CXCL1, CXCL10 and CXCL12 Chemokines are Variously Expressed in Acute Myeloid Leukemia Patients Prior and Post Bone Marrow Transplantation.

AIM: The chemokine-receptor axes play parts in development of leukemia, CXCL1, CXCL10 and CXCL12 are involved in immune responses. Thus, we have examined the serum levels of these chemokines in parallel with their related cognate receptors (CXCR1, CXCR3 and CXCR4) in AML (acute myeloid leukemia) patients prior and post BMT (bone marrow transplantation) therapy. MAIN METHODS: Clinical specimens were collected from 46 AML patients (23 M1 and 23 M3 subtypes) before/after BMT. CXCL1, CXCL10 and CXCL12 concentrations were determined by ELISA. The mRNA levels of the related receptors were detected by QRT_PCR. Data were analyzed by T-test, χ2 and ANOVA statistical methods in SPSS software version 18. A difference was regarded significant if P value < 0.05. KEY FINDINGS: Our results indicated that the elevated levels of CXCL12 in AML patients were remained unchanged after transplantation.  The CXCL10 concentration was decreased in patients. All studied chemokines were elevated in BMT patients with history of 9 times PLT transfusion. In patients who received BMT from siblings CXCL1 and CXCL10 have been elevated, whereby they were compared to patients who received BMT from parents while CXCL12 sustained unchanged in groups. Serum measures of CXCL1 and CXCL10 were induced in acute and chronic GVHD patients in compare to these without GVHD. SIGNIFICANCE: According to the results, it can be concluded that these chemokines play fundamental parts in pathogenesis of both AML and BMT. It is worthy to note that chemokines could be used as diagnostic markers alongside with possible promising therapeutic targets.

A Preliminary Report of the Relationship Between Gene Polymorphism of IL-8 and Its Receptors and Systemic Inflammatory Response Syndrome Caused by Wasp Stings.

The plasma levels of interleukin-8 (IL-8) and its receptors (CXCR1 and CXCR2) play a significant role in the development of systemic inflammatory response syndrome (SIRS), but it is not clear how these proteins are involved in wasp sting patients developing SIRS. To study potential genetic factors predisposing to the risk of SIRS caused by wasp sting injury, we determined the plasma levels of IL-8 and its receptors among SIRS patients with wasp sting injury and investigated the association of single-nucleotide polymorphisms of these genes with SIRS. A total of 225 patients were divided into the SIRS group (n = 62) and non-SIRS group (control, n = 163), and we associated polymorphisms in IL-8 [rs4073 (-251T>A), rs2227532 (-845C>T), rs2227307 (+396G>T), rs2227306 (+781T>C), CXCR1 rs2234671 (+860C>G), CXCR2 [rs2230054 (+811T>C), rs57929613 (+1235C>T), and rs60626131 (+1440A>G)] with SIRS with a linear additive model. In terms of protein expression, the IL-8, CXCR1, and CXCR2 plasma levels were significantly higher in the SIRS group than in the control group (p < 0.001). Significantly higher frequencies were observed for the IL-8 - 251T allele (AT+TT), CXCR2 + 811T allele (CT+TT), and +1235C allele (TC+CC) in the SIRS group, when compared with the control group, with odds ratio (OR) = 3.971 (95% confidence interval [CI], 1.618-9.734), p = 0.003; OR = 4.223 (95% CI, 1.863-9.571), p = 0.001; and OR = 4.012 (95% CI, 1.773-9.079), p = 0.001; respectively. In addition, SIRS is more likely to occur in males, patients with number of wasp stings ≥10 stings, and stings in the limbs. The current study suggests that the IL-8 - 251T allele (AT+TT) and IL-8 receptor CXCR2 + 811C allele (CT+TT) and +1235T allele (TC+CC) could be risk factors among SIRS patients with wasp sting injury.

Reversal of murine alcoholic steatohepatitis by pepducin-based functional blockade of interleukin-8 receptors.

OBJECTIVE: Alcoholic steatohepatitis is a life-threatening condition with short-term mortality up to 40%. It features hepatic neutrophil infiltration and blood neutrophilia, and may evolve from ethanol-induced breakdown of the enteric barrier and consequent bacteraemia. Signalling through CXCR1/2 G-protein-coupled-receptors (GPCRs), the interleukin (IL)-8 receptors, is critical for the recruitment and activation of neutrophils. We have developed short lipopeptides (pepducins), which inhibit post-ligand GPCR activation precisely targeting individual GPCRs. DESIGN: Experimental alcoholic liver disease was induced by administering alcohol and a Lieber-DeCarli high-fat diet. CXCR1/2 GPCRs were blocked via pepducins either from onset of the experiment or after disease was fully established. Hepatic inflammatory infiltration, hepatocyte lipid accumulation and overall survival were assessed as primary outcome parameters. Neutrophil activation was assessed by myeloperoxidase activity and liver cell damage by aspartate aminotransferase and alanine aminotransferase plasma levels. Chemotaxis assays were performed to identify chemoattractant signals derived from alcohol-exposed hepatocytes. RESULTS: Here, we show that experimental alcoholic liver disease is driven by CXCR1/2-dependent activation of neutrophils. CXCR1/2-specific pepducins not only protected mice from liver inflammation, weight loss and mortality associated with experimental alcoholic liver disease, but therapeutic administration cured disease and prevented further mortality in fully established disease. Hepatic neutrophil infiltration and triglyceride accumulation was abrogated by CXCR1/2 blockade. Moreover, CXCL-1 plasma levels were decreased with the pepducin therapy as was the transcription of hepatic IL-1ß mRNA. CONCLUSIONS: We propose that high circulating IL-8 in human alcoholic hepatitis may cause pathogenic overzealous neutrophil activation, and therapeutic blockade via pepducins merits clinical study.

Low expression of CXCR1/2 on neutrophils predicts poor survival in patients with hepatitis B virus-related acute-on-chronic liver failure.

Polymorphonuclear neutrophils (PMNs) and proinflammatory cytokines have been implicated in the pathogenesis of acute-on-chronic liver failure (ACLF). But the utility of CXC chemokine receptor expression on PMNs as a biomarker for prediction of disease severity is still uncertain. In this study, we investigated the dynamic expression of CXCR1 and CXCR2 on neutrophils, and found that patients with hepatitis B virus-related ACLF displayed low expression of CXCR1 and CXCR2 on peripheral neutrophils compared with healthy subjects and patients with chronic hepatitis B. This expression pattern was correlated with disease severity. Additionally, increased production of IL-8 in peripheral blood was significantly associated with reduced CXCR1 and CXCR2 expression, as shown by the decreased CXCR1 and CXCR2 expression on neutrophils after treating neutrophils with plasma from ACLF patients. This effect could be overcomed through IL-8 blockage with an anti-IL-8 antibody. We also found that IL-8 production and neutrophil infiltration were coordinately increased in the liver tissue of HBV-ACLF patients, and this increase was associated with liver inflammation. Overall, increased production of IL-8 associated with neutrophils infiltration into the liver and decreased CXCR1/2 expression on peripheral neutrophils. CXCR1 and CXCR2 expression levels could be served as early markers to predict the severity of ACLF.

Serum IL8 and mRNA level of CD11b in circulating neutrophils are increased in clinically amyopathic dermatomyositis with active interstitial lung disease.

The objective of this study is to assess serum IL8 and the potential activity of circulating neutrophils on relative messenger RNA (mRNA) levels and their relationship with disease activity in clinically amyopathic dermatomyositis (CADM) associated with interstitial lung disease (ILD). We studied 18 CADM patients and compared them with 18 classic dermatomyositis (DM) patients and 18 healthy control subjects. Serum IL8 level and mRNA expressions of neutrophils (chemokine (C-X-C motif) receptor 1 (CXCR1), cluster of differentiation molecule 11b (CD11b), cluster of differentiation 64 (CD64), myeloid cell leukemia 1 (MCL1), interleukin-18 (IL18)) were detected. The overproduction of serum IL8 level was most significant in the CADM group with active period. The mRNA expressions of CD11b, IL18, and MCL1 were greatly increased in the neutrophils in patients with CADM compared with DM or healthy controls. Up-expressions of CD11b, IL18, and MCL1 were detected in the neutrophils in CADM patients of active period compared with remission period. A positive correlation was found between CD11b mRNA level and high-resolution computed tomography (HRCT) score, in CADM associated with ILD. Serum IL8 level and mRNA levels of CD11b, MCL1, and IL18 in circulating neutrophils are related with the disease activity of CADM-ILD. The mRNA level of CD11b is positively correlated with HRCT score in CADM-ILD.

Impaired CXCR1-dependent oxidative defence in active tuberculosis patients.

Much of the pronounced host inflammatory response that occurs in tuberculosis (TB) is related to failed immunity against the invading pathogen. The G-protein coupled receptors CXCR1 and CXCR2 are implicated in important signal transduction pathways in lung inflammatory responses. We investigated the expression and function of these receptors in a simple whole blood model from 24 patients with pulmonary TB and in subjects with latent TB infection (LTBI). Healthy controls were recruited from close contacts to the pulmonary index patients. We found that pulmonary TB patients had significantly increased CXCR1 expression on blood cells compared to LTBI subjects and controls (p < 0.001). In contrast, LTBI subjects had a significant increase in CXCR2 expression compared to pulmonary TB patients (p < 0.001) and controls (p < 0.01). Leukocyte function, measured as oxidative capacity, was decreased in pulmonary TB patients compared to LTBI and controls (p < 0.001) and correlated with the increased CXCR1 expression. Leukocyte recruitment, measured as the expression of microRNA-223 was increased in pulmonary TB patients compared to LTBI (p < 0.05). We found that variations in receptor expression are linked to disease progression and affect the immune response against Mycobacterium tuberculosis (Mtb).

Anchorage independency promoted tumor malignancy of melanoma cells under reattachment through elevated interleukin-8 and CXC chemokine receptor 1 expression.

Metastasis of melanoma cells during the recurrence or the late stage of melanoma has been characterized as the dissemination of tumor cells under anchorage independency. The secreted interleukin-8 (IL-8) and its conical receptors from melanoma cells have been associated with melanoma malignancy. However, their correlations with melanoma cells under anchorage independency were unclear. Suspension of adherent melanoma cells generated the suspended melanoma cell model of anoikis resistance. The in-vivo xenograft experiment, in-vitro cell proliferation/migration assay, microarray, and bioinformatics analysis were used to compare the malignancy and gene expression profiling in adherent and suspended melanoma cells. PCR, enzyme-linked immunosorbent assay, immunohistochemistry, and kinase inhibition assay were adapted to validate the expression and regulation of IL-8 and CXCR1/2. Suspended melanoma cells were anoikis resistant and showed elevated malignancy in vivo and in vitro. Gene expression profiling of adherent and suspended melanoma cells showed extensive alteration associated with cell survival/death, cell signaling, and regulation of gene expression. Microarray and bioinformatics analysis on gene set enrichment analysis further showed elevated IL-8 expression in suspended melanoma cells. The upregulation of IL-8 and the effect on chemotaxis were mediated by MEK/ERK activation upon cell suspension. Change in JNK phosphorylation induced CXCR1 downregulation under cell suspension, but upregulation by cell reattachment. We suggest the possible roles of elevated IL-8 secretion and change in CXCR expression contributing toward elevated melanoma malignancy upon reattachment from cell suspension. We show that the suspension of melanoma cells is critical in promoting melanoma malignancy in vivo and in vitro.

Neutrophil expression of ICAM1, CXCR1, and VEGFR1 in patients with breast cancer before and after adjuvant chemotherapy.

BACKGROUND: Distinct populations of neutrophils have been identified based on the expression of intercellular adhesion molecule 1 (ICAM1, CD54) and chemokine receptor 1 (CXCR1, interleukin 8 receptor α). AIM: We analyzed the expression of vascular endothelial growth factor receptor 1 (VEGFR1), a physiological negative regulator of angiogenesis, on distinct populations of neutrophils from the blood of patients before and after adjuvant chemotherapy for breast cancer. MATERIALS AND METHODS: Neutrophil populations were distinguished as reverse transmigrated (ICAM1(high)/CXCR1(low)), naïve (ICAM1(low)/CXCR1(high)), or tissue-resident neutrophils (ICAM1(low)/CXCR1(low)), and their VEGFR1 expression quantified. RESULTS: Reverse transmigrated ICAM1(high)/CXCR1(low) neutrophilic granulocytes decreased significantly after chemotherapy and these were also the cells with highest mean fluorescence intensity for VEGFR1. CONCLUSION: Chemotherapy mainly reduces the number of reverse transmigrated long-lived ICAM1(high)/CXCR1(low) VEGFR1-expressing neutrophils. The decrease of antiangiogenic VEGFR1 may have a potential impact on tumour angiogenesis in patients undergoing adjuvant chemotherapy.

Elevated expression of the interleukin-8 receptors CXCR1 and CXCR2 in peripheral blood cells in obstructive coronary artery disease.

OBJECTIVES: As accumulation of monocytes and macrophages is a feature of atherosclerotic plaque at all stages, inflammatory gene expression profiling of peripheral blood mononuclear cells may provide a more reliable measure of atherogenesis than systemic inflammatory markers. The aim of this study was to determine whether expression patterns of inflammatory regulators in blood are correlated with severity and progression of coronary artery disease. METHODS: PCR expression arrays were used to profile mRNA levels of 84 candidate genes in blood from three patients with persistent perfusion defects, three with improved perfusion, and two without perfusion defects as measured by serial PET myocardial perfusion imaging. A case-control study compared expression of inflammatory genes in 25 patients with stress-induced perfusion defects and 25 controls using quantitative real-time PCR. RESULTS: Expression array analysis identified IL-8, CXCR1, and CXCR2 as genes showing increased expression in patients with persistent perfusion defects. The case-control study confirmed a significant increase in CXCR1 (P=0.04) and CXCR2 (P=0.002) mRNAs in blood in males with obstructive CAD, but not in females. There was no difference in IL-8 mRNA level between cases and controls (P=0.1). Coordinated expression of CXCR1 and CXCR2 mRNA was more pronounced in controls (r=0.96) than in patients with perfusion defects (r=0.73). CONCLUSIONS: mRNA levels for CXCR1 and CXCR2 are increased in blood in males with obstructive CAD and decreased in patients with improved perfusion, suggesting that these genes may serve as markers of disease severity and progression.

Neutrophil and monocyte activation in chronic kidney disease patients under hemodialysis and its relationship with resistance to recombinant human erythropoietin and to the hemodialysis procedure.

The aim of the present work was to further clarify leukocyte activation due to hemodialysis (HD) procedures and to investigate its relationship with recombinant human erythropoietin resistance. Therefore, we studied the expression of CXCR1 and CD11b on neutrophils, as well as the monocyte expression of CD11b, HLA-DR, and CD14. We studied 34 chronic kidney disease (CKD) patients under HD and recombinant human erythropoietin treatment (26 responders and 8 nonresponders to recombinant human erythropoietin therapy). All CKD patients' blood samples were collected before and immediately after the HD procedure. Eighteen healthy individuals (blood donors) were also studied as a control group. Hematological data, neutrophil (CD11b and CXCR1), and monocyte (CD11b, HLA-DR, and CD14) cell surface markers were measured in all patients (before and after the HD procedure) and controls. When compared with the controls, CKD patients presented a significant decrease in CXCR1 neutrophil expression, and in CD14 monocyte expression, accompanied by a significant increase in HLA-DR monocyte expression. When comparing the 2 groups of patients, we found that nonresponders showed an additional decrease in CXCR1 neutrophil expression. After the HD procedure, a statistically significant increase in CD14 and CD11b monocyte surface markers and a decrease in CXCR1 neutrophil expression and in HLA-DR monocyte expression was found. These data further strengthen our previous studies, showing that neutrophils and monocytes are activated in CKD patients, particularly in nonresponder patients. Moreover, this activation is due, at least in part, to the HD procedure, although we should not exclude that it can also be due to the enhanced inflammatory process observed in nonresponder patients.

Dysregulated chemokine receptor expression and chemokine-mediated cell trafficking in pediatric patients with ESRD.

BACKGROUND AND OBJECTIVES: Children and adolescents with ESRD on dialysis are susceptible to serious bacterial infections (SBI). Chemokines and chemokine receptors play a critical role in modulating macrophage and neutrophil function. This study examined the hypothesis that expression and/or function of these molecules is dysregulated in patients with ESRD, contributing to leukocyte dysfunction. Design setting, participants, & measurements: Pediatric patients, age 6 mo to 18 yr, with ESRD treated with either hemodialysis or peritoneal dialysis were enrolled in this prospective, nontherapeutic study. Blood was collected for plasma chemokine levels, chemokine receptor profiling by flow cytometry, and functional chemotaxis studies on neutrophils and mononuclear cells. RESULTS: ESRD in children was associated with reduced expression of the chemokine receptors CXCR1 and chemokine (C-C motif) receptor 2 (CCR2) on circulating neutrophils and monocytes, respectively. When ESRD patients were divided into two subgroups, those who were infection-free and those who had three or more SBI in the preceding year, the differences in chemokine receptor expression were statistically significant compared with control subjects only in those with recurrent infection. In addition to the effects of ESRD on baseline chemokine receptor expression, the hemodialysis procedure itself acutely lowered neutrophil CXCR1 and monocyte CCR2 expression. Furthermore, neutrophil and monocyte responsiveness to chemokine-mediated trafficking signals was impaired in all ESRD patients studied. This abnormality was independent of the level of chemokine receptor expression on the leukocytes. CONCLUSIONS: The data presented in this study suggest that chemokine receptor dysregulation contributes to leukocyte dysfunction in patients with ESRD. This alteration is especially prominent in ESRD patients with recurrent infection.

Overexpression of CD-11b and CXCR1 on circulating neutrophils: its possible role in COPD.

BACKGROUND: It has been shown that the beta2-integrin molecule is up-regulated in circulating neutrophils in COPD subjects. However, little has been reported about the expression of the cell surface molecules in such patients and their relationship with pulmonary function. The aim of the present study was to investigate the surface expression of molecules in circulating neutrophils and to clarify their possible role in the airflow limitation of COPD. METHODS: The surface expression of Mac-1 cells (ie, CD-11b and CD-18 cells) and CXC chemokine receptor (CXCR) 1 and CXCR2 of circulating neutrophils obtained from COPD patients and healthy subjects (HSs) was measured by flow cytometry analysis. The serum levels of interleukin (IL)-8 were measured by enzyme-linked immunosorbent assay. RESULTS: Both CD-11b and CXCR1 expression were significantly higher in COPD patients than in HSs (mean [+/- SE] CD-11b concentration: HSs, 9.7 +/- 1.0; COPD patients, 14.2 +/- 1.8 [p < 0.05]; mean CXCR1 concentration: HSs, 9.6 +/- 0.5; COPD patients, 11.9 +/- 0.4 [p < 0.01]). Although aging was positively correlated with the expression of CXCR1 (r = 0.440; p < 0.01), none of the other background factors, including smoking and body mass index, showed a correlation with the expression of the molecules. Although serum IL-8 levels were higher in patients with COPD than in HSs, no significant correlation between serum IL-8 levels and the expression of any molecule was seen. The expression of CD-11b (r = -0.317) and CXCR1 (r = -0.383) showed a significant negative correlation with the severity of airflow limitation (both p < 0.05). CONCLUSIONS: The overexpression of CD-11b and CXCR1 in circulating neutrophils may be associated with the development of airflow limitation in COPD patients.

Increased killing activity and decreased cytokine production in NK cells in patients with primary biliary cirrhosis.

Although the pathogenesis of primary biliary cirrhosis (PBC) remains enigmatic, the immune system plays a key role in the initiation and subsequent development of pathology. Previous studies have indicated a critical role of the innate immune system. Importantly, natural killer (NK) cells are abundant in liver where they serve as sentinels of the immune system. In addition, NK cells have significant biologic activity based on their production of immunoregulatory cytokines. To address this issue, we have investigated several qualitative and quantitative activities of NK cells in patients with PBC as well as normal and liver diseased controls. We report herein a marked increase in the frequency and absolute number of blood and liver NK cells in PBC patients. Moreover, the cytotoxic activity and perforin expression by isolated NK cells were significantly increased in PBC patients associated with increased levels of plasma IL-8 and the expression of CD128a (IL-8 receptor) on NK cells. In contrast, the levels of IFN-gamma, IL-6 and IL-8 synthesized by NK cells were significantly decreased in PBC patients as compared to controls. In conclusion, data from this study provide compelling evidence supporting a biologic role of NK cells in the immunopathogenesis of PBC.

Alteration of polymorphonuclear neutrophil surface receptor expression and migratory activity after isolation: comparison of whole blood and isolated PMN preparations from normal and postfracture trauma patients.

BACKGROUND: Post-traumatic MOF results from local tissue injury because of migration and activation of dysfunctional polymorphonuclear leukocytes (PMN). Although fracture surgery exacerbates the postinjury inflammatory response, it is usually beneficial. This study compared changes in PMN receptor expression and migratory activity, in whole blood and following PMN isolation. METHODS: IL-8 mediated PMN migration and expression of CXCR-1, CD11b, and CD18 was studied in isolated and whole blood PMN in normal controls. Migration was studied at admission and day 5 after surgery in trauma patients undergoing fracture surgery. RESULTS: PMN isolation results in increased expression of surface receptors and enhanced migration in normal controls. In trauma patient samples, isolated PMN migration is enhanced after injury, but suppressed when migration from whole blood is studied, both after injury and fracture surgery. CONCLUSION: PMN isolation results in priming for migration, which has a relatively greater impact upon PMN in trauma patients. The observation that PMN activity may decline but priming potential remains enhanced is novel. Further refinements of whole blood and isolated PMN techniques are clearly warranted. This may help to resolve the mismatch in clinical and scientific experience in those patients with major fractures requiring surgical stabilization.

Different subsets of peripheral blood dendritic cells show distinct phenotypic and functional abnormalities in HIV-1 infection.

OBJECTIVES: To analyse the distribution, expression of chemokine receptors and ex vivo production of inflammatory cytokines by peripheral blood (PB) monocytes and DC in HIV-1+ individuals. DESIGN: Dendritic cells (DC) play an important role in the establishment and dissemination of HIV infection. DC interaction with HIV depends on expression of HIV receptors and co-receptors. Accumulating evidence supports the notion that DC functionality is impaired in HIV-1+ patients. METHODS: PB samples from 30 naive-treated HIV-1+ progressors were studied. Additionally, 10 adult healthy volunteers (AHV), seven Hepatitis C virus positive (HCV+)/HIV-1- patients and five long-term non-progressor HIV-1+ patients (HIV-1+LTNP) were included as controls. Flow cytometry immunophenotyping was used for the identification, enumeration and characterization of monocytes and DC. RESULTS: PB myeloid DC (mDC) and plasmacytoid DC (pDC) were significantly decreased in HIV-1+ progressors, while unaltered in HIV-1+LTNP. The expression of CXCR2 and CXCR4 and of CXCR4 and CCR5 were severely altered on PB mDC and pDC from HIV-1+ progressors, respectively. By contrast, both the expression of the chemokine receptors analysed and the numbers of CD16+ DC in HIV-1+ progressors were not different from AHV, while altered in HCV+/HIV-1- and HIV-1+LTNP. Furthermore, PB monocytes and DC from HIV-1+ progressors spontaneously produced inflammatory cytokines, in contrast with AHV. CONCLUSIONS: These results support the existence of a selective interaction between HIV-1 and both mDC and pDC, associated with HCV co-infection-independent spontaneous production of inflammatory cytokines, reflecting the occurrence of in vivo activation of the immune system, which might further contribute to the impaired DC functionality.

CXCR1+CD4+ T cells in human allergic disease.

Chemokine receptors play an important role in the migration of leukocytes to sites of allergic inflammation in humans. In this study, we have identified increased expression of the chemokine receptor CXCR1 on CD4+ T lymphocytes derived from patients with atopic disease compared with normal donors. Enhanced expression of CXCR1 by atopic donors was identified on freshly isolated peripheral blood cells and on expanded cell populations derived from nasal mucosal biopsies and from the periphery. Identification of CXCR1 expression on CD4 cells in the nasal mucosa was confirmed by double immunofluorescence. In addition, expression of CXCR1 was dramatically decreased in patients undergoing successful treatment of allergic rhinitis by specific immunotherapy. CXCR1 provided a functional receptor capable of regulating T cells in the context of allergic disease, since expression of CXC chemokine ligand 8 was up-regulated at the site of allergic inflammation and freshly isolated CXCR1+CD4+ cells from atopic donors showed an enhanced functional response to this ligand. CXCR1 expression on CD4+ T cells was increased in vitro in response to the pro-Th2 cytokine IL-4. Phenotypic analysis reveals that IFN-gamma expression was lower in the CXCR1+CD4+ cells. The identification of CXCR1 as a marker of allergic rhinitis reveals a possible target for therapeutic intervention in atopic disease.

Actin filaments are involved in the regulation of trafficking of two closely related chemokine receptors, CXCR1 and CXCR2.

The ligand-induced internalization and recycling of chemokine receptors play a significant role in their regulation. In this study, we analyzed the involvement of actin filaments and of microtubules in the control of ligand-induced internalization and recycling of CXC chemokine receptor (CXCR)1 and CXCR2, two closely related G protein-coupled receptors that mediate ELR-expressing CXC chemokine-induced cellular responses. Nocodazole, a microtubule-disrupting agent, did not affect the IL-8-induced reduction in cell surface expression of CXCR1 and CXCR2, nor did it affect the recycling of these receptors following ligand removal and cell recovery at 37 degrees C. In contrast, cytochalasin D, an actin filament depolymerizing agent, promoted the IL-8-induced reduction in cell surface expression of both CXCR1 and CXCR2. Cytochalasin D significantly inhibited the recycling of both CXCR1 and CXCR2 following IL-8-induced internalization, the inhibition being more pronounced for CXCR2 than for CXCR1. Potent inhibition of recycling was observed also when internalization of CXCR2 was induced by another ELR-expressing CXC chemokine, granulocyte chemotactic protein-2. By the use of carboxyl terminus-truncated CXCR1 and CXCR2 it was observed that the carboxyl terminus domains of CXCR1 and CXCR2 were partially involved in the regulation of the actin-mediated process of receptor recycling. The cytochalasin D-mediated inhibition of CXCR2 recycling had a functional relevance because it impaired the ability of CXCR2-expressing cells to mediate cellular responses. These results suggest that actin filaments, but not microtubules, are involved in the regulation of the intracellular trafficking of CXCR1 and CXCR2, and that actin filaments may be required to enable cellular resensitization following a desensitized refractory period.