BACKGROUND

Adipokines are bioactive hormones secreted by adipose tissues. Resistin, an adipokine, plays important roles in the regulation of insulin resistance and inflammation. Resistin levels are known to be increased in the serum and synovial fluid of rheumatoid arthritis (RA) patients. However, the pathogenic role of resistin in RA has not yet been elucidated.

METHODS

The expression of resistin and adenylate cyclase-associated protein 1 (CAP1), a receptor for resistin, was examined immunohistochemically in synovial tissue. CAP1 expression in in vitro cultured fibroblast-like synoviocytes (FLSs) was assessed with a reverse transcription-polymerase chain reaction (PCR) and western blotting. The gene expression of resistin-stimulated FLSs was evaluated by RNA sequencing (RNA-Seq) and quantitative real-time PCR. Concentrations of chemokine (C-X-C motif) ligand (CXCL) 8, chemokine (C-C motif) ligand (CCL) 2, interleukin (IL)-1β, IL-6 and IL-32 in culture supernatants were measured by enzyme-linked immunosorbent assay. Small interfering RNA (siRNA) for CAP1 was transfected into FLSs in order to examine inhibitory effects.

RESULTS

The expression of resistin and CAP1 in synovial tissue was stronger in RA than in osteoarthritis (OA). Resistin was expressed by macrophages in the RA synovium, while CAP1 was expressed by macrophages, FLSs and endothelial cells. In vitro cultured RA FLSs also expressed CAP1. RNA-Seq revealed that the expression levels of 18 molecules were more than twofold higher in resistin-stimulated FLSs than in unstimulated FLSs. Seven chemokines, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, and CCL2, were included among the 18 molecules. Increases induced in the expression of CXCL1, CXCL8, and CCL2 by the resistin stimulation were confirmed by real-time PCR. The stimulation with resistin increased the protein levels of CXCL8 and CCL2 produced by RA FLSs, and the upregulated expression of CXCL8 was inhibited by the abrogation of CAP1 by siRNA for CAP1. Production of IL-6 by FLSs was also increased by resistin. Expression of IL-1β and IL-32 was not detected by ELISA.

CONCLUSIONS

Resistin contributes to the pathogenesis of RA by increasing chemokine production by FLSs via CAP1 in synovial tissue.

An indispensable role for oligodendrocytes in the protection of axon function and promotion of neuronal survival is strongly supported by the finding of progressive neuron/axon degeneration in human neurological diseases that affect oligodendrocytes. Imaging and pathological studies of the CNS have shown the presence of neuroaxonal injury in progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the CNS, resulting from destruction of oligodendrocytes upon productive replication of the pathogenic neurotropic polyomavirus JC. Here, we examined the extracellular factors involved in communication between oligodendrocytes and neurons. Culturing cortical neurons with conditioned medium (CM) from rat CG4 oligodendrocytic cells that express the JCV agnoprotein showed that CXCL5/LIX, which is a chemokine closely related to the human CXCL5/ENA78 and CXCL6/GCP-2 chemokines, is essential for neuronal cell survival. We found that in CM from agnoprotein-producing CG-4 cells level of CXC5/LIX is decreased compared to control cells. We also demonstrated that a reduced expression of CXCL5/LIX by CG4 GFP-Agno cells triggered a cascade of signaling events in cortical neurons. Analysis of mitogen-activated protein kinases (MAPK) and glycogen synthase kinase (GSK3) pathways showed that they are involved in mechanisms of neuronal apoptosis in response to the depletion of CXCL5/LIX signaling. These data suggest that agnoprotein-induced dysregulation of chemokine production by oligodendrocytes may contribute to neuronal/axonal injury in the pathogenesis of PML lesions.

OBJECTIVE

To identify disease-specific cytokine profile differences in the aqueous humor (AH) (other than the vascular endothelial growth factor) between patients with dry and treated wet age-related macular degeneration (AMD) and healthy controls.

METHODS

This retrospective study drew on a case-series of patients diagnosed with dry AMD (n = 25) and treated wet AMD (n = 19), as well as on healthy controls (no systemic therapy; n = 20) undergoing phacoemulsification or vitrectomy. Samples of AH and serum were collected in parallel at the beginning of surgery. The levels of 43 cytokines were simultaneously determined using the Bio-Plex® multiplex beads system. Differences between the three groups were statistically compared using the Kruskal-Wallis H-Test after applying the Bonferroni correction for multiple comparisons (p<0.0012).

RESULTS

The concentrations of three cytokines were elevated in the AH of patients with dry AMD (CXCL6; p = 0.00067) and treated wet AMD (CXCL5, CXCL6, MIG/XCXL; all p<0.001) relative to those in the healthy controls. No other differences between the three groups were identified. The AH levels of seven cytokines (16%), including CXCL6, ranged below the lower limit of quantitation of the assay. Without the correction for multiple comparisons (p<0.05), the levels of 31 of the 43 cytokines in the AH of patients with AMD would have differed significantly from those in the control. The systemic cytokine profiles (serum) were similar in all three groups.

CONCLUSIONS

No systematic differences in the AH cytokine environment were identified between patients with dry AMD and those with treated wet AMD. This finding might indicate that AMD is either the result of a persistent imbalance in the physiological tissue milieu, or that the localized process induces no significant change in the cytokine environment of the anterior ocular segment.

Cytokines and chemokines regulate many functions in the body including the brain. The interactions between adipose tissue and the central nervous system (CNS) are important for the regulation of energy balance. CNS function is also influenced by age. The aim of the present study was to investigate the effects of body mass index (BMI) and age on cytokine and chemokine levels in cerebrospinal fluid. Cerebrospinal fluid samples (n=89) were collected from patients undergoing routine surgical procedures. The samples were analyzed using the multiplex proximity extension assay (PEA) in which 92 different cytokines are measured simultaneously using minute sample volume. We found no significant correlations between age and cytokine levels for any of the studied markers. In contrast, at a false discovery rate of 10%, 19 markers were significantly associated with BMI (in decreasing significance: FGF-5, ADA, Beta-NGF, CD40, IL-10RB, CCL19, TGF-alpha, SIRT2, TWEAK, SCF, CSF-1, 4E-BP1, DNER, LIF-R, STAMPB, CXCL10, CXCL6, VEGF-A and CX3CL1). This study reveals a clear effect of BMI on cytokine and chemokine levels in cerebrospinal fluid.

Placental expression of gonadotropin-releasing hormone (GnRH)-I and II, as well as their cognate receptor, coincides with a period of extensive remodeling of the maternal-fetal interface, near the end of the first trimester of pregnancy. To further define the role of GnRH in human placentation, we performed a microarray screen of HTR-8/SVneo trophoblasts to identify GnRH-regulated genes and their roles in placentation. This screen revealed that GnRH regulates the expression of four angiogenic chemokines: CXCL2, CXCL3, CXCL6, and CXCL8. The microarray data were subsequently confirmed by an extensive Q-PCR time-course analysis. CXCL8, a representative chemokine, was selected for further analysis and shown to be strongly expressed by trophoblasts at the maternal-fetal interface of the human placenta, as well as to accumulate in a GnRH-dependent manner in trophoblast-conditioned media in culture. Trophoblasts were subsequently shown to recruit lymphocytes (Jurkat T cells and primary peripheral blood T and uterine natural killer cells) in chemotaxis assays and this was shown to be GnRH dependent. Furthermore, this recruitment was shown to occur via the release of CXCR1/CXCR2 interacting chemokines, such as the CXCLs investigated in this study. This novel regulation of chemokines by GnRH signaling demonstrates the role of GnRH in regulating the recruitment of lymphocytes to the decidua and the possibility of a direct effect on spiral artery remodeling via the release of proangiogenic chemokines and secondary effects via release of angiogenic factors by recruited lymphocytes.

Although many effects of staphylococcal superantigens (SAg) on T cells are well established, less is known about their effects on APC. In this study, bovine PBMC were stimulated with a low dose of staphylococcal enterotoxin C1 (SEC1). The phenotype of adherent cells (Ac) derived from bovine PBMC cultured with SEC1 [SEC1-stimulated Ac (sAc)] for 192 h was CD14(-), CD68(-), CD163(-), dendritic cell (DC)-specific ICAM-3-grabbing nonintegrin(+), MHC class II (MHC II)(high), CD11a(low), CD11b(high), CD11c(high), and CD1b(high), suggesting these cells were dendritic cells (DC). SEC1 also induced transcription of the CXCL1, -2, and -3 family, CXCL6, CCL2, and CCL5 genes in sAc, which increased rapidly but returned to basal levels by 48 h. In contrast, increased transcription of CCL3, CCL8, and CXCL12, responsible for mononuclear cell migration and chronic inflammation, was sustained. In vitro cell migration assays showed vigorous migration of granulocytes, followed by migration of mononuclear cells. The autologous MLR showed that sAc induced a dose-dependent proliferation of CD4(+) T cells and an even stronger proliferation of CD8(+) T cells. This effect was inhibited or reduced by pretreatment with mAb to CD11b, MHC II, or MHC II plus CD18. These results indicate that stimulation of bovine PBMC by SAg induces differentiation of monocytes into DC.

BACKGROUND

Osteosarcoma is the most common malignant bone tumor in children. Despite the advent of chemotherapy, the survival of osteosarcoma patients has not been significantly improved recently. Chemokines are a group of signaling molecules that have been implicated in tumorigenesis and metastasis.

METHODS

The authors used an antibody microarray to identify chemokines that were elevated in the plasma samples of osteosarcoma patients. The results were validated using enzyme-linked immunosorbent assays on an independent set of samples. The tumor expressions of 3 chemokines were examined in 2 sets of osteosarcoma tissue arrays. The authors also evaluated the proliferative effect of the chemokines in 4 osteosarcoma cell lines.

RESULTS

The authors found that the plasma levels of CXCL4, CXCL6, and CXCL12 in the osteosarcoma patients were significantly higher than those in the controls, and the results were validated by an independent osteosarcoma cohort (P < .05). However, CXCL4 (100%) and CXCL6 (91%) were frequently expressed in osteosarcoma, whereas CXCL12 was only expressed in 4%. Survival analysis further showed that higher circulating levels of CXCL4 and CXCL6, but not CXCL12, were associated with a poorer outcome of osteosarcoma patients. Addition of exogenous chemokines significantly promoted the growth of different osteosarcoma cells (P < .05).

CONCLUSIONS

The results demonstrate that CXCL4 and CXCL6 are frequently expressed in osteosarcoma, and that the plasma levels of these 2 chemokines are associated with patient outcomes. Further study of these circulating chemokines may provide a promising approach for prognostication of osteosarcoma. Targeting these chemokines or their receptors may also lead to a novel therapeutic invention.

LPS-induced CXC chemokine (LIX) is a murine chemokine similar to two human chemokines, ENA-78 (CXCL5) and GCP-2 (CXCL6). To clarify the relationship of LIX to human ENA-78 and GCP-2, we cloned and mapped the LIX gene. The organization of the LIX gene ( Scyb5) is similar to those of the human ENA-78 ( SCYB5) and GCP-2 ( SCYB6) genes. The intron-exon boundaries of the three genes are exactly conserved, and the introns have similar sizes. The first 100 bp of the 5' flanking regions are highly similar, with conserved NF-kappaB and GATA sites in identical positions in all three genes. Further 5', the Lix flanking region sequence diverges from those of ENA-78 and GCP-2, which remain highly similar for 350 bp preceding the start sites. Using a (C57BL/6 J x Mus spretus) F1 x C57BL/6J backcross panel, Lix was mapped to a locus near D5Ucla5 at 49.0 cM on Chromosome (Chr) 5. Mapping with the T31 radiation hybrid panel placed Lix between D5Mit360 and D5Mit6. Physical maps of the CXC chemokine clusters on murine Chr 5 and human Chr 21 were constructed using the Celera mouse genome database and the public human genome database. The sequence and mapping data suggest that the human ENA78-PBP-PF4 and GCP2- psi PBP-PF4V1 loci arose from an evolutionarily recent duplication of an ancestral locus related to the murine Lix-Pbp-Pf4 locus.

Increasing evidence suggests that alpha-chemokines serve several important functions in the nervous system, including regulation of neuroimmune responses, neurotransmission, neuronal survival, and central nervous system development. In this study, we first examined the function of two alpha-chemokines, chemokine ligand (CXCL) 6 and CXCL8, and their receptors, CXCR1 and CXCR2, in the developing rat ventral midbrain (VM). We found that CXCR2 and CXCL6 are regulated during VM development and that CXCL6 promotes the differentiation of nurr77-related receptor (Nurr1)+ precursors into dopaminergic (DA) neurons in vitro. Intriguingly, CXCL8, a ligand expressed only in Homo sapiens, enhanced progenitor cell division, neurogenesis, and tyrosine hydroxylase-positive (TH+) cell number in rodent precursor and neurosphere cultures. CXCL1, the murine ortholog of CXCL8, was developmentally regulated in the VM and exhibited activities similar but not identical to those of CXCL8. TH+ cells derived from chemokine-treated VM neurospheres coexpressed Nurr1 and VMAT and were functionally active, as shown by calcium (Ca(2+)) fluxes in response to AMPA. In conclusion, our data demonstrate that CXCL1, CXCL6, and CXCL8 increase the number of DA neurons in VM precursor and neurosphere cultures by diverse mechanisms. Thus, alpha-chemokines may find an application in the preparation of cells for drug development or Parkinson's disease cell replacement therapy.

Vascular endothelial growth factor (VEGF), oncostatin M (OSM), and granulocyte chemotactic protein-2 (GCP-2/CXCL6) are up-regulated in U937 macrophages and peripheral blood macrophages exposed to LPS, beta-adrenergic receptor (beta2-AR) agonists (e.g. zilpaterol, and clenbuterol) and some other agents that induce intracellular cAMP (prostaglandin E2, forskolin, and butyryl cAMP). LPS in combination with beta2-agonists and cAMP elevating agents had an additional effect on the release of VEGF, OSM, and CXCL6. These proteins are up-regulated after 16-24 h of exposure and this is mediated by the beta2-AR, as determined by time course experiments and the use of a specific beta2-AR antagonist (ICI 118551). Beta2-AR agonists are used as bronchodilators in the treatment of asthma, but appear to have no effect on the chronic inflammation of the disease. However, the up-regulation of VEGF, OSM, and CXCL6 may have adverse effects on the inflammatory process of asthma. These mediators are involved in the recruitment of neutrophils, airway remodelling and angiogenesis, known features of chronic inflammatory diseases. We propose that the up-regulation of these proteins could play a role in the adverse effects of prolonged excessive usage of beta2-AR agonists on the airways besides the desensitization of the beta2-AR.

Human bone marrow-derived stromal stem cells (hBMSC) exhibit multiple functions, including differentiation into skeletal cells (progenitor function), hematopoiesis support, and immune regulation (nonprogenitor function). We have previously demonstrated the presence of morphological and functional heterogeneity of hBMSC cultures. In the present study, we characterized in detail two hTERT-BMSC clonal cell populations termed here CL1 and CL2 that represent an opposing phenotype with respect to morphology, markers expression: alkaline phosphatase (ALP) and CD146, and ex vivo differentiation potential. CL1 differentiated readily to osteoblasts, adipocytes, and chondrocytes as shown by expression of lineage specific genes and proteins. Whole genome transcriptome profiling of CL1 versus CL2 revealed enrichment in CL1 of bone-, mineralization-, and skeletal muscle-related genes, for example, ALP, POSTN, IGFBP5 BMP4, and CXCL12. On the other hand, CL2 transcriptome was enriched in immune modulatory genes, for example, CD14, CD99, NOTCH3, CXCL6, CFB, and CFI. Furthermore, gene expression microarray analysis of osteoblast differentiated CL1 versus CL2 showed significant upregulation in CL1 of bone development and osteoblast differentiation genes which included several homeobox genes: TBX15, HOXA2 and HOXA10, and IGF1, FGFR3, BMP6, MCAM, ITGA10, IGFBP5, and ALP. siRNA-based downregulation of the ALP gene in CL1 impaired osteoblastic and adipocytic differentiation. Our studies demonstrate the existence of molecular and functional heterogeneity in cultured hBMSC. ALP can be employed to identify osteoblastic and adipocytic progenitor cells in the heterogeneous hBMSC cultures.

The regulation of hematopoietic stem cell (HSC) survival and self-renewal within the bone marrow (BM) niche is not well understood. We therefore investigated global transcriptomic profiling of normal human HSC/hematopoietic progenitor cells [HPCs], revealing that several chemokine ligands (CXCL1-4, CXCL6, CXCL10, CXCL11, and CXCL13) were upregulated in human quiescent CD34(+)Hoescht(-)Pyronin Y(-) and primitive CD34(+)38(-), as compared with proliferating CD34(+)Hoechst(+)Pyronin Y(+) and CD34(+)38(+) stem/progenitor cells. This suggested that chemokines might play an important role in the homeostasis of HSCs. In human CD34(+) hematopoietic cells, knockdown of CXCL4 or pharmacologic inhibition of the chemokine receptor CXCR2, significantly decreased cell viability and colony forming cell (CFC) potential. Studies on Cxcr2(-/-) mice demonstrated enhanced BM and spleen cellularity, with significantly increased numbers of HSCs, hematopoietic progenitor cell-1 (HPC-1), HPC-2, and Lin(-)Sca-1(+)c-Kit(+) subpopulations. Cxcr2(-/-) stem/progenitor cells showed reduced self-renewal capacity as measured in serial transplantation assays. Parallel studies on Cxcl4 demonstrated reduced numbers of CFC in primary and secondary assays following knockdown in murine c-Kit(+) cells, and Cxcl4(-/-) mice showed a decrease in HSC and reduced self-renewal capacity after secondary transplantation. These data demonstrate that the CXCR2 network and CXCL4 play a role in the maintenance of normal HSC/HPC cell fates, including survival and self-renewal.

Studies in recent years have established that the principal effects in cardiac cell therapy are associated with paracrine/autocrine factors. We combined several complementary techniques to define human cardiac progenitor cell (CPC) secretome constituted by 914 proteins/genes; 51% of these are associated with the exosomal compartment. To define the set of proteins specifically or highly differentially secreted by CPC, we compared human mesenchymal stem cells and dermal fibroblasts; the study defined a group of growth factors, cytokines and chemokines expressed at high to medium levels by CPC. Among them, IL-1, GROa (CXCL1), CXCL6 (GCP2) and IL-8 are examples whose expression was confirmed by most techniques used. ELISA showed that CXCL6 is significantly overexpressed in CPC conditioned medium (CM) (18- to 26-fold) and western blot confirmed expression of its receptors CXCR1 and CXCR2. Addition of anti-CXCL6 completely abolished migration in CPC-CM compared with anti-CXCR2, which promoted partial inhibition, and anti-CXCR1, which was inefficient. Anti-CXCL6 also significantly inhibited CPC CM angiogenic activity. In vivo evaluation also supported a relevant role for angiogenesis. Altogether, these results suggest a notable angiogenic potential in CPC-CM and identify CXCL6 as an important paracrine factor for CPC that signals mainly through CXCR2.

Chemokine plays an important role in lung cancer and CXCL6 is one of chemokine, however, its effect on miRNAs profile and its roles in non-small cell lung cancer cell (NSCLC) is not elucidated. This study is purposed to explore the influence of CXCL6 on miRNA expression profile and found that CXCL6 could reduce the expression of miR-515-5p in NSCLC cells. MiR-515-5p in NSCLC cells could inhibit NSCLC survival and metastasis. MiR-515-5p acted as a tumor suppressor by targeting CXCL6 in NSCLC cells. These data highlighted a novel molecular interaction between miR-515-5p and CXCL6. MiR-515-5p may constitute a potential therapy target for NSCLC.

Despite the high prevalence of histological Benign Prostatic Hypeplasia (BPH) in elderly men, little is known regarding the molecular mechanisms and networks underlying the development and progression of the disease. Here, we explored the effects of a phytotherapeutic agent, Lipidosterolic extract of the dwarf palm plant Serenoa repens (LSESr), on the mRNA gene expression profiles of two representative models of BPH, BPH1 cell line and primary stromal cells derived from BPH. Treatment of these cells with LSESr significantly altered gene expression patterns as assessed by comparative gene expression profiling on gene chip arrays. The expression changes were manifested three hours following in vitro administration of LSESr, suggesting a rapid action for this compound. Among the genes most consistently affected by LSESr treatment, we found numerous genes that were categorized as part of proliferative, apoptotic, and inflammatory pathways. Validation studies using quantitative real-time PCR confirmed the deregulation of genes known to exhibit key roles in these biological processes including IL1B, IL1A, CXCL6, IL1R1, PTGS2, ALOX5, GAS1, PHLDA1, IL6, IL8, NFkBIZ, NFKB1, TFRC, JUN, CDKN1B, and ERBB3. Subsequent analyses also indicated that LSESr treatment can impede the stimulatory effects of certain proinflammatory cytokines such as IL6, IL17, and IL15 in these cells. These results suggest that LSESr may be useful to treat BPH that manifest inflammation characteristics. This also supports a role for inflammation in BPH presumably by mediating the balance between apoptosis and proliferation.

Decidual spiral arteriole (SpA) remodeling is essential to ensure optimal uteroplacental blood flow during human pregnancy, yet very little is known about the regulatory mechanisms. Uterine decidual NK (dNK) cells and macrophages infiltrate the SpAs and are proposed to initiate remodeling before colonization by extravillous trophoblasts (EVTs); however, the trigger for their infiltration is unknown. Using human first trimester placenta, decidua, primary dNK cells, and macrophages, we tested the hypothesis that EVTs activate SpA endothelial cells to secrete chemokines that have the potential to recruit maternal immune cells into SpAs. Gene array, real-time PCR, and ELISA analyses showed that treatment of endothelial cells with EVT conditioned medium significantly increased production of two chemokines, CCL14 and CXCL6. CCL14 induced chemotaxis of both dNK cells and decidual macrophages, whereas CXCL6 also induced dNK cell migration. Analysis of the decidua basalis from early pregnancy demonstrated expression of CCL14 and CXCL6 by endothelial cells in remodeling SpAs, and their cognate receptors are present in both dNK cells and macrophages. Neutralization studies identified IL-6 and CXCL8 as factors secreted by EVTs that induce endothelial cell CCL14 and CXCL6 expression. This study has identified intricate crosstalk between EVTs, SpA cells, and decidual immune cells that governs their recruitment to SpAs in the early stages of remodeling and has identified potential key candidate factors involved. This provides a new understanding of the interactions between maternal and fetal cells during early placentation and highlights novel avenues for research to understand defective SpA remodeling and consequent pregnancy pathology.

Tendinopathy accounts for over 30% of primary care consultations and represents a growing healthcare challenge in an active and increasingly ageing population. Recognising critical cells involved in tendinopathy is essential in developing therapeutics to meet this challenge. Tendon cells are heterogenous and sparsely distributed in a dense collagen matrix; limiting previous methods to investigate cell characteristics ex vivo. We applied next generation CITE-sequencing; combining surface proteomics with in-depth, unbiased gene expression analysis of > 6400 single cells ex vivo from 11 chronically tendinopathic and 8 healthy human tendons. Immunohistochemistry validated the single cell findings. For the first time we show that human tendon harbours at least five distinct COL1A1/2 expressing tenocyte populations in addition to endothelial cells, T-cells, and monocytes. These consist of KRT7/SCX+ cells expressing microfibril associated genes, PTX3+ cells co-expressing high levels of pro-inflammatory markers, APOD+ fibro-adipogenic progenitors, TPPP3/PRG4+ chondrogenic cells, and ITGA7+ smooth muscle-mesenchymal cells. Surface proteomic analysis identified markers by which these sub-classes could be isolated and targeted in future. Chronic tendinopathy was associated with increased expression of pro-inflammatory markers PTX3, CXCL1, CXCL6, CXCL8, and PDPN by microfibril associated tenocytes. Diseased endothelium had increased expression of chemokine and alarmin genes including IL33.

The reproductive tract is continuously challenged by potential pathogens present in the environment. Therefore, robust host defense mechanisms are essential both for the health of the individual and for fertilization. Antibiotic innate immunity peptides possess broad antimicrobial activity. Recently, we found that the CXC chemokine, granulocyte chemotactic protein (GCP)-2/CXCL6, possesses antibacterial activity. In the present study, we investigated, therefore, the presence of GCP-2/CXCL6 in the human male reproductive system. GCP-2/CXCL6 was detected at 19nM (mean; range: 5-47nM; n=14) in seminal plasma of fertile donors, i.e. at levels more than 100 times higher than those previously reported for the related chemokine IL-8/CXCL8. No GCP-2/CXCL6 could be detected in blood plasma of healthy donors, indicating local production in the male reproductive tract. In vasectomized donors, significantly lower levels of GCP-2/CXCL6 were found (mean: 3nM; range 2-7nM; n=7), demonstrating that the testis and epididymis contribute significantly to the GCP-2/CXCL6 content of seminal plasma. Strong expression of GCP-2/CXCL6 was found in the epithelium of the testis, epididymis and seminal vesicles, while the prostate epithelium showed weak expression, as determined by immunohistochemistry. A biological function is suggested, viz. at concentrations of the order of those found in seminal plasma, GCP-2/CXCL6 has antibacterial activity against the urogenital pathogen Neisseria gonorrhoeae. GCP-2/CXCL6 in seminal plasma may play roles in both host defense of the male urogenital tract and during fertilization.

Human milk oligosaccharides (HMO) have been shown to interact directly with immune cells. However, large quantities of HMO are required for intervention or clinical studies, but these are unavailable in most cases. In this respect, bovine milk is potentially an excellent source of commercially viable analogues of these unique molecules. In the present study, we compared the transcriptional response of colonic epithelial cells (HT-29) to the entire pool of HMO and bovine colostrum oligosaccharides (BCO) to determine whether the oligosaccharides from bovine milk had effects on gene expression that were similar to those of their human counterparts. Gene set enrichment analysis of the transcriptional data revealed that there were a number of similar biological processes that may be influenced by both treatments including a response to stimulus, signalling, locomotion, and multicellular, developmental and immune system processes. For a more detailed insight into the effects of milk oligosaccharides, the effect on the expression of immune system-associated glycogenes was chosen as a case study when performing validation studies. Glycogenes in the current context are genes that are directly or indirectly regulated in the presence of glycans and/or glycoconjugates. RT-PCR analysis revealed that HMO and BCO influenced the expression of cytokines (IL-1β, IL-8, colony-stimulating factor 2 (granulocyte-macrophage) (GM-CSF2), IL-17C and platelet factor 4 (PF4)), chemokines (chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine (C-X-C motif) ligand 3 (CXCL3), chemokine (C-C motif) ligand 20 (CCL20), chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand 6 (CXCL6), chemokine (C-C motif) ligand 5 (CCL5), chemokine (C-X3-C motif) ligand 1 (CX3CL1) and CXCL2) and cell surface receptors (interferon γ receptor 1 (IFNGR1), intercellular adhesion molecule-1 (ICAM-1), intercellular adhesion molecule-2 (ICAM-2) and IL-10 receptor α (IL10RA)). The present study suggests that milk oligosaccharides contribute to the development and maturation of the intestinal immune response and that bovine milk may be an attractive commercially viable source of oligosaccharides for such applications.

OBJECTIVE

Mucosal inflammatory responses are orchestrated largely by pro-inflammatory chemokines. The chemokine granulocyte chemotactic protein 2 (CXCL6) is involved in neutrophil recruitment and migration. Previous studies have shown that granulocyte chemotactic protein 2 is up-regulated during mucosal inflammation (e.g. in inflammatory bowel disease), similarly to the functionally and structurally related chemokine interleukin-8. Nevertheless, unlike interleukin-8, a role of granulocyte chemotactic protein 2 in gingival inflammation has not been yet demonstrated. In this study we aimed to evaluate the expression of the chemokine granulocyte chemotactic protein 2 in clinically healthy vs. diseased gingival tissues and to explore possible correlations with clinical and microbiological markers of periodontitis.

METHODS

Gene expression in 184 'diseased' and 63 'healthy' gingival tissue specimens from 90 patients with periodontitis was analyzed using Affymetrix U133Plus2.0 arrays. The expression of granulocyte chemotactic protein 2 was further confirmed by real-time reverse transcription-polymerase chain reaction, western blotting and enzyme-linked immunosorbent assay, while the localization of granulocyte chemotactic protein 2 in gingival tissues was analyzed by immunohistochemistry. Plaque samples from the adjacent periodontal pockets were collected and evaluated for 11 species of periodontal bacteria using checkerboard DNA-DNA hybridizations.

RESULTS

Among all known chemokines, GCP-2 expression was the most up-regulated (3.8-fold, p < 1.1 x 10(-16)), in 'diseased' vs. 'healthy' tissue as compared to a 2.6-fold increased expression of interleukin-8 mRNA (p < 1.2 x 10(-15)). Increased expression of granulocyte chemotactic protein 2 correlated with higher levels of 'red' and 'orange' complex pathogens and with increased probing depth, but not with attachment loss. Immunohistochemistry showed that granulocyte chemotactic protein 2 was expressed in gingival vascular endothelium.

CONCLUSIONS

The level of expression of granulocyte chemotactic protein 2 correlates with the severity of periodontitis and appears to act as a hitherto unrecognized functional adjunct to interleukin-8 in diseased gingival tissues.

Oral cancer kills about 1 person every hour each day in the United States and is the sixth most prevalent cancer worldwide. The pro-inflammatory cytokine 'macrophage migration inhibitory factor' (MIF) has been shown to be expressed in oral cancer patients, yet its precise role in oral carcinogenesis is not clear. In this study, we examined the impact of global Mif deletion on the cellular and molecular process occurring during oral carcinogenesis using a well-established mouse model of oral cancer with the carcinogen 4-nitroquinoline-1-oxide (4NQO). C57BL/6 Wild-type (WT) and Mif knock-out mice were administered with 4NQO in drinking water for 16 weeks, then regular drinking water for 8 weeks. Mif knock-out mice displayed fewer oral tumor incidence and multiplicity, accompanied by a significant reduction in the expression of pro-inflammatory cytokines Il-1β, Tnf-α, chemokines Cxcl1, Cxcl6 and Ccl3 and other molecular biomarkers of oral carcinogenesis Mmp1 and Ptgs2. Further, systemic accumulation of myeloid-derived tumor promoting immune cells was inhibited in Mif knock-out mice. Our results demonstrate that genetic Mif deletion reduces the incidence and severity of oral carcinogenesis, by inhibiting the expression of chronic pro-inflammatory immune mediators. Thus, targeting MIF is a promising strategy for the prevention or therapy of oral cancer.

BACKGROUND

Glucocorticoids (GCs) may affect the expression of hundreds of genes in different cells and tissues from patients with intermittent allergic rhinitis (IAR). It is a formidable challenge to understand these complex changes by studying individual genes. In this study, we aimed to identify (i) pathways affected by local GC treatment and (ii) examine if those pathways could be used to find novel markers of local GC treatment in nasal fluids from patients with IAR.

METHODS

Gene expression microarray- and iTRAQ-based proteomic analyses of nasal fluids, nasal fluid cells and nasal mucosa from patients with IAR were performed to find pathways enriched for differentially expressed genes and proteins. Proteins representing those pathways were analyzed with ELISA in an independent material of nasal fluids from 23 patients with IAR before and after treatment with a local GC.

RESULTS

Transcriptomal and proteomic high-throughput analyses of nasal fluids, nasal fluid cells and nasal mucosal showed that local GC treatment affected a wide variety of pathways in IAR such as the glucocorticoid receptor pathway and the acute phase response pathway. Extracellular proteins encoded by genes in those pathways were analyzed in an independent material of nasal fluids from patients. Proteins that changed significantly in expression included known biomarkers such as eosinophil cationic protein but also proteins that had not been previously described in IAR, namely CCL2, M-CSF, CXCL6 and apoH.

CONCLUSIONS

Pathway-based analyses of genomic and proteomic high-throughput data can be used as a complementary approach to identify novel potential markers of GC treatment in IAR.

CXC chemokines are thought to play an important role at sites of inflammation. Because ELR(+) CXC chemokines are expressed in different types of tonsillitis we investigated the role of the surface/crypt epithelium of human tonsils in producing ELR(+) CXC chemokines: interleukin (IL)-8 (CXCL8), ENA-78 (CXCL5), GRO-alpha (CXCL1) and GCP-2 (CXCL6). Tonsillar tissue was obtained from patients undergoing tonsillectomy and chemokine expression was investigated by means of immunohistochemistry. A549 cells were used as a model to study kinetics of chemokine expression in epithelial cells. Cells were stimulated with tumour necrosis factor (TNF)-alpha, lipopolysaccharide (LPS) and supernatants derived from aerobic/anaerobic Staphylococcus aureus strains. Chemokine expression was measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). We observed epithelial expression of IL-8, GRO-alpha and GCP-2 in different types of tonsillitis, whereas ENA-78 was rarely expressed. In A549 cells abundant expression of ENA-78 was detected. IL-8 and GCP-2 are expressed in an acute type of tonsillitis whereas GRO-alpha was frequently detectable both in chronically and acutely inflamed tonsils. ENA-78 does not seem to play a pivotal role in tonsillitis in vivo.

We assessed the effect of voriconazole (VRC) on the expression and release of selected cytokines and chemokines in the THP-1 human monocytic cell line in response to Aspergillus fumigatus hyphal fragments (HF) by cDNA microarray analysis, reverse transcriptase (RT) PCR, and enzyme-linked immunosorbent assay. Stimulation of THP-1 cells by HF alone caused a significant up-regulation of CCL4 (MIP1B) and CCL16, while CCL2 (MCP1) was down-regulated. By comparison, in the presence of VRC, a large number of genes such as CCL3 (MIP1A), CCL4 (MIP1B), CCL5 (RANTES), CCL7 (MCP3), CCL11 (EOTAXIN), CCL15 (MIP1Delta), CXCL6, and CXCL13 were strongly up-regulated in THP-1 cells challenged by HF, whereas CCL20 (MIP3A) and CCL21 (MIP2) were down-regulated. Among five genes differentially expressed in THP-1 cells, IL12A, IL12B, and IL-16 were down-regulated whereas IL-11 and TGFB1 were significantly up-regulated in the presence of VRC. The inflammation-related genes IFNgamma, IL1R1, and TNFA were also up-regulated in THP-1 cells exposed to HF only in the presence of VRC. RT-PCR of four selected genes validated the results of microarrays. The release of interleukin 1beta (IL-1beta) and IL-12 was significantly increased from monocytes stimulated either by HF alone (P < 0.05) or in the presence of VRC (P < 0.01 and P < 0.05, respectively). In contrast, tumor necrosis factor alpha release from monocytes was enhanced only in the presence of VRC (P < 0.01). The chemokines monocyte chemoattractant protein 1 and macrophage inflammatory protein 1beta were decreased under both conditions (P < 0.01). These results demonstrate that in the presence of VRC, HF induces a more pronounced profile of gene expression in THP-1 cells than HF alone, potentially leading to more-efficient host resistance to A. fumigatus.