Potent Eosinophil Chemoattractant Research Articles

Constitutive and cytokine-stimulated expression of eotaxin by human airway smooth muscle cells.

Airway eosinophilia is a prominent feature of asthma that is believed to be mediated in part through the expression of specific chemokines such as eotaxin, a potent eosinophil chemoattractant that is highly expressed by epithelial cells and inflammatory cells in asthmatic airways. Airway smooth muscle (ASM) has been identified as a potential source of cytokines and chemokines. The aim of the present study was to examine the capacity of human ASM to express eotaxin. We demonstrate that airway myocytes constitutively express eotaxin mRNA as detected by RT-PCR. Treatment of ASM for 24 h with different concentrations of TNF-alpha and IL-1beta alone or in combination enhanced the accumulation of eotaxin transcripts. Maximal mRNA expression of eotaxin was shown at 12 and 24 h following IL-1beta and TNF-alpha stimulation, respectively. The presence of immunoreactive eotaxin was demonstrated by immunocytochemistry, and constitutive and cytokine-stimulated release of eotaxin was confirmed in ASM culture supernatants by ELISA. Strong signals for eotaxin mRNA and immunoreactivity were observed in vivo in smooth muscle in asthmatic airways. In addition, chemotaxis assays demonstrated the presence of chemoattractant activity for eosinophils and PBMCs in ASM supernatants. The chemotactic responses of eosinophils were partly inhibited with antibodies directed against eotaxin or RANTES, and a combined blockade of both chemokines causes > 70% inhibition of eosinophil chemotaxis. The results of this study suggest that ASM may contribute to airway inflammation in asthma through the production and release of eotaxin.

Eotaxin and eotaxin receptor (CCR3) expression in Sephadex particle-induced rat lung inflammation.

The beta chemokine eotaxin is a potent eosinophil activator and chemoattractant. We examined immunohistochemically eotaxin protein expression in a range of normal rat tissues and in rat lung during Sephadex particle-induced pulmonary inflammation. The time course of eotaxin expression in lung at various time points after Sephadex administration was related to the appearance of eosinophils in the bronchoalveolar lavage fluid and tissue distribution of eotaxin receptor (CCR3) positive cells. Results showed that eotaxin protein was constitutively expressed by both lung airway epithelial cells and gut epithelial cells in normal tissues in the absence of inflammation. During Sephadex induced pulmonary inflammation, eotaxin expression increased in alveolar macrophages prior to the major increase in eosinophil numbers which reached a peak at 72 h. The pattern of eotaxin pulmonary expression and the location of CCR3 receptor positive cells suggest a chemoattractant gradient resulting in migration firstly into the tissue and subsequently through the airway epithelium into the airways. Treatment of rats with the glucocorticoid dexamethasone or the immunosuppressant cyclosporin A reduced eosinophil entry into lung tissue and airways but had no apparent effect on eotaxin expression in vivo, indicating that both these drugs inhibit eosinophil recruitment either by an eotaxin-independent mechanism, or by targetting factors that synergise with eotaxin, or an event post eotaxin expression.

Antileukotriene drugs in the management of asthma.

1the mortality and morbidity associated with this disease have not appreciably declined. 2 It continues to be imperative to develop new approaches to asthma therapy. Antileukotriene drugs are a novel form of asthma pharmacotherapy and the first new treatment strategy for chronic asthma in 20 years. In experimental studies, cysteinyl leukotrienes (LTC4, LTD4, and LTE4, together known formerly as slow-reacting substance of anaphylaxis or SRS-A) were found to be extremely potent bronchoconstrictors and to attract inflammatory cells, increase mucous production, and alter vascular permeability. 3 LTB4 was found to be a potent chemoattractant for neutrophils and eosinophils, but not to have a bronchoconstrictive effect. 3 The cysteinyl leukotrienes were also shown to be produced by activated inflammatory cells (eosinophils, basophils, and mast cells) known to be present in the airways of patients with asthma, 3 and were recovered in increased amounts at baseline, after allergen challenge, and during asthma exacerbations from bronchoalveolar lavage fluid and urine of patients with asthma. 3-5 These observations suggested that leukotrienes could be important in the clinical symptoms and physiologic changes of asthma, but confirmation of that concept required the development of drugs that either blocked the synthesis of leukotrienes from arachidonic acid (5-lipoxygenase [5-LO] inhibitors and 5-lipoxygenase activating protein [FLAP] inhibitors) or antagonized the activity of the cysteinyl leukotrienes at their receptor, cysLT1 (leukotriene receptor antagonists). Four oral antileukotriene drugs are now available for the treatment of asthma: 3 leukotriene receptor antagonists, montelukast, zafirlukast, and pranlukast (not available in the United States), and a 5-LO inhibitor, zileuton. Although 5-LO inhibitors inhibit the synthesis of LTB4 as well as the cysteinyl leukotrienes, it remains to be seen whether this broader spectrum of activity produces greater efficacy in the treatment of asthma. These antileukotriene drugs have been extensively studied in clinical trials, but longterm data on effectiveness and adverse effects are lacking. Efficacy Studies indicate that antileukotriene drugs directly inhibit bronchoconstriction and have anti-inflammatory effects as well, a combination of actions that has not been demonstrated as clearly in other classes of asthma drugs. Early in their development, the leukotriene receptor antagonists and FLAP inhibitors, in single doses, were found to almost completely eliminate the immediate bronchoconstriction response to allergen challenge and to reduce the later bronchoconstriction response by approximately 50%. 6 Antileukotriene drugs also abolished the bronchoconstriction response that is observed after the administration of very small doses of aspirin in patients with aspirinsensitive asthma. 7 Further, they reduced exercise-induced bronchoconstriction by 40% to 60%, 8 and recent evidence suggests that this protective effect has no tachyphylaxis, unlike that reported with the use of the long-acting b2-agonist salmeterol. 9,10 Studies of the anti-inflammatory effects of the antileukotriene drugs have shown significant reductions from baseline in the number and proportion of airway and circulating eosinophils in patients with asthma treated with these drugs compared with those who received placebo. 11-13

5-oxo-ETE induces pulmonary eosinophilia in an integrin-dependent manner in Brown Norway rats.

We have shown previously that the 5-lipoxygenase product 5-oxo-6,8, 11,14-eicosatetraenoic acid (5-oxo-ETE) is a highly potent eosinophil chemoattractant in vitro. To determine whether this substance can induce pulmonary eosinophil infiltration in vivo, it was administered to Brown Norway rats by tracheal insufflation. Eosinophils were then counted in lung sections that had been immunostained with an antibody to eosinophil major basic protein. 5-Oxo-ETE induced a dramatic increase in the numbers of eosinophils (ED50, 2.5 microg) around the walls of the airways, which reached maximal levels (five times control levels) between 15 and 24 h after administration, and then declined. LTB4 also induced pulmonary eosinophil infiltration with a similar ED50 but appeared to be somewhat less effective. In contrast, LTD4 and LTE4 were inactive. 5-Oxo-ETE-induced eosinophilia was unaffected by the LTB4 and PAF antagonists LY255283 and WEB 2170, respectively. However, it was inhibited by approximately 75% by monoclonal antibodies to CD49d (VLA-4) or CD11a (LFA-1) but was not significantly affected by an antibody to CD11b (Mac-1). In conclusion, 5-oxo-ETE induces pulmonary eosinophilia in Brown Norway rats, raising the possibility that it may be a physiological mediator of inflammation in asthma.

Eotaxin Modulates Myelopoiesis and Mast Cell Development From Embryonic Hematopoietic Progenitors

Eotaxin is a potent chemoattractant for eosinophils during inflammation and allergic reactions in the adult, but its role in the embryonic development of the hematopoietic system has not been examined. We report here that eotaxin and its receptor, CCR-3, are expressed by embryonic tissues responsible for blood development, such as fetal liver (FL), yolk sac (YS), and peripheral blood. We found that eotaxin acts synergistically with stem cell factor to accelerate the differentiation of embryonic mast cell progenitors, and this response can be suppressed by pertussis toxin, an inhibitor of chemokine-induced signaling through Giα protein and chemotaxis. Eotaxin promotes the differentiation of fetal mast cell progenitors into differentiated mast cells as defined by the expression of mast cell specific proteases. Furthermore, in combination with stem cell factor (SCF), it promotes the growth of Mac-1+myeloid cells from embryonic progenitors. These studies suggest that eotaxin may be involved in the growth of granulocytic progenitors and the differentiation and/or function of mast cells during embryogenesis and/or pathological conditions that induce high levels of eotaxin, such as allergic responses.© 1998 by The American Society of Hematology.

Eotaxin Modulates Myelopoiesis and Mast Cell Development From Embryonic Hematopoietic Progenitors

Eotaxin is a potent chemoattractant for eosinophils during inflammation and allergic reactions in the adult, but its role in the embryonic development of the hematopoietic system has not been examined. We report here that eotaxin and its receptor, CCR-3, are expressed by embryonic tissues responsible for blood development, such as fetal liver (FL), yolk sac (YS), and peripheral blood. We found that eotaxin acts synergistically with stem cell factor to accelerate the differentiation of embryonic mast cell progenitors, and this response can be suppressed by pertussis toxin, an inhibitor of chemokine-induced signaling through Gi protein and chemotaxis. Eotaxin promotes the differentiation of fetal mast cell progenitors into differentiated mast cells as defined by the expression of mast cell specific proteases. Furthermore, in combination with stem cell factor (SCF), it promotes the growth of Mac-1+myeloid cells from embryonic progenitors. These studies suggest that eotaxin may be involved in the growth of granulocytic progenitors and the differentiation and/or function of mast cells during embryogenesis and/or pathological conditions that induce high levels of eotaxin, such as allergic responses.© 1998 by The American Society of Hematology.

Human Ecalectin, a Variant of Human Galectin-9, Is a Novel Eosinophil Chemoattractant Produced by T Lymphocytes

A 1.6-kilobase pair cDNA was isolated from a human T-cell-derived expression library that encodes a novel eosinophil chemoattractant (designated ecalectin) expressed during allergic and parasitic responses. Based on its deduced amino acid sequence, ecalectin is a 36-kDa protein consisting of 323 amino acids. Although ecalectin lacks a hydrophobic signal peptide, it is secreted from mammalian cells. Ecalectin is not related to any known cytokine or chemokine but rather is a variant of human galectin-9, a member of the large family of animal lectins that have affinity for beta-galactosides. Recombinant ecalectin, expressed in COS cells and insect cells, exhibited potent eosinophil chemoattractant activity and attracted eosinophils in vitro and in vivo in a dose-dependent manner but not neutrophils, lymphocytes, or monocytes. The finding that the ecalectin transcript is present in abundance in various lymphatic tissues and that its expression increases substantially in antigen-activated peripheral blood mononuclear cells suggests that ecalectin is an important T-cell-derived regulator of eosinophil recruitment in tissues during inflammatory reactions. We believe that this is the first report of the expression of an immunoregulatory galectin expressed by a T-cell line that is selective for eosinophils.

An appraisal of T cell subsets and the potential for autoimmune injury

The initiation of T cell dependent immune responses requires the T cell to utilize a heterodimeric cell surface antigen receptor to recognize an antigenic peptide in association with major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells (APCs). This is a necessary but insufficient signal for T cell activation. T cells must additionally receive a costimulatory signal that can be delivered through a variety of receptor-ligand pairs. Of these, the best characterized is the CD28-B7 (CD80 and CD86) couple. It has been appreciated for decades that the outcome of the T cell-APC interaction is highly variable and can lead to different forms of immune responses. The past decade has witnessed major advances in the definition of how distinct lymphocyte functions are dictated by expression of distinct cytokines, activation of defined signal transduction pathways, and, most recently, expression of distinct transcription factors. The production of ever increasing numbers of induced mutant mouse strains has created new reagents in which to analyze the role of particular cytokines or other proteins in defined immune responses. While the temptation remains to accomodate emerging information into reductionist models of T cell dependent immunopathology, it is our view that in vivo immune responses are highly complex and regulated events that defy simple categorization.

Eotaxin Induces a Rapid Release of Eosinophils and Their Progenitors From the Bone Marrow

AbstractThe CC-chemokine eotaxin is a potent eosinophil chemoattractant that stimulates recruitment of eosinophils from the blood to sites of allergic inflammation. Mobilization from the bone marrow is an important early step in eosinophil trafficking during the allergic inflammatory response. In this paper we examine the potential of eotaxin to mobilize eosinophils and their progenitors from bone marrow. Eotaxin stimulated selective, dose-dependent chemotaxis of guinea pig bone marrow eosinophils in vitro. Intravenous injection of eotaxin (1 nmol/kg) into guinea pigs in vivo stimulated a rapid blood eosinophilia (from 3.9 ± 1.2 to 28 ± 9.9 × 104eosinophils/mL at 30 minutes) and a corresponding decrease in the number of eosinophils retained in the femoral marrow (from 9.0 ± 0.8 to 4.8 ± 0.8 × 106 eosinophils per femur). To show a direct release of eosinophils from the bone marrow an in situ perfusion system of the guinea pig femoral bone marrow was developed. Infusion of eotaxin into the arterial supply of the perfused femoral marrow stimulated a rapid and selective release of eosinophils into the draining vein. In addition, eotaxin stimulated the release of colony-forming progenitor cells. The cytokine interleukin-5 was chemokinetic for bone marrow eosinophils and exhibited a marked synergism with eotaxin with respect to mobilization of mature eosinophils from the femoral marrow. Thus, eotaxin may be involved in both the mobilization of eosinophils and their progenitors from the bone marrow into the blood and in their subsequent recruitment into sites of allergic inflammation.

Eotaxin Induces a Rapid Release of Eosinophils and Their Progenitors From the Bone Marrow

The CC-chemokine eotaxin is a potent eosinophil chemoattractant that stimulates recruitment of eosinophils from the blood to sites of allergic inflammation. Mobilization from the bone marrow is an important early step in eosinophil trafficking during the allergic inflammatory response. In this paper we examine the potential of eotaxin to mobilize eosinophils and their progenitors from bone marrow. Eotaxin stimulated selective, dose-dependent chemotaxis of guinea pig bone marrow eosinophils in vitro. Intravenous injection of eotaxin (1 nmol/kg) into guinea pigs in vivo stimulated a rapid blood eosinophilia (from 3.9 ± 1.2 to 28 ± 9.9 × 104eosinophils/mL at 30 minutes) and a corresponding decrease in the number of eosinophils retained in the femoral marrow (from 9.0 ± 0.8 to 4.8 ± 0.8 × 106 eosinophils per femur). To show a direct release of eosinophils from the bone marrow an in situ perfusion system of the guinea pig femoral bone marrow was developed. Infusion of eotaxin into the arterial supply of the perfused femoral marrow stimulated a rapid and selective release of eosinophils into the draining vein. In addition, eotaxin stimulated the release of colony-forming progenitor cells. The cytokine interleukin-5 was chemokinetic for bone marrow eosinophils and exhibited a marked synergism with eotaxin with respect to mobilization of mature eosinophils from the femoral marrow. Thus, eotaxin may be involved in both the mobilization of eosinophils and their progenitors from the bone marrow into the blood and in their subsequent recruitment into sites of allergic inflammation.

Expression of RANTES by normal airway epithelial cells after influenza virus A infection.

The chemokine regulated on activation, normal T cells expressed and secreted (RANTES), is a C-C chemokine and a potent chemoattractant for monocytes, T lymphocytes, basophils, and eosinophils. Its expression by human airway epithelium has been demonstrated both in vitro and in vivo. We investigated whether RANTES is expressed by normal human airway epithelial cells after influenza viral infection and examined its bioactivity. Epithelial cells were obtained from bronchial tissue or nasal polyps of patients who had undergone lobectomy for lung cancer or polypectomy for nasal polyps. These cells were cultured by the outgrowth method. Cultured cells were infected with influenza virus A (subtype H3N2) after which the supernatants and the cells were collected 8 to 72 h after infection. RANTES mRNA (messenger RNA) was analyzed by the reverse transcriptase-polymerase chain reaction and Southern blot analysis of its product. Concentrations of RANTES in the supernatants were analyzed by enzyme-linked immunosorbent assay. RANTES protein and mRNA were not detected in the media of uninfected cells. PCR products for RANTES were clearly detected in nasal and bronchial epithelial cells 24 h after infection. Southern blot analysis confirmed that the PCR products were indeed specific for RANTES mRNA. Twenty-four to 72 h after infection, significant levels of RANTES protein were detected in culture media. We also investigated the chemotactic activity of the supernatant of cultured cells. The supernatant of the cells 48 h after infection had potent chemotactic activity for eosinophils, which was attenuated by the addition of anti-RANTES antibodies. These findings suggest that influenza virus infection may induce expression of bioactive RANTES by normal human bronchial and nasal epithelial cells.

Eotaxin mRNA Expression in Bronchial Mucosa of Patients with Asthma

Background: Asthma is a chronic inflammatory disease of the airways characterized by a marked infiltration of eosinophils in the bronchial mucosa. Asthmatic bronchial mucosa produces many factors described as being chemotactic for inflammatory cells. IL-5, RANTES, and MCP-1 alpha are the chemotactic factors for eosinophils, but their roles are controversial. Recently eotaxin that is a potent eosinophil chemoattractant cytokine was detected in a guinea-pig model of allergic airway inflammation, and human eotaxin was cloned. Eotaxin is a specific chemoattractant for eosinophils, but its role in asthma is not confirmed. We examined the in vivo expression of eotaxin in bronchi of asthmatic patients. Methods : 11 asthmatics and 2 normal controls were enrolled. All subjects were underwent bronchoscopy with bronchial biopsies in 2nd or 3rd carina. RNA extraction from biopsy samples was done by acid-guanidium method. Semi-quantitaive RT-PCR was done for evaluation of eotaxin mRNA expression The extent of eosinophil infiltration was evaluated by counting the eosinophils in submucosa in HPF of microscope. Results : Eotaxin mRNA expressed in symptomatic, uncontrolled asthma. Steroid inhibited expression of eotaxin mRNA in asthma. Expression of eotaxin mRNA correlated with eosinophil infiltration in bronchial tissues. Conclusion: Expression of eotaxin mRNA increases in uncontrolled asthma and eotaxin is involved in the recruitment of eosinophils.

Eotaxin influences the development of embryonic hematopoietic progenitors in the mouse.

Eotaxin is a potent chemoattractant for eosinophils during inflammation and allergic reactions in the adult, but its role during development has not been studied. We report that eotaxin and its receptor, CCR-3, are expressed by embryonic tissues responsible for blood development, including the yolk sac, fetal liver, and fetal blood. We also found that eotaxin acts synergistically with stem cell factor (SCF) to accelerate the differentiation of embryonic mast cell progenitors and to promote the growth of Mac-1+/Gr-1- cells from progenitors isolated at 10-12 days of gestation. This response is diminished by Pertussis toxin, the Gi alpha inhibitor. These studies suggest that eotaxin is involved in the growth of myeloid cell progenitors and the differentiation of mast cells during embryogenic development.

Cloning and functional characterization of a novel human CC chemokine that binds to the CCR3 receptor and activates human eosinophils.

Eotaxin has been found to bind exclusively to a single chemokine receptor, CCR3. Using expression sequence tag screening of an activated monocyte library, a second chemokine has been identified; it was expressed and purified from a Drosophila cell culture system and appears to only activate CCR3. Eotaxin-2, MPIF-2, or CKbeta-6, is a human CC chemokine with low amino acid sequence identity to other chemokines. Eotaxin-2 promotes chemotaxis and Ca2+ mobilization in human eosinophils but not in neutrophils or monocytes. Cross-desensitization calcium mobilization experiments using purified eosinophils indicate that eotaxin and MCP-4, but not RANTES, MIP-1alpha, or MCP-3, can completely cross-desensitize the calcium response to eotaxin-2 on these cells, indicating that eotaxin-2 shares the same receptor used by eotaxin and MCP-4. Eotaxin-2 was the most potent eosinophil chemoattractant of all the chemokines tested. Eotaxin-2 also displaced 125I-eotaxin bound to the cloned CCR3 stably expressed in CHO cells (CHO-CCR3) and to freshly isolated human eosinophils with affinities similar to eotaxin and MCP-4. 125I-Eotaxin-2 binds with high affinity to eosinophils and both eotaxin and cold eotaxin-2 displace the ligand with equal affinity. Eotaxin and eotaxin-2 promote a Ca2+ transient in RBL-2H3 cells stably transfected with CCR3 (RBL-2H3-CCR3) and both ligands cross-desensitized the response of the other but not the response to LTD4. The data indicate that eotaxin-2 is a potent eosinophil chemotactic chemokine exerting its activity solely through the CCR3 receptor.

Cutaneous injection of RANTES causes eosinophil recruitment: comparison of nonallergic and allergic human subjects.

RANTES, a member of the C-C chemokine family, is a potent chemoattractant for T lymphocytes and eosinophils, but not neutrophils. To determine the effect of RANTES on cell recruitment in vivo, we injected up to 4 microg of RANTES intradermally into both allergic and nonallergic subjects and obtained biopsies 30 min, 6 h, and 24 h later. A dose- and time-dependent recruitment of eosinophils, CD45RO+ cells, and CD3+ cells was observed, with no effect seen on polymononuclear, cutaneous lymphocyte Ag+, CD68+, or tryptase+ cells. Eosinophil recruitment occurred more rapidly in allergic subjects than in nonallergic subjects. No eosinophil infiltrate was observed in nonallergic biopsies at 30 min and 6 h, whereas significant eosinophil recruitment was observed in allergic subjects by 30 min, reaching near-maximum levels by 6 h. The peak responses at 24 h were similar in both groups (nonallergic, 110 +/- 24 eosinophils/mm2; allergic, 113 +/- 38 eosinophils/mm2). The two groups had comparable numbers of circulating eosinophils. Major basic protein staining demonstrated eosinophil degranulation in both allergic and nonallergic groups. RANTES injection resulted in activation of endothelial E-selectin expression at 24 h. Incubation of cultured HUVECs with RANTES had no effect on adhesion molecule expression, suggesting that the in vivo effect may have been indirect. Our studies demonstrate that RANTES is a potent chemoattractant for eosinophils, CD3+ cells, and CD45RO+ cells in human skin. The accelerated eosinophil recruitment in allergic subjects provides support for the hypothesis that eosinophils from these subjects are primed in vivo.

Expression of eotaxin by human lung epithelial cells: induction by cytokines and inhibition by glucocorticoids.

Eotaxin is a potent and specific eosinophil chemoattractant that is mobilized in the respiratory epithelium after allergic stimulation. Pulmonary levels of eotaxin mRNA are known to increase after allergen exposure in sensitized animals. In this study we demonstrate that TNF alpha and IL-1beta induce the accumulation of eotaxin mRNA in the pulmonary epithelial cell lines A549 and BEAS 2B in a dose-dependent manner. Cytokine-induced A549 cell mRNA accumulation was maximal at 4 h and was significantly enhanced when the cells were costimulated with IFNgamma. TNFalpha- and IL-1beta-induced increases in eotaxin mRNA were diminished in a dose-dependent manner by the glucocorticoid dexamethasone and were augmented by the protein synthesis inhibitor cycloheximide. Cytokine-induced increases in eotaxin mRNA expression correlated with increased eotaxin protein production and secretion, and dexamethasone inhibition of cytokine-induced eotaxin mRNA augmentation was associated with diminished eotaxin protein secretion. These findings, together with the known kinetics of TNF alpha and IL-1beta mobilization in asthmatic airways and the potent eosinophil chemotactic effects of eotaxin, define a mechanism linking inflammatory cytokine mobilization to eosinophil recruitment that may be relevant to the pathogenesis of asthma.

Induction of cerebrospinal fluid eosinophilia in rats by the intraventricular injection of Angiostrongylus cantonensis antigen

Resistance to Angiostrongylus cantonensis is contingent upon the generation of an eosinophilic response in the CSF of infected hosts. We have studied the parameters required for the generation of this CSF eosinophilia in normally permissive rats. We initially induced a marked peripheral eosinophilia in rats by infection with either Mesocestoides corti or Angiostrongylus cantonensis or the surgical transfer of A. cantonensis young adult worms (YA) into their pulmonary arteries. Next, we injected various antigens into the ventricles of these rats. A. cantonensis-preinfected rats demonstrated significant CSF eosinophilia following injection of A. cantonensis egg antigen, 1st-stage larval (L 1) antigen, or M. corti antigen, but not following YA antigen inoculation. A. cantonensis egg and M. corti antigens were potent chemoattractants for eosinophils in an in vitro chemotaxis assay. These data indicate that peripheral eosinophilia, meningeal stimulation by A. cantonensis infection and the presence of potent chemoattractants, e.g., egg and L 1 antigens are prerequisites for CSF eosinophil accumulation in permissive rat hosts.

Human monocyte chemoattractant protein (MCP)-4 is a novel CC chemokine with activities on monocytes, eosinophils, and basophils induced in allergic and nonallergic inflammation that signals through the CC chemokine receptors (CCR)-2 and -3.

The chemokines are a large family of cytokines that regulate the complex and precise recruitment of immune cells into inflammatory foci. To fully appreciate their role in the pathogenesis of human diseases, the entire spectrum of chemokines, their receptors, their cellular targets, and mechanisms of regulation need to be delineated. Using eotaxin as a probe, we isolated a cDNA for a novel human beta (or CC) chemokine that, based on its biological and structural features, we have named monocyte chemoattractant protein (MCP)-4. Purified recombinant MCP-4 protein was a potent chemoattractant for monocytes and eosinophils and stimulated histamine release from basophils. MCP-4 induced a calcium flux in HEK-293 cells transfected with the monocyte selective MCP-1 receptor (CCR-2B) and the eosinophil selective eotaxin receptor (CCR-3), but not in the more widely expressed CCR-1 or CCR-5. This novel chemokine is expressed in TNF-alpha and IL-1 activated epithelial and endothelial cells in vitro, and in the epithelial mucosa of patients with both Th2-type allergic and Th1-type nonallergic sinusitis. Furthermore, both IFN-gamma and IL-4, products of Th1 and Th2 cells, respectively, synergized with TNF-alpha and IL-1 in inducing MCP-4 mRNA accumulation. These properties of MCP-4 offer a molecular explanation for the observed accumulation of monocytes, eosinophils and basophils in both Th1- and Th2-type immune responses.

Expression of RANTES mRNA and protein in airways of patients with mild asthma.

Chemoattractant cytokines (chemokines) such as RANTES, are potent chemoattractants for eosinophils, T lymphocytes, and monocyte/macrophages. We examined RANTES mRNA and protein expression in bronchial biopsies and bronchoalveolar lavage (BAL) cells from patients with mild asthma on beta 2-adrenergic agonist therapy only. Using quantitative polymerase chain reaction (PCR), mean RANTES starting cDNA was 110 +/- 18 fg/pg beta-actin in asthmatics compared with 33.7 +/- 11.0 fg/pg in normals (p < 0.003) but there was no significant difference in RANTES mRNA expression in BAL cells between the two groups. Immunohistochemical staining of cryostat sections of bronchial biopsies with an anti-RANTES antibody using peroxidase antiperoxidase method revealed constitutive staining in airway epithelial cells, airway smooth muscle, and subepithelial cells. However, there was no difference in the number of RANTES-staining cells between normal subjects and asthmatics despite significantly increased numbers of CD4+ T-cells, CD68+ macrophages, and MBP+ eosinophils in mucosal biopsies obtained from asthmatics. Double-staining revealed expression of RANTES in a proportion of CD3+ T lymphocytes. Thus, RANTES is constitutively expressed in the airways and RANTES mRNA is elevated in airways of patients with mild asthma, supporting a role for RANTES in normal and asthmatic airways.

Human RANTES acts as a receptor antagonist for guinea pig eotaxin in vitro and in vivo.

The guinea pig C-C chemokine, eotaxin, is a potent and selective eosinophil chemoattractant in guinea pig airways and skin in vivo, and stimulates both guinea pig and human eosinophils in vitro. The human C-C chemokine RANTES (30% homology with guinea pig eotaxin) stimulates human eosinophils in vitro, but does not stimulate guinea pig eosinophils, even though these cells bind 125I-RANTES. Similar concentrations of eotaxin and unlabeled RANTES competitively inhibit the binding of 125I-RANTES to guinea pig eosinophils, suggesting that eotaxin and RANTES share a common binding site on these cells. In the present study, we investigated the possibility that human RANTES, binding to a putative eotaxin receptor on guinea pig eosinophils, might block functional responses to eotaxin. When fura-2-loaded cells were first exposed to RANTES, which failed to elevate the intracellular calcium concentration, the response to a subsequent challenge with eotaxin was inhibited in a dose-dependent manner. Inhibition was also demonstrated when the two chemokines were added simultaneously. Another human C-C chemokine, MCP-3 (52% homology with guinea pig eotaxin), had similar inhibitory effects on the eotaxin-induced activation of guinea pig eosinophils in vitro. RANTES inhibited (111)In-eosinophil accumulation in response to intradermal eotaxin in vivo. In contrast, RANTES had no significant effect on responses to leukotriene B4 in vitro or in vivo. Thus, these experiments in the guinea pig demonstrate that human RANTES is the first prototypic antagonist of an eotaxin receptor.