Participation of CXCL1 in the glial cells during neuropathic pain.

Neuropathic pain is a chronic pain characterized by injury to the central or peripheral nervous system and that most often causes disability in individuals. Among the mechanisms involved in central sensitization during neuropathic pain are cytokines and chemokines released by spinal glial cells; however, these mechanisms are not well elucidated. Thus, the present study aimed to investigate the involvement of Chemokine (C-X-C motif) ligand 1 (CXCL1) and glial cells in this process. Male Wistar rats weighing 220-240 g were used and underwent a neuropathic pain model induced by chronic constriction injury (CCI). To investigate the involvement of CXCL1, chemokine receptor type 2 (CXCR2), mitogen-activated protein kinases (MAPK) p38, and microglia and astrocytes, the following drugs were used: SB225002, an CXCR2 antagonist; SML0543, a MAPK p38 inhibitor; minocycline, a microglia inhibitor; fluorocitrate, an astrocytes inhibitor; and recombinant CXCL1. The microglia, astrocytes, CXCL1, and MAPK p38 protein levels was evaluated by a Western blot assay. Furthermore, an immunofluorescence assay was performed to localize microglia and astrocytes immunoreactivity in the spinal cord. The results demonstrated that both CCI and CXCL1 induced nociception, and this effect was reversed by SB225002. In addition, minocycline, fluorocitrate, and SML0543 reversed the mechanical allodynia induced by CCI. Furthermore, there was an increase of spinal CXCL1 and microglial marker Iba1 protein levels , which was reversed by SB225002. This antagonist also reduced the Iba1 immunoreactivity in spinal cord. Thus, the present study suggests that the CXCL1 chemokine participates in neuropathic pain through CXCR2 activation in spinal microglia.

Intrathecal Administration of CXCL1 Enhances Potassium Currents in Microglial Cells.

The functioning of microglial cells inside the central nervous system depends on their ion channels expression. Microglia are capable of synthesizing different cytokines and chemokines, including CXCL1, and responding to their action via specific receptors. In this study, we explore the effect of intrathecal injection of CXCL1 on potassium currents, expressed in CX3CR1-Green Fluorescent Protein labeled microglia in transgenic mice. The results showed that CXCL1 hyperpolarized the cells by enhancing inward rectifying potassium currents and increasing the membrane area, suggesting an activating effect on microglia.

Follicle-stimulating hormone peptide-conjugated nanoparticles for targeted shRNA delivery lead to effective gro-α silencing and antitumor activity against ovarian cancer.

The distinct hormone molecules and receptors, such as follicle-stimulating hormone receptor (FSHR) in ovarian cancer, provide opportunities for more precisely targeted therapy. We previously developed FSHR-mediated nanoparticles and found that FSH peptides on the surface of nanoparticles improved the delivery of short interfering RNA (siRNA) into ovarian cancer cells. However, the high toxicity of the nanoparticles and the transient silencing of the siRNA in vivo limited further study. Here, we developed FSH peptide-conjugated nanoparticles with an increased amount of polyethylene glycol (PEG) grafting and encapsulated short hairpin RNA (shRNA) to silence the target gene, growth-regulated oncogene α (gro-α). The nanoparticle complexes exhibited good stability over three weeks. Expression of the target gene, gro-α, was significantly down-regulated by gro-α shRNA-loaded nanoparticles conjugated with FSH peptides (FSH33-G-NP) in FSHR-positive HEY cells. Cell proliferation, migration, and invasion were also inhibited by FSH33-G-NP. Tumor growth was delayed significantly in the mice treated with FSH33-G-NP. No significant loss of body weight or severe toxic effects were observed in any groups. In conclusion, gro-α shRNA-loaded nanoparticles conjugated with FSH peptides overcame the drawbacks of the in vivo application of RNAi therapeutics and polymer-based nanocarriers and showed safe antitumor efficacy. Our study might contribute to the application of FSHR-based targeted therapy and imaging in cancer.

Anti-nociceptive Role of CXCL1 in a Murine Model of Peripheral Nerve Injury-induced Neuropathic Pain.

Both spinal cord infiltrating CD4+ T lymphocytes and microglial CD40 contribute to the maintenance of neuropathic pain-like behaviors induced by spinal nerve L5 transection (L5Tx), a murine model of neuropathic pain. Here, we sought to investigate the involvement of multiple chemokines in microglial CD40-mediated and CD4+ T lymphocytes-mediated L5Tx-induced sensory hypersensitivity. Spinal cord chemokine expression in CD4 knockout (KO), CD40 KO, and wild type (WT) BALB/c mice was determined at the protein level via multiplex assays and at the RNA level via quantitative real-time PCR. In WT mice, L5Tx induced significant increases in CCL2, CCL3, and CCL5 expression (protein and RNA) up to day 21 post-L5Tx, while CD4 KO mice displayed blunted, predominantly non-significant, responses in these chemokines at protein levels post-L5Tx. L5Tx also induced increased expression of these chemokines in CD40 KO mice; however, the overall protein levels of these chemokines were significantly lower than those in WT mice. Further, L5Tx induced a significant increase in CXCL1 at the protein level and in CXCR2 at RNA level only in CD40 KO mice. Intrathecal administration of CXCL1 in WT mice significantly reduced L5Tx-induced mechanical hypersensitivity. CD40 KO mice also displayed higher levels of Ly6G (neutrophil marker) RNA expression in the lumbar spinal cord post-L5Tx. Altogether, our data suggest that CD4+ T lymphocytes and microglial CD40 mediate their pro-nociceptive effects in part by promoting selected chemokine responses, and more importantly, CXCL1 can play an anti-nociceptive role in peripheral nerve injury-induced neuropathic pain, which is possibly mediated by infiltrating neutrophils.

The contribution of the acute phase response to the pathogenesis of relapse in chronic-relapsing experimental autoimmune encephalitis models of multiple sclerosis.

BACKGROUND: Increased relapse rates in multiple sclerosis (MS) as a consequence of peripheral immune system activation, owing to infection for example, have been widely reported, but the mechanism remains unclear. Acute brain injury models can be exacerbated by augmenting the hepatic acute phase response (APR). Here, we explored the contribution of the hepatic APR to relapse in two rodent models of MS. METHODS: Mice with MOG-CFA-induced chronic relapsing experimental autoimmune encephalitis (CR-EAE) were killed before, during and after the first phase of disease, and the brain and liver chemokine, cytokine and acute phase protein (APP) mRNA expression profile was determined. During remission, the APR was reactivated with an intraperitoneal lipopolysaccharide (LPS) and clinical score was monitored throughout. To explore the downstream mediators, CXCL-1, which is induced as part of the APR, was injected into animals with a focal, cytokine/MOG-induced EAE lesion (fEAE) and the cellularity of the lesions was assessed. RESULTS: Compared to CFA control, in a rodent CR-EAE model, an hepatic APR preceded clinical signs and central cytokine production in the initial phase of disease. Compared to administration in naïve animals, an LPS challenge during the asymptomatic remission phase of CR-EAE rodents provoked relapse and resulted in the increased and extended expression of specific peripheral hepatic chemokines. CXCL-1 and several other APPs were markedly elevated. A single intravenous administration of the highly induced chemokine, CXCL-1, was found to be sufficient to reactivate the lesions by increasing microglial activation and the recruitment of T cells in fEAE lesions. CONCLUSIONS: The APR plays a contributing role to the pathology seen in models of chronic brain injury and in translating the effects of peripheral immune system stimulation secondary to trauma or infection into central pathology and behavioural signs. Further elucidation of the exact mechanisms in this process will inform development of more effective, selective therapies in MS that, by suppressing the hepatic chemokine response, may prevent relapse.

CXCL1 microspheres: a novel tool to stimulate arteriogenesis.

CONTEXT: After arterial occlusion, diametrical growth of pre-existing natural bypasses around the obstruction, i.e. arteriogenesis, is the body's main coping mechanism. We have shown before that continuous infusion of chemokine (C-X-C motif) ligand 1 (CXCL1) promotes arteriogenesis in a rodent hind limb ischemia model. OBJECTIVE: For clinical translation of these positive results, we developed a new administration strategy of local and sustained delivery. Here, we investigate the therapeutic potential of CXCL1 in a drug delivery system based on microspheres. MATERIALS AND METHODS: We generated poly(ester amide) (PEA) microspheres loaded with CXCL1 and evaluated them in vitro for cellular toxicity and chemokine release characteristics. In vivo, murine femoral arteries were ligated and CXCL1 was administered either intra-arterially via osmopump or intramuscularly encapsulated in biodegradable microspheres. Perfusion recovery was measured with Laser-Doppler. RESULTS: The developed microspheres were not cytotoxic and displayed a sustained chemokine release up to 28 d in vitro. The amount of released CXCL1 was 100-fold higher than levels in native ligated hind limb. Also, the CXCL1-loaded microspheres significantly enhanced perfusion recovery at day 7 after ligation compared with both saline and non-loaded conditions (55.4 ± 5.0% CXCL1-loaded microspheres versus 43.1 ± 4.5% non-loaded microspheres; n = 8-9; p < 0.05). On day 21 after ligation, the CXCL1-loaded microspheres performed even better than continuous CXCL1 administration (102.1 ± 4.4% CXCL1-loaded microspheres versus 85.7 ± 4.8% CXCL1 osmopump; n = 9; p < 0.05). CONCLUSION: Our results demonstrate a proof of concept that sustained, local delivery of CXCL1 encapsulated in PEA microspheres provides a new tool to stimulate arteriogenesis in vivo.

CXCL1 promotes arteriogenesis through enhanced monocyte recruitment into the peri-collateral space.

AIMS: The mechanisms of monocyte recruitment to arteriogenic collaterals are largely unknown. We investigated the role of chemokine (C-X-C-motif) ligand 1 (CXCL1) and its cognate receptor, chemokine (C-X-C-motif) receptor 2 (CXCR2) in arteriogenesis. METHODS AND RESULTS: After femoral artery ligation in Sprague-Dawley rats, either native collaterals were harvested or placebo, CXCL1 or CXCR2 blocker was administered via an osmopump. Perfusion recovery was measured with Laser Doppler, leukocyte populations were analyzed by fluorescence-activated cell sorting, and hind limb sections were stained for macrophage marker cluster of differentiation 68 (CD68). In vitro, fluorescent CXCL1 or human acute monocytic leukemia cell line (THP-1) monocytic cells were flown over shear-stressed endothelium. CXCL1 mRNA expression in collaterals was dramatically upregulated already 1 h after ligation (ratio ligated/sham 5.73). CD68 mRNA was upregulated from 12 h until 3 days after ligation (peak ratio ligated/sham 2.65). CXCL1 treatment augmented perfusion recovery at 3 and 7 days (p < 0.05) after ligation, and a significant increase in the number of peri-collateral macrophages was evident concomitantly (p < 0.05). Conversely, CXCR2 antagonist treatment caused a decrease in perfusion recovery both at 7 and 10 days postligation (p = 0.01) and also significantly reduced the number of peri-collateral macrophages (p < 0.05). In vitro, CXCL1 tethered to and was taken up by endothelial cells under shear stress conditions and enhanced THP-1 adherence compared to control (p < 0.05). In contrast, CXCR2 antagonist compromised THP-1 adherence to endothelial cells (p < 0.05). CONCLUSION: CXCL1 presented on the luminal endothelial surface leads to an increase in the number of peri-collateral macrophages, thus improving the arteriogenic response after arterial ligation.

Therapeutic administration of the chemokine CXCL1/KC abrogates autoimmune inflammatory heart disease.

Myocarditis, often due to an aberrant immune response to infection, is a major cause of dilated cardiomyopathy. Microbial pattern recognition receptors, such as TLRs, orchestrate the cytokine and chemokine responses that augment or limit the severity of myocarditis. Using the mouse model of experimental autoimmune myocarditis (EAM), in which disease is induced by immunization with a heart-specific self peptide and the agonist to multiple TLRs, complete Freund's adjuvant, we found that increased serum concentrations of the chemokine CXCL1/KC correlated directly with decreased severity of myocarditis. To directly test whether CXCL1/KC caused the amelioration of myocarditis, we treated mice, after challenge with heart-specific self peptide, with exogenous recombinant CXCL1/KC. We found that the administration of recombinant mouse CXCL1/KC completely abrogated heart inflammatory infiltration and cardiomyocyte damage. Moreover, we show that TLR4 signaling is required to increase serum protein concentrations of CXCL1/KC in EAM, and we demonstrate that the administration of the TLR4 agonist LPS significantly decreased severity and prevalence of EAM and reduced the number of heart-specific self peptide reactive effector T cells. These findings reveal a novel function of CXCL1/KC in the context of organ-specific autoimmune disease that may prove useful for the treatment of inflammatory conditions that underlie human heart disease.

CXCR2 knockout mice are protected against DSS-colitis-induced acute kidney injury and inflammation.

Organ cross talk exists in many diseases of the human and animal models of human diseases. A recent study demonstrated that inflammatory mediators can cause acute kidney injury and neutrophil infiltration in a mouse model of dextran sodium sulfate (DSS)-colitis. However, the chemokines and their receptors that may mediate distant organ effects in colitis are unknown. We hypothesized that keratinocyte chemoattractant (KC)/IL-8 receptor chemokine (C-X-C motif) ligand 2 (CXCL2) mediates DSS-colitis-induced acute kidney injury. Consistent with our hypothesis, wild-type (WT) mice developed severe colitis with DSS treatment, which was associated with inflammatory cytokine and chemokine expression and neutrophil infiltration in the colon. DSS-colitis in WT was accompanied by acute kidney injury and enhanced expression of inflammatory cytokines in the kidney. However, CXCR2 knockout mice were protected against DSS-colitis as well as acute kidney injury. Moreover, the expression of cytokines and chemokines and neutrophil infiltration was blunted in CXCR2 knockout mice in the colon and kidney. Administration of recombinant KC exacerbated DSS-colitis-induced acute kidney injury. Our results suggest that KC/IL-8 and its receptor CXCR2 are critical and major mediators of organ cross talk in DSS colitis and neutralization of CXCR2 will help to reduce the incidence of acute kidney injury due to ulcerative colitis and Crohn's disease in humans.

CXCR2 antagonists block the N-Ac-PGP-induced neutrophil influx in the airways of mice, but not the production of the chemokine CXCL1.

Neutrophils are innate immune cells in chronic inflammatory diseases including chronic obstructive pulmonary disease (COPD) and can be attracted to the site of inflammation via the collagen breakdown product N-acetyl Proline-Glycine-Proline (N-Ac-PGP). To elucidate whether CXCR2 is involved in N-Ac-PGP-induced neutrophil migration and activation, studies using specific antagonists were performed in vivo. N-Ac-PGP and keratinocyte cell-derived chemokine (KC; CXCL1) were administered in C57Bl/6 mice via oropharyngeal aspiration. Intraperitoneal applications of CXCR2 antagonist SB225002 or SB332235 were administered 1h prior and 1h after oropharyngeal aspiration. Six hours after oropharyngeal aspiration mice were sacrificed. Neutrophil counts and CXCL1 levels were determined in bronchoalveolar lavage fluid, myleoperoxidase (MPO) levels were measured in lung tissue homogenates and an immunohistological staining for neutrophils was performed on lung tissue. N-Ac-PGP and CXCL1 induced a neutrophil influx in the bronchoalveolar lavage fluid and lung tissue, which was also reflected by increased MPO levels in lung tissue. The N-Ac-PGP- and CXCL1-induced neutrophil influx and the increased pulmonary tissue MPO levels were inhibited by the CXCR2 antagonists SB225002 and SB332235. Moreover, N-Ac-PGP administration enhanced the CXCL1 levels in bronchoalveolar lavage fluid, which could not be attenuated by both CXCR2 antagonists. In conclusion, neutrophil migration induced by N-Ac-PGP is mediated via direct CXCR2 interaction. The N-Ac-PGP-induced release of CXCL1 is independent of CXCR2. Related to the maximal effect of CXCL1, N-Ac-PGP is more potent at inducing neutrophil migration in the pulmonary tissue than into the bronchoalveolar lavage fluid, or N-ac-PGP may be more potent at inducing MPO levels in the lung tissue.

CXCL1 and its receptor, CXCR2, mediate murine sickle cell vaso-occlusion during hemolytic transfusion reactions.

Hemolytic transfusion reactions (HTRs) can produce serious and potentially life-threatening complications in sickle cell disease (SCD) patients; however, the mechanisms underlying these complications remain undetermined. We established a model of alloimmune, IgG-mediated HTRs in a well-characterized humanized murine model of SCD. HTRs induced acute vaso-occlusive crisis (VOC), resulting in shortened survival of SCD mice. Acute VOC was associated with elevated circulating inflammatory chemokine levels, including striking elevation of the levels of the neutrophil chemoattractant CXCL1. Recombinant CXCL1 administration was sufficient to induce acute VOC in SCD mice, characterized by leukocyte recruitment in venules, capture of circulating red blood cells, reduction of venular flow, and shortened survival. In contrast, blockade of the CXCL1 receptor, CXCR2, prevented HTR-elicited acute VOC and prolonged survival in SCD mice. These results indicate that CXCL1 is a key inflammatory mediator of acute VOC in SCD mice. Targeted inhibition of CXCL1 and/or CXCR2 may therefore represent a new therapeutic approach for acute VOC in SCD patients.

Cytokine-induced neutrophil chemoattractant (CINC)-1 induces fever by a prostaglandin-dependent mechanism in rats.

Cytokine-induced neutrophil chemoattractant-1 (CINC-1), a member of the ELR+CXC subfamily [ELR motif (glutamic acid-leucine-arginine) adjacent to the cysteine-X-cysteine (CXC) motif located at the N-terminus of the protein], is an acute-phase protein and its synthesis is induced by endogenous and exogenous pyrogens. However, there are no studies on the pyrogenic property of CINC-1. Therefore, the present study evaluates whether centrally administered CINC-1 promotes an integrated febrile response along with an increase in the prostaglandin (PG)E2 content of the cerebrospinal fluid (CSF) of rats. The effects of antipyretic drugs on fever and on the PGE2 content of the CSF as well as the effectiveness of a neutralizing anti-CINC-1 antibody on the fever induced by CINC-1 have also been investigated. Intracerebroventricular (i.c.v.) injection of CINC-1 induced a dose-dependent bell-shaped rise on body temperature and increased PGE2 concentration in the CSF of conscious rats. Injected into the preoptic area of the anterior hypothalamus (AH/POA) (i.h.), CINC-1 also induced a dose-dependent bell-shaped increase in body temperature along with a decrease on tail skin temperature. Indomethacin (INDO, 2 mg kg(-1), i.p.) and ibuprofen (IBU, 10 mg kg(-1), i.p.) markedly reduced the fever evoked by i.c.v. injection of CINC-1 (25 ng/site). Orally given celecoxib (5 mg kg(-1), 30 min. before) abolished the fever induced by CINC-1 i.c.v. or i.h. (50 pg) injection. The antipyretic drugs also blocked the PGE(2) increase after CINC-1 i.c.v. injection. Co-injected anti-CINC antibody (10 ng/site) strongly reduced the febrile response induced by CINC-1 (50 pg/site) injected intrahypothalamically. This is the first time that centrally injected CINC-1 has been reported to act directly on the pyrogen-sensitive neurons of AH/POA, promoting a thermoregulatory response that seems to depend on other endogenous pyrogens synthesis and, as seen here, on PGE2.

Tissue- and stimulus-dependent role of phosphatidylinositol 3-kinase isoforms for neutrophil recruitment induced by chemoattractants in vivo.

PI3K plays a fundamental role in regulating neutrophil recruitment into sites of inflammation but the role of the different isoforms of PI3K remains unclear. In this study, we evaluated the role of PI3Kgamma and PI3Kdelta for neutrophil influx induced by the exogenous administration or the endogenous generation of the chemokine CXCL1. Administration of CXCL1 in PI3Kgamma(-/-) or wild-type (WT) mice induced similar increases in leukocyte rolling, adhesion, and emigration in the cremaster muscle when examined by intravital microscopy. The induction of neutrophil recruitment into the pleural cavity or the tibia-femoral joint induced by the injection of CXCL1 was not significantly different in PI3Kgamma(-/-) or WT mice. Neutrophil influx was not altered by treatment of WT mice with a specific PI3Kdelta inhibitor, IC87114, or a specific PI3Kgamma inhibitor, AS605240. The administration of IC87114 prevented CXCL1-induced neutrophil recruitment only in presence of the PI3Kgamma inhibitor or in PI3Kgamma(-/-) mice. Ag challenge of immunized mice induced CXCR2-dependent neutrophil recruitment that was inhibited by wortmannin or by blockade of and PI3Kdelta in PI3Kgamma(-/-) mice. Neutrophil recruitment to bronchoalveolar lavage induced by exogenously added or endogenous production of CXCL1 was prevented in PI3Kgamma(-/-) mice. The accumulation of the neutrophils in lung tissues was significantly inhibited only in PI3Kgamma(-/-) mice treated with IC87114. Neutrophil recruitment induced by exogenous administration of C5a or fMLP appeared to rely solely on PI3Kgamma. Altogether, our data demonstrate that there is a tissue- and stimulus-dependent role of PI3Kgamma and PI3Kdelta for neutrophil recruitment induced by different chemoattractants in vivo.

Growth-related oncogene-alpha induces endothelial dysfunction through oxidative stress and downregulation of eNOS in porcine coronary arteries.

Growth-related oncogene-alpha (GRO-alpha) is a member of the CXC chemokine family, which is involved in the inflammatory process including atherosclerosis. We hypothesized that GRO-alpha may affect endothelial functions in both porcine coronary arteries and human coronary artery endothelial cells (HCAECs). Vasomotor function was analyzed in response to thromboxane A2 analog U-46619 for contraction, bradykinin for endothelium-dependent vasorelaxation, and sodium nitroprusside (SNP) for endothelium-independent vasorelaxation. In response to 10(-6) M bradykinin, GRO-alpha (50 and 100 ng/ml) significantly reduced endothelium-dependent vasorelaxation by 34.73 and 48.8%, respectively, compared with controls (P < 0.05). There were no changes in response to U-46619 or SNP between treated and control groups. With the lucigenin-enhanced chemiluminescence assay, superoxide anion production in GRO-alpha-treated vessels (50 and 100 ng/ml) was significantly increased by 50 and 86%, respectively, compared with controls (P < 0.05). With real-time PCR analysis, endothelial nitric oxide synthase (eNOS) mRNA levels in porcine coronary arteries and HCAECs after GRO-alpha treatment were significantly decreased compared with controls (P < 0.05). The eNOS protein levels by both immunohistochemistry and Western blot analyses were also decreased in GRO-alpha-treated vessels. Antioxidant seleno-l-methionine and anti-GRO-alpha antibody effectively blocked these effects of GRO-alpha on both porcine coronary arteries and HCAECs. In addition, GRO-alpha immunoreactivity was substantially increased in the atherosclerotic regions compared with nonatherosclerotic regions in human coronary arteries. Thus GRO-alpha impairs endothelium-dependent vasorelaxation in porcine coronary arteries through a mechanism of overproduction of superoxide anion and downregulation of eNOS. GRO-alpha may contribute to human coronary artery disease.