16 patients completed a total of 37 weeks of assessment (15 'well' weeks; 22 self-assessed 'sick' weeks). Viral infections were detected in 3 of the 'well' weeks and 17 of the 'sick' weeks (10 rhinovirus, three coronavirus, two influenza A, two influenza B, two respiratory syncytial virus, one parainfluenza). Compared to virus-negative well weeks, nasal aspirate IFN-γ, CXCL8/IL-8, CXCL10/IP-10, CCL5/RANTES, CCL11/eotaxin-1, CCL2/MCP-1, CCL4/MIP-1β, CCL7/MCP-3, and CCL20/MIP3α protein levels increased during virus-positive sick weeks. Only a subset of cytokines (IFN-γ, CXCL8, CCL2, CCL4, CCL5, and CCL20) correlated with self-reported respiratory tract symptoms. While many aspirates were dilute and showed no mRNA signal, viral infection significantly increased the number of samples that were positive for IFN-λ1, IFN-λ2/3, TLR3, RIG-I, and IRF7 mRNA.

CONCLUSIONS

We conclude that in children with asthma, naturally occurring viral infections apparently induce a robust innate immune response including expression of specific chemokines, IFNs, and IFN-responsive genes.

Th17 is a newly identified lineage of effector T cells involved in autoimmunity and immune responses to pathogens. We demonstrate in this study the pathogenic role of IL-17-producing CD4(+) T lymphocytes in allergic contact dermatitis (ACD) to skin-applied chemicals. IL-17(+) T cells infiltrate ACD reactions and predominantly distribute at the site of heavy spongiosis. Skin IL-17(+) T cells were functionally and phenotypically heterogeneous: although pure Th17 prevailed in ACD skin, hapten responsiveness was restricted to Th1/IL-17 (IFN-gamma(+)IL-17(+)) and Th0/IL-17 (IFN-gamma(+)IL-17(+)IL-4(+)) fractions, and to lesser extent Th2/IL-17 cells. In the IFN-gamma-dominated ACD environment, IL-17-releasing T cells affect immune function of keratinocytes by promoting CXCL8, IL-6, and HBD-2 production. In addition, compared with Th1, supernatants from Th1/IL-17 T cells were much more efficient in inducing ICAM-1 expression on keratinocytes and keratinocyte-T cell adhesiveness in vitro. As a consequence, exposure to combined IFN-gamma and IL-17 rendered keratinocytes susceptible to ICAM-1-dependent Ag nonspecific T cell killing. Thus, IL-17 efficiently amplifies the allergic reaction by rendering virtually all of the T lymphocytes recruited at the site of skin inflammation capable to directly contribute to tissue damage.

Extracellular ATP is a signaling molecule that often serves as a danger signal to alert the immune system of tissue damage. This molecule activates P2 nucleotide receptors, that include the ionotropic P2X receptors and metabotropic P2Y receptors. Recently, it has been reported that ATP accumulates in the airways of both asthmatic patients and sensitized mice after allergen challenge. The role and function of ATP in the pathogenesis of chronic obstructive pulmonary diseases (COPD) are not well understood. In this study we investigated the effect of cigarette smoke on purinergic receptors and ATP release by neutrophils. Neutrophils and their mediators are key players in the pathogenesis of lung emphysema. Here we demonstrated that in an in vivo model of cigarette smoke-induced lung emphysema, the amount of ATP was increased in the bronchoalveolar lavage fluid. Moreover, activation of neutrophils with cigarette smoke extract induced ATP release. Treatment of neutrophils with apyrase (catalyses the hydrolysis of ATP to yield AMP) and suramin (P2-receptor antagonist) abrogated the release of CXCL8 and elastase induced by cigarette smoke extract and exogenous ATP. These observations indicate that activation of purinergic signaling by cigarette smoke may take part in the pathogenesis of lung emphysema.

There are a large number of stable pancreatic ductal carcinoma cell lines (PDCL) that are used by researchers worldwide. Detailed data about their differentiation status and genetic alterations are present in the literature, but a systematic correlation with cell biological behavior is often lacking. PDCL ( n=12) were clustered by source of tumor cell (ascites, primary tumor, metastasis), and the data of functional cell biology were correlated with the reported structural and genetic profiles. Major histocompatibility complex expression, chemosensitivity and aneuploidia appeared to be related to the source of PDCL, and proliferative capacity appeared to be related to the grade of differentiation. No correlation between genetic/structural features of PDCL and biological behavior was found. All the cell lines appeared generally insensitive to in vitro treatment with 5-fluorouracil and showed variable degrees of susceptibility to gemcitabine, raltitrexed and oxaliplatin. All the PDCL showed resistance to Fas-mediated apoptosis but were significantly sensitive to the pro-apoptotic effect of inflammatory cytokines [interleukin (IL)-1beta, tumor necrosis factor (TNF)alpha and interferon gamma]. PDCL were characterized for the secretion of several factors relevant to the tumor-immune cross talk. Vascular endothelial growth factor, CCL2, CCL5 and transforming growth factor beta were the factors most frequently released; less frequent was the secretion of CXCL8, CCL22, IL-6 and sporadically CXCL12, IL-10 and hepatocyte growth factor. The cytokines IL-1beta and TNFalpha were always undetectable. In conclusion, a clear correlation between structural/genetic features and function could not be detected, suggesting the weakness of a "morphological" classification for the in vitro studies of pancreatic cancer.

Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) that are characterized by chronic intestinal inflammation and a constant influx of leukocytes mediated by pro-inflammatory cytokines and chemokines. The intestinal expression of the CXCR1-binding chemokines IL-8/CXCL8 and GCP-2/CXCL6 and the participation of immunocompetent cells in IBD were evaluated. IL-8 production by peripheral blood mononuclear cells (PBMC) from IBD patients, stimulated with endotoxin, plant lectin or double-stranded RNA, was significantly lowered in patients with CD, but not in UC patients or healthy subjects. The reduced chemokine production by PBMC from IBD patients was both IL-8 and CD specific, but not inducer dependent. In serum, most chemokines remained undetectable, while the levels of those that were measurable remained unaltered in IBD patients. GCP-2, but not ENA-78/CXCL5, nor IL-8, were highly expressed by endothelial cells in inflamed intestinal tissue of IBD patients. In contrast, stimulated endothelial cell cultures produced more IL-8 than GCP-2. The selective GCP-2 staining of endothelial cells at sites of ulcerations suggests that GCP-2, despite its low production capacity in vitro, plays a role in IBD that is different from that of structurally (ENA-78) and functionally (IL-8) related ELR(+) CXC chemokines. Thus, the chemokine network shows complementarity rather than redundancy.

In neutrophils, growth-related protein-alpha (CXCL1) and interleukin-8 (CXCL8), are potent chemoattractants (Cytokine 14:27-36, 2001; Biochemistry 42:2874-2886, 2003) and can stimulate myeloperoxidase release via activation of the G protein-coupled receptors CXCR1 and CXCR2. The role of CXCR1 and CXCR2 in the pathogenesis of inflammatory responses has encouraged the development of small molecule antagonists for these receptors. The data presented herein describe the pharmacology of 2-hydroxy-N,N-dimethyl-3-{2-[[(R)-1-(5-methyl-furan-2-yl)-propyl]amino]-3,4-dioxo-cyclobut-1-enylamino}-benzamide (Sch527123), a novel antagonist of both CXCR1 and CXCR2. Sch527123 inhibited chemokine binding to (and activation of) these receptors in an insurmountable manner and, as such, is categorized as an allosteric antagonist. Sch527123 inhibited neutrophil chemotaxis and myeloperoxidase release in response to CXCL1 and CXCL8 but had no effect on the response of these cells to C5a or formyl-methionyl-leucyl-phenylalanine. The pharmacological specificity of Sch527123 was confirmed by testing in a diversity profile against a panel of enzymes, channels, and receptors. To measure compound affinity, we characterized [(3)H]Sch527123 in both equilibrium and nonequilibrium binding analyses. Sch527123 binding to CXCR1 and CXCR2 was both saturable and reversible. Although Sch527123 bound to CXCR1 with good affinity (K(d) = 3.9 +/- 0.3 nM), the compound is CXCR2-selective (K(d) = 0.049 +/- 0.004 nM). Taken together, our data show that Sch527123 represents a novel, potent, and specific CXCR2 antagonist with potential therapeutic utility in a variety of inflammatory conditions.

Pro-inflammatory mediators, like prostaglandin (PG) and chemokines, promote tumourigenesis by enhancing cell proliferation, migration of immune cells and recruitment of blood vessels. Recently we showed elevated expression of the chemokine (C-X-C motif) receptor 2 (CXCR2) in endometrial adenocarcinomas localized to neutrophils and neoplastic epithelial and vascular cells. Furthermore we found that PGF(2alpha)-F-prostanoid (FP) receptor regulates the expression of the CXCR2 ligand CXCL1, to promote neutrophil chemotaxis in endometrial adenocarcinomas. In the present study we identified another CXCR2 ligand, CXCL8 as a target for PGF(2alpha)-FP receptor signalling which enhances epithelial cell proliferation in endometrial adenocarcinoma cells in vitro and in nude mice in vivo. We found that PGF(2alpha)-FP receptor interaction induces CXCL8 expression in endometrial adenocarcinoma cells via the protein kinase C-calcium-calcineurin-NFAT signaling pathway. Promoter analysis revealed that CXCL8 transcriptional activation by PGF(2alpha) signaling is mediated by cooperative interactions between the AP1 and NFAT binding sites. Furthermore, PGF(2alpha) via the FP receptor induced the expression of the regulator of calcineurin 1 isoform 4 (RCAN1-4) via the calcineurin/NFAT pathway in a reciprocal manner to CXCL8. Using an adenovirus to overexpress RCAN1-4, we found that RCAN1-4 is a negative regulator of CXCL8 expression in endometrial adenocarcinoma cells. Taken together our data have elucidated the molecular and cellular mechanism whereby PGF(2alpha) regulates CXCL8 expression via the FP receptor in endometrial adenocarcinomas and have highlighted RCAN1-4 as a negative regulator of CXCL8 expression which may be exploited therapeutically to inhibit CXCL8-mediated tumour development.

Macrophage migration inhibitory factor (MIF) has been defined as an important chemokine-like function (CLF) chemokine with an essential role in monocyte recruitment and arrest. Adhesion of monocytes to the vessel wall and their transendothelial migration are critical in atherogenesis and many other inflammatory diseases. Chemokines carefully control all steps of the monocyte recruitment process. Those chemokines specialized in controlling arrest are typically immobilized on the endothelial surface, mediating the arrest of rolling monocytes by chemokine receptor-triggered pathways. The chemokine receptor CXCR2 functions as an important arrest receptor on monocytes. An arrest function has been revealed for the bona fide CXCR2 ligands CXCL1 and CXCL8, but genetic studies also suggested that additional arrest chemokines are likely to be involved in atherogenic leukocyte recruitment. While CXCR2 is known to interact with numerous CXC chemokine ligands, the CLF chemokine MIF, which structurally does not belong to the CXC chemokine sub-family, was surprisingly identified as a non-cognate ligand of CXCR2, responsible for critical arrest functions during the atherogenic process. MIF was originally identified as macrophage migration inhibitory factor (this function being eponymous), but is now known as a potent inflammatory cytokine with CLFs including chemotaxis and leukocyte arrest. This review will cover the mechanisms underlying these functions, including MIF's effects on LFA1 integrin activity and signal transduction, and will discuss the structural similarities between MIF and the bona fide CXCR2 ligand CXCL8 while emphasizing the structural differences. As MIF also interacts with CXCR4, a chemokine receptor implicated in CXCL12-elicited lymphocyte arrest, the arrest potential of the MIF/CXCR4 axis will also be scrutinized as well as the recently identified role of pericyte MIF in attracting leukocytes exiting through venules as part of the pericyte "motility instruction program."

Type I IFNs are used for treating viral, neoplastic, and inflammatory disorders. The protein products encoded by IFN-stimulated genes (ISGs) likely mediate clinical effects of IFN in patients. Macroarray assays, used for studying ISG induction in IFN-treated patients, comprise genes identified predominantly through analysis of long-term cell lines. To discover genes induced selectively by IFN-beta in PBMC, we exposed whole blood to physiological concentrations of IFN-beta. PBMC were prepared, and RNA was extracted, reverse-transcribed, and hybridized to cDNA microarrays, and microarray analysis identified 39 ISGs and 20 IFN-repressed genes (IRGs). Thirty-three ISGs were known previously, and six ISGs were novel. New ISGs included GTP cyclohydrolase 1; hypothetical protein LOC129607; hypothetical protein FLJ38348; leucine aminopeptidase 3; squalene epoxidase; and GTP-binding protein overexpressed in skeletal muscle. Twenty IRGs included IL-1beta and CXCL8, which had been identified earlier. CXCL1 was a novel IRG identified in the current study. PCR analysis demonstrated the regulation of six novel ISGs and CXCL1 as an IRG in PBMC and astrocytoma cells. Results were validated using RNA obtained ex vivo from blood of patients after injection with IFN-beta. Identification of new ISGs and IRGs in primary PBMC will enhance macroarray assays for monitoring IFN responsiveness.

Ceramide is recognized as an antiproliferative and proapoptotic sphingolipid metabolite; however, the role of ceramide in inflammation is not well understood. To determine the role of C6-ceramide in regulating inflammatory responses, human corneal epithelial cells were treated with C6-ceramide in 80 nm diameter nanoliposome bilayer formulation (Lip-C6) prior to stimulation with UV-killed Staphylococcus aureus. Lip-C6 (5 muM) inhibited the phosphorylation of proinflammatory and proapoptotic MAP kinases JNK and p38 and production of neutrophil chemotactic cytokines CXCL1, CXCL5, and CXCL8. Lip-C6 also blocked CXC chemokine production by human and murine neutrophils. To determine the effect of Lip-C6 in vivo, a murine model of corneal inflammation was used in which LPS or S. aureus added to the abraded corneal surface induces neutrophil infiltration to the corneal stroma, resulting in increased corneal haze. Mice were treated topically with 2 nMoles (811 ng) Lip-C6 or with control liposomes prior to, or following, LPS or S. aureus stimulation. We found that corneal inflammation was significantly inhibited by Lip-C6 but not control liposomes given prior to, or following, activation by LPS or S. aureus. Furthermore, Lip-C6 did not induce apoptosis of corneal epithelial cells in vitro or in vivo, nor did it inhibit corneal wound healing. Together, these findings demonstrate a novel, anti-inflammatory, nontoxic, therapeutic role for liposomally delivered short-chain ceramide.

Dendritic cells (DCs) initiate adaptive immunity and regulate the inflammatory response by producing inflammatory chemokines. This study was aimed to elucidate their role in the pathogenesis of the suppurative granuloma induced by Bartonella henselae infection, which characterizes cat scratch disease (CSD). In vitro DC infection by B. henselae results in internalization of bacteria, phenotypic maturation with increased expression of HLA-DR and CD86, and induction of CD83, CD208, and CCR7. In comparison to LPS-activated DCs, B henselae-infected DCs produce higher amounts of IL-10, whereas the production of IL-12p70 is reduced. Infected DCs also produce high levels of CXCL8 and CXCL13, 2 chemokines active respectively on neutrophils and B lymphocytes. These results provide the molecular basis for the morphogenesis of CSD granuloma, which typically contains high numbers of neutrophils and B cells. Remarkably, CSD granulomas in vivo contain CXCL13-producing DCs. We further demonstrate that the B cells in CSD granulomas are represented by monocytoid B cells and, worth noting, they express T-bet, a transcription factor able to induce a T-independent immunoglobulin (Ig) class switch in B lymphocytes. These findings suggest that the humoral immune response to B henselae initiates in the extrafollicular areas of infected lymph nodes and is regulated by DCs.

BACKGROUND

Mast cell microlocalisation within the airway smooth muscle (ASM) bundle is an important determinant of the asthmatic phenotype. We hypothesised that mast cells migrate towards ASM in response to ASM derived chemokines.

METHODS

Primary ASM cultures from subjects with and without asthma were stimulated with interleukin (IL)-1beta, IL-4, and IL-13 alone and in combination. Mast cell chemotaxis towards these ASM supernatants was investigated, and the chemotaxins mediating migration by using specific blocking antibodies for stem cell factor (SCF) and the chemokine receptors CCR3, CXCR1, 3 and 4 as well as the Gi inhibitor pertussis toxin and the tyrosine kinase inhibitor genistein were defined. The concentrations of CCL11, CXCL8, CXCL10, TGF-beta, and SCF in the supernatants were measured and the effect of non-asthmatic ASM supernatants on the mast cell chemotactic activity of asthmatic ASM was examined.

RESULTS

Human lung mast cells and HMC-1 cells migrated towards Th2 stimulated ASM from asthmatics but not non-asthmatics. Mast cell migration was mediated through the combined activation of CCR3 and CXCR1. CCL11 and CXCL8 expression by ASM increased markedly after stimulation, but was similar in those with and without asthma. ASM supernatants from non-asthmatics inhibited mast cell migration towards the asthmatic ASM supernatant.

CONCLUSIONS

Th2 stimulated ASM from asthmatics is chemotactic for mast cells. Non-asthmatic ASM releases a mediator or mediators that inhibit mast cell migration towards stimulated asthmatic ASM. Specifically targeting mast cell migration into the ASM bundle may provide a novel treatment for asthma.

The CXC chemokine CXCL8/IL-8 plays a major role in the activation and recruitment of polymorphonuclear (PMN) cells at inflammatory sites. CXCL8 activates PMNs by binding the seven-transmembrane (7-TM) G-protein-coupled receptors CXC chemokine receptor 1 (CXCR1) and CXC chemokine receptor 2 (CXCR2). (R)-Ketoprofen (1) was previously reported to be a potent and specific noncompetitive inhibitor of CXCL8-induced human PMNs chemotaxis. We report here molecular modeling studies showing a putative interaction site of 1 in the TM region of CXCR1. The binding model was confirmed by alanine scanning mutagenesis and photoaffinity labeling experiments. The molecular model driven medicinal chemistry optimization of 1 led to a new class of potent and specific inhibitors of CXCL8 biological activity. Among these, repertaxin (13) was selected as a clinical candidate drug for prevention of post-ischemia reperfusion injury.

Cryptosporidium parvum causes a zoonotic infection with worldwide distribution. Besides humans, cryptosporidiosis affects a wide range of animals leading to significant economic losses due to severe enteritis in neonatal livestock. Neutrophil extracellular trap (NET) formation has been demonstrated as an important host effector mechanism of PMN acting against several invading pathogens. In the present study, C. parvum-mediated NET formation was investigated in human and bovine PMN in vitro. We here demonstrate that C. parvum sporozoites indeed trigger NET formation in a time-dependent manner. Thereby, the classical characteristics of NETs were demonstrated by co-localization of extracellular DNA with histones, neutrophil elastase (NE) and myeloperoxidase (MPO). A significant reduction of NET formation was measured following treatments of PMN with NADPH oxidase-, NE- and MPO-inhibitors, confirming the key role of these enzymes in C. parvum-induced NETs. Additionally, sporozoite-triggered NETosis revealed as dependent on intracellular Ca(++) concentration and the ERK 1/2 and p38 MAPK-mediated signaling pathway. Moreover, sporozoite-triggered NET formation led to significant parasite entrapment since 15% of the parasites were immobilized in NET structures. Consequently, PMN-pre-exposed sporozoites showed significantly reduced infectivity for epithelial host cells confirming the capability of NETs to prevent active parasite invasion. Besides NETs, we here show that C. parvum significantly up-regulated CXCL8, IL6, TNF-α and of GM-CSF gene transcription upon sporozoite confrontation, indicating a pivotal role of PMN not only in the bovine and human system but most probably in other final hosts for C. parvum.

Inflammatory illness is associated with depression. Preclinical work has shown that chemokines are linked with peripheral-central crosstalk and may be important in mediating depressive behaviours. We sought to establish what evidence exists that differences in blood or cerebrospinal fluid chemokine concentration discriminate between individuals with depression and those without. Following PRISMA guidelines, we systematically searched Embase, PsycINFO and Medline databases. We included participants with physical illness for subgroup analysis, and excluded participants with comorbid psychiatric diagnoses. Seventy-three studies met the inclusion criteria for the meta-analysis. Individuals with depression had higher levels of blood CXCL4 and CXCL7 and lower levels of blood CCL4. Sensitivity analysis of studies with only physically healthy participants identified higher blood levels of CCL2, CCL3, CCL11, CXCL7 and CXCL8 and lower blood levels of CCL4. All other chemokines examined did not reveal significant differences (blood CCL5, CCL7, CXCL9, CXCL10 and cerebrospinal fluid CXCL8 and CXCL10). Analysis of the clinical utility of the effect size of plasma CXCL8 in healthy individuals found a negative predictive value 93.5%, given the population prevalence of depression of 10%. Overall, our meta-analysis finds evidence linking abnormalities of blood chemokines with depression in humans. Furthermore, we have demonstrated the possibility of classifying individuals with depression based on their inflammatory biomarker profile. Future research should explore putative mechanisms underlying this association, attempt to replicate existing findings in larger populations and aim to develop new diagnostic and therapeutic strategies.

OBJECTIVE

Local interaction between soluble mediators within the inflamed synovium is a key factor that governs the pathologic outcome of inflammatory arthritides. Our aim was to investigate the interplay between the Th1 lymphokine interferon-gamma (IFNgamma) and pivotal cytokines that drive rheumatoid arthritis (RA) pathology (interleukin-1beta [IL-1beta] and tumor necrosis factor alpha [TNFalpha]) in modulating inflammation and arthritis in vitro and in vivo.

METHODS

Monarticular antigen-induced arthritis (AIA) was initiated in IFNgamma-deficient (IFNgamma(-/-)) mice and age-matched wild-type (IFNgamma(+/+)) mice. Joint swelling was measured and histologic analysis was performed in order to assess changes in both inflammatory and degenerative parameters in vivo. In vitro, the influence of IFNgamma in regulating IL-1beta- and TNFalpha-driven CXCL8 and CCL2 production was quantified by enzyme-linked immunosorbent assay.

RESULTS

In murine AIA, both inflammatory and degenerative arthritis parameters were significantly exacerbated in the absence of IFNgamma. IFNgamma appeared to be a crucial factor in regulating CXCR2+ neutrophil influx in the joint. In in vitro studies using RA fibroblast-like synoviocytes, IFNgamma modulated both IL-1beta- and TNFalpha-driven chemokine synthesis, resulting in the down-regulation of CXCL8 production.

CONCLUSIONS

IFNgamma exerts antiinflammatory, chondroprotective, and antiosteoclastogenic effects in murine AIA through a mechanism that involves the regulation of chemokine synthesis and local neutrophil recruitment. These studies suggest a potential therapeutic role of modulating IFNgamma signaling in the treatment of inflammatory arthritides.

Chemokines play an important role in leukocyte mobilization, hematopoiesis, and angiogenesis. Tissue-specific expression of particular chemokines also influences tumor growth and metastasis. Here, the CC chemokine pulmonary and activation-regulated chemokine (PARC)/CCL18 was measured in pediatric patients with acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML). Surprisingly, PARC immunoreactivity was consistently detected in plasma from healthy donors. After purification to homogeneity, the presence of intact PARC (1-69) and processed PARC (1-68) in normal human plasma was confirmed by sequence and mass spectrometry analysis. Furthermore, PARC serum levels were significantly increased in children with T-ALL and prepreB-ALL compared to control serum samples, whereas serum levels in AML and preB-ALL patients were not significantly different from controls. In contrast, the hemofiltrate CC chemokine-1 (HCC-1)/CCL14 was not found to be a biomarker in any of these patients' strata, whereas the cytokine interleukin-6 (IL-6) was significantly decreased in AML and prepreB-ALL. Stimulated leukocytic cell lines or lymphoblasts from patients produced IL-8/CXCL8 or macrophage inflammatory protein-1alpha (MIP-1alpha/CCL3) but not PARC, not even after IL-4 or IL-10 treatment. However, PARC was produced by superantigen or IL-4 stimulated monocytes co-cultured with lymphocytes or lymphoblastic cells. Serum PARC levels thus constitute a novel leukemia marker, possibly reflecting tumor/host cell interactions in the circulation.

Adiponectin is believed to exert hepatoprotective effects and induces CXCL8, a chemokine that functions as a survival factor, in vascular cells. In the current study, it is demonstrated that adiponectin also induces CXCL8 expression in primary human hepatocytes but not in hepatocellular carcinoma cell lines. Knock down of the adiponectin receptor (AdipoR) 1 or AdipoR2 by small-interfering RNA indicates that AdipoR1 is involved in adiponectin-stimulated CXCL8 release. Adiponectin activates nuclear factor (NF)-kappaB in primary hepatocytes and pharmacological inhibition of NF-kappaB, the p38 mitogen-activated protein kinase, and extracellular signal-regulated kinase (ERK) 1/ERK2 reduces adiponectin-mediated CXCL8 secretion. Furthermore, adiponectin also activates STAT3 involved in interleukin (IL)-6 and leptin-mediated CXCL8 induction in primary hepatocytes. Inhibition of JAK2 by AG-490 does not abolish adiponectin-stimulated CXCL8, indicating that this kinase is not involved. Pretreatment of primary cells with "STAT3 Inhibitor VI," however, elevates hepatocytic CXCL8 secretion, demonstrating that STAT3 is a negative regulator of CXCL8 in these cells. In accordance with this assumption, IL-6, a well-characterized activator of STAT3, reduces hepatocytic CXCL8. Therefore, adiponectin-stimulated induction of CXCL8 seems to be tightly controlled in primary human hepatocytes, whereas neither NF-kappaB, STAT3, nor CXCL8 are influenced in hepatocytic cell lines. CXCL8 is a survival factor, and its upregulation by adiponectin may contribute to the hepatoprotective effects of this adipokine.

Epidermal keratinocytes produce proinflammatory cytokines/chemokines upon stimulation with cytokine milieus and Toll-like receptor ligands, which are considered to reflect epidermal environments in inflamed skin. The human antimicrobial peptide LL-37, besides having microbicidal functions, plays multiple roles as a "host defense peptide" in the immune system. Here, we examined the effect of LL-37 on proinflammatory responses induced by double-stranded RNA (dsRNA) and cytokines in primary human keratinocytes. LL-37 inhibited dsRNA-induced production of thymic stromal lymphopoietin (TSLP), CCL5/RANTES, CXCL10/IP-10, and CXCL8/IL-8, which was attributable to interaction between LL-37 and dsRNA, although LL-37 upregulated CXCL8 expression at an earlier time point (8 h). LL-37 inhibited the increase of CXCL10 and CCL5 induced by TNF-α- and/or IFN-γ but enhanced that of CXCL8. LL-37 and Th17 cytokines (IL-17 and IL-22) synergistically upregulated the expression of CXCL8 and IL-6. LL-37 showed the effects above at a high concentration (25 μg/ml, 5.6 μM). We also examined effects of a peptide with a scrambled LL-37 sequence, which has been frequently used as a negative control, and those of another peptide with the reversed LL-37 sequence, activities of which have not been well investigated. Interestingly, the reversed LL-37 had effects similar to LL-37 but the scrambled LL-37 did not. The modulation by LL-37 of the keratinocyte proinflammatory responses induced by cytokine milieus and dsRNA suggests novel roles for LL-37 in skin inflammation such as the promotion of IL17/IL-22/IL-6-associated psoriasis and suppression of TSLP-associated atopic dermatitis.

Chemokine IL-8 (CXCL8) binds to its cognate receptors CXCR1 and CXCR2 to induce inflammatory responses, wound healing, tumorogenesis, and neuronal survival. Here we identify the N-loop residues in IL-8 (H18 and F21) and the receptor N-termini as the major structural determinants regulating the rate of receptor internalization, which in turn controlled the activation profile of ERK1/2, a central component of the receptor/ERK signaling pathway that dictates signal specificity. Our data further support the idea that the chemokine receptor core acts as a plastic scaffold. Thus, the diversity and intensity of inflammatory and noninflammatory responses mediated by chemokine receptors appear to be primarily determined by the initial interaction between the receptor N-terminus and the N-loop of chemokines.

The response of psoriasis to antibodies targeting the interleukin (IL)-23/IL-17A pathway suggests a prominent role of T-helper type-17 (Th17) cells in this disease. We examined the clinical and immunological response patterns of 100 subjects with moderate-to-severe psoriasis receiving 3 different intravenous dosing regimens of the anti-IL-17A antibody secukinumab (1 × 3 mg/kg or 1 × 10 mg/kg on Day 1, or 3 × 10 mg/kg on Days 1, 15 and 29) or placebo in a phase 2 trial. Baseline biopsies revealed typical features of active psoriasis, including epidermal accumulation of neutrophils and formation of microabscesses in >60% of cases. Neutrophils were the numerically largest fraction of infiltrating cells containing IL-17 and may store the cytokine preformed, as IL-17A mRNA was not detectable in neutrophils isolated from active plaques. Significant clinical responses to secukinumab were observed 2 weeks after a single infusion, associated with extensive clearance of cutaneous neutrophils parallel to the normalization of keratinocyte abnormalities and reduction of IL-17-inducible neutrophil chemoattractants (e.g. CXCL1, CXCL8); effects on numbers of T cells and CD11c-positive dendritic cells were more delayed. Histological and immunological improvements were generally dose dependent and not observed in the placebo group. In the lowest-dose group, a recurrence of neutrophils was seen in some subjects at Week 12; these subjects relapsed faster than those without microabscesses. Our findings are indicative of a neutrophil-keratinocyte axis in psoriasis that may involve neutrophil-derived IL-17 and is an early target of IL-17A-directed therapies such as secukinumab.

Macrophage migration inhibitory factor (MIF) and CXCL8 (also named IL-8) are strongly expressed in the tissues of nasopharyngeal carcinoma (NPC). However, their role in the growth of NPC has not been fully examined. This study aims to evaluate the functions of MIF and CXCL8 on the growth of NPC tumor spheres. The elevated expression of CXCL8 in tumor over normal tissues was confirmed in 37 pairs of biopsies from NPC patients. In the in vitro study, all the poorly differentiated NPC cell lines, including the EBV-positive C666-1, and the EBV-negative CNE-1, CNE-2, SUNE-1, HNE-1 and HONE-1 cells, were found to express CXCL8 and MIF. Therefore, the EBV-positive C666-1 cell was selected to examine for the role of MIF and CXCL8 in the growth of the NPC tumor spheres. Functional study showed that the growth of C666-1 tumor spheres, under the nutrient poor or growth factor supplemented culture conditions, could be inhibited by the CXCL8 specific peptide inhibitor. The growth of the tumor spheres could also be reduced by the CXCR2 specific inhibitor SB225002 or the PI3K/AKT inhibitor LY294002, indicating that the endogenously produced CXCL8 plays an autocrine role in the growth of the tumor spheres. Further mechanistic studies revealed that the gene expression of CXCL8 could be reduced by the MIF specific small interfering RNA (siRNA) or NF-κB inhibitor parthenolide, and the growth of tumor spheres was also reduced after MIF siRNA transfection. Taken together, the present study highlights the role of MIF/CXCL8/CXCR2 axis in the growth of NPC tumor spheres. Chemotherapeutic interference of this signaling pathway may help to control the growth of the NPC tumor.

The present study aimed at profiling inflammatory cytokines for neurological and psychiatric diseases. A total of 86 patients with meningitis, multiple sclerosis, tension-type headache, idiopathic facial nerve palsy (IFNP), affective and schizophrenic disorders were tested for both, serum and cerebrospinal fluid (CSF) using a multiplexed cytokine ELISA for IFN-γ, TNF-α, IL-1β, IL-2, IL-4, IL-5, IL-8/CXCL8, IL-10, IL12p70, IL-13 and IL-17. Cases with viral and bacterial meningitis had unequivocally higher cytokine concentrations in the CSF when compared with serum. Bacterial meningitis was unique by extremely elevated IL-17, TNF-α and IL-1β, indicating a plethora of inflammatory pathways, selectively activated in the CSF. In relapsing multiple sclerosis, IFN-γ and IL-10 were elevated in both, serum and CSF, but IL-12p70, IL-5, IL-13, and TNF-α were more prominent in serum than in CSF. Qualitatively similar biomarker patterns were detected in patients with idiopathic facial nerve palsy and tension-type cephalgia. Affective and schizophrenic disorders clearly present with an inflammatory phenotype in the CSF and also serum, the cytokines determined were in general higher in schizophrenia. Except IFN-γ, schizophrenic patients had higher IL-12p70 and a trend of higher IL-10 and IL-13 in serum suggesting a more prominent TH2-type counter regulatory immune response than in affective disorders. These differences were also mirrored in the CSF. Elevated IL-8 appears to be the most sensitive marker for inflammation in the CSF of all diseases studied, whereas TNF-α was restricted to peripheral blood. With the exception of IL-8, all but viral and bacterial meningitis, studied, displayed higher means of elevated lymphokine concentrations in the serum than in the CSF. This observation supports the concept of immunological crosstalk between periphery and intrathecal immunity in neurological and psychiatric diseases.

We have previously demonstrated that chloroquine may evoke inflammatory responses in the central nervous system by inducing expression of pro-inflammatory cytokines by astroglial cells. In this study, we further examined the molecular mechanism responsible for chloroquine-induced activation of NF-kappaB and subsequent expression of chemokines by astroglial cells. We observed that (1) chloroquine induced expression of chemokines such as CCL2 and CXCL8 in a dose- and time-dependent manner in human astroglial cells; (2) other lysosomotropic agents such as ammonium chloride and bafilomycin A1 had minimal effects on chemokine expression; (3) inhibition of NF-kappaB by MG-132 and TPCK suppressed chloroquine-induced mRNA expression of chemokines; (4) chloroquine increased the intracellular level of reactive oxygen species (ROS) in a dose- and time-dependent manner by human astroglial cells, but not by monocytic/microglial cells; (5) chloroquine-induced increase of intracellular ROS level was suppressed by pre-incubation with diphenyl iodonium (DPI) and N-acetyl cysteine (NAC); and (6) inhibition of chloroquine-induced ROS production by DPI or NAC suppressed chloroquine-mediated activation of NF-kappaB and subsequent mRNA expression of chemokines in astroglial cells. These results collectively suggest that chloroquine generates ROS, which is responsible for NF-kappaB activation and subsequent expression of pro-inflammatory chemokines in human astroglial cells.