Small cell lung cancer (SCLC) is characterised by early and widespread metastasis. However, SCLC cells have so far been found to produce low levels of known pro-angiogenic factors. We speculated that SCLC cells might produce alternative pro-angiogenic factors. Here, we report that a panel of SCLC cell lines constitutively secrete granulocyte chemotactic protein-2 (GCP-2)/CXCL6, a CXC ELR+ chemokine. In contrast, none of the three tested NSCLC cell lines secreted GCP-2. Production of GCP-2 in vivo was also confirmed in seven out of nine specimens with SCLC. We demonstrate that expression of GCP-2 is mediated by NF-kappaB as ALLN, an NF-kappaB pathway inhibitor, almost completely abolished GCP-2 production in SCLC cell lines. We also demonstrate that GCP-2 can be significantly upregulated by IL-1beta and hypoxia in SCLC cell lines. This result suggests a role for GCP-2 in promoting tumour progression in vivo under unfavourable conditions such as oxygen deprivation. As SCLC cells express both GCP-2 and its receptors CXCR1 and CXCR2, their biological significance in SCLC progression was further studied. We demonstrate that GCP-2 is an autocrine growth factor. Cell proliferation was significantly inhibited by anti-GCP-2 neutralising antibody in two high-GCP-2-producing cell lines. In addition, expression of the proliferation marker PCNA was upregulated by exogenous GCP-2 in two low-GCP-2-producing cell lines. Taken together, these results suggest an important role for GCP-2 as an autocrine mitogen in the growth and metastasis of SCLC.

Genome-wide analysis of vitamin D receptor (VDR) binding sites in THP-1 human monocyte-like cells highlighted the interleukin 8 gene, also known as chemokine CXC motif ligand 8 (CXCL8). CXCL8 is a chemotactic cytokine with important functions during acute inflammation as well as in the context of various cancers. The nine genes of the CXCL cluster and the strong VDR binding site close to the CXCL8 gene are insulated from neighboring genes by CCCTC-binding factor (CTCF) binding sites. Only CXCL8, CXCL6 and CXCL1 are expressed in THP-1 cells, but all three are up-regulated primary 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) target genes. Formaldehyde-assisted isolation of regulatory elements sequencing analysis of the whole CXCL cluster demonstrated 1,25(OH)2D3-dependent chromatin opening exclusively for the VDR binding site. In differentiated THP-1 cells the CXCL8 gene showed a 33-fold higher basal expression, but is together with CXCL6 and CXCL1 still a primary 1,25(OH)2D3 target under the control of the same genomic VDR binding site. In summary, both in undifferentiated and differentiated THP-1 cells the genes CXCL8, CXCL6 and CXCL1 are under the primary control of 1,25(OH)2D3 and its receptor VDR. Our observation provides further evidence for the immune-related functions of vitamin D.

Post-translational modification of chemokines is an essential regulatory mechanism to enhance or dampen the inflammatory response. CD26/dipeptidylpeptidase IV, ubiquitously expressed in tissues and blood, removes NH2-terminal dipeptides from proteins with a penultimate Pro or Ala. A large number of human chemokines, including CXCL2, CXCL6, CXCL9, CXCL10, CXCL11, CXCL12, CCL3L1, CCL4, CCL5, CCL11, CCL14, and CCL22, are cleaved by CD26; however, the efficiency is clearly influenced by the amino acids surrounding the cleavage site and although not yet proven, potentially affected by the chemokine concentration and interactions with third molecules. NH2-terminal cleavage of chemokines by CD26 has prominent effects on their receptor binding, signaling, and hence, in vitro and in vivo biologic activities. However, rather than having a similar result, the outcome of NH2-terminal truncation is highly diverse. Either no difference in activity or drastic alterations in receptor recognition/specificity and hence, chemotactic activity are observed. Analogously, chemokine-dependent inhibition of HIV infection is enhanced (for CCL3L1 and CCL5) or decreased (for CXCL12) by CD26 cleavage. The occurrence of CD26-processed chemokine isoforms in plasma underscores the importance of the in vitro-observed CD26 cleavages. Through modulation of chemokine activity, CD26 regulates leukocyte/tumor cell migration and progenitor cell release from the bone marrow, as shown by use of mice treated with CD26 inhibitors or CD26 knockout mice. As chemokine processing by CD26 has a significant impact on physiologic and pathologic processes, application of CD26 inhibitors to affect chemokine function is currently explored, e.g., as add-on therapy in viral infection and cancer.

OBJECTIVE

To determine the transcriptional responses of human corneal epithelial cells (HCECs) after herpes simplex virus type (HSV)-1 infection and to identify the critical inflammatory element(s).

METHODS

Immortalized HCECs were infected with HSV-1, and the global transcriptional profile determined. Molecular signaling networks were constructed from the HSV-1-induced transcriptomes. The relationships of the identified networks were confirmed by real-time-PCR and ELISA. Contributions of the critical network nodes were further evaluated by protein array analyses as candidates for inflammatory element induction.

RESULTS

HSV-1 infection induced a global transcriptional response, with 412 genes significantly activated or suppressed compared with mock-infected HCECs (P < 0.05, 2< or 0.5> threshold). Infection by UV-inactivated HSV-1 did not induce significant transcriptional activity. Network analysis showed that the HSV-1-induced transcriptomes were associated with JUN N-terminal kinase, p38, extracellular signal-regulated kinase, and nuclear factor kappa-B signaling pathways. These findings indicate that interleukin (IL)-6 and vascular endothelial growth factor (VEGF) probably serve as critical nodes of signaling events. ELISA and protein array analyses verified the induction of the inflammatory elements by HSV infection. Blocking the induction of IL-6 significantly reduced the expression of 21 cytokines, including CCL7, CCL8, CXCL6, transforming growth factor-beta2, platelet-derived growth factor, interferon-gamma, IL-2, and VEGF, thus confirming the critical role of IL-6.

CONCLUSIONS

HCECs respond to HSV-1 infection by initiating mitogen-activated protein kinase-related transcriptional events, and IL-6 may serve to induce expression of an array of inflammatory mediators.

OBJECTIVE

To investigate whether di-(2-ethylhexyl) phthalate (DEHP) affects the production of inflammatory cytokines by human macrophages.

METHODS

Differentiated macrophage-like THP-1 cells were exposed to 200 μM DEHP for 3 h, followed by incubation in the presence or absence of opsonized zymosan A, and the concentrations of TNF-α, IL-1β, IL-8, and IL-6 in the culture media were determined by ELISA. DNA microarray and quantitative real-time RT-PCR analyses were performed to identify genes that showed changes in expression in response to DEHP.

RESULTS

DEHP treatment increased the concentrations of TNF-α, IL-1β, IL-8, and IL-6 in the media, regardless of whether the cells phagocytosed zymosan. DNA microarray analysis showed that DEHP increased the levels of expression of IL-8, CXCL1, CXCL2, CXCL3, CXCL6, CCL3, MMP3, MMP10, MMP14, and CSF2 mRNA, and real-time RT-PCR showed that DEHP significantly enhanced the levels of expression of IL-8, CXCL1, CXCL2, CXCL3, CXCL6, CCL3, MMP10, CSF2, TNF-α, IL-1β, and IL-6 mRNA in THP-1 cells. DEHP significantly induced translocation of p65 NF-κB into the nucleus.

CONCLUSIONS

DEHP enhances the production of inflammatory cytokines and chemokines by macrophages, and exacerbates their inflammatory response.

BACKGROUND

Rhinoviruses (RVs) are the major triggers of asthma exacerbations. We have shown previously that lower respiratory tract symptoms, airflow obstruction, and neutrophilic airway inflammation were increased in experimental RV-induced asthma exacerbations.

OBJECTIVE

We hypothesized that neutrophil-related CXC chemokines and antimicrobial peptides are increased and related to clinical, virologic, and pathologic outcomes in RV-induced exacerbations of asthma.

METHODS

Protein levels of antimicrobial peptides (SLPI, HNP 1-3, elafin, and LL-37) and neutrophil chemokines (CXCL1/GRO-α, CXCL2/GRO-β, CXCL5/ENA-78, CXCL6/GCP-2, CXCL7/NAP-2, and CXCL8/IL-8) were determined in bronchoalveolar lavage (BAL) fluid of 10 asthmatics and 15 normal controls taken before, at day four during and 6 weeks post-experimental infection.

RESULTS

BAL HNP 1-3 and Elafin were higher, CXCL7/NAP-2 was lower in asthmatics compared with controls at day 4 (P = 0.035, P = 0.048, and P = 0.025, respectively). BAL HNP 1-3 and CXCL8/IL-8 were increased during infection (P = 0.003 and P = 0.011, respectively). There was a trend to increased BAL neutrophils at day 4 compared with baseline (P = 0.076). BAL HNP 1-3 was positively correlated with BAL neutrophil numbers at day 4. There were no correlations between clinical parameters and HNP1-3 or IL-8 levels.

CONCLUSIONS

We propose that RV infection in asthma leads to increased release of CXCL8/IL-8, attracting neutrophils into the airways where they release HNP 1-3, which further enhances airway neutrophilia. Strategies to inhibit CXCL8/IL-8 may be useful in treatment of virus-induced asthma exacerbations.

Open reading frame 74 (ORF74) of many gamma(2)-herpesviruses encodes a CXC chemokine receptor. The molecular pharmacological profile of ORF74 from herpesvirus saimiri, ECRF3, is characterized here and compared with that of the well known ORF74 from human herpesvirus 8 (HHV8). The ECRF3 receptor bound the so-called ELR (Glu-Leu-Arg) CXC chemokines (125)I-CXCL1/GRO alpha, (125)I-CXCL6/GCP-2, and (125)I-CXCL8/interleukin-8 with high affinity; but in contrast to ORF74 from HHV8, it did not bind the non-ELR CXC chemokine (125)I-CXCL10/IP10. Interestingly, the B(max) value for CXCL6/GCP-2 was 3-fold higher than the capacity for maximal binding of CXCL1/GRO alpha to ECRF3 and 85-fold higher than that of CXCL8/interleukin-8, despite similar affinities. Like ORF74 from HHV8, ECRF3 activated a broad range of pathways (G(q), G(i), and G(12/13) as well as the cAMP response element-binding protein, NF-kappa B, NFAT, and serum response element transcription factors) in a ligand-regulated manner, with CXCL6/GCP-2 being the most potent and efficacious agonist. ECRF3 signaled constitutively through G(i) and G(12/13), but surprisingly not through G(q). At the level of transcription factor activation, the serum response element was activated constitutively by ECRF3, whereas cAMP response element-binding protein, NFAT, and NF-kappa B were only ligand-regulated. The maximal signaling capacities were similar for the two receptors; however, the ligand-regulated signaling was responsible for the major part of the total ECRF3 signaling and only for a minor part of the total HHV8 ORF74 signaling. The activation pattern of ECRF3 with constitutive activation of some (but not all) of the employed pathways has not been seen before in endogenous or virus-encoded chemokine receptors. The results suggest that the unique ligand selectivity of ECRF3 among ORF74 receptors could reflect differences in the cellular tropism of the gamma(2)-herpesviruses.

We explore the association of the inflammatory gene expression profile observed in the chronic inflammatory autoimmune disorder Sjögren's syndrome (SS) with changes in TNF-α converting enzyme (TACE), tumor necrosis factor (TNF)-α and nuclear factor (NF)-κB levels showing that pathways that include TNF-α signaling converge on NF-κB contributing to exacerbate the diseases. The treatment of human salivary gland epithelial cells (SGECs) with SS anti-Ro/SSA autoantibodies (Abs) result in a progressive increase in NF-κB-DNA binding, that includes a marked enhancement in NF-κB subunit p65 protein-DNA binding. A human cytokine multi-analyte array demonstrated that the NF-κB proinflammatory target genes, increased by anti-Ro/SSA Abs treatment, includes CXC chemokines (CXCL1, CXCL6 and CXCL9), CC chemokines (CCL2, CCL13 and CCL20), interleukins (IL-1α, IL-1β, IL-1F8, IL-6, IL-8, IL-9, IL-13, IL-17 and IL-22) and their receptors (IL-1RN, IL-10Rα, IL-13Rα, CCR1, CCR2, CCR3, CCR4 and CXCR1). Blockade of TACE through the use of the specific inhibitor TAPI-1 regulates proinflammatory cytokines production in SGEC treated with anti-Ro/SSA Abs inhibiting NF-κB nuclear translocation and activation. To further investigate the role of NF-κB on anti-Ro/SSA Abs-determined proinflammatory gene expression, we used the inhibitory protein IκB-α dominant negative super-repressor as inhibitor of NF-κB-DNA binding, demonstrating that transfection with dominant-negative IκB-α in anti-Ro/SSA-treated SGEC determined a marked reduction of proinflammatory cytokines gene expression. Although further studies are needed to clarify the mechanisms underlying SS, our results demonstrate that SS Abs exert their pathogenic effects via triggering the TACE/TNF-α/NF-κB axis.

We have recently reported that the human lymphatic endothelium has toll-like receptor 4 (TLR4)-mediated lipopolysaccharide recognition mechanisms that induce the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Although ligand engagement with TLR2 enables activation of the MyD88-dependent pathway similarly to TLR4, whether TLR2 ligands such as lipoteichoic acid (LTA) trigger the activation of lymphatic endothelium remains unclear. This study has been designed to investigate the expression dynamics of LTA-induced leukocyte adhesion molecules and chemokines in cultured human lymphatic endothelium (LEC). Reverse transcription/polymerase chain reaction (RT-PCR) and real-time quantitative PCR analyses have shown that LEC usually expresses TLR2 and increases TLR2 gene expression on LTA treatment. Indeed, LTA-treated LEC increases the expression of E-selectin, ICAM-1, and VCAM-1 but does not alter the gene expression of ICAM-2, ICAM-3, junctional adhesion molecule-1 (JAM-1), JAM-3, or platelet endothelial cell adhesion molecule-1 (PECAM-1). The expression of LTA-induced E-selectin, ICAM-1, and VCAM-1 in LEC is suppressed by anti-TLR2 but not by anti-TLR4 and is also suppressed by TLR2-specific short interfering RNA (siRNA) but not by siRNA for TLR4. The expression of CCL2, CCL5, and CCL20 (Cys-Cys motif chemokines) and of CXCL1, CXCL3, CXCL5, CXCL6, and CXCL8 (Cys-X-Cys motif chemokines) was induced in LEC with LTA. These data suggest that the human lymphatic endothelial phenotype has TLR2-mediated LTA-recognition mechanisms, resulting in increased expression of inflammatory leukocyte adhesion molecules and phagocyte-attractive chemokines. The human lymphatic endothelium may thus function to collect leukocytes from tissues into lymphatic vessels by means of immunologically functional molecules.

Chemokines play crucial roles in combating microbial infection and initiating tissue repair by recruiting neutrophils in a timely and coordinated manner. In humans, no less than seven chemokines (CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8) and two receptors (CXCR1 and CXCR2) mediate neutrophil functions but in a context dependent manner. Neutrophil-activating chemokines reversibly exist as monomers and dimers, and their receptor binding triggers conformational changes that are coupled to G-protein and β-arrestin signaling pathways. G-protein signaling activates a variety of effectors including Ca2+ channels and phospholipase C. β-arrestin serves as a multifunctional adaptor and is coupled to several signaling hubs including MAP kinase and tyrosine kinase pathways. Both G-protein and β-arrestin signaling pathways play important non-overlapping roles in neutrophil trafficking and activation. Functional studies have established many similarities but distinct differences for a given chemokine and between chemokines at the level of monomer vs. dimer, CXCR1 vs. CXCR2 activation, and G-protein vs. β-arrestin pathways. We propose that two forms of the ligand binding two receptors and activating two signaling pathways enables fine-tuned neutrophil function compared to a single form, a single receptor, or a single pathway. We summarize the current knowledge on the molecular mechanisms by which chemokine monomers/dimers activate CXCR1/CXCR2 and how these interactions trigger G-protein/β-arrestin-coupled signaling pathways. We also discuss current challenges and knowledge gaps, and likely advances in the near future that will lead to a better understanding of the relationship between the chemokine-CXCR1/CXCR2-G-protein/β-arrestin axis and neutrophil function.

Animal models suggest that chemokines are important mediators in the pathophysiology of neuropathic pain. Indeed, these substances have been called "gliotransmitters," a term that illustrates the close interplay between glial cells and neurons in the context of neuroinflammation and pain. However, evidence in humans is scarce. The aim of the study was to determine a comprehensive cerebrospinal fluid (CSF) inflammatory profile of patients with neuropathic pain. Our hypothesis was that we would thereby find indications of a postulated on-going process of central neuroinflammation. Samples of CSF were collected from 2 cohorts of patients with neuropathic pain (n = 11 and n = 16, respectively) and healthy control subjects (n = 11). The samples were analyzed with a multiplex proximity extension assay in which 92 inflammation-related proteins were measured simultaneously (Proseek Multiplex Inflammation I; Olink Bioscience, Uppsala, Sweden). Univariate testing with control of false discovery rate, as well as orthogonal partial least squares discriminant analysis, were used for statistical analyses. Levels of chemokines CXCL6, CXCL10, CCL8, CCL11, CCL23 in CSF, as well as protein LAPTGF-beta-1, were significantly higher in both neuropathic pain cohorts compared with healthy controls, pointing to neuroinflammation in patients. These 6 proteins were also major results in a recent similar study in patients with fibromyalgia. The findings need to be confirmed in larger cohorts, and the question of causality remains to be settled. Because it has been suggested that prevalent comorbidities to chronic pain (eg, depression, anxiety, poor sleep, and tiredness) also are associated with neuroinflammation, it will be important to determine whether neuroinflammation is a common mediator.

The chemokine granulocyte chemotactic protein (GCP)-2/CXCL6 promotes tumor growth as angiogenesis inducer and neutrophil chemoattractant. The neutralizing capacity and specificity of monoclonal mouse anti-murine (mu)GCP-2/CXCL6 antibodies were evidenced by granulocyte chemotaxis and signaling assays. The half-life of the non-antigenic antibody in the blood circulation was approximately 15 days. The titers remained constant upon weekly injection. Tumor growth and lymphogenic metastases of human melanoma over-expressing muGCP-2 were reduced in mice treated with anti-muGCP-2. Moreover, the drop in muGCP-2 antibody titer correlated with the melanoma tumor size. Taken together, we show that functional blocking of GCP-2 inhibits tumor growth and metastases.

BACKGROUND

Clinical and translational research on lung cancer patients undergoing surgical treatment can provide valuable scientific data and unique opportunity to study tumor microenvironment. CXC chemokines, which are members of a big family of cytokines, are undoubtedly involved in tumor growth regulation and metastasizing pathways. For better understanding of CXC chemokine involvement in the process of carcinogenesis we have studied the cohort of early stage non-small cell lung cancer patients undergoing surgery with curative intent. Our aim was to assess CXC chemokine ligand (CXCL) levels in patient blood samples representing systemic circulation and tumor microenvironment; assess CXC chemokine receptor (CXCR) expression in tumor tissue; and measure tumor infiltrating immune cell subpopulations.

METHODS

A total of 54 patients with NSCLC had radical lung resection were enrolled in a single center prospective study and were followed-up annually for up to six years. During surgical procedure peripheral and tumor draining blood samples were taken. CXCL1, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11 and CXCL12 levels were determined by ELISA, and chemokine concentration gradient was calculated. Tumor infiltrating immune cells (T helper cells, T cytotoxic cells, macrophages, B cells, plasma cells) and expression of CXCR1, CXCR2, CXCR3 and CXCR4 in tumor tissue were assessed by immunohistochemistry.

RESULTS

Statistically significant decrease in chemokine concentration was found for CXCL4 (P=0.002) and CXCL5 (P=0.011), and statistically significant concentration increase was found for CXCL7 (P=0.001) in total cohort. We have found statistically significant CXC chemokine concentration change for majority of chemokines-CXCL1 (P=0.002), CXCL4 (P=0.001), CXCL5 (P=0.013), CXCL7 (P=0.036), CXCL8 (P=0.026), CXCL9 (P=0.034) and CXCL10 (P=0.032) in a group of patients who had good clinical result after surgery with no evidence of relapse, on the other hand patients with cancer recurrence including local and systemic cancer spread did not show any change of chemokine concentration in blood except for CXCL1 (P=0.041). We have also found that chemokine levels and gradients correlate with CXC receptor expression and number of tumor infiltrating immune cell subpopulations.

CONCLUSIONS

Assessment of tumor microcirculation is useful for evaluation of different types of circulating biomarkers and application of our method can be very wide, integrating thoracic surgeons into translational cancer research.

We established co-cultures of invasive or non-invasive NSCLC cell lines and various types of fibroblasts (FBs) to more precisely characterize the molecular mechanism of tumor-stroma crosstalk in lung cancer. The HGF-MET-ERK1/2-CREB-axis was shown to contribute to the onset of the invasive phenotype of Calu-1 with HGF being secreted by FBs. Differential expression analysis of the respective mono- and co-cultures revealed an upregulation of NFκB-related genes exclusively in co-cultures with Calu-1. Cytokine Array- and ELISA-based characterization of the "cytokine fingerprints" identified CSF2 (GM-CSF), CXCL1, CXCL6, VEGF, IL6, RANTES and IL8 as being specifically upregulated in various co-cultures. Whilst CXCL6 exhibited a strictly FB-type-specific induction profile regardless of the invasiveness of the tumor cell line, CSF2 was only induced in co-cultures of invasive cell lines regardless of the partnered FB type. These cultures revealed a clear link between the induction of CSF2 and the EMT signature of the cancer cell line. The canonical NFκB signaling in FBs, but not in tumor cells, was shown to be responsible for the induced and constitutive CSF2 expression. In addition to CSF2, cytokine IL6, IL8 and IL1B, and chemokine CXCL1 and CXCL6 transcripts were also shown to be increased in co-cultured FBs. In contrast, their induction was not strictly dependent on the invasiveness of the co-cultured tumor cell. In a multi-reporter assay, additional signaling pathways (AP-1, HIF1-α, KLF4, SP-1 and ELK-1) were found to be induced in FBs co-cultured with Calu-1. Most importantly, no difference was observed in the level of inducibility of these six signaling pathways with regard to the type of FBs used. Finally, upon tumor fibroblast interaction the massive induction of chemokines such as CXCL1 and CXCL6 in FBs might be responsible for increased recruitment of a monocytic cell line (THP-1) in a transwell assay.

OBJECTIVE

To identify differentially expressed genes in synovial fibroblasts and examine the effect on gene expression of exposure to TNF-alpha and IL-1beta.

METHODS

Restriction fragment differential display was used to isolate genes using degenerate primers complementary to the lysophosphatidic acid acyl transferase gene family. Differential gene expression was confirmed by reverse transcription-polymerase chain reaction and immunohistochemistry using a variety of synovial fibroblasts, including cells from patients with osteoarthritis and self-limiting parvovirus arthritis.

RESULTS

Irrespective of disease process, synovial fibroblasts constitutively produced higher levels of IL-6 and monocyte chemoattractant protein 1 (MCP-1) (CCL2) than skin fibroblasts. Seven genes were differentially expressed in synovial fibroblasts compared with skin fibroblasts. Of these genes, four [tissue factor pathway inhibitor 2 (TFPI2), growth regulatory oncogene beta (GRObeta), manganese superoxide dismutase (MnSOD) and granulocyte chemotactic protein 2 (GCP-2)] were all found to be constitutively overexpressed in synoviocytes derived from patients with osteoarthritis. These four genes were only weakly expressed in other synovial fibroblasts (rheumatoid and self-limiting parvovirus infection). However, expression in all types of fibroblasts was increased after stimulation with TNF-alpha and IL-1beta. Three other genes (aggrecan, biglycan and caldesmon) were expressed at higher levels in all types of synovial fibroblasts compared with skin fibroblasts even after stimulation with TNF-alpha and IL-1.

CONCLUSIONS

Seven genes have been identified with differential expression patterns in terms of disease process (osteoarthritis vs rheumatoid arthritis), state of activation (resting vs cytokine activation) and anatomical location (synovium vs skin). Four of these genes, TFPI2, GRObeta (CXCL2), MnSOD and GCP-2 (CXCL6), were selectively overexpressed in osteoarthritis fibroblasts rather than rheumatoid fibroblasts. While these differences may represent differential behaviour of synovial fibroblasts in in vitro culture, these observations suggest that TFPI2, GRObeta (CXCL2), MnSOD and GCP-2 (CXCL6) may represent new targets for treatments specifically tailored to osteoarthritis.

Chemokines have been suggested to play a role during development of left ventricular failure, but little is known about their role during right ventricular (RV) remodeling and dysfunction. We have previously shown that the chemokine (C-X-C motif) ligand 13 (CXCL13) regulates small leucine-rich proteoglycans (SLRPs). We hypothesized that chemokines are upregulated in the pressure-overloaded RV, and that they regulate SLRPs. Mice with RV pressure overload following pulmonary banding (PB) had a significant increase in RV weight and an increase in liver weight after 1 wk. Microarray analysis (Affymetrix) of RV tissue from mice with PB revealed that CXCL10, CXCL6, chemokine (C-X3-C motif) ligand 1 (CX3CL1), chemokine (C-C motif) ligand 5 (CCL5), CXCL16, and CCL2 were the most upregulated chemokines. Stimulation of cardiac fibroblasts with these same chemokines showed that CXCL16 increased the expression of the four SLRPs: decorin, lumican, biglycan, and fibromodulin. CCL5 increased the same SLRPs, except decorin, whereas CX3CL1 increased the expression of decorin and lumican. CXCL16, CX3CL1, and CCL5 were also shown to increase the levels of glycosylated decorin and lumican in the medium after stimulation of fibroblasts. In the pressure-overloaded RV tissue, Western blotting revealed an increase in the total protein level of lumican and a glycosylated form of decorin with a higher molecular weight compared with control mice. Both mice with PB and patients with pulmonary stenosis had significantly increased circulating levels of CXCL16 compared with healthy controls measured by enzyme immunoassay. In conclusion, we have found that chemokines are upregulated in the pressure-overloaded RV and that CXCL16, CX3CL1, and CCL5 regulate expression and posttranslational modifications of SLRPs in cardiac fibroblasts. In the pressure-overloaded RV, protein levels of lumican were increased, and a glycosylated form of decorin with a high molecular weight appeared.

Atopic dermatitis (AD) is a common chronic inflammatory skin disease. Although the pathogenesis of AD is not fully understood, we and others have shown that IL-4 plays a key role. In this study we aimed to identify keratinocyte genes regulated by IL-4 that may play important roles in the pathophysiology of AD. HaCat cells were treated with IL-4 at various concentrations for 24h, and PCR gene array on inflammation/autoimmunity was performed three times for analysis of differential gene expression. Of all the 370 genes examined, 32 and 53 genes are up- and down-regulated, respectively. Specifically related to AD, chemokines CCL3L1, CCL8, CCL24, CCL25, CCL26, CXCL6 and CXCL16 are up-regulated by IL-4. Pro-inflammatory factors, such as IL-19, IL-20, IL-1α, IL-12Rβ2, IL-25, IL-31RA, OSMR and nitric oxide synthase 2, are also up-regulated. In addition, IL-4 up-regulates VEGFA, a pro-angiogenic factor. In contrast, antimicrobial peptides (AMPs) or factors involved in APM production, such as IFN-κ, S100s, Toll-like receptors, and several chemokines are down-regulated. Similarly IL-4 also down-regulates TNF-α, lymphotoxin-β, an IgE suppressor, TNFSF18, a T-cells function regulator, and the glucocorticoid receptor. On the in vivo level, real-time RT-PCR on the selected genes confirmed that IL-4 up-regulates chemokines, proinflammatory cytokines while it suppresses AMP production related genes in the skin obtained from IL-4 Tg mice. Detailed examination of these genes will delineate their specific roles in chemotaxis, inflammation, angiogenesis and AMP production, all of which may contribute to the development and progression of AD.

Acute myeloid leukemia is a haematopoietic malignancy originating from the transformation of myeloid progenitors that proliferate and accumulate in the bone marrow. In AML patients the survival rate at 5 years is 40-50% highlighting the need for novel therapies. In this study we have asked whether IL-12, an immuno-modulatory cytokine with anti-tumor activity, may inhibit directly AML cell growth. We show that the human AML cell lines U937, K562 and THP-1 expressed both chains of the IL-12 receptor (R), i.e. IL-12Rβ1 and IL-12Rβ2. IL-12 inhibited the angiogenic potential of AML cells in vitro, but did not affect their survival or proliferation. In vivo experiments were performed using SCID-NOD mice injected intraperitoneally (i.p.) with the human U937 AML cell line and subsequently treated with human recombinant IL-12 or PBS i.p. Histological, immunohistochemical and flow cytometric analyses on explanted tumors revealed that IL-12 reduced new vessel formation, induced apoptosis and inhibited tumor cell proliferation. Studies on a panel of angiogenesis related genes in explanted tumors using PCR arrays showed significantly down-regulated expression of numerous pro-angiogenic genes including VEGF-C, IL-6, IL-8, CXCL1, CXCL6 and alanyl aminopeptidase in IL-12 vs PBS treated mice. This study shows for the first time that IL-12 targets directly AML cell growth and paves the way to further investigation of IL-12 as potential drug for AML treatment.

Neutrophil recruitment to inflammatory sites is mediated by two related receptors: CXC chemokine receptors 1 (CXCR1) and 2 (CXCR2). Both receptors share two ligands, interleukin-8 (CXCL8) and GCP-2 (CXCL6), whereas several chemokines, including growth related oncogen alpha (CXCL1) and a non-peptide antagonist (SB 225002) are specific for CXCR2. The objective of this study was to map the different amino acids involved in the binding and activation/inhibition of human CXCR2. This was performed by exchanging non-conserved amino acids of CXCR2 with their counterparts in CXCR1. The mutants generated showed that: (a) for CXCL8 binding, the N-terminus of CXCR1 and the second extra-cellular loop of CXCR2 are determinant, the N-terminus of CXCR2 is not sufficient and the transmembrane domain seven is probably involved; (b) for CXCL1, the N-terminus of CXCR2 is necessary but not sufficient for binding. The activation study indicated that amino acids critical for activation are not necessarily involved in binding process. Finally, the mechanism of binding of a non-peptide antagonist on CXCR2 was investigated: it occurred through epitopes (a) which were disseminated within the receptor, (b) which differed according to the use of CXCL8 or CXCL1 as a competitor and (c) which did not necessarily overlap with agonist binding sites. We also showed that inhibition of binding and inhibition of activation involved different amino acids.

BACKGROUND

Ionizing radiation has been demonstrated to result in degranulation of dermal mast cells. Chemokines are thought to play a crucial role in the early phase of the cutaneous radiation reaction. In human skin, mast cells are located in close proximity to dermal fibroblasts, which thus are a potential target for the action of mast cell mediators.

OBJECTIVE

In this study, we evaluated the effects of mast cell-derived histamine, serotonin, tumour necrosis factor (TNF)-α and tryptase on chemokine release from dermal fibroblasts.

METHODS

Human mast cells (HMC-1) were investigated for histamine release and cytokine production after ionizing radiation using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Receptor expression on human fetal foreskin fibroblasts (HFFF2) and human adult skin fibroblasts (HDFa) was examined by flow cytometry. Chemokine mRNA and protein expression were analyzed by gene array and ELISA, respectively.

RESULTS

Ionizing radiation significantly increased histamine release and cytokine expression by HMC-1 cells. Receptors for histamine, serotonin, TNF-α and tryptase were detected both in HFFF2 and in HDFa cells. Dermal fibroblasts constitutively expressed distinct sets of chemokine mRNA. Mast cell mediators differentially affected the release of chemokines CCL8, CCL13, CXCL4 and CXCL6 by fibroblasts.

CONCLUSIONS

Our data suggest that radiation-induced mast cell mediators have a tremendous impact on inflammatory cell recruitment into irradiated skin. We postulate the activation of mast cells to be an initial key event in the cutaneous radiation reaction, which might offer promising targets for treatment of both normal tissue side effects in radiation therapy and radiation injuries.

OBJECTIVE

Can the chemokine CXCL6 affect trophoblast cell migration and invasion in human first-trimester placenta?

CONCLUSIONS

Chemokine CXCL6 inhibits trophoblast cell migration and invasion by suppressing matrix metalloproteinase (MMP)-2 activity in human first-trimester placenta.

BACKGROUND

Several chemokines including CXCL8, CXCL12, CXCL14, CXCL16, CX3CL1, CCL14 and CCL4 can promote or inhibit trophoblast cell migration and invasion in human first-trimester placenta.

METHODS

We used the trophoblast cell line HTR8/SVneo cells, primary trophoblast cells and villi explants to investigate the effect of rhCXCL6 on trophoblast cell migration and invasion.

METHODS

First, the CXCL6 RNA transcript level was detected in HTR8/SVneo cells derived from human first-trimester, second-trimester and third-trimester placenta by RT-PCR. Protein expression of CXCL6 and its receptors was tested in first-trimester placenta by immunohistochemistry. Secreted CXCL6 protein was detected in HTR8/SVneo cell supernatants by enzyme-linked immunosorbent assay. Secondly, the effect of rhCXCL6 on HTR8/SVneo cell proliferation was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Thirdly, the effect of rhCXCL6 on cell migration and invasion of HTR8/SVneo cells, primary trophoblast cells and villi explants was tested by transwell migration and invasion assays, respectively. Last, MMP-2 and MMP-9 activity in the supernatants of HTR8/SVneo and primary trophoblast cells treated by rhCXCL6 in the invasion assay was assessed by gelatin zymography.

RESULTS

Abundance of the CXCL6 RNA transcript increased with pregnancy development. CXCL6 and its receptor were expressed in several cells at the human maternal-fetal interface. RhCXCL6 inhibited trophoblast cell migration and invasion by suppressing MMP-2 activity.

CONCLUSIONS

These experiments are only in vitro.

CONCLUSIONS

According to the literature, CXCL6 could promote tumour cell migration and invasion by accelerating MMP-9 activity. However, CXCL6 inhibited trophoblast cell migration and invasion by suppressing MMP-2 activity in human first-trimester interface. These data suggest that strict regulation of CXCL6 is required for normal migration and invasion of cells, such as those involved at the maternal-fetal interface.

OBJECTIVE

To identify genes whose expressions in primary human trabecular meshwork (TM) cell cultures are affected by the transcription factor pituitary homeobox 2 (PITX2) and to identify genes that may have roles in glaucoma. Known glaucoma causing genes account for disease in a small fraction of patients, and we aimed at identification of other genes that may have subtle and accumulative effects not easily identifiable by a genetic approach.

METHODS

Expression profiles derived using microarrays were compared between TM control cells and cells treated with PITX2 siRNAs using three protocols so as to minimize false positive and negative results. The first protocol was based on the commonly used B statistic. The second and third protocols were based on fold change in expression. The second protocol used a threshold of at least 2 fold change in expression, whereas the third protocol used ranking in fold change without setting a threshold. The likelihood of a selected gene being a true positive was considered to correlate with the number of protocols by which it was selected. By considering all genes that were selected by at least one protocol, the likelihood of false negatives was expected to decrease. Effects on a subset of selected genes were verified by real time PCR, western blots, and immunocytochemistry. Effects on ALDH1A1, were further pursued because its protein product, aldehyde dehydrogenase 1 family, member A1, has roles in oxidative stress and because oxidative stress is known to be relevant to the etiology of glaucoma.

RESULTS

The expression level of 41 genes was assessed by to be possibly affected by PITX2 knockdown. Twenty one genes were down-regulated and twenty were upregulated. The expression of five genes was assessed to be altered by all three analysis protocols. The five genes were DIRAS3 (DIRAS family, GTP-binding RAS-like 3), CXCL6 (chemokine (C-X-C motif) ligand 6), SAMD5 (sterile alpha motif domain containing 5), CBFB (core-binding factor, beta subunit), and MEIS2 (meis homeobox 2). Real time PCR experiments verified results on a subset of genes tested. Notably, the results were also confirmed in two independent TMs. Effects on CXCL6 and ALDH1A1 were also confirmed by western blots, and effects on ALDH1A1 were further shown by immunocytochemistry. Data consistent with PITX2 involvement in ALDH1A1 mediated response to oxidative stress were presented.

CONCLUSIONS

Bioinformatics tools revealed that the genes identified affect functions and pathways relevant to glaucoma. Involvement of PITX2 in expression of some of the genes and in some of the pathways is being reported here for the first time. As many of the genes identified have not been studied vis-à-vis glaucoma, we feel they introduce new candidates for understanding this devastating disease.

BACKGROUND

Studies in mice have shown that PPARα is an important regulator of lipid metabolism in liver and key transcription factor involved in the adaptive response to fasting. However, much less is known about the role of PPARα in human liver.

METHODS

Here we set out to study the function of PPARα in human liver via analysis of whole genome gene regulation in human liver slices treated with the PPARα agonist Wy14643.

RESULTS

Quantitative PCR indicated that PPARα is well expressed in human liver and human liver slices and that the classical PPARα targets PLIN2, VLDLR, ANGPTL4, CPT1A and PDK4 are robustly induced by PPARα activation. Transcriptomics analysis indicated that 617 genes were upregulated and 665 genes were downregulated by PPARα activation (q value < 0.05). Many genes induced by PPARα activation were involved in lipid metabolism (ACSL5, AGPAT9, FADS1, SLC27A4), xenobiotic metabolism (POR, ABCC2, CYP3A5) or the unfolded protein response, whereas most of the downregulated genes were involved in immune-related pathways. Among the most highly repressed genes upon PPARα activation were several chemokines (e.g. CXCL9-11, CCL8, CX3CL1, CXCL6), interferon γ-induced genes (e.g. IFITM1, IFIT1, IFIT2, IFIT3) and numerous other immune-related genes (e.g. TLR3, NOS2, and LCN2). Comparative analysis of gene regulation by Wy14643 between human liver slices and primary human hepatocytes showed that down-regulation of gene expression by PPARα is much better captured by liver slices as compared to primary hepatocytes. In particular, PPARα activation markedly suppressed immunity/inflammation-related genes in human liver slices but not in primary hepatocytes. Finally, several putative new target genes of PPARα were identified that were commonly induced by PPARα activation in the two human liver model systems, including TSKU, RHOF, CA12 and VSIG10L.

CONCLUSIONS

Our paper demonstrates the suitability and superiority of human liver slices over primary hepatocytes for studying the functional role of PPARα in human liver. Our data underscore the major role of PPARα in regulation of hepatic lipid and xenobiotic metabolism in human liver and reveal a marked immuno-suppressive/anti-inflammatory effect of PPARα in human liver slices that may be therapeutically relevant for non-alcoholic fatty liver disease.

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.