RESUMO
CC chemokine receptor 3 (CCR3), also known as CD193, belongs to class A of G protein-coupled receptors and is present in high levels in eosinophils, basophils, and airway epithelial cells. CCR3 is considered the therapeutic target for human immunodeficiency virus (HIV) infections and allergic diseases; therefore, the development of sensitive monoclonal antibodies (mAbs) for CCR3 has been desired. This study aimed to establish a specific and sensitive mAb against mouse CCR3 (mCCR3) useful for flow cytometry analysis by employing the Cell-Based Immunization and Screening (CBIS) method. The generated anti-mCCR3 mAb, C3Mab-2 (rat IgG2b, kappa), was found to react with mCCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCCR3) cells, according to flow cytometric analysis. Also, it reacted with P388 (mouse lymphoid neoplasm) or J774-1 (mouse macrophage-like) cells, which express endogenous mCCR3. Taken together, C3Mab-2, generated by the CBIS method, can be a valuable tool for detecting mCCR3 on the surface of mouse cells.
Assuntos
Anticorpos Anti-Idiotípicos/farmacologia , Anticorpos Monoclonais/farmacologia , Infecções por HIV/imunologia , Receptores CCR3/imunologia , Animais , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Células CHO , Cricetulus , Citometria de Fluxo , Infecções por HIV/terapia , Infecções por HIV/virologia , Humanos , Hipersensibilidade/imunologia , Hipersensibilidade/terapia , Macrófagos/imunologia , Camundongos , Receptores CCR3/antagonistas & inibidoresRESUMO
Platelet activation and pulmonary recruitment occur in patients with asthma and in animal models of allergic asthma, in which leukocyte infiltration, airway remodeling, and hyperresponsiveness are suppressed by experimental platelet depletion. These observations suggest the importance of platelets to various characteristics of allergic disease, but the mechanisms of platelet migration and location are not understood. The aim of this study was to assess the mechanism of platelet recruitment to extravascular compartments of lungs from patients with asthma and after allergen challenge in mice sensitized to house dust mite (HDM) extract (contains the DerP1 [Dermatophagoides pteronyssinus extract peptidase 1] allergen); in addition, we assessed the role of chemokines in this process. Lung sections were immunohistochemically stained for CD42b+ platelets. Intravital microscopy in allergic mice was used to visualize platelets tagged with an anti-mouse CD49b-PE (phycoerythrin) antibody. Platelet-endothelial interactions were measured in response to HDM (DerP1) exposure in the presence of antagonists to CCR3, CCR4, and CXCR4. Extravascular CD42b+ platelets were detected in the epithelium and submucosa in bronchial biopsy specimens taken from subjects with steroid-naive mild asthma. Platelets were significantly raised in the lung parenchyma from patients with fatal asthma compared with postmortem control-lung tissue. Furthermore, in DerP1-sensitized mice, subsequent HDM exposure induced endothelial rolling, endothelial adhesion, and recruitment of platelets into airway walls, compared with sham-sensitized mice, via a CCR3-dependent mechanism in the absence of aggregation or interactions with leukocytes. Localization of singular, nonaggregated platelets occurs in lungs of patients with asthma. In allergic mice, platelet recruitment occurs via recognized vascular adhesive and migratory events, independently of leukocytes via a CCR3-dependent mechanism.
Assuntos
Asma/imunologia , Plaquetas/imunologia , Hiper-Reatividade Brônquica/imunologia , Pulmão/imunologia , Ativação Plaquetária/imunologia , Receptores CCR3/imunologia , Adolescente , Adulto , Idoso , Alérgenos/administração & dosagem , Animais , Antígenos de Dermatophagoides/administração & dosagem , Proteínas de Artrópodes/administração & dosagem , Asma/genética , Asma/mortalidade , Asma/patologia , Plaquetas/efeitos dos fármacos , Hiper-Reatividade Brônquica/induzido quimicamente , Hiper-Reatividade Brônquica/genética , Hiper-Reatividade Brônquica/patologia , Criança , Cisteína Endopeptidases/administração & dosagem , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Ativação Plaquetária/efeitos dos fármacos , Pyroglyphidae/química , Pyroglyphidae/imunologia , Receptores CCR3/genética , Receptores CCR4/genética , Receptores CCR4/imunologia , Receptores CXCR4/genética , Receptores CXCR4/imunologia , Transdução de Sinais , Análise de SobrevidaRESUMO
Helminth infections are accompanied by eosinophilia in parasitized tissues. Eosinophils are effectors of immunity to tissue helminths. We previously reported that in the context of experimental filarial nematode infection, optimum tissue eosinophil recruitment was coordinated by local macrophage populations following IL-4R-dependent in situ proliferation and alternative activation. However, in the current study, we identify that control of chronic adult filarial worm infection is evident in IL-4Rα-deficient (IL-4Rα-/-) mice, whereby the majority of infections do not achieve patency. An associated residual eosinophilia was apparent in infected IL-4Rα-/- mice. By treating IL-4Rα-/- mice serially with anti-CCR3 Ab or introducing a compound deficiency in CCR3 within IL-4Rα-/- mice, residual eosinophilia was ablated, and susceptibility to chronic adult Brugia malayi infection was established, promoting a functional role for CCR3-dependent eosinophil influx in immune control in the absence of IL-4/IL-13-dependent immune mechanisms. We investigated additional cytokine signals involved in residual eosinophilia in the absence IL-4Rα signaling and defined that IL-4Rα-/-/IL-5-/- double-knockout mice displayed significant eosinophil deficiency compared with IL-4Rα-/- mice and were susceptible to chronic fecund adult filarial infections. Contrastingly, there was no evidence that either IL-4R-dependent or IL-4R-independent/CCR3/IL-5-dependent immunity influenced B. malayi microfilarial loads in the blood. Our data demonstrate multiplicity of Th2-cytokine control of eosinophil tissue recruitment during chronic filarial infection and that IL-4R-independent/IL-5- and CCR3-dependent pathways are sufficient to control filarial adult infection via an eosinophil-dependent effector response prior to patency.
Assuntos
Brugia Malayi/imunologia , Eosinófilos/imunologia , Filariose/imunologia , Receptores de Superfície Celular/imunologia , Células Th2/imunologia , Animais , Eosinófilos/patologia , Filariose/genética , Filariose/patologia , Gerbillinae , Interleucina-13/genética , Interleucina-13/imunologia , Interleucina-4/genética , Interleucina-4/imunologia , Interleucina-5/genética , Interleucina-5/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptores CCR3/genética , Receptores CCR3/imunologia , Receptores de Superfície Celular/genética , Células Th2/patologiaRESUMO
The CC chemokine receptor 3 (CCR3) expressed by eosinophils, mast cells and Th2 cells is closely related to allergic diseases. The objective of this study was to explore whether silencing of CCR3 with short hairpin RNAs (shRNAs) delivered by a lentiviral vector could impact the function of mast cells in a murine model of allergic rhinitis (AR) in vivo. The murine model of allergic rhinitis (AR) inducing by ovalbumin (OVA) was constructed, and the BALB/c mice were divided into normal control group, AR group, controlshRNA treated group and lentiviral CCR3-shRNA treated group. The recombinant lentivirus vectors which express a short hairpin RNA (shRNA) targeting the CCR3 were dropped into the nasal cavity of OVA-sensitized mice before the challenges. Real-time fluorescence quantitative PCR and western blotting were performed to observe inhibitory effect of CCR3 gene. Nasal symptoms of mice and OVA-specific IgE in each group were assessed. Concentrations of histamine, tryptase and Prostaglandin D2 (PGD2) in bone marrow, peripheral blood and nasal mucosa were analyzed. Furthermore, histological analysis and electron microscopy analysis were applied to detect the histology changes of nasal mucosa and the infiltration of mast cells in nasal mucosa. The results showed that administration of CCR3shRNA could effectively inhibit the expression of the CCR3 gene in bone marrow, peripheral blood and nasal mucosa, which reduced the nasal symptoms, the level of OVA-specific IgE, the inflammatory cells and mast cells infiltration into nasal cavity, and relieved the histopathological changes of nasal mucosa. In addition, intervention of CCR3shRNA could reduce the levels of the histamine, tryptase and PGD2 in bone marrow, peripheral blood and nasal mucosa. These results suggest that inhibition of CCR3 gene expression by shRNAs lentiviral vectors can effectively attenuate migration, infiltration and degranulation of mast cells in local tissues and alleviate the inflammation of allergic rhinitis mice.
Assuntos
Mastócitos/imunologia , Mucosa Nasal/imunologia , Receptores CCR3/genética , Rinite Alérgica/terapia , Adjuvantes Imunológicos/administração & dosagem , Hidróxido de Alumínio/administração & dosagem , Hidróxido de Alumínio/imunologia , Animais , Medula Óssea/patologia , Degranulação Celular/genética , Degranulação Celular/imunologia , Movimento Celular/genética , Movimento Celular/imunologia , Modelos Animais de Doenças , Humanos , Lentivirus/genética , Masculino , Mastócitos/metabolismo , Camundongos , Microscopia Eletrônica , Mucosa Nasal/patologia , Mucosa Nasal/ultraestrutura , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores CCR3/imunologia , Receptores CCR3/metabolismo , Rinite Alérgica/imunologia , Rinite Alérgica/patologiaRESUMO
BACKGROUND: The diagnostic and prognostic role of Th-1 chemokine receptor and Th-2 chemokine receptor in patients with primary immune thrombocytopenia has not been investigated extensively so far. In this study, our goal is to explore the diagnostic and prognostic role of C-C chemokine receptor 3 (CCR3) and C-C chemokine receptor 5 (CCR5) in patients with primary immune thrombocytopenia. METHODS: The expression levels of CCR3 and CCR5 were measured in peripheral blood mononuclear cells of pa-tients with primary immune thrombocytopenia and healthy subjects. The relationship between the expression levels of CCR3 and CCR5 and clinicopathological characteristics was analyzed. The diagnostic accuracy of CCR3 and CCR5 as biomarkers to discriminate primary immune thrombocytopenia patients from healthy subjects was determined. Univariate and multivariate Cox regression analysis were performed to determine the prognosis value of CCR3 and CCR5 in primary immune thrombocytopenia. The outcome of primary immune thrombocytopenia patients was also evaluated. RESULTS: Compared to healthy subjects, the expression level of CCR3 was significantly downregulated and CCR5 was significantly upregulated (p < 0.05). The expression levels of CCR3 and CCR5 were significantly correlated with bleeding times and platelet counts at diagnosis (p < 0.05). CCR3 and CCR5 could act as a suitable biomarker for differentiating the primary immune thrombocytopenia patients from healthy subjects. CCR3 and CCR5 were independent prognostic factors. Overexpression of CCR5 and low expression of CCR3 lead to poor clinical benefits and indicated poor prognosis of primary immune thrombocytopenia. CONCLUSIONS: To summarize, our results suggested that CCR3 and CCR5 could act as suitable biomarkers and indicated poor prognosis of primary immune thrombocytopenia.
Assuntos
Púrpura Trombocitopênica Idiopática/imunologia , Receptores CCR/imunologia , Células Th1/imunologia , Células Th2/imunologia , Biomarcadores/metabolismo , Quimiocinas CC/imunologia , Quimiocinas CC/metabolismo , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Púrpura Trombocitopênica Idiopática/diagnóstico , Púrpura Trombocitopênica Idiopática/metabolismo , Receptores CCR/metabolismo , Receptores CCR3/imunologia , Receptores CCR3/metabolismo , Receptores CCR5/imunologia , Receptores CCR5/metabolismo , Células Th1/metabolismo , Células Th2/metabolismoRESUMO
Currently, we lack an understanding of the individual and combinatorial roles for chemokine receptors in the inflammatory process. We report studies on mice with a compound deletion of Ccr1, Ccr2, Ccr3, and Ccr5, which together control monocytic and eosinophilic recruitment to resting and inflamed sites. Analysis of resting tissues from these mice, and mice deficient in each individual receptor, provides clear evidence for redundant use of these receptors in establishing tissue-resident monocytic cell populations. In contrast, analysis of cellular recruitment to inflamed sites provides evidence of specificity of receptor use for distinct leukocyte subtypes and no indication of comprehensive redundancy. We find no evidence of involvement of any of these receptors in the recruitment of neutrophils or lymphocytes to resting or acutely inflamed tissues. Our data shed important light on combinatorial inflammatory chemokine receptor function and highlight Ccr2 as the primary driver of myelomonocytic cell recruitment in acutely inflamed contexts.
Assuntos
Eosinófilos/imunologia , Inflamação/imunologia , Monócitos/imunologia , Receptores CCR/imunologia , Animais , Quimiocinas/imunologia , Quimiocinas/metabolismo , Eosinófilos/metabolismo , Perfilação da Expressão Gênica/métodos , Inflamação/genética , Inflamação/metabolismo , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Receptores CCR/genética , Receptores CCR/metabolismo , Receptores CCR1/imunologia , Receptores CCR1/metabolismo , Receptores CCR2/imunologia , Receptores CCR2/metabolismo , Receptores CCR3/imunologia , Receptores CCR3/metabolismo , Receptores CCR5/imunologia , Receptores CCR5/metabolismoRESUMO
Leptin is a cytokine, produced mainly by mature adipocytes, that regulates the central nervous system, mainly to suppress appetite and stimulate energy expenditure. Leptin also regulates the immune response by controlling activation of immunomodulatory cells, including eosinophils. While emerging as immune regulatory cells with roles in adipose tissue homeostasis, eosinophils have a well-established ability to synthesize pro-inflammatory molecules such as lipid mediators, a key event in several inflammatory pathologies. Here, we investigated the impact and mechanisms involved in leptin-driven activation of eicosanoid-synthesizing machinery within eosinophils. Direct in vitro activation of human or mouse eosinophils with leptin elicited synthesis of lipoxygenase as well as cyclooxygenase products. Displaying selectivity, leptin triggered synthesis of LTC4 and PGD2, but not PGE2, in parallel to dose-dependent induction of lipid body/lipid droplets biogenesis. While dependent on PI3K activation, leptin-driven eosinophil activation was also sensitive to pertussis toxin, indicating the involvement of G-protein coupled receptors on leptin effects. Leptin-induced lipid body-driven LTC4 synthesis appeared to be mediated through autocrine activation of G-coupled CCR3 receptors by eosinophil-derived CCL5, inasmuch as leptin was able to trigger rapid CCL5 secretion, and neutralizing anti-RANTES or anti-CCR3 antibodies blocked lipid body assembly and LTC4 synthesis induced by leptin. Remarkably, autocrine activation of PGD2 G-coupled receptors DP1 and DP2 also contributes to leptin-elicited lipid body-driven LTC4 synthesis by eosinophils in a PGD2-dependent fashion. Blockade of leptin-induced PGD2 autocrine/paracrine activity by a specific synthesis inhibitor or DP1 and DP2 receptor antagonists, inhibited both lipid body biogenesis and LTC4 synthesis induced by leptin stimulation within eosinophils. In addition, CCL5-driven CCR3 activation appears to precede PGD2 receptor activation within eosinophils, since neutralizing anti-CCL5 or anti-CCR3 antibodies inhibited leptin-induced PGD2 secretion, while it failed to alter PGD2-induced LTC4 synthesis. Altogether, sequential activation of CCR3 and then PGD2 receptors by autocrine ligands in response to leptin stimulation of eosinophils culminates with eosinophil activation, characterized here by assembly of lipidic cytoplasmic platforms synthesis and secretion of the pleiotropic lipid mediators, PGD2, and LTC4.
Assuntos
Eosinófilos/imunologia , Leptina/metabolismo , Leucotrieno C4/biossíntese , Receptores CCR3/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Animais , Células Cultivadas , Quimiocina CCL5/antagonistas & inibidores , Quimiocina CCL5/metabolismo , Eosinófilos/citologia , Eosinófilos/efeitos dos fármacos , Eosinófilos/metabolismo , Feminino , Humanos , Hidantoínas/farmacologia , Oxirredutases Intramoleculares/antagonistas & inibidores , Oxirredutases Intramoleculares/metabolismo , Leptina/imunologia , Leucotrieno C4/imunologia , Gotículas Lipídicas/imunologia , Gotículas Lipídicas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Piperidinas/farmacologia , Cultura Primária de Células , Prostaglandina D2/metabolismo , Receptores CCR3/antagonistas & inibidores , Receptores CCR3/imunologia , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/imunologia , Receptores de Prostaglandina/antagonistas & inibidores , Receptores de Prostaglandina/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
Asparaginyl-tRNA synthetase (NRS) is not only essential in protein translation but also associated with autoimmune diseases. Particularly, patients with antibodies that recognize NRS often develop interstitial lung disease (ILD). However, the underlying mechanism of how NRS is recognized by immune cells and provokes inflammatory responses is not well-understood. Here, we found that the crystal structure of the unique N-terminal extension domain of human NRS (named as UNE-N, where -N denotes NRS) resembles that of the chemotactic N-terminal domain of NRS from a filarial nematode, Brugia malayi, which recruits and activates specific immune cells by interacting with CXC chemokine receptor 1 and 2. UNE-N induced migration of CC chemokine receptor 3 (CCR3)-expressing cells. The chemokine activity of UNE-N was significantly reduced by suppressing CCR3 expression with CCR3-targeting siRNA, and the loop3 region of UNE-N was shown to interact mainly with the extracellular domains of CCR3 in nuclear magnetic resonance perturbation experiments. Based on these results, evolutionarily acquired UNE-N elicits chemokine activities that would promote NRS-CCR3-mediated proinflammatory signaling in ILD.
Assuntos
Aspartato-tRNA Ligase/química , Inflamação/genética , Doenças Pulmonares Intersticiais/genética , Aminoacil-RNA de Transferência/química , Receptores CCR3/química , Animais , Aspartato-tRNA Ligase/genética , Aspartato-tRNA Ligase/imunologia , Brugia Malayi/química , Brugia Malayi/patogenicidade , Quimiocinas/química , Quimiocinas/genética , Quimiocinas/imunologia , Cristalografia por Raios X , Humanos , Inflamação/imunologia , Inflamação/patologia , Doenças Pulmonares Intersticiais/imunologia , Doenças Pulmonares Intersticiais/patologia , Domínios Proteicos , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/imunologia , Receptores CCR3/genética , Receptores CCR3/imunologiaRESUMO
The present study aimed to investigate the effects of CC chemokine receptor type 3 (CCR3) gene knockout on allergic rhinitis (AR) in mice, as well as the underlying molecular mechanisms. Ovalbumin was administrated to CCR3+/+ and CCR3/ BALB/c mice to establish an AR model. The mice were divided into four groups: i) Normal control (CG), ii) AR model (AR), iii) CCR3 knockout CG (CCR3/CG) and iv) AR model with CCR3 knockout (CCR3/AR). Histological sections of nasal mucosae were examined by hematoxylin and eosin staining, which revealed that CCR3 knockout suppressed the invasion of inflammatory cells and relieved the damage of nasal mucosae. Peripheral blood smear and nasalwashing smears were evaluated by Wright's staining. Eosinophil (EOS) numbers in nasal mucosae, peripheral blood, and nasal washings of the various groups were ranked in the order: AR>CCR3/AR>CG>CCR3/. mRNA expression levels of CCR3, EOS peroxidase (EPO), EOS cationic protein (ECP), and major basic protein (MBP) in the peripheral serum and nasal washings were detected by reverse transcriptionpolymerase chain reaction. Interferonγ (IFNγ), interleukin (IL)4, IL10, and immunoglobulin E (IgE) protein levels in the peripheral serum and nasal washings were investigated by ELISA. CCR3 mRNA expression was not detected in the CCR3/ and CCR3/AR groups, whereas expression levels in the AR group were markedly higher compared with expression in the CG group. Compared with the CGassociated groups (i.e., the CG and CCR3/CG groups), the levels of EPO, ECP, MBP, IL4, and IgE were significantly increased in the ARassociated groups (that is, R and CCR3/AR). In addition, the CCR3/AR group mice produced significantly lower levels of EPO, ECP, MBP, IL4 and IgE compared with the AR group, whereas the expression levels of IFNγ and IL10 were increased. CCR3 gene knockout may alleviate EOS invasion and the inflammatory response in AR model mice by reducing the expression levels of EPO, ECP, MBP, IL4, and IgE, and increasing the expression of IL10 and IFNγ.
Assuntos
Proteínas Granulares de Eosinófilos/imunologia , Fatores Imunológicos/imunologia , Inflamação/imunologia , Receptores CCR3/imunologia , Rinite Alérgica/imunologia , Animais , Proteínas Granulares de Eosinófilos/genética , Feminino , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Fatores Imunológicos/genética , Inflamação/genética , Inflamação/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Knockout , Mucosa Nasal/imunologia , Mucosa Nasal/metabolismo , Mucosa Nasal/patologia , Receptores CCR3/genética , Rinite Alérgica/genética , Rinite Alérgica/patologiaRESUMO
Inflammation can occur via different mechanisms, such as via acute and chronic responses, on numerous occasions and function accordingly through various roles. There are more than five subsets of neutrophils; neutrophilic heterogeneity is modulated by the inflammatory condition. To understand the characteristics of inflammation, identification of atypical neutrophils is important. In this study, we found that the expression of eotaxin receptor (CD193) on atypical neutrophils in the duodenum is augmented in IL-21 isoform transgenic (Tg) mice. In a series of studies, we have established a Tg mouse strain to further investigate the functions of IL-21 in vivo. Interestingly, Tg mice immunized with ovalbumin (OVA) were more sensitive to OVA-induced systemic anaphylaxis as compared with wild type mice with duodenal and splenic gross congestion. Further analysis conducted in the duodenum of Tg mice revealed that only the number of neutrophils migrating into the duodenum was significantly increased prior to immunization. Previous studies have shown that the gastrointestinal compartment and the spleen constantly produce eotaxin, which regulates basal levels of tissue eosinophils. Therefore, we analyzed CD193 expression on neutrophils and eosinophils. As expected, its expression by duodenal neutrophils was upregulated in Tg mice. Furthermore, the addition of IL-21 into bone marrow cell culture increased the number of CD193+ neutrophils, which easily migrated into the duodenum. These observations suggested that CD193+ neutrophils increase in number under inflammatory conditions due to chronic IL-21 production.
Assuntos
Duodeno/imunologia , Inflamação/imunologia , Interleucinas/imunologia , Neutrófilos/imunologia , Receptores CCR3/imunologia , Animais , Medula Óssea/imunologia , Células Cultivadas , Eosinófilos/imunologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina/imunologia , Isoformas de Proteínas/imunologia , Baço/imunologia , Regulação para Cima/imunologiaRESUMO
Eotaxins are C-C motif chemokines first identified as potent eosinophil chemoattractants. They facilitate eosinophil recruitment to sites of inflammation in response to parasitic infections as well as allergic and autoimmune diseases such as asthma, atopic dermatitis, and inflammatory bowel disease. The eotaxin family currently includes three members: eotaxin-1 (CCL11), eotaxin-2 (CCL24), and eotaxin-3 (CCL26). Despite having only ~30% sequence homology to one another, each was identified based on its ability to bind the chemokine receptor, CCR3. Beyond their role in innate immunity, recent studies have shown that CCL11 and related molecules may directly contribute to degenerative processes in the central nervous system (CNS). CCL11 levels increase in the plasma and cerebrospinal fluid of both mice and humans as part of normal aging. In mice, these increases are associated with declining neurogenesis and impaired cognition and memory. In humans, elevated plasma levels of CCL11 have been observed in Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, and secondary progressive multiple sclerosis when compared to age-matched, healthy controls. Since CCL11 is capable of crossing the blood-brain barrier of normal mice, it is plausible that eotaxins generated in the periphery may exert physiological and pathological actions in the CNS. Here, we briefly review known functions of eotaxin family members during innate immunity, and then focus on whether and how these molecules might participate in the progression of neurodegenerative diseases.
Assuntos
Quimiocina CCL11/imunologia , Quimiocina CCL24/imunologia , Quimiocina CCL26/imunologia , Imunidade Inata/imunologia , Doenças Neurodegenerativas/imunologia , Envelhecimento/imunologia , Animais , Quimiocina CCL11/sangue , Quimiocina CCL11/líquido cefalorraquidiano , Quimiocina CCL24/sangue , Quimiocina CCL24/líquido cefalorraquidiano , Quimiocina CCL26/sangue , Quimiocina CCL26/líquido cefalorraquidiano , Humanos , Doenças Neurodegenerativas/sangue , Doenças Neurodegenerativas/líquido cefalorraquidiano , Receptores CCR3/imunologia , Receptores CCR3/metabolismoRESUMO
Flow cytometry protocols designed to identify mouse eosinophils typically target Siglec F, an α-2,3-sialic acid binding transmembrane protein expressed universally on cells of this lineage. While a convenient target, antibody-mediated ligation of Siglec F induces eosinophil apoptosis, which limits its usefulness for isolations that are to be followed by functional and/or gene expression studies. We present here a method for FACS isolation which does not target Siglec F and likewise utilizes no antibodies targeting IL5Rα (CD125) or CCR3. Single cell suspensions are prepared from lungs of mice that were sensitized and challenged with Aspergillus fumigatus antigens; eosinophils were identified and isolated by FACS as live SSChi/FSChi CD11c-Gr1-/loMHCII- cells. This strategy was also effective for eosinophil isolation from the lungs of IL5tg mice. Purity by visual inspection of stained cytospin preparations and by Siglec F-diagnostic flow cytometry was 98-99% and 97-99%, respectively. Eosinophils isolated by this method (yield, ~4×106/mouse) generated high-quality RNA suitable for gene expression analysis.
Assuntos
Anticorpos/química , Eosinófilos/citologia , Citometria de Fluxo/métodos , Pulmão/química , Animais , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/imunologia , Eosinófilos/imunologia , Subunidade alfa de Receptor de Interleucina-5/genética , Subunidade alfa de Receptor de Interleucina-5/imunologia , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Receptores CCR3/genética , Receptores CCR3/imunologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido SiálicoRESUMO
Pyoderma gangrenosum (PG) is a rare, immune-mediated skin disease classified into the group of neutrophilic dermatoses. Although a number of studies confirmed the central role of innate immunity, only few studies have investigated the possible contributing role of acquired immunity. In particular, no reports concerning T helper type 1 (Th1) and Th2 cells are available as yet. Therefore, 15 patients with PG, five with Sweet's syndrome (SS) and nine skin specimens from healthy controls (HC) were investigated, evaluating the expression of Th1-related markers interleukin (IL)-12, interferon (IFN)-γ, C-X-C motif chemokine receptor 3 (CXCR3) and C-C motif chemokine receptor 5 (CCR5), of the Th2-related molecules IL-4, IL-5, IL-13 and CCR3, of the co-stimulatory axis CD40/CD40 ligand, of IL-15 and the natural killer (NK) cell marker CD56 in skin lesions by immunohistochemistry. Patients with PG and SS showed a higher expression of Th1 markers than HC. Conversely, IL-5- and CCR3-expressing cells were less numerous in PG skin lesions compared to SS (P = 0·0157 and < 0·0001, respectively). Both CD40 and CD40L were expressed more in PG than in SS and HC (P < 0·0001 for both). Finally, the number of IL-15+ and CD56+ cells was higher in the skin of patients with PG than in those of SS and HC (P < 0·0001 for both). Our results suggest that Th2 cells are down-regulated in PG. At the same time, over-expression of the co-stimulatory axis CD40/CD40L amplifies the impairment of the Th1/Th2 balance. Both these findings might explain the most aggressive behaviour of PG in comparison to SS. Moreover, over-expression of IL-15+ and CD56+ cells may suggest a possible role of NK cells in the pathogenesis of the disease.
Assuntos
Interleucina-15/genética , Pioderma Gangrenoso/imunologia , Pele/imunologia , Células Th1/imunologia , Células Th2/imunologia , Adulto , Idoso , Ligante de CD40/genética , Ligante de CD40/imunologia , Antígeno CD56/genética , Antígeno CD56/imunologia , Feminino , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-13/genética , Interleucina-13/imunologia , Interleucina-15/imunologia , Interleucina-4/genética , Interleucina-4/imunologia , Interleucina-5/genética , Interleucina-5/imunologia , Masculino , Pessoa de Meia-Idade , Pioderma Gangrenoso/fisiopatologia , Receptores CCR3/genética , Receptores CCR3/imunologia , Receptores CCR5/genética , Receptores CCR5/imunologia , Receptores CXCR3/genética , Receptores CXCR3/imunologia , Síndrome de Sweet/imunologia , Síndrome de Sweet/fisiopatologia , Equilíbrio Th1-Th2RESUMO
BACKGROUND AND OBJECTIVE: Acute eosinophilic pneumonia (AEP) is characterized by a massive pulmonary infiltration of eosinophils. Mechanisms regulating the selective accumulation of eosinophils in AEP have not been fully established. The objective of this study was to evaluate the mechanisms of eosinophil accumulation in alveolar spaces through examination of bronchoalveolar lavage fluid (BALF) from AEP patients (AEP-BALF). METHODS: Eosinophils were isolated from the blood of healthy subjects and were placed on a human pulmonary microvascular endothelial cell monolayer cultured on Transwell filters (Coster, Cambridge, MA, USA). A saline control solution or BALF from patients with AEP, sarcoidosis or hypersensitivity pneumonitis was applied to the lower compartment, and the transendothelial migration of the eosinophils was evaluated. The concentrations of cytokines and chemokines in BALF were also measured. RESULTS: Transmigration of eosinophils across endothelial cells was only induced by the AEP-BALF. This transmigration was blocked by anti-ß2 integrin mAb. The concentrations of eotaxin-2 and monocyte chemotactic protein (MCP)-4, which are CC chemokine receptor (CCR) 3 ligands, were elevated in the AEP-BALF, and anti-CCR3 mAb or anti-MCP-4 mAb inhibited the AEP-BALF-induced transmigration of eosinophils. Furthermore, the concentration of leukotriene (LT) B4 was increased in the AEP-BALF, and an LTB4 receptor antagonist partially suppressed the AEP-BALF-induced transmigration of eosinophils. CONCLUSION: These findings suggest that CCR3 ligands including eotaxin-2 and MCP-4, and LTB4 play a role in the accumulation of eosinophils in AEP.
Assuntos
Citocinas/imunologia , Eosinófilos/imunologia , Eosinofilia Pulmonar/imunologia , Migração Transendotelial e Transepitelial/imunologia , Adolescente , Adulto , Idoso , Alveolite Alérgica Extrínseca/imunologia , Líquido da Lavagem Broncoalveolar , Estudos de Casos e Controles , Quimiocina CCL24/imunologia , Quimiocinas/imunologia , Feminino , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Proteínas Quimioatraentes de Monócitos/imunologia , Receptores CCR3/imunologia , Sarcoidose Pulmonar/imunologia , Adulto JovemRESUMO
IL-5-induced chemotaxis of eosinophils is an important feature of allergic airway inflammatory diseases. Simvastatin, a lipid lowering agent, has been shown to exhibit anti-inflammatory and anti-allergic effects. Our aim was to investigate the effect of simvastatin on IL-5-induced eosinophil chemotaxis and its regulatory mechanisms. Eosinophils were derived by treating HL-60 clone 15 (HC15) cells with butyric acid (BA) in an alkaline condition or through direct isolation from human peripheral blood. The expressions of CC chemokine receptor 3 (CCR3) and interleukin (IL)-5 receptors (IL5Rα and ß) were analyzed using RT/real-time PCR. The granular proteins were stained using fast green. Eotaxin-induced chemotaxis was measured using a transwell migration assay. CCR3 protein expression was revealed by immunocytochemistry. An animal model of allergic rhinitis was established by challenging Sprague-Dawley® rats repeatedly with ovalbumin. Butyric acid significantly increased the expression of IL5Rα and IL5Rß, CCR3 and granular proteins in HC15 cells, indicating the maturation of eosinophils (BA-E cells). IL-5 further enhanced the CCR3 expression at both the mRNA and protein levels and the eotaxin-induced chemotaxis of BA-E cells. Simvastatin inhibited the effects of IL-5 on BA-E cells, but not in the presence of mevalonate. Similar results were also exhibited in human primary eosinophils. In vivo animal studies further confirmed that oral simvastatin could significantly suppress the infiltration of eosinophils into turbinate tissues of allergic rats. Therefore, simvastatin was demonstrated to inhibit IL-5-induced CCR3 expression and chemotaxis of eosinophils mediated via the mevalonate pathway. We confirmed that simvastatin also reduced eosinophilic infiltration in allergic rhinitis.
Assuntos
Quimiotaxia/efeitos dos fármacos , Eosinófilos/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-5/imunologia , Receptores CCR3/imunologia , Sinvastatina/farmacologia , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica/imunologia , Células HL-60 , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Rinite Alérgica/induzido quimicamente , Rinite Alérgica/imunologia , Rinite Alérgica/patologia , Sinvastatina/efeitos adversosRESUMO
BACKGROUND: Flowcytometric identification of basophils is a prerequisite for measuring activation of basophils with IgE-dependent or IgE-independent stimuli. Aim of this study was to compare different marker combinations in a simultaneous multicolor flowcytometric measurement. METHODS: Ten patients with a grass pollen allergy and three controls were included in the study. Basophilic cells were gated by using anti-CCR3, anti-IgE, anti-CRTH2, anti-CD203c, and anti-CD3. Cells were activated by a monoclonal anti-FcεRI antibody, N-formyl-methionyl-leucyl-phenylalanine (fMLP), and the allergen extract Phleum pratense. The activation marker anti-CD63 was used. RESULTS: The highest relative number of basophils was found with anti-CCR3+ cells, anti-IgE+ and anti-IgE+ /anti-CD203c+ cells, the lowest with CRTH2+/CD203c+/CD3- cells. A very good and good concordance of CCR3+ cells was seen with CCR3+/CD3- cells and CRTH2+/CD203c+/CD3- cells in all experiments. The contamination of the CCR3+ population with CD3+ cells and the contamination of the IgE+-population with CCR3- cells and CD203- cells were the lowest compared to all other marker combinations. CONCLUSIONS: As the highest relative number of basophils was identified by anti-CCR3 followed by the anti-IgE and anti-IgE/antiCD203c positive population in most cases, these markers can generally be recommended for identification of basophils. If a basophil population with very high purity is needed, anti-IgE should be chosen.
Assuntos
Anticorpos Anti-Idiotípicos/química , Basófilos/imunologia , Imunoglobulina E/sangue , Imunofenotipagem/métodos , Hipersensibilidade Respiratória/diagnóstico , Adulto , Alérgenos/química , Alérgenos/imunologia , Anticorpos Monoclonais/farmacologia , Teste de Degranulação de Basófilos , Basófilos/efeitos dos fármacos , Basófilos/patologia , Complexo CD3/genética , Complexo CD3/imunologia , Estudos de Casos e Controles , Células Cultivadas , Feminino , Citometria de Fluxo/métodos , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/imunologia , Extratos Vegetais/química , Extratos Vegetais/imunologia , Extratos Vegetais/farmacologia , Pólen/química , Pólen/imunologia , Pirofosfatases/genética , Pirofosfatases/imunologia , Receptores CCR3/genética , Receptores CCR3/imunologia , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/imunologia , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/patologia , Tetraspanina 30/genética , Tetraspanina 30/imunologiaRESUMO
Murine zymosan-induced peritonitis is the model most frequently used to study resolution of inflammation. However, the antigen-induced peritonitis model may be better suited for studying resolution of inflammation and the adaptive phase that follows. The objective of this study was to provide an evaluation of the kinetics of cells and mediators during induction, resolution and the adaptive immune phases of a murine antigen-induced inflammation. Female C57BL/6 mice were immunized twice subcutaneously with mBSA and three weeks after the initial immunization they were injected intraperitoneally (i.p.) with mBSA, which induced peritonitis. Peritoneal cells were counted and expression of surface molecules and chemokine receptors analyzed with flow cytometry. Chemokine and cytokine concentrations in peritoneal fluid were determined by ELISA. Two neutrophil populations, differing in size and granularity and slightly in expression of surface molecules, were observed in the peritoneal cavity after induction of inflammation. Macrophages disappeared from the peritoneal cavity following i.p. administration of mBSA but appeared again as they differentiated from recruited monocytes and peaked in numbers at 48 h. At that time point, two distinct populations of macrophages were present in the peritoneal cavity; one with high expression of F4/80, also expressing the atypical chemokine receptor D6 as well as CCR7; the other expressing low levels of F4/80 and also expressing CD11c and CD138. Eosinophils appeared in the peritoneum 3h following i.p. administration of mBSA and peaked at 48 h. At that time point they had upregulated their expression of CCR3 but decreased their expression of CD11b. Peritoneal levels of CCL11 peaked at 6h and may have led to recruitment of the eosinophils. NK cells and T cells peaked at 48 h, whereas B cells peaked at 5 days, with the majority being B1 cells. Peritoneal concentrations of pro-inflammatory cytokines (IL-ß and IL-6) and chemokines (CCL2 and CCL3) peaked at 3h, whereas IL-1ra peaked at 6h, sTNF-R at 24h and sIL-6R and TGF-ß at 48 h. The results show kinetic alterations in cell populations and mediators in a murine model that may be an excellent model to study initiation and resolution of inflammation and the following adaptive phase.
Assuntos
Citocinas/biossíntese , Macrófagos Peritoneais/imunologia , Neutrófilos/imunologia , Peritonite/imunologia , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Linfócitos B/imunologia , Linfócitos B/patologia , Antígeno CD11c/genética , Antígeno CD11c/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Eosinófilos/imunologia , Eosinófilos/patologia , Feminino , Expressão Gênica , Injeções Intraperitoneais , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/patologia , Cavidade Peritoneal/patologia , Peritonite/induzido quimicamente , Peritonite/genética , Peritonite/patologia , Receptores CCR3/genética , Receptores CCR3/imunologia , Soroalbumina Bovina , Sindecana-1/genética , Sindecana-1/imunologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
The innate immune system can recognize non-self through pattern recognition receptors and provides a first line of antimicrobial host defense. Thus innate immunity plays a very important role in resistance against major bacterial disease in vertebrates. In the innate immune responses, the chemokine receptors act as the main receptors of chemokines which are released at the sites of infection, inflammation and injury. In this study, the Miichthys miiuy CCR3 and CCR9 genes were cloned and characterized, showing that MIMI-CCR3 possesses a highly conserved DRYLA motif similar to that of other fishes. MIMI-CCR3 and CCR9 were ubiquitously expressed in all tested tissues and the expressions were significantly up-regulated after infection with Vibrio anguillarum except that of CCR9 in spleen. Evolutionary analysis showed that all the ancestral lineages of CCR3 and CCR9 in mammals and teleosts underwent positive selection, indicating that the ancestor of terrestrial animals further evolved to adapt to terrestrial environments and the continuous intrusion of microbes stimulated the evolution of CCR genes in the ancestor of teleost. Multiple ML methods were used to detect the robust candidates for sites under positive selection. In total, 11 and 8 positively selected sites were found in the subsets of current mammal and teleost CCR3 genes, and 3 and 2 sites were detected in the subsets of current mammals and teleosts in CCR9. Interestingly, for mammal CCR3 and CCR9 genes, the robust candidates of positively selected sites were mainly located in the extracellular domains which were the ligand binding and pathogen interaction regions. For teleost CCR3 and CCR9 genes, the positively selected sites were not only located in the extracellular domains but also in the C-terminal and intracellular domains, indicating mammals and teleosts experienced different selection pressures upon their N-terminus, C-terminus and intracellular loops of CCRs.
Assuntos
Perciformes/genética , Receptores CCR3/genética , Receptores CCR/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular/métodos , Peixes , Expressão Gênica , Mamíferos , Dados de Sequência Molecular , Perciformes/imunologia , Filogenia , Estrutura Terciária de Proteína , Receptores CCR/imunologia , Receptores CCR3/imunologia , Seleção Genética , Alinhamento de Sequência , Vibrio/imunologia , Vibrioses/imunologiaRESUMO
BACKGROUND: Eosinophils have the capacity to secrete varied cytotoxic proteins. Among the proteins are the eosinophil-associated RNases (EARs): the human eosinophil-derived neurotoxin and eosinophilic cationic protein, and their murine ortholog EARs, which have been shown to be involved in host defense, tissue remodeling, and immunity regulation. However, the signal transduction that regulates EARs secretion in response to physiological stimuli, such as chemokines, has been little studied in human and scarcely in mouse eosinophils, the foremost animal model for eosinophil-associated human diseases. OBJECTIVE: In this study, we aimed to understand the signal transduction involved in the secretion of enzymatically active EARs following chemokine stimulation. METHODS: Fresh mouse and human eosinophils were stimulated with CCL11 and CCL24, and the secretion of enzymatically active EARs was detected using an RNase activity assay. The involvement of signaling factors or integrins was probed using specific inhibitors and blocking antibodies. Adhesion was evaluated by microscopy. RESULTS: We found that secretion of mouse EARs in response to CCL11 and CCL24 was Gαi -dependent. Both mouse and human eosinophils required the activation of PI3K, ERK, and p38 MAPK. In addition, the adhesion molecules ß1 and ß2 integrins were found to be crucial for EAR secretion, and we suggest a mechanism in which spreading is obligatory for EAR secretion. CONCLUSIONS: Collectively, these data suggest a common CCR3-mediated signaling pathway that leads to EAR secretion in both mouse and human eosinophils. These findings are applicable for eosinophil-mediated host defense and eosinophil-associated diseases.
Assuntos
Eosinófilos/imunologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Integrinas/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Ribonucleases/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Animais , Western Blotting , Células Cultivadas , Quimiocina CCL11/imunologia , Quimiocina CCL11/metabolismo , Eosinófilos/citologia , Eosinófilos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Citometria de Fluxo , Humanos , Integrinas/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Receptores CCR3/imunologia , Receptores CCR3/metabolismo , Ribonucleases/imunologia , Sensibilidade e Especificidade , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Bone marrow (BM) eosinopoiesis is a common feature during allergen exposure in atopic individuals. Airway exposure to staphylococcal superantigens aggravates allergic airway disease and increases the output of BM eosinophils. However, the exact mechanisms regulating eosinophil mobilization and trafficking to the peripheral circulation and airways remain to be elucidated. Therefore, this study aimed to investigate the mechanisms determining the BM eosinopoiesis in allergic mice under exposure to staphylococcal enterotoxin A (SEA). Ovalbumin (OVA)-sensitized male BALB/C mice were intranasally exposed to SEA (1 µg), and at 4, 12, 24, and 48 h later animals were challenged with OVA (10 µg, twice a day). Measurement of IL-5, eotaxin, and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels, flow cytometry for CCR3(+), VLA4(+), and CCR3(+)VLA4(+), as well as adhesion assays to VCAM-1 were performed in BM. Prior airway exposure to SEA time dependently increased the BM eosinophil number in OVA-challenged mice. Eosinophils gradually disappear from peripheral blood, being recruited over time to the airways, where they achieve a maximal infiltration at 24 h. SEA exposure increased the levels of IL-5 and eotaxin (but not GM-CSF) in BM of OVA-challenged mice. Marked increases in CCR3(+) and CCR3(+)VLA4(+) expressions in BM eosinophils of OVA-challenged mice were observed, an effect largely reduced by prior exposure to SEA. Adhesion of BM eosinophils to VCAM-1 was increased in OVA-challenged mice, but prior SEA exposure abrogated this enhanced cell adhesion. Accumulation of BM eosinophils by airway SEA exposure takes place through IL-5- and CCR3-dependent mechanisms, along with downregulation of CCR3/VL4 and impaired cell adhesion to VCAM-1.