Eotaxins and C-C chemokine receptor type 3 in Parkinson's disease.

Parkinson's disease (PD) is one of the most common neuroinflammatory disorders and inflammatory processes seem to play an important role in the pathogenesis of PD. Chemokines as inflammatory mediators, which are involved in the recruitment of leukocytes, can play a role in the pathogenesis of PD. The aim of this study was to examine the serum level of eotaxins (CCL11, CCL24, and CCL26) and the expression of C-C chemokine receptor type 3 (CCR3) in patients with PD compared with healthy subjects. In this study, we measured the serum levels of CCL11, CCL24, and CCL26 with ELISA. In addition, gene and protein expression of CCR3 were measured by RT-PCR and flow cytometry techniques in PD patients (n = 30) and age- and sex-matched healthy subjects (n = 30). All patients suffering from PD were assessed clinically through Unified Parkinson's Disease Rating Scale, Motor Examination (UPDRS ME). The results of this study showed that there was no significant alteration in the serum level of these chemokines and also their receptor among patients with PD and healthy subjects. No significant correlation was observed between the eotaxins serum levels and the clinical measures of PD severity. Based on the results, it can be concluded that eotaxins cannot be considered as appropriate targets for the diagnosis or treatment of PD.

Upregulated expression of CCR3 in rheumatoid arthritis and CCR3-dependent activation of fibroblast-like synoviocytes.

It is recognized that CC chemokine receptor 3 (CCR3) is associated with numerous inflammatory conditions and fibroblast-like synoviocyte (FLS) invasiveness correlates with articular damage in rheumatoid arthritis (RA). However, little is known of the expression and action of CCR3 on FLS in RA. In the present study, we investigated the expression of CCR3 on dispersed synovial tissue and peripheral blood cells in RA and influence of eotaxin-1 on FLS functions by using flow cytometry analysis, FLS challenge, and real-time PCR techniques. The results showed that approximately 7.0 % dispersed synovial cells are CCR3+ cells. Among those CCR3+ cells, 38.1, 23.8, and 20.6 % cells are CD90+CD14-CD3- (representing FLS), CD14+, and CD8+ cells, respectively, indicating that FLS is one of the major populations of CCR3+ cells in the synovial tissue of RA. In peripheral blood, CD14+ CCR3+ cells are elevated, but CD8+CCR3+ cells are reduced in RA. It was found that eotaxin-1 induced upregulated expression of CCR3 and matrix metalloproteinase (MMP)-9 messenger RNAs (mRNAs) in FLS. Since an antagonist of CCR3 suppressed the action of eotaxin-1, the event appeared CCR3 dependent. Moreover, we observed that interleukin (IL)-1ß induced markedly enhanced eotaxin-1 release from FLS, but TNF-α reduced eotaxin-1 release at 12 and 24 h following incubation. In conclusion, enhanced expression of CCR3 on synovial cells and increased levels of eotaxin-1 in plasma and synovial fluid (SF) of RA indicate that CCR3-mediated mechanisms may play an important role in RA. Blockage of eotaxin-1 provoked CCR3 and MMP-9 expression in FLS by antagonist of CCR3, implicating that anti-CCR3 agents may have therapeutic use for RA.

Basophil markers for identification and activation in the indirect basophil activation test by flow cytometry for diagnosis of autoimmune urticaria.

BACKGROUND: The indirect basophil activation test using flow cytometry is a promising tool for autoimmune urticaria diagnosis. We aimed to identify better donor basophils (from atopic vs. non-atopic donors and interleukin-3 primed vs. unprimed basophils) and improve basophil identification and activation markers (eotaxin CC chemokine receptor-3 [CCR3] vs. CD123 and CD63 vs. CD203c). METHODS: Donor basophils were obtained from non-atopic and atopic group O donors. Positive control sera were artificially prepared to simulate autoimmune urticaria patients' sera. Patient sera were obtained from nine children with chronic urticaria. Assay sensitivity was compared among each variation by using positive control sera (n=21), applying cutoff values defined from negative control sera (n=20). RESULTS: For basophil identification, a combination of CCR3 and CD123 markers revealed a higher correlation with automated complete blood count (r=0.530) compared with that observed using CD123 (r=0.498) or CCR3 alone (r=0.195). Three activation markers on the atopic donor basophils attained 100% assay sensitivity: CD203c on unprimed basophils, CD63+CD203+ or CD63 alone on primed basophils; however, these markers on the non-atopic donor basophils attained lower assay sensitivity. CONCLUSIONS: For basophil identification markers, a combination of CD123 and CCR3 is recommended, while CD123 alone may be used as an alternative. Donor basophils should be obtained from an atopic donor. For basophil activation markers, either CD203c alone on unprimed basophils or CD203c and CD63 on primed basophils are recommended, while CD63 alone on primed basophils may be used as an alternative.

Blood fibrocytes are recruited during acute exacerbations of chronic obstructive pulmonary disease through a CXCR4-dependent pathway.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by peribronchial fibrosis. The chronic course of COPD is worsened by recurrent acute exacerbations. OBJECTIVE: The aim of the study was to evaluate the recruitment of blood fibrocytes in patients with COPD during exacerbations and, subsequently, to identify potential mechanisms implicated in such recruitment. METHODS: Using flow cytometry, we quantified circulating fibrocytes and characterized their chemokine receptor expression in 54 patients with COPD examined during an acute exacerbation (V1) and 2 months afterward (V2) and in 40 control subjects. The role of the chemokines CXCL12 and CCL11 in fibrocyte migration was investigated by using a chemotaxis assay. Patients were followed for up to 3 years after V1. RESULTS: We demonstrated a significantly increased number of circulating fibrocytes at V1 compared with control subjects. The number of circulating fibrocytes decreased at V2. A high percentage of circulating fibrocytes during exacerbation was associated with increased risk of death. The percentage of fibrocytes at V2 was negatively correlated with FEV1, forced vital capacity, FEV1/forced vital capacity ratio, transfer lung capacity of carbon monoxide, and Pao2. Fibrocytes highly expressed CXCR4 and CCR3, the chemokine receptors for CXCL12 and CCL11, respectively. Fibrocytes collected from patients with COPD at V1 had increased chemotactic migration in response to CXCL12 but not to CCL11 compared with those from control subjects. Plerixafor, a CXCR4 antagonist, decreased fibrocyte migration to plasma from patients with exacerbating COPD. CONCLUSION: Blood fibrocytes are recruited during COPD exacerbations and related to mortality and low lung function. The CXCL12/CXCR4 axis is involved in such fibrocyte recruitment (Firebrob study; ClinicalTrials NCT01196832).

Basophil Markers for Identification and Activation in the Indirect Basophil Activation Test by Flow Cytometry for Diagnosis of Autoimmune Urticaria

BACKGROUND: The indirect basophil activation test using flow cytometry is a promising tool for autoimmune urticaria diagnosis. We aimed to identify better donor basophils (from atopic vs. non-atopic donors and interleukin-3 primed vs. unprimed basophils) and improve basophil identification and activation markers (eotaxin CC chemokine receptor-3 [CCR3] vs. CD123 and CD63 vs. CD203c). METHODS: Donor basophils were obtained from non-atopic and atopic group O donors. Positive control sera were artificially prepared to simulate autoimmune urticaria patients' sera. Patient sera were obtained from nine children with chronic urticaria. Assay sensitivity was compared among each variation by using positive control sera (n=21), applying cutoff values defined from negative control sera (n=20). RESULTS: For basophil identification, a combination of CCR3 and CD123 markers revealed a higher correlation with automated complete blood count (r=0.530) compared with that observed using CD123 (r=0.498) or CCR3 alone (r=0.195). Three activation markers on the atopic donor basophils attained 100% assay sensitivity: CD203c on unprimed basophils, CD63+CD203+ or CD63 alone on primed basophils; however, these markers on the non-atopic donor basophils attained lower assay sensitivity. CONCLUSIONS: For basophil identification markers, a combination of CD123 and CCR3 is recommended, while CD123 alone may be used as an alternative. Donor basophils should be obtained from an atopic donor. For basophil activation markers, either CD203c alone on unprimed basophils or CD203c and CD63 on primed basophils are recommended, while CD63 alone on primed basophils may be used as an alternative.

Pregnancy and malaria exposure are associated with changes in the B cell pool and in plasma eotaxin levels.

Pregnancy triggers immunological changes aimed to tolerate the fetus, but its impact on B lymphocytes is poorly understood. In addition, exposure to the Plasmodium parasite is associated with altered distribution of peripheral memory B cell (MBC) subsets. To study the combined impact of high malaria exposure and pregnancy in B cell subpopulations, we analyzed PBMCs from pregnant and nonpregnant individuals from a malaria-nonendemic country (Spain) and from a high malaria-endemic country (Papua New Guinea). In the malaria-naive cohorts, pregnancy was associated with a significant expansion of all switched (IgD(-)) MBC and a decrease of naive B cells. Malaria-exposed women had more atypical MBC and fewer marginal zone-like MBC, and their levels correlated with both Plasmodium vivax- and Plasmodium falciparum-specific plasma IgG levels. Classical but not atypical MBC were increased in P. falciparum infections. Moreover, active atypical MBC positively correlated with proinflammatory cytokine plasma concentrations and had lower surface IgG levels than the average. Decreased plasma eotaxin (CCL11) levels were associated with pregnancy and malaria exposure and also correlated with B cell subset frequencies. Additionally, active atypical and active classical MBC expressed higher levels of eotaxin receptor CCR3 than the other B cell subsets, suggesting a chemotactic effect of eotaxin on these B cell subsets. These findings are important to understand immunity to infections like malaria that result in negative outcomes for both the mother and the newborn and may have important implications on vaccine development.

Blood expression levels of chemokine receptor CCR3 and chemokine CCL11 in age-related macular degeneration: a case-control study.

BACKGROUND: Dysregulation of the CCR3/CCL11 pathway has been implicated in the pathogenesis of choroidal neovascularisation, a common feature of late age-related macular degeneration (AMD). The aim of this study was to investigate the expression of CCR3 and its ligand CCL11 in peripheral blood in patients with neovascular AMD. METHODS: Patients with neovascular AMD and healthy controls were included. Blood samples were obtained and prepared for flow cytometry to investigate the expression of CCR3. Levels of CCL11 were measured in plasma using Cytometric Bead Array. Differences between the groups were tested using Kruskal-Wallis test and Mann-Whitney U test. RESULTS: Patients (n = 83) with neovascular AMD and healthy control persons (n = 114) were included in the study. No significant difference in the expression of CCR3 was found on CD9+ granulocytes when comparing patients suffering from neovascular AMD with any of the control groups. We did not find any alteration in CCL11 levels in patients among the age matched groups. There was no correlation between expression of CCR3/CCL11 and clinical response to treatment with anti-vascular endothelial growth factor (VEGF). CONCLUSION: Our results do not suggest a systemic alteration of the CCR3/CCL11 receptor/ligand complex in patients with neovascular AMD.

Enhanced CCR5+/CCR3+ T helper cell ratio in patients with active cutaneous lupus erythematosus.

Cutaneous lupus erythematosus (CLE) is characterized by enhanced interferon α (IFNα) levels in serum and in tissue. Since IFNα promotes a Th1-biased immune response, we hypothesized that a Th1-associated chemokine receptor profile should be a typical finding in patients with active CLE. Therefore, peripheral blood mononuclear cells were isolated from patients with different CLE subsets (n = 15), healthy controls (n = 13) and patients under immunotherapy with IFNα (n = 7). T helper cells were analysed by flow cytometry for the expression of the chemokines receptor CCR5, indicative for Th1 cells, and of CCR3, indicating Th2. In addition, intracellular levels of the type I IFN-inducible MxA protein were measured. Patients with widespread active CLE skin lesions had a significantly increased expression of CCR5, whereas expression of CCR3 was decreased when compared with healthy controls. MxA expression was significantly enhanced in all investigated CLE subtypes, with the highest levels in patients with widespread skin lesions. The enhanced CCR5/CCR3 ratio closely correlated with the MxA levels in peripheral lymphocytes and with disease activity. Our analyses revealed that active CLE is associated with a systemic type I IFN effect that appears to induce a shift towards a Th1-associated chemokine receptor profile. The CCR5/CCR3 T-helper cell ratio might therefore represent an indirect marker for the disease activity in CLE.

HIV-1 Tat-induced platelet activation and release of CD154 contribute to HIV-1-associated autoimmune thrombocytopenia.

BACKGROUND: Enhanced platelet activation in human immunodeficiency virus (HIV)-1-infected patients has been reported and shown to strongly correlate with plasma viral load. Activated platelets are known to express and to release a variety of proteins that can modulate the immune system. Specifically, platelet-derived CD154 has been shown to be directly involved in the development of autoimmune thrombocytopenia (ITP). The mechanism by which HIV-1 infection leads to platelet activation and the effect of this activation on the development of HIV-1 ITP, however, is not fully understood. OBJECTIVE: We have investigated the effect of HIV-1 Trans activating factor (Tat) on platelet activation. RESULTS: We report that HIV-1 Tat directly interacts with platelets and induces platelet activation resulting in platelet micro-particle release. This activation by Tat requires the chemokine receptor CCR3 and ß3-integrin expression on platelets, as well as calcium flux. Tat-induced activation of platelets releases platelet CD154, an immune modulator. Enhanced B-cell activity is found in mouse spleen B cells co-cultured with platelets treated with Tat in vitro. An early antibody response against adenovirus is found in Tat-injected mouse immunized with adenovirus, suggesting an enhanced immune response in vivo. CONCLUSIONS: We have described a role of Tat-induced platelet activation in the modulation of the immune system, with implications for the development of HIV-1-associated thrombocytopenia.

Genome profiles in maternal blood during early onset preeclampsia and towards term.

AIMS: inflammatory processes are present during preeclampsia and in normal pregnancy. Maternal inflammatory reactions may change towards term. Our objective was to evaluate genome signaling in blood during preeclampsia and towards term using microarrays. METHODS: RNA microarrays (Illumina) were conducted on blood from preeclamptic pregnancies delivered preterm, normal pregnancies at term and normal pregnancies at gestational week 31. Two statistical methods (Q-value cut-off 1%) identified data structures in the three groups and retrieved activated genes along a time axis and a diseased-healthy axis. Signaling genes were localized within known pathways and gene sets, and genes associated with inflammation were identified. RESULTS: early onset preeclampsia and term pregnancies both showed distinct expression patterns when compared to normal pregnancy at gestational week 31. In preeclampsia, 19 genes were differentially expressed, including a down-regulation of CC-chemokine receptor 3 (CCR3). Among the 183 differentially expressed genes towards term, tumor necrosis factor superfamily member 15 (TNFSF15) was up-regulated and interferon-γ receptor 2 (IFNGR2) and CXC-chemokine receptor type 4 (CXCR4) were down-regulated. Seven of the genes were similarly changed during preeclampsia and towards term. CONCLUSIONS: a possible type 1 immune response was identified both during preeclampsia and towards term. In pre-eclampsia a premature activation of leucocytes might be present.

Decreased levels of CCR3 in CD4+ lymphocytes of rheumatoid arthritis patients.

OBJECTIVES: To evaluate the expression of CCR3 receptors as well as CCR3 agonists, including eotaxin-2 and RANTES, among patients suffering from rheumatoid arthritis and healthy controls, as a possible pathogenetic mechanism in inflammatory joint disease. METHODS: Twenty-two patients and 13 healthy controls were recruited and clinically evaluated. CCR3 expression on CD4+ lymphocytes and mononuclear cells was evaluated by FACS analysis after staining with human CD4 APC (bioscience) and human CCR3 (CD193)PE. Levels of eotaxin-2 and RANTES were analysed by ELISA. RESULTS: A significant decrease was observed in the level of CD4+ cells expressing the CCR3 receptor in serum of RA patients (0.96+/-0.5) as compared with healthy controls (1.48+/-0.6) (p<0.05). A significant decrease in serum eotaxin-2 levels was evident among RA patients suffering from active disease, defined by a DAS-28 score above 5.5, compared with RA patients with lower activity scores (2.1+/-1.6 vs. 7.0+/-5.1; p=0.01). A significant decrease was evident in the number of CCR3 expressing Monocytes among RA patients treated with steroids and anti TNF-a medications as compared with RA patients not receiving such treatment. CONCLUSIONS: CCR3 is differentially expressed on inflammatory cells in RA, while eotaxin-2, a potent CCR3 agonist, is differentially expressed in active disease. Anti-inflammatory medications may down-regulate CCR3 expression in RA. The CCR3-CCR3 agonist pathway may thus have a pathogenic role in RA and may be a future target for novel treatment modalities.

[Correlation of chemokines and chemokine receptors with clinical features in newly diagnosed systemic lupus erythematosus].

OBJECTIVE: To explore the correlation of chemokines and chemokine receptors with clinical features of newly diagnosed systemic lupus erythematosus (SLE). METHODS: Samples of venous blood were collected from 37 newly diagnosed SLE patients, 2 males and 35 females, aged 32.5 (15-54), and 20 healthy controls. The serum concentration of macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta and reduced upon activation normal T cell expressed and secreted (RANTES) were measured by ELISA. Samples of anticoagulative serum were collected from 18 of the 37 newly diagnosed SLE patients and 10 healthy controls. The expression rates of CCR1, CCR3, and CCR5 on CD4+ T cells were detected by flow cytometry. Indirect immunofluorescence method was used to detect the anti-ds-DNA antibody, and blot immunoassay was used to detect the anti-RNP and anti-SSA antibodies. The associations of chemokines and chemokine receptors with the clinical features were analyzed. RESULTS: The serum concentration of MIP-1alpha of the SLE patients was (37 +/- 25) ng/L, significantly higher than that of the controls (8 +/- 9) ng/L (P < 0.001). The serum concentration of MIP-1beta of the SLE patients was (160 +/- 140) ng/L, significantly higher than that of the controls (76 +/- 41) ng/L (P = 0.003). The serum concentration of RANTES of the SLE patients was (184 +/- 22) ng/L, not significantly different from that of the controls (144 +/- 79) ng/L (P > 0.05). The serum concentration of MIP-1alpha of the SLE patients with fever was 52 ng/L +/- 27 ng/L, significantly higher than that of the SLE patients without fever (28 ng/L +/- 19 ng/L, P = 0.006). The serum concentration of MIP-1beta of the SLE patients with arthritis was 221 ng/L +/- 158 ng/L, significantly higher than that of the SLE patients without arthritis (95 ng/L +/- 83 ng/L, P = 0.008). The serum concentration of RANTES of the SLE patients with low blood platelet count was 130 ng/L +/- 122 ng/L, significantly lower than that of the SLE patients with normal blood platelet count (212 ng/L +/- 114 ng/L, P = 0.049). The percentage of CD4+ CCR3+ T cell subgroup in CD4+ T cell in peripheral blood of the SLE patients positive in anti-RNP antibody was 14.8% +/- 3.0%, significantly lower than that of the SLE patients negative in anti-RNP antibody (11.3% +/- 2.6%, P = 0.018). CONCLUSION: Abnormality of different chemokines and chemokine receptors may concern with different clinical features of SLE.