Tryptase-Chymase Double-Positive Human Mast Cells Express the Eotaxin Receptor CCR3 and Are Attracted by CCR3-Binding Chemokines
1999; Elsevier BV; Volume: 155; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)65222-4
ISSN1525-2191
AutoresPaola Romagnani, Amato de Paulis, Chiara Beltrame, Francesco Annunziato, Valeria Dente, Enrico Maggi, S Romagnani, Gianni Marone,
Tópico(s)Allergic Rhinitis and Sensitization
ResumoEosinophils, basophils, and Th2 cells express the chemokine receptor CCR3, which binds eotaxin, RANTES, and some other chemokines. Using immunohistochemistry and flow cytometry, we demonstrate that CCR3 is also expressed by a variable proportion of human mast cells in gut, skin, and lung tissue. By contrast, with the same anti-CCR3 antibody (B711), CCR3 was poorly if at all detectable on human Th2 cells in vitro and in vivo. Eotaxin neither induced histamine release from purified human mast cells nor increased anti-IgE-stimulated histamine secretion. However, both eotaxin and RANTES elicited mast cell migration in vitro with a similar efficacy. High percentages of CCR3-expressing mast cells were present in the skin and in the intestinal submucosa; much lower percentages were found in the intestinal mucosa and in lung interstitium. Double immunostaining with anti-CCR3 and anti-chymase antibody showed that the vast majority of CCR3-expressing mast cells in the various tissues examined were tryptase-chymase double-positive. Therefore, tryptase-chymase double-positive mast cells express CCR3 and are attracted by CCR3-binding chemokines, eotaxin, and RANTES. Our findings indicate that these chemokines may play an important role in the differentiation and/or migration of this mast cell subset in connective tissues, as well as in sites of allergic inflammation. Eosinophils, basophils, and Th2 cells express the chemokine receptor CCR3, which binds eotaxin, RANTES, and some other chemokines. Using immunohistochemistry and flow cytometry, we demonstrate that CCR3 is also expressed by a variable proportion of human mast cells in gut, skin, and lung tissue. By contrast, with the same anti-CCR3 antibody (B711), CCR3 was poorly if at all detectable on human Th2 cells in vitro and in vivo. Eotaxin neither induced histamine release from purified human mast cells nor increased anti-IgE-stimulated histamine secretion. However, both eotaxin and RANTES elicited mast cell migration in vitro with a similar efficacy. High percentages of CCR3-expressing mast cells were present in the skin and in the intestinal submucosa; much lower percentages were found in the intestinal mucosa and in lung interstitium. Double immunostaining with anti-CCR3 and anti-chymase antibody showed that the vast majority of CCR3-expressing mast cells in the various tissues examined were tryptase-chymase double-positive. Therefore, tryptase-chymase double-positive mast cells express CCR3 and are attracted by CCR3-binding chemokines, eotaxin, and RANTES. Our findings indicate that these chemokines may play an important role in the differentiation and/or migration of this mast cell subset in connective tissues, as well as in sites of allergic inflammation. The attraction of leukocytes to tissues is a basic mechanism for inflammation and for the host response to infections. This process is controlled by a large number of chemokines, which are chemotactic cytokines.1Baggiolini MB Dewald B Moser B Human chemokines: an update.Ann Rev Immunol. 1997; 15: 675-705Crossref PubMed Scopus (1994) Google Scholar, 2Mantovani A Allavena P Vecchi A Sozzani S Chemokines and chemokine receptors during activation and deactivation of monocytes and dendritic cells and in amplification of Th1 versus Th2 responses.Int J Clin Lab Res. 1998; 28: 77-82Crossref PubMed Scopus (54) Google Scholar Chemokines are 8- to 10-kd proteins with 20 to 70 percent homology in amino acid sequences. They have been subdivided into families on the basis of the relative position of their cysteine residues.1Baggiolini MB Dewald B Moser B Human chemokines: an update.Ann Rev Immunol. 1997; 15: 675-705Crossref PubMed Scopus (1994) Google Scholar, 3Luster AD Chemokines: chemotactic cytokines that mediate inflammation.New Engl J Med. 1998; 338: 436-445Crossref PubMed Scopus (3272) Google Scholar There are at least four families of chemokines, but only two have been extensively characterized. The α and β chemokines, which contain four cysteines, appear to be the largest families. In the α-chemokines, one amino acid separates the first two cysteines residues (CXC), whereas in the β-chemokines, the first two cysteine residues are adjacent to each other (CC). The α-chemokines containing the sequence glutamic acid-leucine-arginine near the N terminal (preceding the CXC sequence) are chemotactic for neutrophils; those not containing the sequence act on lymphocytes. The β-chemokines, in general, do not act on neutrophils but attract monocytes, eosinophils, basophils, and lymphocytes with variable selectivity.1Baggiolini MB Dewald B Moser B Human chemokines: an update.Ann Rev Immunol. 1997; 15: 675-705Crossref PubMed Scopus (1994) Google Scholar, 3Luster AD Chemokines: chemotactic cytokines that mediate inflammation.New Engl J Med. 1998; 338: 436-445Crossref PubMed Scopus (3272) Google Scholar Chemokines induce cell migration and activation by binding to specific G protein-coupled cell-surface CXCR and CCR receptors on target cells.1Baggiolini MB Dewald B Moser B Human chemokines: an update.Ann Rev Immunol. 1997; 15: 675-705Crossref PubMed Scopus (1994) Google Scholar, 3Luster AD Chemokines: chemotactic cytokines that mediate inflammation.New Engl J Med. 1998; 338: 436-445Crossref PubMed Scopus (3272) Google Scholar Four human CXC chemokine receptors, CXCR1 through CXCR4, and nine human CC chemokine receptors, CCR1 through CCR9, have been identified. Chemokine receptors are expressed on different types of leukocytes, some being restricted to certain cells, others being more widely expressed not only on leukocytes but also on nonhemopoietic cells. Although most chemokine receptors bind more than one chemokine, CC receptors bind only CC chemokines and CXC receptors bind only CXC chemokines.1Baggiolini MB Dewald B Moser B Human chemokines: an update.Ann Rev Immunol. 1997; 15: 675-705Crossref PubMed Scopus (1994) Google Scholar, 3Luster AD Chemokines: chemotactic cytokines that mediate inflammation.New Engl J Med. 1998; 338: 436-445Crossref PubMed Scopus (3272) Google Scholar The chemokine receptor CCR3, which binds eotaxin, RANTES, monocyte chemotactic protein (MCP)-3, MCP-4, and some other chemokines, is expressed in human eosinophils,4Kitamura M Nakajima T Imai T Harada S Combadiere C Tiffany HL Murphy PM Yoshie O Molecular cloning of human eotaxin, an eosinophil-selective CC chemokine and identification of a specific eosinophil eotaxin receptor.J Biol Chem. 1996; 271: 7725-7730Crossref PubMed Scopus (372) Google Scholar, 5Ponath PD Qin SX Post TW Wang J Wu L Gerard NP Newman W Gerard C Mackay CR Molecular cloning and characterization of a human eotaxin receptor expressed selectively on eosinophils.J Exp Med. 1997; 183: 2437-2448Crossref Scopus (553) Google Scholar, 6Daugherty BL Siciliano SJ DeMartino JA Malkowitz L Sirotina A Springer MS Cloning, expression, and characterization of the human eosinophil eotaxin receptor.J Exp Med. 1996; 183: 2349-2354Crossref PubMed Scopus (502) Google Scholar basophils,7Uguccioni M Mackay CR Ochensberger B Loetscher P Rhis S LaRosa GJ Rao P Ponath PD Baggiolini M Dahinden CA High expression of the chemokine receptor CCR3 in human blood basophils. Role in activation by eotaxin, MCP-4 and other chemokines.J Clin Invest. 1997; 100: 1137-1143Crossref PubMed Scopus (438) Google Scholar and type 2 T helper (Th2) cells.8Sallusto F Lanzavecchia A Selective expression of the eotaxin receptor CCR3 by human T helper 2 cells.Science. 1997; 277: 2005-2007Crossref PubMed Scopus (942) Google Scholar, 9Gerber BO Zanni MP Uguccioni M Loetscher P Mackay CR Pichler WJ Yawalkar N Baggiolini B Moser B Functional expression of the eotaxin receptor CCR3 in T lymphocytes co-localizing with eosinophils.Curr Biol. 1997; 7: 836-843Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar In this study, by using immunohistochemistry and flow cytometry, we did not detect CCR3 expression on human Th2 cells in vitro or in vivo, whereas we found that CCR3 is expressed by basophils and, at the tissue level, by eosinophils. In addition, we demonstrate CCR3 expression on remarkable proportions of mast cells (MC) in different human tissues and on isolated cells. Although eotaxin apparently did not induce histamine release by purified human MC, both eotaxin and the CCR3 ligand, RANTES, exerted chemotactic activity on these cells. Interestingly, CCR3 expression was virtually limited to tryptase-chymase double-positive mast cells (MCTC), suggesting that migration and persistence of this MC subset into non-inflamed and inflamed tissues may depend largely on its CCR3 expression. Anti-CCR3 monoclonal antibody (mAb) (7B11; IgG2a) was kindly provided by Leukosite (Boston, MA); anti-human eosinophil cationic protein (ECP; clone EG2) and anti-tryptase mAb10Schwartz LB Sakai K Bradford TR Ren S Zweiman B Worobec AS Metcalfe DD The α form of human tryptase is the predominant type present in blood at baseline in normal subjects and is elevated in those with systemic mastocytosis.J Clin Invest. 1995; 96: 2702-2710Crossref PubMed Scopus (335) Google Scholar by Pharmacia & Upjohn (Uppsala, Sweden); anti-macrophage associated antigen mannose receptor (anti-PAM-1) mAb11Uccini S Sirianni MC Vincenzi V Stopacciaro A Lesnoni LA Parola L Capuana M Cerimele D Cells M Lanzavecchia A Allavena P Mantovani A Baroni CD Ruco LP Kaposi's sarcoma cells express the macrophage associated antigen mannose receptor and develop in peripheral blood cultures of Kaposi's sarcoma patients.Am J Pathol. 1997; 150: 929-938PubMed Google Scholar by A. Mantovani (Istituto Mario Negri, Milano, Italy); anti-FcɛRI α chain mAb (IgG1) by J. Hakimi (Hoffman-La Roche Inc., Nutley, NJ); rabbit anti-human-Fcɛ Ab by T. and K. Ishizaka (La Jolla, CA). Anti-CD3 mAb was purchased from Ancell (Bayport, MN); anti-chymase mAb from Chemicon International Inc. (Temecula, CA); eotaxin, RANTES, and MIP-1α from PeproTech EC LTD (London, UK); irrelevant anti-E-selectin mAb from R & D Systems (Minneapolis, MN); BSA, α-chymotrypsin, piperazine-N,N′-bis (2-ethanesulfonic acid) hyaluronidase, chymopapain, collagenase, elastase type I, and rabbit and sheep polyclonal nonimmune IgG from Sigma Chemical Co. (St. Louis, MO); FCS from Gibco (Grand Island, NY); deoxyribonuclease I and pronase from Calbiochem (La Jolla, CA); 60% HClO4 from Baker Chemical Co. (Deventer, The Netherlands); RPMI 1640 with 25 mmol/L Hepes buffer, Eagle's minimum essential medium (MEM) from Flow Laboratories (Irvine, Scotland); Dextran 70 and Percoll from Pharmacia Fine Chemicals (Uppsala, Sweden); Alcian Blue 8GX from Carlo Erba (Milan, Italy); recombinant human stem cell factor (SCF) from Amgen (Thousand Oaks, CA). PMA, ionomycin, brefeldin A, anti-glycophorin A and B mAbs, and saponin were purchased from Sigma. The PE-conjugated anti-IL-4 (3010.211, IgG1), FITC-conjugated anti-IFN-γ (25723.11, IgG2b), PerCP- and PE-conjugated anti-CD4 as well as the purified anti-CD8, anti-CD14, anti-CD20, anti-CD56 and anti-CD45Ro mAbs were purchased from Becton Dickinson (San Jose, CA). The purified IgG1 and IgG2a isotype control mAbs, as well as the FITC-conjugated anti-IgG2a and PE-conjugated IgG1 Abs, were purchased from Southern Biotechnology Associates (Birmingham, AL). The goat anti-mouse IgG Abs conjugated with magnetic beads (MACS) were purchased from Miltenyi Biotec (Bisley, Germany). The PIPES buffer used in the histamine release and chemotaxis experiments was made up of 24 mmol/L PIPES, pH 7.37, 110 mmol/L NaCl, 5 mmol/L KCl (P). The mixture which is referred to as P2CG contains in addition to P, human serum albumin (HSA) 3%, 2 mmol/L CaCl2, 1 g/l dextrose. pH was titrated to 7.4 with sodium bicarbonate. PACGM contains in addition to P, HSA 3%, 1 mmol/L CaCl2, 1 g/l dextrose and 0.25 g/l MgCl2×6H2O, pH 7.4; PGMD contains 0.25 g/l MgCl2×6H2O, 10 mg/l DNase, and 1 g/l gelatin in addition to P, pH 7.37.12De Paulis A Marinò I Ciccarelli A de Crescenzo G Concardi M Verga L Arbustini E Marone G Human synovial mast cells. I. Ultrastructural in situ and in vitro immunologic characterization.Arthritis Rheum. 1996; 39: 1222-1233Crossref PubMed Scopus (72) Google Scholar, 13Patella V Casolaro V Ciccarelli A Petit GR Columbo M Marone G The antineoplastic bryostatins affect differently human basophils and mast cells.Blood. 1995; 85: 1272-1281PubMed Google Scholar Immunohistochemical analysis was performed on skin, gut, and lung tissues. Skin specimens were obtained from four patients with systemic sclerosis, one with lichen ruber planus and one with systemic mastocytosis, undergoing skin biopsy for diagnostic purpose. Gut specimens were obtained from biopsies of four patients with ulcerative colitis and from fragments of normal gut tissue of three patients undergoing colectomy because of colon cancer. Lung specimens were obtained from fragments of normal lung of three patients subjected to pneumectomy because of lung cancer. The procedures used in this study were in accordance with the criteria of the regional ethical committee on human experimentation. Immunohistochemical staining was performed according to a technique previously described.14Romagnani P Annunziato F Manetti R Mavilia C Lasagni L Manuelli C Vannelli GB Maggi E Pupilli C Romagnani S High CD30 ligand expression by epithelial cells and Hassal's corpuscles in the medulla of human thymus.Blood. 1998; 91: 3323-3332PubMed Google Scholar To this end, 10-μm cryostat sections were fixed in 4% paraformaldehyde for 20 minutes and subsequently exposed to 0.3% hydrogen peroxide-methanol solution to quench endogenous peroxidase activity. After a 30-minute preincubation with normal horse serum (Vectastain ABC kit; Vector Laboratories, DBA, Milan, Italy), sections were layered for 30 minutes with anti-CCR3 (0.2 μg/ml), anti-CD3, (5 μg/ml), anti-PAM-1 (5 μg/ml), anti-ECP (2 μg/ml), anti-tryptase (0.4 μg/ml) or anti-chymase (0.5 μg/ml) Abs, followed by biotinylated anti-mouse IgG Ab, and the avidin-biotin-peroxidase complex (Vectastain ABC kit), as described.13Patella V Casolaro V Ciccarelli A Petit GR Columbo M Marone G The antineoplastic bryostatins affect differently human basophils and mast cells.Blood. 1995; 85: 1272-1281PubMed Google Scholar 3-amino-9-ethylcarbazole (AEC; Vector) or Vector SG were used as peroxidase substrates. Finally, sections were counterstained with Gill's hematoxylin or Methyl Green and mounted with Kaiser's glycerol gelatin. All incubations were performed at room temperature. As negative control, primary mAb was replaced with an isotype-matched antibody with irrelevant specificity or mouse ascites fluid. Double immunostaining was performed with anti-CCR3 and anti-CD3, anti-CCR3 and anti-ECP, anti-CCR3 and anti-tryptase, and anti-CCR3 and anti-chymase Ab by using the avidin-biotin-peroxidase system with two different substrates, as described.14Romagnani P Annunziato F Manetti R Mavilia C Lasagni L Manuelli C Vannelli GB Maggi E Pupilli C Romagnani S High CD30 ligand expression by epithelial cells and Hassal's corpuscles in the medulla of human thymus.Blood. 1998; 91: 3323-3332PubMed Google Scholar To identify the two molecules on the same specimen, the Vector SG (bluish-grey color) and the AEC (red color) substrates were used, respectively. Polyclonal CD4+ T cell lines were generated from UCB mononuclear cell (MNC) suspensions of four donors, as described.15Galli G Annunziato F Mavilia C Romagnani P Cosmi L Manetti R Pupilli C Maggi E Romagnani S Enhanced HIV expression during Th2-oriented responses explained by the opposite regulatory effect of IL-4 and IFN-γ on fusin/CXCR4.Eur J Immunol. 1998; 28: 3280-3290Crossref PubMed Scopus (70) Google Scholar Briefly, CD4+ CD45RA+ T cells were purified by negative magnetic selection using the MACS system after a two-step incubation with a mixture of anti-CD8, anti-CD14, anti-CD20, anti-CD56, antiCD45R0, anti-glycophorin A and B mAbs, followed by goat anti-mouse IgG conjugated with magnetic beads. Recovered cells (>99% CD3+ CD4+ CD45RA+) were then stimulated with PHA (0.1% vol/vol) and IL-2 (20 U/ml) in the presence of IL-4 (100 U/ml) in RPMI medium containing 10% heat-inactivated FCS (primary stimulation). On day 7, cells were washed and restimulated with PHA (0.1% vol/vol), IL-2 (20 U/ml), and IL-4 (100 U/ml) for 4 more days (secondary stimulation). Intracytofluorimetric analysis of IFN-γ and IL-4 synthesis at the single-cell level was performed as described elsewhere.15Galli G Annunziato F Mavilia C Romagnani P Cosmi L Manetti R Pupilli C Maggi E Romagnani S Enhanced HIV expression during Th2-oriented responses explained by the opposite regulatory effect of IL-4 and IFN-γ on fusin/CXCR4.Eur J Immunol. 1998; 28: 3280-3290Crossref PubMed Scopus (70) Google Scholar Briefly, T cell blasts were stimulated with PMA (10 ng/ml) plus ionomycin (1 μmol/L) for 4 hours, the last 2 of which were in the presence of brefeldin A (5 μg/ml). After incubation, cells were washed twice with PBS, pH 7.2, fixed 15 minutes with formaldehyde (2% in PBS, pH 7.2), washed twice with PBS, pH 7.2, permeabilized with PBS, pH 7.2, containing 0.5% BSA and 0.5% saponin, then incubated with the appropriate mAb. Cells were analyzed on a FACSCalibur cytofluorimeter using CellQuest software (Becton Dickinson). The area of positivity was determined using an isotype-matched Ab. A total of 104 events for each sample were acquired in all cytofluorimetric analyses. Basophils were purified from PB cells of normal subjects undergoing hemapheresis. “Buffy coat” cell packs from healthy volunteers, provided by the Immunohematology Service at the University of Naples Federico II, were reconstituted in PBS containing 0.5 g/l HSA and 3.42 g/l sodium citrate, and loaded onto a countercurrent elutriator (model J2-21, Beckman Instruments, Fullerton, CA). Several fractions were collected, and fractions containing basophils in large numbers (>20 × 106 basophils) and of good purity (>15%) were further enriched by discontinuous Percoll gradients.16de Paulis A Ciccarelli A de Crescenzo G Cirillo R Patella V Marone G Cyclosporin H is a potent and selective competitive antagonist of human basophil activation by FMLP.J Allergy Clin Immunol. 1996; 98: 152-164Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar Yields ranged from 3 to 10 × 106 basophils with purity usually higher than 65%, as assessed by basophil staining with Alcian Blue and counting in a Spiers-Levy eosinophil counter.16de Paulis A Ciccarelli A de Crescenzo G Cirillo R Patella V Marone G Cyclosporin H is a potent and selective competitive antagonist of human basophil activation by FMLP.J Allergy Clin Immunol. 1996; 98: 152-164Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar Lung tissue was obtained from 12 patients undergoing thoracotomy and lung resection. These samples were different from those used for immunohistochemical analysis. Macroscopically normal parenchyma was dissected free from pleura, bronchi, and blood vessels, and minced into a single-cell suspension, as previously described.12De Paulis A Marinò I Ciccarelli A de Crescenzo G Concardi M Verga L Arbustini E Marone G Human synovial mast cells. I. Ultrastructural in situ and in vitro immunologic characterization.Arthritis Rheum. 1996; 39: 1222-1233Crossref PubMed Scopus (72) Google Scholar Yields with this technique ranged between 3 × 106 and 18 × 106 MC, and purities were between 1% and 8%. Human lung MC were purified by countercurrent elutriation (J2-21, Beckman Instruments) and then by discontinuous Percoll density gradient, as previously described.12De Paulis A Marinò I Ciccarelli A de Crescenzo G Concardi M Verga L Arbustini E Marone G Human synovial mast cells. I. Ultrastructural in situ and in vitro immunologic characterization.Arthritis Rheum. 1996; 39: 1222-1233Crossref PubMed Scopus (72) Google Scholar The final preparations contained >95% viable cells, as assessed by trypan blue exclusion method, and consisted of 25 to 96% MC. Flow cytometric analysis of cell surface molecules was performed as described elsewhere.15Galli G Annunziato F Mavilia C Romagnani P Cosmi L Manetti R Pupilli C Maggi E Romagnani S Enhanced HIV expression during Th2-oriented responses explained by the opposite regulatory effect of IL-4 and IFN-γ on fusin/CXCR4.Eur J Immunol. 1998; 28: 3280-3290Crossref PubMed Scopus (70) Google Scholar Briefly, after saturation of nonspecific binding sites with total rabbit IgG, cells were incubated for 20 minutes on ice with specific or isotype control Abs. For indirect staining this step was followed by a second incubation on ice with an appropriate anti-isotype-conjugated Ab. Finally, cells were washed and analyzed on a FACSCalibur cytofluorimeter using CellQuest software (Becton Dickinson). A total of 104 events for each sample were acquired in all cytofluorimetric analyses. Cells (∼3 × 104 MC per tube) were resuspended in P2CG, and 0.3 ml of the cell suspensions were placed in 12 × 75 mm polyethylene tubes (Sarstadt Inc., Princeton, NJ) and warmed to 37°C; 0.2 ml of each prewarmed releasing stimulus was added, and incubation was continued at 37°C.16de Paulis A Ciccarelli A de Crescenzo G Cirillo R Patella V Marone G Cyclosporin H is a potent and selective competitive antagonist of human basophil activation by FMLP.J Allergy Clin Immunol. 1996; 98: 152-164Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar The P2CG and PGMD buffers12De Paulis A Marinò I Ciccarelli A de Crescenzo G Concardi M Verga L Arbustini E Marone G Human synovial mast cells. I. Ultrastructural in situ and in vitro immunologic characterization.Arthritis Rheum. 1996; 39: 1222-1233Crossref PubMed Scopus (72) Google Scholar, 13Patella V Casolaro V Ciccarelli A Petit GR Columbo M Marone G The antineoplastic bryostatins affect differently human basophils and mast cells.Blood. 1995; 85: 1272-1281PubMed Google Scholar were used in these experiments. At the end of this step, the reaction was stopped by centrifugation (1,000 × g, 22°C, 1 minute), and the cell-free supernatants were stored at −20°C for subsequent assay of histamine content, with an automated fluorimetric technique.17Siraganian RP An automated continuous-flow system for the extraction and fluorometric analysis of histamine.Anal Biochem. 1974; 57: 383-394Crossref PubMed Scopus (664) Google Scholar Total histamine content was assessed by lysis induced by incubating the cells with 2% HClO4 before centrifugation. To calculate histamine release as a percentage of total cellular histamine, the spontaneous release of histamine from mast cells (2% to 14% of the total cellular histamine) was subtracted from both numerator and denominator.13Patella V Casolaro V Ciccarelli A Petit GR Columbo M Marone G The antineoplastic bryostatins affect differently human basophils and mast cells.Blood. 1995; 85: 1272-1281PubMed Google Scholar The percentage of histamine release was calculated according to the following equation: A-BT-B×100where A is the sample, B is the spontaneous histamine release, and T is the total histamine content. All values are based on means of duplicate or triplicate determinations. Replicated differed in histamine content by less than 10%. MC chemotaxis was performed using a modified Boyden chamber technique, as described previously.18Schleimer RP Freeland HS Peters SP Brown KE Perse CP An assessment of the effects of glucocorticoids on degranulation, chemotaxis to vascular endothelial cells and formation of leukotriene B4 by purified human neutrophils.J Pharmacol Exp Ther. 1992; 250: 598-604Google Scholar Briefly, 25 μl of PACGM buffer or various concentrations of the stimuli in the same buffer were placed in triplicate in the lower compartment of a 48-well micro-chemotaxis chamber (Neuroprobe, Cabin John, MD). The lower compartments were covered with a two-filter sandwich, a lower 5-μm pore size and an upper 8-μm pore size polycarbonate membranes (Nucleopore Corp., Pleasanton, CA), and then 50 μl of the cell suspension (5 × 104/well) resuspended in PACGM were pipetted into the upper compartments. The chemotactic chamber was then incubated for 3 hours at 37°C in a humified incubator with 5% CO2 (Automatic CO2 Incubator, Model 160 IR, ICN Flow). After the incubation period, the upper polycarbonate filter was discarded, while the lower nitrate cellulose filters were fixed in methanol, stained with Alcian Blue,19Stead RH Tomioka M Quinonez G Simon GT Felten SY Bienenstock J Intestinal mucosal mast cells in normal and nematode-infected rat intestines are in intimate contact with peptidergic nerves.Proc Natl Acad Sci USA. 1987; 84: 2975-2979Crossref PubMed Scopus (493) Google Scholar and then mounted on a microscope slide with Cytoseal (Stephen Scientific, Springfield, NJ). MC chemotaxis was quantitated microscopically by counting the number of cells that had traversed the upper 8-μm polycarbonate filter and were attached to the surface of the 5-μm cellulose nitrate filter. In each experiment, ten fields per triplicate filter were measured at 40× magnification. The results were compared with buffer controls. To discriminate between chemotaxis and nondirected migration (chemokinesis) of MC, checkerboard analyses were performed. In the experiments, MC were placed in the upper chambers and various concentrations of SCF (10 to 100 ng/ml), eotaxin (10 to 100 ng/ml), RANTES (10 to 100 ng/ml) or PACGM buffer was added into either the upper or lower wells or into both. Spontaneous migration (chemochinesis) was determined in the absence of chemoattractant or when stimuli were added into either the lower and upper chambers. The MC migratory response to SCF, RANTES, and eotaxin was largely due to chemotaxis and not to chemokinesis. Indeed, a checkerboard analysis, in which chemoattractants above and below the filter were varied, resulted in significant migration only when there was a gradient of the factor below the filters (data not shown). The results are expressed as means ± SE. Statistical significance was analyzed by one-way analysis of variance and, when the F value was significant, by Duncan's multiple range test. Differences were considered significant when P < 0.05. We used immunohistochemistry to investigate CCR3 expression in human skin, gut, and lung tissues. Several CCR3+ cells were detected in all tissues, but were more abundant in the skin and in the intestinal submucosa than in intestinal mucosa or in the lung (Figure 1 A–D). To identify the nature of CCR3+ cells in the various tissues, we used double immunostaining with anti-CCR3 and anti-CD3 Ab (for T cells) or CCR3 and anti-ECP Ab (for eosinophils). Cells staining for both CCR3 and ECP were absent from normal tissues, but they were clearly detectable in the gut of patients with ulcerative colitis. However, several CCR3+ cells in the gut of these patients, as well as the vast majority of those present in the other tissues tested, were ECP-negative (Figure 1E). Cells staining for both CCR3 and CD3 were virtually absent from every tissue examined, including the gut of patients with ulcerative colitis (data not shown), where CCR3+ T cells have been reported,9Gerber BO Zanni MP Uguccioni M Loetscher P Mackay CR Pichler WJ Yawalkar N Baggiolini B Moser B Functional expression of the eotaxin receptor CCR3 in T lymphocytes co-localizing with eosinophils.Curr Biol. 1997; 7: 836-843Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar and the skin of patients with systemic sclerosis (Figure 1F), where the majority of infiltrating T cells express a Th2-type cytokine profile.20Mavilia C Scaletti C Romagnani P Carossino AM Pignone A Emmi L Pupilli C Pizzolo G Maggi E Romagnani S Type 2 helper T (Th2) cell predominance and high CD30 expression in systemic sclerosis.Am J Pathol. 1997; 151: 1751-1758PubMed Google Scholar Morphologically, the CCR3+ ECP− CD3− cells present in all tissues examined were usually larger than lymphocytes and eosinophils. In an attempt to identify these cells, we double-stained them with CCR3 and PAM-1, which is expressed by macrophages and dendritic cells.11Uccini S Sirianni MC Vincenzi V Stopacciaro A Lesnoni LA Parola L Capuana M Cerimele D Cells M Lanzavecchia A Allavena P Mantovani A Baroni CD Ruco LP Kaposi's sarcoma cells express the macrophage associated antigen mannose receptor and develop in peripheral blood cultures of Kaposi's sarcoma patients.Am J Pathol. 1997; 150: 929-938PubMed Google Scholar As shown in Figure 1G, CCR3 and PAM-1 staining was clearly distinct, indicating that these cells do not belong to the macrophage lineage. We then verified whether CCR3-expressing cells present in different tissues were MC. To this end, we first examined a skin biopsy specimen from a patient with systemic mastocytosis for CCR3-positive cells. CCR3 expression was found in a remarkable proportion of tryptase-positive cells in the skin of this patient (Figure 2, A and B). Therefore, we performed double immunostaining for tryptase and CCR3 on normal lung, skin, and gut specimens. In all tissues examined, variable proportions of tryptase-positive cells showed costaining for CCR3. A representative experiment showing double immunostaining for tryptase and CCR3 in the submucosa of normal gut is shown in Figures 2, C and D. MC-enriched suspensions were then prepared from normal lung tissue and assessed for CCR3 expression by flow cytometry. As controls, CCR3 expression was also assessed in basophil-enriched suspensions obtained from normal PB and in Th2-polarized short-term T cell lines generated from normal UCB CD4+ T lymphocytes. CCR3 expression occurred in the vast majority of FcɛRI+ cells in basophil-enriched PB suspensions (Figure 3A) and in a remarkable proportion of FcRɛl+ cells (25%) present in MC-enriched lung suspensions (Figure 3B). By contrast, despite their clearly polarized Th2 profile of cytokine production, short-term T cell lines generated from UC
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