Hypertension is a major risk factor for cardiovascular disease, which is a serious health problem in the highly industrialized countries. In more than 95% of the cases, the etiology of hypertension remains unknown. A key role in the etiology of hypertension is played by endothelial dysfunction and the inflammatory reaction in the vascular wall, in which the low molecular weight proteins so called chemokines are involved. The chemokines involved in the pathogenesis of hypertension include monocyte chemoattractant protein-1, MCP-1, CCL2, interferon-inducible protein (IP-10; CXCL10), interleukin-8 (IL-8; CXCL8), RANTES (CCL5), fractalkine (CX3CL1) and their receptors CCR2, CCR5, CXCR1, CXCR2, CXCR3 and CX3CR1. The mechanisms involving chemokines and their receptors in the pathogenesis of hypertension are complex and not fully understood. They include the impact of the migration of macrophages and monocytes to the vascular wall, endothelial dysfunction, effects on nitric oxide and endothelin-1 and smooth muscle cell proliferation. Chemokines are also involved in the pathogenesis of complications of hypertension, such as atherosclerosis, myocardial and renal fibrosis. In Poland, only about 26% of patients are effectively treated with antihypertensive drugs. The use of new therapeutic methods based on the inhibition of the inflammatory process in the vascular wall, including the impact on the function of chemokines and their receptors, could improve the effectiveness of the treatment of hypertension.

Chemotaxis is fundamental to the directional migration of neutrophils toward endogenous and exogenous chemoattractants. Recent studies have demonstrated that ADF/cofilin superfamily members play important roles in reorganizing the actin cytoskeleton by disassembling actin filaments. GMFG, a novel ADF/cofilin superfamily protein that is expressed in inflammatory cells, has been implicated in regulating actin reorganization in microendothelial cells, but its function in neutrophils remains unclear. Here, we show that GMFG is an important regulator for cell migration and polarity in neutrophils. Knockdown of endogenous GMFG impaired fMLF- and IL-8 (CXCL8)-induced chemotaxis in dHL-60 cells. GMFG knockdown attenuated the formation of lamellipodia at the leading edge of cells exposed to fMLF or CXCL8, as well as the phosphorylation of p38 and PAK1/2 in response to fMLF or CXCL8. Live cell imaging revealed that GMFG was recruited to the leading edge of cells in response to fMLF, as well as CXCL8. Overexpression of GMFG enhanced phosphorylation of p38 but not of PAK1/2 in dHL-60 cells. In addition, we found that GMFG is associated with WAVE2. Taken together, our findings suggest that GMFG is a novel factor in regulating neutrophil chemotaxis by modulating actin cytoskeleton reorganization.

Fatty liver is closely associated with obesity and sensitizes the liver to further insults. The aims of the current study are (1) to identify lipid species changed in rodent fatty liver, (2) to analyze for possible associations of these lipids with triglycerides, cholesterol or CXCL8 which is elevated in the steatotic liver and (3) to find out whether systemic levels of these lipids are concordantly altered. Lipidomic analysis has confirmed an already reported reduction of phosphatidylcholine in the steatotic liver. Phosphatidylserine is lower and phosphatidylethanolamine tends to be diminished. Sphingomyelin levels are normal while monounsaturated ceramides and hexosylceramides are reduced. Sixteen of the 20 fatty acid species measured in the total lipid fraction are elevated while α-linolenic acid is diminished. Of note, medium chain saturated fatty acids are markedly decreased. Plasmalogen 18:0 and 18:1 species are strongly increased in the steatotic liver. None of the markedly changed individual lipid species strongly correlates with hepatic CXCL8 mRNA, triglycerides or cholesterol. About 60% of the lipids altered in fatty liver are congruently altered in serum. These data show that there are multiple changes in lipid composition in fatty liver and part of the lipids may be monitored by serum analysis.

Recent experiments show that vascular endothelial growth factor (VEGF) is the crucial mediator of downstream events that ultimately lead to enhanced endothelial cell survival and increased vascular density within many tumors. The newly discovered pathway involves up-regulation of the anti-apoptotic protein Bcl-2, which in turn leads to increased production of interleukin-8 (CXCL8). The VEGF-Bcl-2-CXCL8 pathway suggests new targets for the development of anti-angiogenic strategies including short interfering RNA (siRNA) that silence the CXCL8 gene and small molecule inhibitors of Bcl-2. In this paper, we present and validate a mathematical model designed to predict the effect of the therapeutic blockage of VEGF, CXCL8, and Bcl-2 at different stages of tumor progression. In agreement with experimental observations, the model predicts that curtailing the production of CXCL8 early in development can result in a delay in tumor growth and vascular development; however, it has little effect when applied at late stages of tumor progression. Numerical simulations also show that blocking Bcl-2 up-regulation, either at early stages or after the tumor has fully developed, ensures that both microvascular and tumor cell density stabilize at low values representing growth control. These results provide insight into those aspects of the VEGF-Bcl-2-CXCL8 pathway, which independently and in combination, are crucial mediators of tumor growth and vascular development. Continued quantitative modeling in this direction may have profound implications for the development of novel therapies directed against specific proteins and chemokines to alter tumor progression.

Influenza A viruses commonly cause pancreatitis in naturally and experimentally infected animals. In this study, we report the results of in vivo investigations carried out to establish whether influenza virus infection could cause metabolic disorders linked to pancreatic infection. In addition, in vitro tests in human pancreatic islets and in human pancreatic cell lines were performed to evaluate viral growth and cell damage. Infection of an avian model with two low-pathogenicity avian influenza isolates caused pancreatic damage resulting in hyperlipasemia in over 50% of subjects, which evolved into hyperglycemia and subsequently diabetes. Histopathology of the pancreas showed signs of an acute infection resulting in severe fibrosis and disruption of the structure of the organ. Influenza virus nucleoprotein was detected by immunohistochemistry (IHC) in the acinar tissue. Human seasonal H1N1 and H3N2 viruses and avian H7N1 and H7N3 influenza virus isolates were able to infect a selection of human pancreatic cell lines. Human viruses were also shown to be able to infect human pancreatic islets. In situ hybridization assays indicated that viral nucleoprotein could be detected in beta cells. The cytokine activation profile indicated a significant increase of MIG/CXCL9, IP-10/CXCL10, RANTES/CCL5, MIP1b/CCL4, Groa/CXCL1, interleukin 8 (IL-8)/CXCL8, tumor necrosis factor alpha (TNF-α), and IL-6. Our findings indicate that influenza virus infection may play a role as a causative agent of pancreatitis and diabetes in humans and other mammals.

Sepsis is a clinical syndrome characterized by non-specific inflammatory response with evidence of profound changes in the function and structure of endothelium. Recent evidence suggests that vascular maintenance, repair and angiogenesis are in part mediated by recruitment from bone marrow (BM) of endothelial progenitor cells (EPCs). In this study we were interested in whether EPCs are increasingly mobilized during sepsis and if this mobilization is associated with sepsis severity. Our flow cytometry data demonstrate that in the CD34+ cell gate the number of EPCs in the blood of patients with sepsis had a four-fold increase (45 +/- 4.5% p < 0.001) compared to healthy controls (12 +/- 3.6%) and that this increase was already evident at 6 hours from diagnosis (40.6 +/- 4.2 percent), reaching its maximum at 72 hours. Also the percentage of cEPCs identified in the patients with sepsis (35 +/- 4.6% of the CD34+ cell) was statistically different (p < 0.001) compared to that found in the blood of patients with severe sepsis (75 +/- 4.9%). In addition, we proved that at six hours after sepsis diagnosis, VEGF, CXCL8 and CXCL12 serum levels were significantly higher in septic patients compared to healthy volunteers 559 +/- 82.14 pg/ml vs 2.9 +/- 0.6 (p < 0.0001), 189.8 +/- 67.3 pg/ml 15 vs 11.9 +/- 1.6 (p = 0.014) and 780.5 +/- 106.5 pg/ml; vs 190.2 +/- 71.4 (p < 0.001). Our data suggest that the cEPC evaluation in peripheral blood, even at early times of diagnosis, in patients with sepsis can be envisaged as a valuable parameter to confirm diagnosis and suggest further prognosis.

BACKGROUND

Chemotaxis is essential for a number of physiological processes including leukocyte recruitment. Chemokines initiate intracellular signaling pathways necessary for chemotaxis through binding seven transmembrane G protein-couple receptors. Little is known about the proteins that interact with the intracellular domains of chemokine receptors to initiate cellular signaling upon ligand binding. CXCR2 is a major chemokine receptor expressed on several cell types, including endothelial cells and neutrophils. We hypothesize that multiple proteins interact with the intracellular domains of CXCR2 upon ligand stimulation and these interactions comprise a "chemosynapse", and play important roles in transducing CXCR2 mediated signaling processes.

RESULTS

In an effort to define the complex of proteins that assemble upon CXCR2 activation to relay signals from activated chemokine receptors, a proteomics approach was employed to identify proteins that co-associate with CXCR2 with or without ligand stimulation. The components of the CXCR2 "chemosynapse" are involved in processes ranging from intracellular trafficking to cytoskeletal modification. IQ motif containing GTPase activating protein 1 (IQGAP1) was among the novel proteins identified to interact directly with CXCR2. Herein, we demonstrate that CXCR2 co-localizes with IQGAP1 at the leading edge of polarized human neutrophils and CXCR2 expressing differentiated HL-60 cells. Moreover, amino acids 1-160 of IQGAP1 directly interact with the carboxyl-terminal domain of CXCR2 and stimulation with CXCL8 enhances IQGAP1 association with Cdc42.

CONCLUSIONS

Our studies indicate that IQGAP1 is a novel essential component of the CXCR2 "chemosynapse".

Unlike mammals, bony fish have two type II interferons, IFNgamma and IFNgammarel, whose pro-inflammatory functions have not been fully characterized. To elucidate the distinct roles of these type II interferons of bony fish, we examined the effects of recombinant goldfish (rg) IFNgamma and IFNgammarel on the macrophage antimicrobial responses, immune gene expression, and their signaling pathways. Our findings indicate that rgIFNgamma and rgIFNgammarel possess unique capacities to mediate each of the above processes. Q-PCR analysis revealed similar expression of both cytokines in tissues and immune cell populations of the goldfish, although IFNgamma mRNA levels were generally higher in most tissues and cell types. Whereas rgIFNgamma had long-lasting effects on the priming of goldfish monocyte ROI production, the rgIFNgammarel had relatively short-lived ROI priming potential and eventually down-regulated the priming of ROI production induced by rgIFNgamma or rgTNFalpha2. Whereas rgIFNgamma induced relatively modest phagocytic and nitric oxide responses of goldfish macrophages, rgIFNgammarel induced significantly higher phagocytosis, iNOSA and iNOSB gene expression and nitric oxide production compared with rgIFNgamma. The rgIFNgamma and rgIFNgammarel induced different gene expression profiles in goldfish monocytes. These differences included significantly higher induction of TNFalpha2, CXCL8, ceruloplasmin, and interferon regulatory factor (IRFs) expression after activation of monocytes with rgIFNgammarel. The rgIFNgammarel was more abundant in whole cell lysates compared with rgIFNgamma. Both cytokines induced the phosphorylation of Stat1, while the nuclear localization of Stat1 was only observed following treatment of monocytes with rgIFNgamma. Our findings suggest the presence of functional segregation of the induction of macrophage antimicrobial functions by type II interferons of bony fish.

OBJECTIVE

To investigate the involvement of the chemokines CXCL10/IP-10, CXCL11/I-TAC, CXCL8/1L-8, CXCL6/GCP-2, CCL3/MIP-1alpha, and CCL18/PARC, and gelatinases A and B in uveitis.

METHODS

Prospective, experimental, case-control study.

METHODS

Aqueous humor samples from 30 patients with active uveitis, and 14 control patients and paired serum samples were assayed for chemokines with specific enzyme-linked immunosorbent assays (ELISAs) and for gelatinase levels by quantitative zymography.

RESULTS

In control AH, none of the chemokines was detected. Gelatinase A was detected in all samples, and gelatinase B was detected in only one sample. In patients with uveitis, IP-10 was detected in all AH samples, whereas I-TAC, IL-8, GCP-2, MIP-1alpha, and PARC were detected in three, 16, six, two, and 12 samples, respectively. IP-10 levels were significantly higher in AH samples than those of serum (P =.006). Gelatinase A was detected in 29 AH samples and gelatinase B was detected in 26 samples. Gelatinase A levels were significantly higher in AH samples from patients than those of controls (P <.0001). In 11 AH samples, gelatinase B was detected in complex with lipocalin (NGAL). Disease activity correlated significantly with the levels of IP-10 (r =.627; P <.0001), gelatinase A (r =.508; P =.002), gelatinase B (r =.685; P <.0001), and NGAL-gelatinase B complex (r =.595; P <.0001).

CONCLUSIONS

These data suggest a pathogenic role of the T lymphocyte chemoattractant IP-10 and gelatinases in the recruitment and activity of T cells into the eye in patients with uveitis and in the pathogenesis of uveitis.

All chemokines share a common structural scaffold that mediate a remarkable variety of functions from immune surveillance to organogenesis. Chemokines are classified as CXC or CC on the basis of conserved cysteines, and the two subclasses bind distinct sets of GPCR class of receptors and also have markedly different quaternary structures, suggesting that the CXC/CC motif plays a prominent role in both structure and function. For both classes, receptor activation involves interactions between chemokine N-loop and receptor N-domain residues (Site-I), and between chemokine N-terminal and receptor extracellular/transmembrane residues (Site-II). We engineered a CC variant (labeled as CC-CXCL8) of the chemokine CXCL8 by deleting residue X (CXC → CC), and found its structure is essentially similar to WT. In stark contrast, CC-CXCL8 bound poorly to its cognate receptors CXCR1 and CXCR2 (K(i)>> 1 μm). Further, CC-CXCL8 failed to mobilize Ca(2+) in CXCR2-expressing HL-60 cells or recruit neutrophils in a mouse lung model. However, most interestingly, CC-CXCL8 mobilizes Ca(2+) in neutrophils and in CXCR1-expressing HL-60 cells. Compared with the WT, CC-CXCL8 binds CXCR1 N-domain with only ∼5-fold lower affinity indicating that the weak binding to intact CXCR1 must be due to its weak binding at Site-II. Nevertheless, this level of binding is sufficient for receptor activation indicating that affinity and activity are separable functions. We propose that the CXC motif functions as a conformational switch that couples Site-I and Site-II interactions for both receptors, and that this coupling is critical for high affinity binding but differentially regulates activation.

Acute myeloid leukemia (AML) is a heterogeneous and aggressive malignancy with poor overall survival. Constitutive as well as cytokine-initiated activation of PI3K/Akt/mTOR signaling is a common feature of AML patients, and inhibition of this pathway is considered as a possible therapeutic strategy in AML. Human AML cells and different stromal cell populations were cultured under highly standardized in vitro conditions. We investigated the effects of mTOR inhibitors (rapamycin and temsirolimus) and PI3K inhibitors (GDC-0941 and 3-methyladenin (3-MA)) on cell proliferation and the constitutive release of angioregulatory mediators by AML and stromal cells. Primary human AML cells were heterogeneous, though most patients showed high CXCL8 levels and detectable release of CXCL10, Ang-1, HGF and MMP-9. Hierarchical clustering analysis showed that disruption of PI3K/Akt/mTOR pathways decreased AML cell release of CXCL8-11 for a large subset of patients, whereas the effects on other mediators were divergent. Various stromal cells (endothelial cells, fibroblasts, cells with osteoblastic phenotype) also showed constitutive release of angioregulatory mediators, and inhibitors of both the PI3K and mTOR pathway had anti-proliferative effects on stromal cells and resulted in decreased release of these angioregulatory mediators. PI3K and mTOR inhibitors can decrease constitutive cytokine release both by AML and stromal cells, suggesting potential direct and indirect antileukemic effects.

OBJECTIVE

Vasoactive intestinal peptide (VIP) has demonstrated beneficial effects in several murine models of immune-mediated inflammation by inhibiting both the inflammatory and the autoimmune components of the disease. We investigate its potential to modulate the release of proinflammatory cytokines and chemokines by human synovial cells from patients with rheumatoid arthritis (RA).

METHODS

Fresh suspensions of synovial tissue cells (STC) or cultured fibroblast-like synoviocytes (FLS) were obtained from patients with RA or osteoarthritis (OA). The effects of VIP on basal or tumour necrosis factor alpha (TNF-alpha)-stimulated production of CCL2 (MCP-1, monocyte chemotactic protein 1), CXCL8 [interleukin (IL)-8], IL-6 and TNF-alpha were studied by specific ELISAs (enzyme-linked immunosorbent assays). The mRNAs for CCL2, CXCL8 and IL-6 in FLS were analysed by real-time reverse transcription-polymerase chain reaction.

RESULTS

VIP at 10 nm down-regulated chemokine production by STC and FLS from RA and OA patients. VIP also down-regulated the expression of mRNAs for CCL2, CXCL8 and IL-6. The effects of VIP were more clearly detected in RA samples and after stimulation with TNF-alpha.

CONCLUSIONS

Our observations confirm that the proposed anti-inflammatory actions of VIP in murine models also apply to human synovial cells ex vivo. Further studies are encouraged to evaluate the use of VIP as a potential therapy for chronic inflammatory joint diseases.

OBJECTIVE

To determine the effects of proinflammatory cytokines on differential gene expression profiles in the human corneal endothelium (HCE), by using a cDNA expression array.

METHODS

A human cDNA expression array technology was used to study the simultaneous expression of HCE incubated with interleukin (IL)-1alpha and tumor necrosis factor-(TNF)-alpha. Gene-specific semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were performed to examine the gene and protein expression patterns revealed by the cDNA expression array, in the presence and absence of proinflammatory cytokines. Moreover, the expression of these genes was studied in ex vivo HCE of donor cornea by RT-PCR.

RESULTS

IL-1alpha and TNF-alpha upregulated the expression of 46 of 268 genes for cytokines, chemokines, and their receptors in stimulated HCE. The most upregulated genes in the cDNA expression array, those of monocyte chemotactic protein (MCP)-1 (CCL2), IL-8 (CXCL8), IL-6, and growth-related beta (GRObeta, CXCL2), were studied. Semiquantitative RT-PCR and ELISA analyses revealed the proinflammatory cytokine-mediated changes in the respective gene transcription and protein expression levels. The mRNAs were detected in ex vivo HCE of donor cornea stimulated with proinflammatory cytokines.

CONCLUSIONS

HCE can abundantly express cytokines and chemokines through the stimulation of proinflammatory cytokines. The detected genes, those of CCL2, CXCL8, IL-6, and CXCL2, in HCE could facilitate understanding of the inflammatory responses, including the production of keratic precipitates and the correlation between CE and an inflamed cornea or aqueous humor.

BACKGROUND

During the first line defense of an infected host, circulating neutrophils invade the inflamed tissue, whereas mature neutrophils from the bone marrow pool migrate into the blood circulation and from there reinforce tissue infiltration. The CXC chemokine CXCL8, also know as interleukin-8, is a potent attractant of neutrophils. Recently, we discovered a new natural post-translational modification of CXCL8, i.e. the deimination of arginine into citrulline by peptidylarginine deiminases.

METHODS

The ability to provoke leukocytosis was assessed by intravenous administration of citrullinated CXCL8 in rabbits. Adsorption of citrullinated CXCL8 to the Duffy antigen/receptor for chemokines on human or rabbit erythrocytes was evaluated using a competitive binding assay. Finally, surface expression of adhesion molecules was studied after stimulating neutrophils with citrullinated CXCL8.

RESULTS

Citrullination of CXCL8 significantly increased this chemokine's ability to recruit neutrophils into the blood circulation. In addition, the competitive binding properties of CXCL8 for the Duffy antigen/receptor for chemokines were impaired upon citrullination. Since the Duffy antigen/receptor for chemokines is an important scavenging receptor for CXCL8 in the blood stream, citrullination may delay CXCL8 clearance from the circulation. Furthermore, the shedding of CD62L (L-selectin) and the upregulation of CD11b (beta2-integrin) protein expression on CXCL8-induced neutrophils were improved by deimination of CXCL8, possibly contributing to the neutrophil egress from the bone marrow. Conversely, surface expression of CD15, the neutrophilic ligand of endothelial selectins, was equally well upregulated by intact and citrullinated CXCL8.

CONCLUSIONS

These data show that citrullination of CXCL8 enhances leukocytosis, possibly through impaired chemokine clearance from the blood circulation and prolonged presentation to the bone marrow.

Human natural killer (NK) cell development is a step-by-step process characterized by phenotypically identified stages. CD161 is a marker informative of the NK cell lineage commitment, whereas CD56, CD117, and CD94/NKG2A contribute to define discrete differentiation stages. In cells undergoing in vitro differentiation from CD34(+) umbilical cord blood (UCB) progenitors, LFA-1 expression allowed to discriminate between immature noncytolytic CD161(+)CD56(+)LFA-1(-) and more differentiated cytolytic CD161(+)CD56(+)LFA-1(+) NK cells. CD161(+)CD56(+)LFA-1(-) NK cells produce large amounts of CXCL8 after phorbol myristate acetate (PMA) or cytokine treatment. Remarkably, CXCL8 mRNA expression was also detected in fresh stage III immature NK cells isolated from tonsils and these cells expressed CXCL8 protein on PMA stimulation. Within in vitro UCB-derived CD161(+)CD56(+)LFA-1(-) NK cells, CXCL8 release was also induced on antibody-mediated cross-linking of NKp44 and CD161. Such unexpected activating function of CD161 was confined to the CD161(+)CD56(+)LFA-1(-) subset, because it did not induce cytokine release or CD107a expression in CD161(+)CD56(+)LFA-1(+) cells or in mature peripheral blood NK cells. Anti-CXCL8 neutralizing antibody induced a partial inhibition of NK cell differentiation, which suggests a regulatory role of CXCL8 during early NK cell differentiation. Altogether, these data provide novel information that may offer clues to optimize NK cell maturation in hematopoietic stem cell transplantation.

Airway smooth muscle (ASM) mass is increased in asthma, and ASM cells from patients with asthma are hyperproliferative and release more IL-6 and CXCL8. The BET (bromo- and extra-terminal) family of proteins (Brd2, Brd3, and Brd4) govern the assembly of histone acetylation-dependent chromatin complexes. We have examined whether they modulate proliferation and cytokine expression in asthmatic ASM cells by studying the effect of BET bromodomain mimics JQ1/SGCBD01 and I-BET762. ASM cells from healthy individuals and nonsevere and severe asthmatics were pretreated with JQ1/SGCBD01 and I-BET762 prior to stimulation with FCS and TGF-β. Proliferation was measured by BrdU incorporation. IL-6 and CXCL8 release was measured by ELISA, and mRNA expression was measured by quantitative RT-PCR. ChIP using a specific anti-Brd4 antibody and PCR primers directed against the transcriptional start site of IL-6 and CXCL8 gene promoters was performed. Neither JQ1/SGCBD01 nor I-BET762 had any effect on ASM cell viability. JQ1/SGCBD01 and I-BET762 inhibited FCS+TGF-β-induced ASM cell proliferation and IL-6 and CXCL8 release in healthy individuals (≥ 30 nM) and in nonsevere and severe asthma patients (≥100 nM), with the latter requiring higher concentrations of these mimics. JQ1/SGCBD01 reduced Brd4 binding to IL8 and IL6 promoters induced by FCS+TGF-β. Mimics of BET bromodomains inhibit aberrant ASM cell proliferation and inflammation with lesser efficiency in those from asthmatic patients. They may be effective in reducing airway remodeling in asthma.

BACKGROUND

The aryl hydrocarbon receptor (AhR) has gradually emerged as a regulator of inflammation in the lung and other tissues. AhR may interact with the p65-subunit of the nuclear factor (NF)-κB transcription factors, but reported outcomes of AhR/NF-κB-interactions are conflicting. Some studies suggest that AhR possess pro-inflammatory activities while others suggest that AhR may be anti-inflammatory. The present study explored the impact of AhR and its binding partner AhR nuclear translocator (Arnt) on p65-activation and two differentially regulated chemokines, CXCL8 (IL-8) and CCL5 (RANTES), in human bronchial epithelial cells (BEAS-2B).

RESULTS

Cells were exposed to CXCL8- and CCL5-inducing chemicals, 1-nitropyrene (1-NP) and 1-aminopyrene (1-AP) respectively, or the synthetic double-stranded RNA analogue, polyinosinic-polycytidylic acid (Poly I:C) which induced both chemokines. Only CXCL8, and not CCL5, appeared to be p65-dependent. Yet, constitutively active unligated AhR suppressed both CXCL8 and CCL5, as shown by siRNA knock-down and the AhR antagonist α-naphthoflavone. Moreover, AhR suppressed activation of p65 by TNF-α and Poly I:C as assessed by luciferase-assay and p65-phosphorylation at serine 536, without affecting basal p65-activity. In contrast, Arnt suppressed only CXCL8, but did not prevent the p65-activation directly. However, Arnt suppressed expression of the NF-κB-subunit RelB which is under transcriptional regulation by p65. Furthermore, AhR-ligands alone at high concentrations induced a moderate CXCL8-response, without affecting CCL5, but suppressed both CXCL8 and CCL5-responses by Poly I:C.

CONCLUSIONS

AhR and Arnt may differentially and independently regulate chemokine-responses induced by both inhaled pollutants and pulmonary infections. Constitutively active, unligated AhR suppressed the activation of p65, while Arnt may possibly interfere with the action of activated p65. Moreover, ligand-activated AhR suppressed CXCL8 and CCL5 responses by other agents, but AhR ligands alone induced CXCL8 responses when given at sufficiently high concentrations, thus underscoring the duality of AhR in regulation of inflammation. We propose that AhR-signaling may be a weak activator of p65-signaling that suppresses p65-activity induced by strong activators of NF-κB, but that its anti-inflammatory properties also are due to interference with additional pathways.

Bovine milk is known to exert a potent chemotactic activity on neutrophils, but the responsible agent has not been identified. The objective of the study was to characterize the main biochemical component responsible for this chemotactic activity. A neutrophil shape change assay was used to locate active milk fractions separated by chromatography. A single protein was isolated and identified by amino acid sequencing and mass spectrometry as CXCL3. Recombinant bovine chemokines and specific antibodies were used to show that normal milk contains active concentrations of CXCL1 (1-5ng/ml) and CXCL3 (100-500ng/ml), whereas CXCL2 and CXCL8/IL-8 were not detected. Depletion experiments with antibodies showed that CXCL3 was the main chemotaxin for neutrophils in normal (non-mastitic) milk. The chemokine CXCL3 was located by immunohistochemistry in mammary epithelial cells, and abundant mRNA was found in uninflamed mammary tissue, suggesting constitutive secretion by the lactating mammary epithelium. These results indicate that CXCL3/GRO-gamma is the major chemotactic factor for neutrophils in bovine milk in the absence of inflammation, and that it is secreted constitutively in milk by mammary epithelial cells. This finding prompts the question of the biological significance of permanent high concentrations of a CXC chemokine in milk.

Hepatitis C virus (HCV) encodes for several proteins that can interfere with host cell signaling and antiviral response. Previously, serine protease NS3/4A was shown to block host cell interferon (IFN) production by proteolytic cleavage of MAVS and TRIF, the adaptor molecules of the RIG-I and TLR3 signaling pathways, respectively. This study shows that another HCV protease, NS2 can interfere efficiently with cytokine gene expression. NS2 and its proteolytically inactive mutant forms were able to inhibit type I and type III IFN, CCL5 and CXCL10 gene promoters activated by Sendai virus infection. However, the CXCL8 gene promoter was not inhibited by NS2. In addition, constitutively active RIG-I (ΔRIG-I), MAVS, TRIF, IKKε, and TBK1-induced activation of IFN-β promoter was inhibited by NS2. Cotransfection experiments with IKKε or TBK1 together with interferon regulatory factor 3 (IRF3) and HCV expression constructs revealed that NS2 in a dose-dependent manner inhibited IKKε and especially TBK1-induced IRF3 phosphorylation. GST pull-down experiments with GST-NS2 and in vitro-translated and cell-expressed IKKε and TBK1 demonstrated direct physical interactions of the kinases with NS2. Further evidence that the IKKε/TBK1 kinase complex is the target for NS2 was obtained from the observation that the constitutively active form of IRF3 (IRF3-5D) activated readily IFN-β promoter in the presence of NS2. The present study identified HCV NS2 as a potent interferon antagonist, and describes an explanation of how NS2 downregulates the major signaling pathways involved in the development of host innate antiviral responses.

OBJECTIVE

Interleukin (IL)-6, a multifunctional cytokine with regulatory functions in wound healing, and several chemokines have been implicated in the pathogenesis of proliferative vitreoretinopathy (PVR) after rhegmatogenous retinal detachment (RRD). The exact role of these chemokines, their correlation with IL-6 after primary RRD, and their association with the future development of PVR are not yet known.

METHODS

A multiplex immunoassay was used to determine levels of 15 different chemokines and IL-6 in subretinal fluid samples obtained during scleral buckling surgery for primary RRD. Samples from patients with preoperative uveitis, preoperative trauma, or preoperative vitreous hemorrhage were excluded. Patients who developed a redetachment due to postsurgical PVR within 2.5 months (n = 21) were compared with control subjects who had an uncomplicated retinal detachment during the overall follow-up period (n = 54). Control subjects were matched for sex, age, and storage time.

RESULTS

Levels of IL-6 (P = 0.001), MIF (P = 0.016), CCL2 (P = 0.041), CCL11 (P = 0.012), CCL17 (P = 0.003), CCL18 (P = 0.007), CCL19 (P < 0.001), CCL22 (P < 0.001), CXCL8 (P = 0.027), CXCL9 (P = 0.007), and CXCL10 (P = 0.002) were significantly higher in patients who developed postoperative PVR after primary RRD than in patients with uncomplicated retinal detachment. A significant positive correlation was observed between IL-6 and both CCL22 (r = 0.538; P < 0.0001) and CXCL8 (r = 0.645; P < 0.0001).

CONCLUSIONS

Various chemokines and IL-6 are upregulated in patients in whom fibrotic membranes develop after primary RRD repair and may therefore be involved in the future development of postoperative PVR.

The endocervical epithelium is a major reservoir for Chlamydia trachomatis in women, and genital infections are extended in their duration. Epithelial cells act as mucosal sentinels by secreting cytokines and chemokines in response to pathogen challenge and infection. We therefore determined the signature cytokine and chemokine response of primary-like endocervix-derived epithelial cells in response to a common genital serovar (D) of C. trachomatis. For these studies, we used a recently-established polarized, immortalized, endocervical epithelial cell model (polA2EN) that maintains, in vitro, the architectural and functional characteristics of endocervical epithelial cells in vivo including the production of pro-inflammatory cytokines. PolA2EN cells were susceptible to C. trachomatis infection, and chlamydiae in these cells underwent a normal developmental cycle as determined by a one-step growth curve. IL1α protein levels were increased in both apical and basolateral secretions of C. trachomatis infected polA2EN cells, but this response did not occur until 72h after infection. Furthermore, protein levels of the pro-inflammatory cytokines and chemokines IL6, TNFα and CXCL8 were not significantly different between C. trachomatis infected polA2EN cells and mock infected cells at any time during the chlamydial developmental cycle up to 120h post-infection. Intriguingly, C. trachomatis infection resulted in a significant decrease in the constitutive secretion of T cell chemokines IP10 and RANTES, and this required a productive C. trachomatis infection. Examination of anti-inflammatory cytokines revealed a high constitutive apical secretion of IL1ra from polA2EN cells that was not significantly modulated by C. trachomatis infection. IL-11 was induced by C. trachomatis, although only from the basolateral membrane. These results suggest that C. trachomatis can use evasion strategies to circumvent a robust pro-inflammatory cytokine and chemokine response. These evasion strategies, together with the inherent immune repertoire of endocervical epithelial cells, may aid chlamydiae in establishing, and possibly sustaining, an intracellular niche in microenvironments of the endocervix in vivo.

OBJECTIVE

Evaluation of the efficacy of green tea extract (GTE) in regulating chemokine production and chemokine receptor expression in human RA synovial fibroblasts and rat adjuvant-induced arthritis (AIA).

METHODS

Fibroblasts isolated from human RA synovium were used in the study. Regulated upon activation normal T cell expressed and secreted (RANTES)/CCL5, monocyte chemoattractant protein (MCP)-1/CCL2, growth-regulated oncogene (GRO)alpha/CXCL1 and IL-8/CXCL8 production was measured by ELISA. Western blotting was used to study the phosphorylation of protein kinase C (PKC)delta and c-Jun N-terminal kinases (JNK). Chemokine and chemokine receptor expression was determined by quantitative RT-PCR. The benefit of GTE administration in rat AIA was determined.

RESULTS

GTE (2.5-40 microg/ml) inhibited IL-1beta-induced MCP-1/CCL2 (10 ng/ml), RANTES/CCL5, GROalpha/CXCL1 and IL-8/CXCL8 production in human RA synovial fibroblasts (P < 0.05). However, GTE inhibited MCP-1/CCL2 and GROalpha/CXCL1 mRNA synthesis in RA synovial fibroblasts. Furthermore, GTE also inhibited IL-1beta-induced phosphorylation of PKCdelta, the signalling pathway mediating IL-1beta-induced chemokine production. Interestingly, GTE preincubation enhanced constitutive and IL-1beta-induced CCR1, CCR2b, CCR5, CXCR1 and CXCR2 receptor expression. GTE administration (200 mg/kg/day p.o.) modestly ameliorated rat AIA, which was accompanied by a decrease in MCP-1/CCL2 and GROalpha/CXCL1 levels and enhanced CCR-1, -2, -5 and CXCR1 receptor expression in the joints of GTE administered rats.

CONCLUSIONS

Chemokine receptor overexpression with reduced chemokine production by GTE may be one potential mechanism to limit the overall inflammation and joint destruction in RA.

The use of electronic cigarettes (e-cigs) is increasing and there is widespread perception that e-cigs are safe. E-cigs contain harmful chemicals; more research is needed to evaluate the safety of e-cig use. Our aim was to investigate the effects of e-cigs on the inflammatory response of human neutrophils.

Neutrophils were exposed to e-cig vapour extract (ECVE) and the expression of CD11b and CD66b was measured by flow cytometry and MMP-9 and CXCL8 by ELISA. We also measured the activity of neutrophil elastase (NE) and MMP-9, along with the activation of inflammatory signalling pathways. Finally we analysed the biochemical composition of ECVE by ultra-high performance liquid chromatography mass spectrometry.

ECVE caused an increase in the expression of CD11b and CD66b, and increased the release of MMP-9 and CXCL8. Furthermore, there was an increase in NE and MMP-9 activity and an increase in p38 MAPK activation. We also identified several harmful chemicals in ECVE, including known carcinogens.

ECVE causes a pro-inflammatory response from human neutrophils. This raises concerns over the safety of e-cig use.

Drug resistance is a major impediment in medical oncology. Recent studies have emphasized the importance of the tumour microenvironment (TME) to innate resistance, to molecularly targeted therapies. In this study, we investigate the role of TME in resistance to cixutumumab, an anti-IGF-1R monoclonal antibody that has shown limited clinical efficacy. We show that treatment with cixutumumab accelerates tumour infiltration of stromal cells and metastatic tumour growth, and decreases overall survival of mice. Cixutumumab treatment stimulates STAT3-dependent transcriptional upregulation of IGF-2 in cancer cells and recruitment of macrophages and fibroblasts via paracrine IGF-2/IGF-2R activation, resulting in the stroma-derived CXCL8 production, and thus angiogenic and metastatic environment. Silencing IGF-2 or STAT3 expression in cancer cells or IGF-2R or CXCL8 expression in stromal cells significantly inhibits the cancer-stroma communication and vascular endothelial cells' angiogenic activities. These findings suggest that blocking the STAT3/IGF-2/IGF-2R intercellular signalling loop may overcome the adverse consequences of anti-IGF-1R monoclonal antibody-based therapies.