Chemokine receptor expression on human eosinophils from peripheral blood and bronchoalveolar lavage fluid after segmental antigen challenge

BACKGROUND: The recruitment of circulating eosinophils to the lung is a characteristic feature of allergic airway inflammation. Chemokine receptors likely play a role in this complex process. However, reports of chemokine receptor expression on human eosinophils are conflicting. OBJECTIVE: The aim of this study was to determine whether the chemokine receptor profile of human eosinophils change when these cells are recruited to the airway after an antigen challenge and development of an allergic inflammatory response. METHODS: Blood and bronchoalveolar lavage (BAL) cells were obtained from 13 allergic subjects 48 hours after segmental bronchoprovocation with antigen. The CC chemokine receptor (CCR) 1 to 7, 9, and CXC chemokine receptor (CXCR) 1 to 4 were determined by flow cytometric analysis of whole blood and unseparated BAL cells. RESULTS: Compared with their circulating counterparts, airway eosinophils had decreased CCR3 and increased CCR4, CCR9, and CXCR3 expression on their cell surface. Furthermore, expression of CCR3, CCR4, and CXCR3 was significantly correlated with the percentage of eosinophils in BAL fluid at 48 hours. Eosinophils also expressed CXCR4, but this receptor did not change after antigen-induced recruitment to the airway. In contrast, the expression of CCR1, CCR2, CCR5, CCR6, CCR7, CXCR1, and CXCR2 remained undetectable on either blood or BAL eosinophils. CONCLUSIONS: Our data suggest that recruitment of eosinophils to the airway is associated with a modulation of their chemokine receptor profiles. These changes in chemokine receptors could be involved in determining eosinophil function and antigen-induced airway inflammation.     

Switch in chemokine receptor expression upon TCR stimulation reveals novel homing potential for recently activated T cells.

When naive T lymphocytes are activated and differentiate into memory/effector cells, they down-regulate receptors for constitutive chemokines such as CXCR4 and CCR7 and acquire receptors for inflammatory chemokines such as CCR3, CCR5 and CXCR3, depending on the Th1/Th2 polarization. This switch in chemokine receptor usage leads to the acquisition of the capacity to migrate into inflamed tissues. Using RNase protection assays, staining with specific antibodies, and response to recombinant chemokines, we now show that following TCR stimulation, memory/effector T cells undergo a further and transient switch in receptor expression. CCR1, CCR2, CCR3, CCR5, CCR6 and CXCR3 are down-regulated within 6 h, while CCR7, CCR4, CCR8 and CXCR5 are up-regulated for 2 to 3 days. Up-regulation of CCR7 following TCR stimulation was observed also among resting peripheral blood T cells and required neither co-stimulation nor exogenous IL-2. On the other hand IL-2 down-regulated CXCR5, up-regulated CCR8 and facilitated the recovery of CCR3 and CCR5. Upon TCR stimulation, Th1 and Th2 cells produced comparable sets of chemokines, including RANTES, macrophage inflammatory protein-1beta, I-309, IL-8 and macrophage-derived chemokine, which may modulate surface chemokine receptors and contribute to cell recruitment at sites of antigenic recognition. Altogether these results show that following TCR stimulation effector/memory T cells transiently acquire responsiveness to constitutive chemokines. As a result, T cells that are activated in tissues may either recirculate to draining lymph nodes or migrate to nearby sites of organized ectopic lymphoid tissues.     

Surface expression of CC- and CXC-chemokine receptors on leucocyte subsets in inflammatory joint diseases.

Chemokine receptors play a crucial role in the recruitment of leucocyte subsets into inflamed tissue. Using FACS analysis we have studied the surface expression of different CC- and CXC-chemokine receptors on synovial fluid (SF) and peripheral blood leucocytes from 20 patients with various forms of arthritis. In the SF the majority T cells stained positive for CCR5 (93%) and CCR2 (57%), compared to the peripheral blood (36% and 25%). In addition, most of the T cells expressed CXCR4 in both compartments, with a somewhat higher percentage in the SF (90%) versus peripheral blood (83%). To date little information is available on chemokine receptor expression on monocytes in arthritis. We report a marked increase of CCR5(+) monocytes in the SF (87%) compared to the peripheral blood (22%). In contrast, the frequency of CXCR1(+), CXCR2(+), CXCR4(+) and CCR1(+) monocytes was considerably lower in the SF than in the peripheral blood. Moreover, we report the expression CXCR4 on neutrophils in the SF. Approximately 60% of neutrophils stained positive for CXCR4 in the SF, while in the peripheral blood the number of CXCR4(+) neutrophils was low (24%). Surface expression of CXCR1 and CXCR2 was significantly reduced on SF neutrophils (53% and 68%) compared to the peripheral blood. Chemokine receptors are differentially expressed on leucocyte subsets in arthritis. The identification of their pattern of expression might help to identify suitable targets for therapeutic intervention.     

Accumulation of CXCR3-expressing eosinophils and increased concentration of its ligands (IP10 and Mig) in bronchoalveolar lavage fluid of patients with chronic eosinophilic pneumonia

BACKGROUND: Since human peripheral eosinophils have been shown to migrate to the CXC chemokine receptor 3 (CXCR3) ligands IFN-gamma-inducible protein 10 (IP10) and monokine induced by IFN-gamma (Mig), this confirms that CXCR3 is functionally expressed on these cells. IP10 expression has been shown to be increased in the airways of asthmatics. Eosinophil accumulations are found in bronchoalveolar lavage fluid (BALF) from patients with chronic eosinophilic pneumonia (CEP). To examine the contribution of IP10 and Mig in the pathogenesis of CEP, we measured the concentration of IP10 and Mig, and evaluated the expression of CXCR3 on eosinophils in BALF taken from patients with CEP. METHODS: The concentrations of IP10 and Mig in BALF were measured by ELISA. The proportion of CXCR3-expressing CD4+ T cells and CD16-negative eosinophils was determined by flow cytometry. RESULTS: The BALF concentrations of IP10 and Mig were higher in patients with CEP, as well as in patients with sarcoidosis, when compared to healthy controls. The absolute number of CXCR3+ CD4+ T cells was significantly higher in the BALF of patients with sarcoidosis, but not in the patients with CEP, when compared to healthy volunteers. There were higher percentages of CXCR3-expressing eosinophils in the BALF than in the peripheral blood of patients with CEP. CONCLUSIONS: Our findings suggest that IP10 and Mig contribute to the accumulation of CXCR3-expressing eosinophils in the lungs of patients with CEP, and modulate the eosinophilic inflammation of the lung.     

Chemokines induce eosinophil degranulation through CCR-3

BACKGROUND: Such CC chemokines as eotaxin and RANTES induce preferential eosinophil recruitment in allergic inflammation. They also elicit proinflammatory effector functions of eosinophils, such as enhanced adhesion and superoxide generation. Eosinophil degranulation by chemokines, however, has not been studied in detail. OBJECTIVE: The purpose of this study was to identify chemokines and their corresponding receptors that induce eosinophil degranulation by using a panel of chemokines and blocking antibodies to candidate receptors. METHODS: Highly purified eosinophils were preloaded with Fura-2 and stimulated with a panel of chemokine ligands for 14 known chemokine receptors: CCR1 to CCR8, CXCR1 to CXCR4, CX3CR1, and XCR1. Calcium influx was measured with fluorescence spectrometry. Eosinophils were also stimulated with the chemokines in the presence or absence of IL-5, and levels of eosinophil-derived neurotoxin were measured in the supernatant with RIA. Specific antibodies to chemokine receptors were used to block degranulation. RESULTS: Calcium influx was induced by monocyte chemotactic protein (MCP) 1, MCP-3, MCP-4, RANTES, eotaxin, IL-8, and stromal cell-derived factor 1alpha, which are chemokines that bind several chemokine receptors. However, degranulation was induced only by CCR3 ligands, including MCP-3, MCP-4, RANTES, and eotaxin. Priming of eosinophils with IL-5 enhanced CCR3 ligand-induced degranulation but did not cause non-CCR3 ligands to induce eosinophil-derived neurotoxin release. An antibody against CCR3 significantly inhibited degranulation induced by CCR3 ligands, eotaxin, or RANTES. CONCLUSION: These results suggest that chemokine-induced eosinophil degranulation, a major effector of eosinophil functions, is mediated through only CCR3, although some non-CCR3 ligands induce calcium influx in eosinophils. CCR3 may be an important target in the treatment of eosinophilic inflammation.     

Expression of the chemokine receptors CCR4, CCR5, and CXCR3 by human tissue-infiltrating lymphocytes.

Differential expression of adhesion molecules and chemokine receptors has been useful for identification of peripheral blood memory lymphocyte subsets with distinct tissue and microenvironmental tropisms. Expression of CCR4 by circulating memory CD4(+) lymphocytes is associated with cutaneous and other systemic populations while expression of CCR9 is associated with a small intestine-homing subset. CCR5 and CXCR3 are also expressed by discrete memory CD4(+) populations in blood, as well as by tissue-infiltrating lymphocytes from a number of sites. To characterize the similarities and differences among tissue-infiltrating lymphocytes, and to shed light on the specialization of lymphocyte subsets that mediate inflammation and immune surveillance in particular tissues, we have examined the expression of CCR4, CXCR3, and CCR5 on CD4(+) lymphocytes directly isolated from a wide variety of normal and inflamed tissues. Extra-lymphoid tissues contained only memory lymphocytes, many of which were activated (CD69(+)). As predicted by classical studies, skin lymphocytes were enriched in CLA expression whereas intestinal lymphocytes were enriched in alpha(4)beta(7) expression. CCR4 was expressed at high levels by skin-infiltrating lymphocytes, at lower levels by lung and synovial fluid lymphocytes, but never by intestinal lymphocytes. Only the high CCR4 levels characteristic of skin lymphocytes were associated with robust chemotactic and adhesive responses to TARC, consistent with a selective role for CCR4 in skin lymphocyte homing. In contrast, CXCR3 and CCR5 were present on the majority of lymphocytes from each non-lymphoid tissue examined, suggesting that these receptors are unlikely to determine tissue specificity, but rather, may play a wider role in tissue inflammation.     

Expression and functional activity of chemokine receptors in glatiramer acetate-specific T cells isolated from multiple sclerosis patient receiving the drug glatiramer acetate

The purpose of the current study is to examine the surface expression of chemokine receptors and the chemotaxis toward the respective chemokines of glatiramer acetate (GA)-specific CD4(+) T cells isolated from the blood and the cerebrospinal fluid (CSF) of a multiple sclerosis (MS) patient. Four clones were selected, two isolated from the peripheral blood and two from the CSF. CCR4 and CXCR3 were expressed on all four clones. Both blood-derived clones also expressed CCR5 and, to a lesser extent, CCR6. Similarly, one CSF clone expressed CCR5 and CCR6. In contrast, CCR1, CCR2, CCR3, CCR7, CCR9, CCR10, CXCR1, CXCR4, CXCR5, CXCR6, and CCR6 were either expressed on few cells or were not expressed at all on all four clones examined. The expression of chemokine receptors was corroborated with the ability of the cells to respond chemotactically to the corresponding chemokines, CCL5/RANTES, CCL20/MIP-3α, CCL22/MDC and CXCL10/IP-10. Both the receptor expression and chemotaxis were reduced upon activation with PMA and ionomycin. The shared expression of chemokine receptors and the migration patterns suggest that GA-reactive cells have migrated from the blood into the CSF, and that local reactivation within the inflamed CSF may downregulate the expression of chemokine receptors and hence impede their migration intrathecally. The results may also explain the beneficial synergistic effects of combining immunosuppressive drugs with GA in MS patients.     

Rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation

Dendritic cells (DC) migrate into inflamed peripheral tissues where they capture antigens and, following maturation, to lymph nodes where they stimulate T cells. To gain insight into this process we compared chemokine receptor expression in immature and mature DC. Immature DC expressed CCR1, CCR2, CCR5 and CXCR1 and responded to their respective ligands, which are chemokines produced at inflammatory sites. Following stimulation with LPS or TNF-α maturing DC expressed high levels of CCR7 mRNA and acquired responsiveness to the CCR7 ligand EBI1 ligand chemokine (ELC), a chemokine produced in lymphoid organs. Maturation also resulted in up-regulation of CXCR4 and down-regulation of CXCR1 mRNA, while CCR1 and CCR5 mRNA were only marginally affected for up to 40 h. However, CCR1 and CCR5 were lost from the cell surface within 3 h, due to receptor down-regulation mediated by chemokines produced by maturing DC. A complete down-regulation of CCR1 and CCR5 mRNA was observed only after stimulation with CD40 ligand of DC induced to mature by LPS treatment. These different patterns of chemokine receptors are consistent with “inflammatory” and “primary response” phases of DC function.     

Granule protein changes and membrane receptor phenotype in maturing human eosinophils cultured from CD34+ progenitors

BACKGROUND: Eosinophils are now recognized as major effector cells in allergic and asthmatic disease with a potent armoury of mediators whose release makes a major contribution to the inflammation underlying these conditions. OBJECTIVES: The purpose of this study was to compare cultured eosinophils (CE) with normal-density peripheral blood eosinophils (PBE) in terms of their membrane receptor expression and to analyse the expression and storage of the eosinophil granule proteins major basic protein (MBP) and eosinophil cationic protein (ECP) during eosinophil maturation in vitro. METHODS: Purified human peripheral blood CD34+ cells were cultured in the presence of recombinant human IL-3, IL-5, rhGM-CSF, SCF, and FLT-3 ligand. PBE were isolated by density gradient centrifugation and negative immunomagnetic selection. Expression of CD11b, CD18, CD45, CD45RA, CD45RB, CD45RO, CD69, CD95, IL-5Ralpha, IL-9Ralpha, CCR1, CCR3, and CXCR4 by CE as they matured in culture were assessed by immunostaining and flow cytometry and expression of these receptors compared with freshly isolated PBE. Immunohistochemical staining and labophot-2TM light microscopy determined expression of MBP, ECP, and CD69 during eosinophil maturation. RESULTS: Positive immunostaining for MBP and ECP was detectable in a proportion (15-20%) of CE as early as 3 days of culture even though these cells were mononuclear in appearance. The numbers of CE positive for both granule proteins increased in rhIL-3 and rhIL-5 treated cells to a maximum of approximately 80% by day 28. Maturing eosinophils exhibited positive immunostaining for CD69 after 14, 21 and 28 days of culture. Compared with PBE, CE had lower expression of pan-CD45 and CD45 isoforms, CD95 and CD11b. In contrast, the specific mean fluorescence for CD69, CD18, IL-5Ralpha, and IL-9Ralpha was significantly elevated for CE compared with PBE. CCR3 expression by CE and PBE was similar with no expression of CXCR4 detected by either CE or PBE. No significant difference in expression of CCR1 was found between CE and PBE. CONCLUSION: These data suggest that CE and PBE share many phenotypic properties and both MBP and ECP appear early in eosinophil development in vitro. However, there are quantitative differences that may be a consequence of their immaturity and/or the influence of the cytokines used in their culture.     

Functional expression of chemokine receptor 2 by normal human eosinophils

BACKGROUND: Within the granulocytes, the CC chemokines preferentially activate basophils and eosinophils on binding to chemokine receptors (CCRs). In vivo administration of neutralizing anti-monocyte chemoattractant protein 1 (MCP-1) antibodies can block accumulation of eosinophils in the lungs of antigen-challenged animals. OBJECTIVE: We studied a panel of chemokines for chemotactic activity in normal human eosinophils from healthy donors with a special focus on MCP-1, identified the respective receptor required for the biological response of eosinophils, and investigated mediators used for signal transduction. METHODS: Cells were enriched by magnetic cell sorting. Receptor expression in eosinophils was shown by RT-PCR and fluorescence-activated cell sorting. The biological response was tested in chemotaxis and calcium mobilization assays. RESULTS: Eosinophils have detectable mRNA for CCR2, and the receptor protein is expressed on cell surfaces. MCP-1 induces chemotaxis and calcium mobilization in eosinophils. The chemotactic activity of MCP-1 revealed a double-peaked dose-response curve; one of the peaks is abolished by addition of a blocking antibody to CCR2, but it is insensitive to blocking of CCR1 or CCR3. Specific enzyme inhibitors ruled out signaling characteristics of CCR2 in eosinophils. CONCLUSION: Normal human eosinophils express functional CCR2 on cell surfaces.     

T-cells in the cerebrospinal fluid express a similar repertoire of inflammatory chemokine receptors in the absence or presence of CNS inflammation: implications for CNS trafficking

It is believed that chemokines and their receptors are involved in trafficking of T-cells to the central nervous system (CNS). The aim of the current study was to define the expression on cerebrospinal fluid (CSF) T-cells of six chemokine receptors associated with trafficking to sites of inflammation. Flow cytometry was used to detect chemokine receptor expression. We observed that CD3+T-cells in the CSF express a restricted array of inflammatory chemokine receptors, specifically CXCR3, CCR5 and CCR6, but little CCR1-3. This repertoire was independent of the presence of CNS inflammation, since comparable findings were obtained in patients with multiple sclerosis (MS) and individuals with non-inflammatory neurological diseases. The enrichment of CCR5+T-cells in the CSF could largely be explained by higher frequency of CD4+/CD45RO+T-cells in this compartment. In contrast, CD4+/CD45RO+T-cells expressing CXCR3 were significantly enriched in CSF as compared with blood. Similar levels of CCR6+/CD3+T-cells were observed in blood and CSF, while levels of CCR2+/CD3+T-cells were lower in CSF than in blood. The CSF was virtually devoid of CCR5+/CXCR3- T-cells, suggesting that the expression of CCR5 alone is not sufficient for the trafficking of CD3+T-cells to the CSF. We hypothesize that CXCR3 is the principal inflammatory chemokine receptor involved in intrathecal accumulation of T-cells in MS. Through interactions with its ligands, CXCR3 is proposed to mediate retention of T-cells in the inflamed CNS.     

Abnormal expression of chemokine receptors in Behçet's disease: relationship to intracellular Th1/Th2 cytokines and to clinical manifestations

Dynamic interplay between cytokines and chemokines directs trafficking of peripheral blood mononuclear cells to tissues in autoimmune and/or viral diseases. The aim of the current study was to define the expression on CD3+ T cells of six chemokine receptors associated with inflammatory sites and the expression of intracellular cytokines, such as interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), in Behcet's disease (BD). Flow cytometry was used to detect chemokine receptor and intracytoplasmic cytokines' expression. We observed that CD3+ T cells in the peripheral blood express a restricted array of inflammatory chemokine receptors, specifically, CCR5, CCR6 and CXCR3, but little CCR1-3. The highest expression of CXCR3 on CD3+ T cells is associated with the presence of central nervous system (CNS) manifestations or pulmonary involvement. CXCR3 is the principal inflammatory chemokine receptor involved in BD. CCR5 chemokine receptor is increased in BD regardless of clinical manifestations. The frequency of IFN-gamma-producing cells expressing CXCR3+ CD3+ cells is significantly increased in patients with BD compared with normal controls. IL-4-producing cells are decreased in BD. These results demonstrate the predominance of type 1 cytokine producing cells in CXCR3+ CD3+ T cells during BD. We hypothesize that CXCR3 is the principal inflammatory chemokine receptor involved in BD, particularly during CNS and pulmonary manifestations.     

Selective recruitment of CXCR3+ and CCR5+ CCR4+ T cells into synovial tissue in patients with rheumatoid arthritis

The inflamed synovial tissue (ST) of rheumatoid arthritis (RA) is characterized by the selective accumulation of interferon gamma-producing Th1-type CD4+ T cells. In this study, we investigated whether the predominance of Th1-type CD4+ cells in the ST lesion is mediated by their selective recruitment through Th1 cell-associated chemokine receptors CXCR3 and CCR5. The lymphocyte aggregates in the ST of RA contained a large number of CD4+ T cells, which mostly expressed both CXCR3 and CCR5, but not CCR4. In contrast, the frequencies of CD4+ and CD8+ T cells expressing CXCR3 and CCR5 in the blood were significantly decreased in RA patients, compared with healthy controls (HC), although there was no difference in the frequencies of CCR4-expressing CD4+ and CD8+ T cells between RA and HC. CXCR3, CCR5, and CCR4 expression in blood CD4 + T cells and CXCR3 expression in CD8+ T cells were increased after interleukin-15 (IL-15) stimulation. Therefore, the distribution of Th1-type CD4+ T cells into the ST from the blood in RA may be associated with the local expression of chemokines, both CXCR3 and CCR5 ligands, and IL-15 may play a role in enhancing these chemokine receptors on CD4+ T cell infiltrates.     

Chemokine-receptor expression on T cells in lung compartments of challenged asthmatic patients

BACKGROUND: The interaction of chemokines with their receptors strongly influences the migration of leucocytes. OBJECTIVE: In order to assess the contribution of these molecules to the local recruitment of T cells in bronchial asthma, we analysed the expression of 14 chemokine receptors on lung-derived T cells. METHODS: Chemokine-receptor expression by T cells derived from the peripheral blood, the bronchoalveolar lavage fluid and the bronchial mucosa was analysed by flow cytometry and immunohistochemistry. Expression profiles in healthy and mildly asthmatic individuals were compared, the latter prior and after segmental allergen provocation. RESULTS: Compared with peripheral blood, alveolar T cells expressed significantly more CCR2, CCR5, CCR6, CXCR3 and CCR4. However, no differences were observed between healthy controls and unchallenged asthmatics. In patients developing significant inflammatory responses following specific allergen challenge, a marked increase in the percentage of CCR4+ and CCR7+, and reduced numbers of CXCR3-bearing alveolar T cells were detected. Following specific allergen challenge, chemokine-receptor expression profiles of T cells from the alveolar space and the mucosa or the submucosa were similar, excluding a particular subcompartmentalization of the chemokine/chemokine-receptor system. CONCLUSION: The expression of certain chemokine receptors by lung T cells suggests a contribution to the physiological recruitment of T cells to the lungs, both in healthy controls and unchallenged mild asthmatics. However, strong allergen-induced airway responses were associated with a specific chemokine-receptor profile, suggesting the involvement of certain chemokine receptors in the pathogenesis of allergic bronchial inflammation.     

Lack of correlation between chemokine receptor and T(h)1/T(h)2 cytokine expression by individual memory T cells

Chemokine and chemokine receptor interactions may have important roles in leukocyte migration to specific immune reaction sites. Recently, it has been reported that CXC chemokine receptor (CXCR) 3 and CC chemokine receptor (CCR) 5 were preferentially expressed on T(h)1 cells, and CCR3 and CCR4 were preferentially expressed on T(h)2 cells. To investigate chemokine receptor expression by T(h) subsets in vivo, we analyzed cytokine (IL-2, IL-4 and IFN-gamma) and chemokine receptor (CXCR3, CXCR4, CCR3, CCR4 and CCR5) mRNA expression by individual peripheral CD4(+) memory T cells after short-term stimulation, employing a single-cell RT-PCR method. This ex vivo analysis shows that the frequencies of cells expressing chemokine receptor mRNA were not significantly different between T(h)1 and T(h)2 cells in normal peripheral blood. To assess a potential role of in vivo stimulation, we also analyzed unstimulated rheumatoid arthritis synovial CD4(+) memory T cells. CXCR3, CXCR4, CCR3 and CCR5 expression was detected by individual synovial T cells, but the frequencies of chemokine receptor mRNA were not clearly different between T(h)1 and non-T(h)1 cells defined by expression of IFN-gamma or lymphotoxin-alpha mRNA in all RA patients. These data suggest that chemokine receptor expression does not identify individual memory T cells producing T(h)-defining cytokines and therefore chemokine receptor expression cannot be a marker for T(h)1 or T(h)2 cells in vivo.