Chemoattractant Function Research Articles

High Mobility Group Box 1 as an Autocrine Chemoattractant for Oligodendrocyte Lineage Cells in White Matter Stroke.

The migration of oligodendrocyte precursor cells (OPC) is a key process of remyelination, which is essential for the treatment of white matter stroke. This study aimed to investigate the role of HMGB1 (high mobility group box 1), a damage-associated molecular pattern released from dying oligodendrocytes, as an autocrine chemoattractant that promotes OPC migration. The migratory capacity of primary cultured OPCs was measured using the Boyden chamber assay. The downstream pathway of HMGB1-mediated OPC migration was specified by siRNA-induced knockdown or pharmacological blockade of TLR2 (toll-like receptor 2), RAGE (receptor for advanced glycation end product), Src, ERK1/2 (extracellular signal-regulated kinase1/2), and FAK (focal adhesion kinase). Conditioned media were collected from oxygen-glucose deprivation-treated oligodendrocytes, and the impact on OPC migration was assessed. Lesion size and number of intralesional Olig2(+) cells were analyzed in an in vivo model of white matter stroke with N5-(1-iminoethyl)-L-ornithine (L-NIO). HMGB1 treatment promoted OPC migration. HMGB1 antagonism reversed such effects to untreated levels. Among the candidates for the downstream signal of HMGB1-mediated migration, the knockdown of TLR2 rather than that of RAGE attenuated the migration-promoting effect of HMGB1. Further specification of the HMGB1-TLR2 axis revealed that the phosphorylation of ERK1/2 and its downstream molecule FAK, rather than of Src, was decreased in TLR2-knockdown OPCs, and pharmacological inhibition of ERK1/2 and FAK led to decreased OPC migration. Oxygen-glucose deprivation-conditioned media promoted OPC migration, suggesting the autocrine chemoattractant function of HMGB1. In vivo, TLR2(-/-)-mice showed lesser intralesional Olig2(+) cells compared to wild-type controls in response to L-NIO induced ischemic injury regardless of HMGB1 administration. HMGB1, through the TLR2-ERK1/2-FAK axis, functions as an autocrine chemoattractant to promote OPC migration, which is an initial and indispensable step in remyelination. Thus, a novel treatment strategy for white matter stroke based on the HMGB1-TLR2 axis in the oligodendrocyte lineage could be feasible.

High Mobility Group Box 1 as an Autocrine Chemoattractant for Oligodendrocyte Lineage Cells in White Matter Stroke.

The migration of oligodendrocyte precursor cells (OPC) is a key process of remyelination, which is essential for the treatment of white matter stroke. This study aimed to investigate the role of HMGB1 (high mobility group box 1), a damage-associated molecular pattern released from dying oligodendrocytes, as an autocrine chemoattractant that promotes OPC migration. The migratory capacity of primary cultured OPCs was measured using the Boyden chamber assay. The downstream pathway of HMGB1-mediated OPC migration was specified by siRNA-induced knockdown or pharmacological blockade of TLR2 (toll-like receptor 2), RAGE (receptor for advanced glycation end product), Src, ERK1/2 (extracellular signal-regulated kinase1/2), and FAK (focal adhesion kinase). Conditioned media were collected from oxygen-glucose deprivation-treated oligodendrocytes, and the impact on OPC migration was assessed. Lesion size and number of intralesional Olig2(+) cells were analyzed in an in vivo model of white matter stroke with N5-(1-iminoethyl)-L-ornithine (L-NIO). HMGB1 treatment promoted OPC migration. HMGB1 antagonism reversed such effects to untreated levels. Among the candidates for the downstream signal of HMGB1-mediated migration, the knockdown of TLR2 rather than that of RAGE attenuated the migration-promoting effect of HMGB1. Further specification of the HMGB1-TLR2 axis revealed that the phosphorylation of ERK1/2 and its downstream molecule FAK, rather than of Src, was decreased in TLR2-knockdown OPCs, and pharmacological inhibition of ERK1/2 and FAK led to decreased OPC migration. Oxygen-glucose deprivation-conditioned media promoted OPC migration, suggesting the autocrine chemoattractant function of HMGB1. In vivo, TLR2(-/-)-mice showed lesser intralesional Olig2(+) cells compared to wild-type controls in response to L-NIO induced ischemic injury regardless of HMGB1 administration. HMGB1, through the TLR2-ERK1/2-FAK axis, functions as an autocrine chemoattractant to promote OPC migration, which is an initial and indispensable step in remyelination. Thus, a novel treatment strategy for white matter stroke based on the HMGB1-TLR2 axis in the oligodendrocyte lineage could be feasible.

MicroRNA-424 regulates the expression of CX3CL1 (fractalkine) in human microvascular endothelial cells during Rickettsia rickettsii infection

Cytokines and chemokines trigger complex intracellular signaling through specific receptors to mediate immune cell recruitment and activation at the sites of infection. CX3CL1 (Fractalkine), a membrane-bound chemokine also capable of facilitating intercellular interactions as an adhesion molecule, contributes to host immune responses by virtue of its chemoattractant functions. Published studies have documented increased CX3CL1 expression in target tissues in a murine model of spotted fever rickettsiosis temporally corresponding to infiltration of macrophages and recovery from infection. Because pathogenic rickettsiae primarily target vascular endothelium in the mammalian hosts, we have now determined CX3CL1 mRNA and protein expression in cultured human microvascular endothelial cells (HMECs) infected in vitro with Rickettsia rickettsii. Our findings reveal 15.5 ± 4.0-fold and 12.3 ± 2.3-fold increase in Cx3cl1 mRNA expression at 3 h and 24 h post-infection, coinciding with higher steady-state levels of the corresponding protein in comparison to uninfected HMECs. Since CX3CL1 is a validated target of microRNA (miR)-424-5p (miR-424) and our earlier findings demonstrated robust down-regulation of miR-424 in R. rickettsii-infected HMECs, we further explored the possibility of regulation of CX3CL1 expression during rickettsial infection by miR-424. As expected, R. rickettsii infection resulted in 87 ± 5% reduction in miR-424 expression in host HMECs. Interestingly, a miR-424 mimic downregulated R. rickettsii-induced expression of CX3CL1, whereas an inhibitor of miR-424 yielded a converse up-regulatory effect, suggesting miR-424-mediated regulation of CX3CL1 during infection. Together, these findings provide the first evidence for the roles of a host microRNA in the regulation of an important bifunctional chemokine governing innate immune responses to pathogenic rickettsiae.

Mitochondrial DNA Is a Pro-Inflammatory Damage-Associated Molecular Pattern Released During Active IBD.

Due to common evolutionary origins, mitochondrial DNA (mtDNA) shares many similarities with immunogenic bacterial DNA. MtDNA is recognized as a pro-inflammatory damage-associated molecular pattern (DAMP) with a pathogenic role in several inflammatory diseases. We hypothesised that mtDNA is released during active disease, serving as a key pro-inflammatory factor in inflammatory bowel disease (IBD). Between 2014 and 2015, we collected plasma separated within 2 hours of sampling from 97 prospectively recruited IBD patients (67 ulcerative colitis [UC] and 30 Crohn's disease [CD]) and 40 non-IBD controls. We measured circulating mtDNA using quantitative polymerase chain reaction (amplifying mitochondria COXIII/ND2 genes) and also in mouse colitis induced by dextran sulfate-sodium (DSS). We used a mass spectometry approach to detect free plasma mitochondrial formylated peptides. Furthermore, we examined for mitochondrial damage using electron microscopy (EM) and TLR9 expression, the target for mtDNA, in human intestinal IBD mucosa. Plasma mtDNA levels were increased in UC and CD (both P < 0.0001) compared with non-IBD controls. These levels were significantly correlated to blood (C-reactive protein, albumin, white cell count), clinical and endoscopic markers of severity, and disease activity. In active UC, we identified 5 mitochondrial formylated peptides (the most abundant being fMMYALF with known chemoattractant function) in plasma. We observed mitochondrial damage in inflamed UC mucosa and significantly higher fecal MtDNA levels (vs non-IBD controls [P < 0.0001]), which supports gut mucosal mitochondrial DAMP release as the primary source. In parallel, plasma mtDNA levels increased during induction of acute DSS colitis and were associated with more severe colitis (P < 0.05). In active IBD, TLR9+ lamina propria inflammatory cells were significantly higher in UC and CD compared with controls (P < 0.05). We present the first evidence to show that mtDNA is released during active IBD. MtDNA is a potential mechanistic biomarker, and our data point to mtDNA-TLR9 as a therapeutic target in IBD. 10.1093/ibd/izy095_videoizy095.video5776747659001.

Low CCL-21 expression associates with unfavorable postoperative prognosis of patients with metastatic renal cell carcinoma.

BackgroundChemokine (C-C motif) ligand 21 (CCL21), a ligand of the chemokine (C-C motif) receptor 7, has recently been identified as an immuno-based anti-cancer molecule for its dendritic cells and T lymphocytes chemoattractant function. The aim of this study was to investigate prognostic values of CCL21 expression in metastatic renal cell carcinoma patients treated with targeted therapy.MethodsThis study included 111 patients with metastatic renal cell carcinoma receiving targeted therapy. CCL21 expression was analyzed by immunohistochemistry on tissue microarrays. Prognostic value of tumoral CCL21 expression and patients clinical outcomes were evaluated.RESULTSKaplan-Meier method showed that low CCL21 expression was associated with shorter patient overall survival and progression-free survival (overall survival, P = 0.005; progression-free survival, P = 0.044). Further stratified analysis showed that low CCL21 expression was significantly associated with shorter overall survival in clear cell renal cell carcinoma patients (P = 0.017) and patients treated with sorafenib (P = 0.009). Low CCL21 expression was also an adverse independent risk factor for overall survival (hazard ratio, 2.106; 95% CI, 1.286-3.450; P = 0.003) and progression-free survival (hazard ratio 1.617; 95%CI 1.060-2.465; P = 0.026) in multivariate analyses. CCL21 expression was significantly associated with treatment best response to targeted therapy (P = 0.009). This molecule could also be combined with Heng risk model to increase its overall survival predictive accuracy.ConclusionLow CCL21 expression was a potential independent adverse prognostic biomarker for overall survival and progression-free survival for metastatic renal cell carcinoma patients treated with targeted therapy.

Cathelicidins and defensins regulate mast cell antimicrobial activity.

Cathelicidins and defensins are the multifunctional host defense molecules essential for immune response to infection. In recent years they have been shown to be natural, broad-spectrum antimicrobials against both Gram-positive and Gram-negative bacteria, enveloped viruses, and fungi. These small peptides kill the invaded pathogens by destroying their cell membranes and can neutralize biological activities of endotoxin. Apart from exerting direct antimicrobial effects, cathelicidins and defensins can also trigger innate and adaptive defense responses in the host. The functions of the host derived peptides in immunomodulation have been also investigated. Reported activities of these peptides include chemoattractant function, inhibition of neutrophil apoptosis, and ROS production. These peptides directly activate inflammatory cells to production and release of different pro-inflammatory and immunoregulatory mediators, cytokines, and chemokines, however cathelicidins and defensins might mediate the generation of anti-inflammatory cytokines, as well. Insights into the miscellaneous functions of mast cells have exposed that they possess the ability to respond to pathogens and modulate immune response. These immune sentinel cells play a pivotal role in defense mechanisms mainly through the presence of pattern recognition receptors and by release different preformed and newly synthesized mediators and cytokines. The present review provides an introduction to the field of cathelicidins and defensins in general and discusses their impact on mast cells.

N-acetyl proline-glycine-proline: implications for neurological disorders.

N-acetyl proline-glycine-proline: implications for neurological disorders.

Leukocyte chemoattractant receptors in human disease pathogenesis.

Combinations of leukocyte attractant ligands and cognate heptahelical receptors specify the systemic recruitment of circulating cells by triggering integrin-dependent adhesion to endothelial cells, supporting extravasation, and directing specific intratissue localization via gradient-driven chemotaxis. Chemoattractant receptors also control leukocyte egress from lymphoid organs and peripheral tissues. In this article, we summarize the fundamental mechanics of leukocyte trafficking, from the evolution of multistep models of leukocyte recruitment and navigation to the regulation of chemoattractant availability and function by atypical heptahelical receptors. To provide a more complete picture of the migratory circuits involved in leukocyte trafficking, we integrate a number of nonchemokine chemoattractant receptors into our discussion. Leukocyte chemoattractant receptors play key roles in the pathogenesis of autoimmune diseases, allergy, inflammatory disorders, and cancer. We review recent advances in our understanding of chemoattractant receptors in disease pathogenesis, with a focus on genome-wide association studies in humans and the translational implications of mechanistic studies in animal disease models.

Effects of chemoattractant function of regulated upon activation normal T cell expressed and secreted with adenovirus-mediated gene transfer of S24F of the human umbilical vein endothelial cells

Objective To investigate the effects of chemoattractant function of regulated upon activation normal T cell expressed and secreted (RANTES) with adenovirus-mediated gene transfer of S24F of the human umbilical vein endothelial cells (HUVECs).Methods Construction of the recombinant Adenoviruses Vector pAV-MCMV/S24F-GFP-3FLAG (Ad-S24F),an adenoviral vector containing no transgene pAV-MCMV-GFP (Ad-Null) was used as a control.HUVECs were isolated and cultured in vitro,and were transfected with Ad-S24F (Ad-S24F group),Ad-Null (Ad-Null group),or transfected with no virus (control group).The expression of reconstructive protein after transfection was detected by using Western blotting and fluorescence microscopy in each group.The in vitro transendothelialchemotaxis assay was used to compare RANTES-induced transmigration of peripheral blood mononuclear cells (PBMCs) across HUVECs cultured on the upper Transwell chamber.Results The successful construction of recombinant adenoviral vector carrying S24F gene.RANTES-induced PBMC transendothelialchemotaxis is inhibited by S24F [Ad-S24F:(9.20 ± 0.02) ％ ; Ad-Null:(17.70 ± 0.02) ％ ; control:(15.10 ± 0.01) ％].Conclusion Adenovirus-mediated gene transfer of S24F of the human umbilical vein endothelial cells may inhibit RANTES-induced PBMC transendothelialchemotaxis. Key words: S24F gene; Regulated upon activation normal T cell expressed and secreted; Adenovirus vector; Gene transfer

CXCL12 modulation of CXCR4 and CXCR7 activity in human glioblastoma stem-like cells and regulation of the tumor microenvironment.

Chemokines are crucial autocrine and paracrine players in tumor development. In particular, CXCL12, through its receptors CXCR4 and CXCR7, affects tumor progression by controlling cancer cell survival, proliferation and migration, and, indirectly, via angiogenesis or recruiting immune cells. Glioblastoma (GBM) is the most prevalent primary malignant brain tumor in adults and despite current multimodal therapies it remains almost incurable. The aggressive and recurrent phenotype of GBM is ascribed to high growth rate, invasiveness to normal brain, marked angiogenesis, ability to escape the immune system and resistance to standard of care therapies. Tumor molecular and cellular heterogeneity severely hinders GBM therapeutic improvement. In particular, a subpopulation of chemo- and radio-therapy resistant tumorigenic cancer stem–like cells (CSCs) is believed to be the main responsible for tumor cell dissemination to the brain. GBM cells display heterogeneous expression levels of CXCR4 and CXCR7 that are overexpressed in CSCs, representing a molecular correlate for the invasive potential of GBM. The microenvironment contribution in GBM development is increasingly emphasized. An interplay exists between CSCs, differentiated GBM cells, and the microenvironment, mainly through secreted chemokines (e.g., CXCL12) causing recruitment of fibroblasts, endothelial, mesenchymal and inflammatory cells to the tumor, via specific receptors such as CXCR4. This review covers recent developments on the role of CXCL12/CXCR4–CXCR7 networks in GBM progression and the potential translational impact of their targeting. The biological and molecular understanding of the heterogeneous GBM cell behavior, phenotype and signaling is still limited. Progress in the identification of chemokine-dependent mechanisms that affect GBM cell survival, trafficking and chemo-attractive functions, opens new perspectives for development of more specific therapeutic approaches that include chemokine-based drugs.

Desmoglein 2 Depletion Leads to Increased Migration and Upregulation of the Chemoattractant Secretoneurin in Melanoma Cells

During development and progression of malignant melanoma, an important role has been attributed to alterations of cell-cell adhesions, in particular, to a “cadherin switch” from E- to N-cadherin. We have previously shown that a subtype of melanoma cells express the desmosomal cadherin desmoglein 2 as non-junction-bound cell surface protein in addition to classical cadherins. To study the role of desmoglein 2 in melanoma cells, melanoma lines containing high endogenous amounts of desmoglein 2 were depleted of the protein by RNA interference. Transwell migration and scratch wounding assays showed markedly increased migration upon desmoglein 2 suppression whereas proliferation and viability remained unaltered. In gene expression profiles, desmoglein 2 depletion was associated with overexpression of migration-related genes. Strongest overexpression was found for secretogranin II which has not been reported in melanoma cells before. The bioactive peptide derived from secretogranin II, secretoneurin, is known to exert chemoattractive functions and was demonstrated here to stimulate melanoma cell migration. In summary, we show that desmoglein 2 expression attenuates migration of melanoma cells. The mechanism of desmoglein 2 impaired cell migration is mediated by downregulation of secretogranin II. Loss of desmoglein 2 increases expression of secretogranin II, followed by an enhanced migratory activity of melanoma cells. Our data add a new pathway of regulating melanoma cell migration related to a desmoglein 2 – secretogranin II axis.

PC3-Secreted Microprotein Is a Novel Chemoattractant Protein and Functions as a High-Affinity Ligand for CC Chemokine Receptor 2

PC3-secreted microprotein (PSMP) or microseminoprotein is a newly discovered secreted protein whose function is currently unknown. In this study, PSMP was found to possess chemotactic ability toward monocytes and lymphocytes, and its functional receptor was identified as CCR2B. PSMP was identified as a chemoattractant protein from a PBMC chemoattractant platform screen that we established. The mature secreted PSMP was able to chemoattract human peripheral blood monocytes, PBLs, and CCR2B-expressing THP-1 cells, but not peripheral blood neutrophils, even though it does not contain the classical structure of chemokines. CCR2B was identified as one receptor for PSMP-mediated chemotaxis by screening HEK293 cells that transiently expressed classical chemokine receptors; results obtained from the chemotaxis, calcium flux, receptor internalization, and radioligand-binding assays all confirmed this finding. To further identify the major function of PSMP, we analyzed its expression profile in tissues. PSMP is highly expressed in benign prostatic hyperplasia and in some prostate cancers, and can also be detected in breast tumor tissue. In response to PSMP stimulation, phosphorylated ERK levels downstream of CCR2B signaling were upregulated in the PC3 cell line. Taken together, our data collectively suggest that PSMP is a chemoattractant protein acting as a novel CCR2 ligand that may influence inflammation and cancer development.

CX3CL1 expression is associated with poor outcome in breast cancer patients

The significance of chemokines in cancer biology has been widely recognized in recent years. CX3CL1 is a unique subclass of chemokine with complex functions, including recruitment of anti-tumor leukocytes and promoting cancer survival, thus affecting cancer progression in both the directions. It is not clear how these different functions interact in breast cancers. This is further complicated by the heterogeneity of breast cancer, and differential association of CX3CL1 with different subgroups could be present. There is only limited knowledge of CX3CL1 expression profile, its relationship with different biological features, subtypes, and outcomes in breast cancers. In this study, CX3CL1 expression was examined in a large cohort of breast cancers by immunohistochemistry and its association with clinicopathological factors, biomarker expression, and impact on patients' survival was assessed. High CX3CL1 expression was detected in 33.3% (252/757) of primary invasive cancers. In line with its chemo-attractant function, CX3CL1 expression correlated positively with increased tumor infiltrating lymphocytes (TIL) (p=0.005). In addition, CX3CL1 also correlated positively with adverse features in breast cancers, including lymph node involvement (p=0.007), high Ki67 (p=0.002), α-B crystallin expression (p=0.008), and luminal B (worse prognosis luminal cancers) subtype (p=0.024). Consistently, breast cancers with high expression of CX3CL1 were found to have a poorer overall survival (χ(2)=4.797, p=0.029). Interestingly, the adverse effect of CX3CL1 on outcome appeared to be more prominent in cancers with low TIL. These findings indicated that CX3CL1 could also have a pro-tumor role in breast cancer, despite its previously suggested role in enhancing anti-tumor immunity. The results highlighted the complicated functions of CX3CL1 in breast carcinogenesis. Further studies are needed to clarify the relative contribution of these anti- and pro-tumor functions in order to understand the true prognostic and potential therapeutic values of CX3CL1.

Elevated fractalkine in patients with obstructive sleep apnea hypopnea syndrome

Obstructive sleep apnea hypopnea syndrome (OSAHS) has been increasingly linked to cardiovascular disease. Inflammatory processes associated with OSAHS may contribute to atherosclerosis in these patients. Fractalkine is a unique chemokine which has both adhesive and chemoattractant functions. We tested the hypothesis that OSAHS patients have increased fractalkine. We studied 20 patients (18 males and 2 females) with newly diagnosed OSAHS, who were free of other diseases, had never been treated for OSAHS, and were taking no medications. We compared fractalkine measurements in these patients to measurements obtained in 15 control subjects (14 males and 1 female) who were matched for age and body mass index, and in whom occult OSAHS was excluded. Plasma fractalkine levels were significantly higher in patients with OSAHS than in controls (463.15 ± 110.78 versus 364.67 ± 64.81 pg/mL, F = 2.58, P = 0.004). Fractalkine were associated with AHI (r = 0.756, P < 0.0001), lowest oxygen saturation (r = -0.466, P = 0.005), and mean oxygen saturation (r = -0.344, P = 0.043). Plasma fractalkine levels were significantly decreased in patients with OSAHS after four nights nCPAP (463.15 ± 110.78 versus 416.75 ± 97.67 pg/mL, P = 0.001). OSAHS is associated with elevated levels of fractalkine, a marker of inflammation related to atherosclerosis. The severity of OSAHS is proportional to the fractalkine level.

Production of bioactive sheep β-defensin-1 in Pichia pastoris

Previous research has shown that sheep β-defensin-1 (sBD-1), a small cationic peptide with a broad range of antimicrobial activities, could inhibit the growth of both Gram-positive and Gram-negative bacteria as well as that of fungi. In order to increase the yield of current ovine defensin purification methods, mature sBD-1 (msBD-1) was added with a 6-His tag on the C-terminus (msBD-1-T) and expressed in Pichia pastoris in the presented work. The msBD-1 and msBD-1-T were expressed in the Pichia pastoris. Both msBD-1 and msBD-1-T were purification, and the two peptides were used to inhibit Escherichia coli, Staphylococcus aureus, Proteus vulgaris, Pseudomonas aeruginosa, and Shigella flexneri. The antimicrobial activity of the 6-His tagged msBD-1-T peptide was not significantly different from that of the native msBD-1 peptide. The two peptides could inhibit the growth of Escherichia coli, Staphylococcus aureus, Proteus vulgaris, Pseudomonas aeruginosa, and Shigella flexneri with equal efficiency as well as chemoattractant function. In addition, the yield of purified 6-His-tagged msBD-1 was greater than that of msBD-1. The presented method might be a more efficient approach to produce bioactive sBD-1.

Inhibitory effects of C4a on chemoattractant and secretagogue functions of the other anaphylatoxins via Gi protein-adenylyl cyclase inhibition pathway in mast cells

A recombinant complement anaphylatoxin, C4a, inhibited chemotaxis, respiratory burst and histamine release in mast cell-like HMC-1 cells that were treated with recombinant C5a anaphylatoxin. C4a also inhibited histamine release from HMC-1 cells that were induced by recombinant C3a. The inhibition of C5a- and C3a-induced leukocyte reactions by C4a was recapitulated in peripheral blood CD133 + cell-derived differentiated mast cells. In HMC-1 cells, C4a inhibited cytoplasmic Ca 2+ influx, an event that precedes anaphylatoxin-induced chemotactic and secretary responses. A conditioned medium of HMC-1 cells after shortly treated with C4a also inhibited the anaphylatoxin-induced Ca 2+ influx even after removal of C4a, indicating that the effect of C4a is to liberate an autocrine inhibitor from the mast cells. The inhibitor secretion by C4a was prevented with pertussis toxin or with a phosphodiesterase inhibitor. Conversely, an adenylyl cyclase inhibitor reproduced the effect of C4a. C4a decreased the intracellular cyclic AMP concentration of HMC-1 cells, indicating that C4a elicited the Gi protein-adenylyl cyclase inhibition pathway. Neither C4a nor the conditioned medium, however, inhibited Ca 2+ influx and respiratory burst in C5a- or C3a-stimulated peripheral neutrophils, suggesting that these cells lack this inhibitory system. Additionally, in HMC-1 cells, C4a did not inhibit Ca 2+-independent, Leu72Gln-C5a-stimulated chemotactic response. In agreement with this finding, C4a treatment inhibited ERK1/2 phosphorylation in HMC-1 cells stimulated with other anaphylatoxins but did not inhibit p38MAPK phosphorylation in cells stimulated with Leu72Gln-C5a. Taken together, these findings suggest that the autocrine inhibitory effect elicited by C4a is attributed to interruption of Ca 2+-dependent intracellular signaling pathway.

Down-regulation of CXCL1 inhibits tumor growth in colorectal liver metastasis

As part of ongoing studies to obtain a global picture of invasion related events in colorectal liver metastases, here, we report our findings on gene expression of the pro-angiogenic subgroup of chemokines, the CXCL-ELR+ chemokines. Apart from their pro-angiogenic and chemoattractant function, these chemokines appear to also contribute to tumor cell transformation, growth and invasion. In our nude mouse model of colorectal liver metastases, we found CXCL1,2,3,5 and 8 (IL-8) to be up-regulated in the tumor cells of the invasion front as compared to the tumor cells in the inner parts of the tumor. ShRNA mediated down-regulation of the most prominently up-regulated group member, CXCL1/gro-alpha resulted in inhibition of cell viability, invasion and proliferation. In vivo, down-regulation of CXCL1 resulted in a nearly complete prevention of tumor growth in nude mice. Mechanistically, auto-regulatory mechanisms involving NF-kappaB and Akt appear to be involved in pro-tumorigenic functions of CXCL1.

Direct Gene Transfer with IP-10 Mutant Ameliorates Mouse CVB3-Induced Myocarditis by Blunting Th1 Immune Responses

BackgroundMyocarditis is an inflammation of the myocardium that often follows the enterovirus infections, with coxsackievirus B3 (CVB3) being the most dominant etiologic agent. We and other groups previously reported that chemokine IP-10 was significantly induced in the heart tissue of CVB3-infected mice and contributed to the migration of massive inflammatory cells into the myocardium, which represents one of the most important mechanisms of viral myocarditis. To evaluate the direct effect of IP-10 on the inflammatory responses in CVB3 myocarditis, herein an IP-10 mutant deprived of chemo-attractant function was introduced into mice to antagonize the endogenous IP-10 activity, and its therapeutic effect on CVB3-induced myocarditis was evaluated.Methodology/Principal FindingsThe depletion mutant pIP-10-AT, with an additional methionine after removal of the 5 N-terminal amino acids, was genetically constructed and intramuscularly injected into BALB/c mice after CVB3 infection. Compared with vector or no treatment, pIP-10-AT treatment had significantly reduced heart/body weight ratio and serum CK-MB level, increased survival rate and improved heart histopathology, suggesting an ameliorated myocarditis. This therapeutic effect was not attributable to an enhanced viral clearance, but to a blunted Th1 immune response, as evidenced by significantly decreased splenic CD4+/CD8+IFN-γ+ T cell percentages and reduced myocardial Th1 cytokine levels.Conclusion/SignificanceOur findings constitute the first preclinical data indicating that interfering in vivo IP-10 activity could ameliorate CVB3 induced myocarditis. This strategy may represent as a new therapeutic approach in treating viral myocarditis.

CCL5 induces a pro-inflammatory profile in microglia in vitro

The chemokine receptors CCR1, CCR2, CCR3, CCR5, and CXCR2 have been found to be expressed on microglia in many neurodegenerative diseases, such as multiple sclerosis and Alzheimer’s disease. There is emerging evidence that chemokines, besides chemoattraction, might directly modulate reactive profiles of microglia. To address this hypothesis we have investigated the effects of CCL2, CCL3, CCL5, and CXCL1 on cytokine and growth factor production, NO synthesis, and phagocytosis in non-stimulated and lipopolysaccharide-stimulated primary rat microglia. The respective receptors CCR1, CCR5, and CXCR2 were shown to be functionally expressed on microglia. All tested chemokines stimulated chemotaxis whereas only CCL5 increased NO secretion and attenuated IL-10 as well as IGF-1 production in activated microglia. Based on these findings we propose that besides its chemoattractant function CCL5 has a modulatory effect on activated microglia.

A Common Polymorphism in the Monocyte Chemoattractant Protein-1 (MCP-1) Gene Regulatory Region Influences MCP-1 Expression and Function of Isolated Human Pancreatic Islets

Background and aims Islet transplantation is an attractive approach to treat type 1 diabetic patients. However, suboptimal islet engraftment still represents an unsolved problem. It has been shown that human islets release monocyte chemoattractant protein-1 (MCP-1), one of the most powerful macrophage chemokines, which may impair the fate of the transplant. The aim of this study was to evaluate the presence and role of MCP-1 in isolated human islets, including genotyping for a common polymorphism. Methods Pancreatic islets were isolated by enzymatic digestion and gradient purification from 41 nondiabetic multiorgan donors. We measured MCP-1 mRNA expression by quantitative real- time reverse-transcriptase polymerization chain reaction, analyzed the MCP-1 single nucleotide polymorphism, −2518 G/A (SNP, rs 1024611) and evaluated glucose-stimulated insulin release (IR; μU/islet/min). Results MCP-1 mRNA expression was found in all studied batches of islets. Overall, IR was significantly higher at 16.7 mmol/L than 3.3 mmol/L glucose. We observed a significant negative correlation between MCP-1 mRNA expression and stimulation index (SI). We found that MCP-1 mRNA expression was significantly higher in CC and CT compared with TT genotype groups. Finally, SI was significant lower in the CC with respect to the TT genotype group. Conclusions These data show that MCP-1 gene expression regulated by the −2518 G/A polymorphism, is correlated with glucose-stimulated insulin release. The study of MCP-1 expression and genotype on isolated islets before transplantation may be useful to understand the inflammatory response after infusion of human islets into patients with type 1 diabetes mellitus.