CXCR3 and Its Ligand CXCL10 Are Expressed by Inflammatory Cells Infiltrating Lung Allografts and Mediate Chemotaxis of T Cells at Sites of Rejection
2001; Elsevier BV; Volume: 158; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)64126-0
ISSN1525-2191
AutoresCarlo Agostini, Fiorella Calabrese, Federico Rea, Monica Facco, Alicia Tosoni, Monica Loy, Gianni Binotto, Marialuisa Valente, Livio Trentin, Gianpietro Semenzato,
Tópico(s)Immune Cell Function and Interaction
ResumoThe attraction of T lymphocytes into the pulmonary parenchyma represents an essential step in mechanisms ultimately leading to lung allograft rejection. In this study we evaluated whether IP-10 (CXCL10), a chemokine that is induced by interferon-γ and stimulates the directional migration of activated T cells, plays a role in regulating the trafficking of effector T cells during lung allograft rejection episodes. Immunohistochemical examination showed that areas characterized by acute cellular rejection (grades 1 to 4) and active obliterative bronchiolitis (chronic rejection, Ca) were infiltrated by T cells expressing CXCR3, ie, the specific receptor for CXCL10. In parallel, T cells accumulating in the bronchoalveolar lavage of lung transplant recipients with rejection episodes were CXCR3+ and exhibited a strong in vitro migratory capability in response to CXCL10. In lung biopsies, CXCL10 was abundantly expressed by graft-infiltrating macrophages and occasionally by epithelial cells. Alveolar macrophages expressed and secreted definite levels of CXCL10 capable of inducing chemotaxis of the CXCR3+ T-cell line 300-19; the secretory capability of alveolar macrophages was up-regulated by preincubation with interferon-γ. Interestingly, striking levels of CXCR3 ligands could be demonstrated in the fluid component of the bronchoalveolar lavage in individuals with rejection episodes. These data indicate the role of the CXCR3/CXCL10 interactions in the recruitment of lymphocytes at sites of lung rejection and provide a rationale for the use of agents that block the CXCR3/CXCL10 axis in the treatment of lung allograft rejection. The attraction of T lymphocytes into the pulmonary parenchyma represents an essential step in mechanisms ultimately leading to lung allograft rejection. In this study we evaluated whether IP-10 (CXCL10), a chemokine that is induced by interferon-γ and stimulates the directional migration of activated T cells, plays a role in regulating the trafficking of effector T cells during lung allograft rejection episodes. Immunohistochemical examination showed that areas characterized by acute cellular rejection (grades 1 to 4) and active obliterative bronchiolitis (chronic rejection, Ca) were infiltrated by T cells expressing CXCR3, ie, the specific receptor for CXCL10. In parallel, T cells accumulating in the bronchoalveolar lavage of lung transplant recipients with rejection episodes were CXCR3+ and exhibited a strong in vitro migratory capability in response to CXCL10. In lung biopsies, CXCL10 was abundantly expressed by graft-infiltrating macrophages and occasionally by epithelial cells. Alveolar macrophages expressed and secreted definite levels of CXCL10 capable of inducing chemotaxis of the CXCR3+ T-cell line 300-19; the secretory capability of alveolar macrophages was up-regulated by preincubation with interferon-γ. Interestingly, striking levels of CXCR3 ligands could be demonstrated in the fluid component of the bronchoalveolar lavage in individuals with rejection episodes. These data indicate the role of the CXCR3/CXCL10 interactions in the recruitment of lymphocytes at sites of lung rejection and provide a rationale for the use of agents that block the CXCR3/CXCL10 axis in the treatment of lung allograft rejection. The bronchiolitis obliterans syndrome (BOS) is a fibroproliferative process sustained by the migration and proliferation of mesenchymal cells in the vascular and airway lumen that manifests clinically as an unexplained chronic graft deterioration.1Arcasoy SM Kotloff RM Lung transplantation.N Engl J Med. 1999; 340: 1081-1091Crossref PubMed Scopus (459) Google Scholar, 2Trulock EP Lung transplantation.Am J Respir Crit Care Med. 1997; 155: 789-818Crossref PubMed Scopus (536) Google Scholar, 3Heng D Sharples LD McNeil K Stewart S Wreghitt T Wallwork J Bronchiolitis obliterans syndrome: incidence, natural history, prognosis, and risk factors.J Heart Lung Transplant. 1998; 17: 1255-1263PubMed Google Scholar Although BOS represents an important cause of morbidity and mortality among lung allograft recipients,1Arcasoy SM Kotloff RM Lung transplantation.N Engl J Med. 1999; 340: 1081-1091Crossref PubMed Scopus (459) Google Scholar, 2Trulock EP Lung transplantation.Am J Respir Crit Care Med. 1997; 155: 789-818Crossref PubMed Scopus (536) Google Scholar, 3Heng D Sharples LD McNeil K Stewart S Wreghitt T Wallwork J Bronchiolitis obliterans syndrome: incidence, natural history, prognosis, and risk factors.J Heart Lung Transplant. 1998; 17: 1255-1263PubMed Google Scholar effector mechanisms triggering the local fibroproliferation are still debated. Because the acute cell-mediated rejection has been identified as an important risk factor for the development of BOS, it has been suggested that BOS is an immune-mediated complication of lung transplantation.3Heng D Sharples LD McNeil K Stewart S Wreghitt T Wallwork J Bronchiolitis obliterans syndrome: incidence, natural history, prognosis, and risk factors.J Heart Lung Transplant. 1998; 17: 1255-1263PubMed Google Scholar, 4Kelly K Hertz MI Obliterative bronchiolitis.Clin Chest Med. 1997; 18: 319-338Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar In particular, as in other pulmonary-immunomediated disorders characterized by a fibroproliferative response, activated macrophages and T cells are likely to play a pathogenetic role in mediating an early hypersensitivity reaction, perhaps triggered by epithelial expression of donor-specific major histocompatibility complex molecules.2Trulock EP Lung transplantation.Am J Respir Crit Care Med. 1997; 155: 789-818Crossref PubMed Scopus (536) Google Scholar, 4Kelly K Hertz MI Obliterative bronchiolitis.Clin Chest Med. 1997; 18: 319-338Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar Despite immunosuppressive therapy, the presence of perivascular, interstitial, and intra-alveolar lymphocytic infiltrate may in fact persist in approximately a third of rejecting patients.1Arcasoy SM Kotloff RM Lung transplantation.N Engl J Med. 1999; 340: 1081-1091Crossref PubMed Scopus (459) Google Scholar Hypersensitivity reaction against alloantigens is followed by local injury, uncontrolled fibroproliferative repair processes, and the consequent filling of the airway lumen.5Kelly KE Hertz MI Mueller DL T-cell and major histocompatibility complex requirements for obliterative airway disease in heterotopically transplanted murine tracheas.Transplantation. 1998; 66: 764-771Crossref PubMed Scopus (58) Google Scholar Given the above, it is accepted that the severity and persistence of lymphocyte infiltrates represent the limiting factors determining the extension of the tissue involvement by local hypersensitivity reactions and driving the permanent structural alterations of the respiratory tract. The central event in the pathogenesis of pulmonary T cell responses is the organ-specific traffic of T lymphocytes.6Semenzato G Chemotactic cytokines: from the molecular level to clinical use.Sarcoidosis Vasc Diffuse Lung Dis. 1998; 15: 131-133PubMed Google Scholar Recently, a number of chemoattractants for leukocytes have been described. These molecules belong to the superfamily of chemokines that can be divided into four groups (C, CC, CXC, and CX3C), according to the positioning of the cysteine residues in the mature protein.7Kim CH Broxmeyer HE Chemokines: signal lamps for trafficking of T and B cells for development and effector function.J Leukoc Biol. 1999; 65: 6-15PubMed Google Scholar, 8Baggiolini M Chemokines and leukocyte traffic.Nature. 1998; 392: 565-568Crossref PubMed Scopus (2421) Google Scholar, 9Luster A Chemokines-chemotactic cytokines that mediate inflammation.N Engl J Med. 1998; 228: 436-445Google Scholar, 10Zlotnik A Morales J Hedrick JA Recent advances in chemokines and chemokine receptors.Crit Rev Immunol. 1999; 19: 1-47Crossref PubMed Google Scholar Expression of chemokine receptors on the cell surface of pulmonary immunocompetent cells and interactions with their specific ligands are involved in the mechanisms accounting for the accumulation of inflammatory cells within the lung microenvironment and the establishment of local hypersensitivity reactions.6Semenzato G Chemotactic cytokines: from the molecular level to clinical use.Sarcoidosis Vasc Diffuse Lung Dis. 1998; 15: 131-133PubMed Google Scholar Although the interactions of chemokine receptors are often characterized by considerable promiscuity, a human chemokine receptor that is selectively involved in the recruitment of T cells is CXCR3.11Farber JM Mig and IP-10: CXC chemokines that target lymphocytes.J Leukoc Biol. 1997; 61: 246-257Crossref PubMed Scopus (693) Google Scholar, 12Loetscher M Gerber B Loetscher P Jones SA Piali L Clark-Lewis I Baggiolini M Moser B Chemokine receptor specific for IP-10 and Mig: structure, function and expression in activated T-lymphocytes.J Exp Med. 1996; 184: 963-969Crossref PubMed Scopus (1074) Google Scholar CXCR3 is selectively expressed by activated T lymphocytes, and, in turn, its ligands interferon (IFN)-γ-inducible protein-10 (IP-10, CXCL10 according to the new nomenclature of chemokine and chemokine receptors,13Murphy PM Baggiolini M Charo IF Hebert CA Horuk R Matsushima K Miller LH Oppenheim JJ Power CA International union of pharmacology. XXII. Nomenclature for chemokine receptors.Pharmacol Rev. 2000; 52: 145-176PubMed Google Scholar), monokine induced by IFN-γ (Mig/CXCL9), interferon-inducible T-cell α-chemoattractant (I-TAC/CXCL11) are specifically chemotactic for activated T cells. In this study, using cells recovered from the bronchoalveolar lavage (BAL) and related biopsy specimens, we have evaluated the role of local CXCR3/CXCL10 receptor interactions in the immunological events ultimately leading to lung allograft rejection. We have shown that T cells accumulating in the lower respiratory tract of patients with acute lung allograft rejection, as well as in individuals suffering from BOS, express a functional CXCR3 because they are able to migrate in response to CXCL10. Furthermore, the specific CXCR ligand CXCL10 was abundantly expressed by graft-infiltrating macrophages of patients showing T cell infiltrate. According to our protocol, 56 consecutive transbronchial biopsies and BALs were performed during the follow-up of 24 lung allograft recipients (14 men and 10 women) who underwent surveillance bronchoscopy for clinical suspicion of infection or rejection. All patients underwent immunosuppression with rabbit anti-thymocyte globulin postoperatively, followed by a triple immunosuppression regimen of cyclosporin A or tacrolimus, azathioprine or RAD, and prednisone. Transbronchial biopsies (average, 6.1; range, 5.0 to 11.0) were taken from each lobe and serial sections were used for histological diagnosis and immunohistochemical analysis. Biopsy samples were evaluated according to the revised International Society for Heart and Lung Transplantation working formulation.14Yousem SA Berry GJ Cagle PT Chamberlain D Husain AN Hruban RH Marchevsky A Ohori NP Ritter J Stewart S Tazelaar HD Revision of the 1990 working formulation for the classification of pulmonary allograft rejection: Lung Rejection Study Group.J Heart Lung Transplant. 1996; 15: 1-15PubMed Google Scholar Overall, 47 of 58 biopsy samples showed no histological signs of rejection and eight showed acute rejection ISHLT grade A1 (n = 3), A2 (n = 3), and A3 (n = 2). Three patients were classified as suffering from BOS. BAL was performed according to the technical recommendations and guidelines for the standardization of BAL procedures.15Costabel U Danel C Haslam P Higgenbottam T Klech H Pohl W Rennard S Rossi G Rust M Semenzato G Technical recommendations and guidelines for bronchoalveolar lavage (BAL). Report of the European Society of Pneumology Task Group on BAL.Eur Respir J. 1989; 2: 561-585PubMed Google Scholar Briefly, a total of 200 ml of saline solution was injected in 25-ml aliquots via fiberoptic bronchoscopy, with immediate vacuum aspiration after each aliquot. Immediately after the BAL, the fluid was filtered through gauze and the volume measured. A volume of 100 to 200 ml of BAL recovery and a sample of 50% of the instilled volume with a minimum of 50 ml was considered acceptable. The percentage of BAL recovery was 53.7 ± 4.2% and 55.1 ± 3.7% of the injected fluid in allograft recipients and control patients, respectively. Cells recovered from the BAL were washed three times with phosphate-buffered saline (PBS), resuspended in endotoxin tested RPMI 1640 (Sigma Chemical Co., St. Louis, MO) supplemented with 20 mmol/L HEPES andl-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal calf serum (ICN Flow, Costa Mesa, CA), and then counted. A standard morphological and immunological analysis of BAL cellular components was performed and included cell recovery; differential count of macrophages, lymphocytes, neutrophils, and eosinophils; and flow cytometry analysis of CD4/CD8 BAL T cell ratio. AMs and T cells were enriched from the BAL cell suspensions by rosetting with neuraminidase-treated sheep red blood cells followed by F/H gradient separations and removing residual CD3+ lymphocytes using high-gradient magnetic separation columns (Mini MACS; Miltenyi Biotec, Germany).16Agostini C Cassatella M Zambello R Trentin L Gasperini S Perin A Piazza F Siviero M Facco M Dziejman M Chilosi M Qin S Luster AD Semenzato G Involvement of the IP-10 chemokine in sarcoid granulomatous reactions.J Immunol. 1998; 161: 6413-6420PubMed Google Scholar After this multistep selection procedure >95% of the above cells were viable, as judged by the trypan blue exclusion test. Staining with mAb showed that >99% of purified lymphocytes were CD3+ T cells. The commercially available conjugated or unconjugated mAbs used belonged to the Becton Dickinson and PharMingen series and included: CD3, CD4, CD8, CD45R0, CD45RA, and isotype-matched controls. Anti-IL-4 and anti-IFN-γ mAbs were purchased from PharMingen (San Diego, CA). Purified rabbit anti-human CXCL10 polyclonal antibody (R&D Systems Inc., Minneapolis, MN) and anti-hCXCR3 mAb (R&D Systems Inc.) were also used. Expression of CXCR3 and CXCL10 was measured by permanent section immunohistochemistry with anti-CXCR3 and anti-IP10 antibodies. Four-μm thick paraffin-embedded sections were used for immunostaining using the standard avidin-biotin complex method (Vectastain ABC Kit; Vector Laboratories, Burlingame, CA). The reliability of both antibodies in paraffin sections was compared with cryostatic (frozen) lung sections of two patients who died from persistent acute airway rejection (A3) and active obliterative bronchiolitis (Ca), respectively. Sections were deparaffinized in xylene (5 minutes, three times) and rehydrated through graded ethanol (twice for 5 minutes in 100% ethanol, 3 minutes in 95% ethanol, 3 minutes in 70% ethanol, and 5 minutes in distilled H20). For the microwave antigen retrieval procedure, slides were placed in a 2-L glass beaker containing 0.01 mol/L citrate buffer, pH 5.9, and microwaved at full power (800 W for 5 minutes, three times) before cooling and equilibration in PBS. To neutralize endogenous peroxidase activity, slides were pretreated with 3% hydrogen peroxide for 5 minutes. Primary antibodies were applied at the following concentrations: anti-hCXCR3 mAb 1:100 and anti-hIP-10/CXCL10 polyclonal 1:100 for 1 hour in a humidified chamber at 37°C. Immunoreactivity was detected using biotinylated secondary antibodies (1:50 rabbit anti-goat and 1:1,000 dilution goat anti-rabbit in PBS-bovine serum albumin buffer) incubated for 45 minutes followed by a 30-min incubation with avidin-peroxidase (1:200) and visualized by a 7-minute incubation with the use of 0.1% 3,3′-diaminobenzidene tetrahydrochloride as the chromogen. The intensity of antibody staining was classified in three groups: strong, weak, and negative. Parallel control slides were prepared either lacking primary antibody or lacking primary and secondary antibodies, or stained with normal sera to control for background reactivity. The frequency of BAL cells positive for the above reagents was determined by overlaying the flow cytometry histograms of the samples stained with the different reagents as previously reported.16Agostini C Cassatella M Zambello R Trentin L Gasperini S Perin A Piazza F Siviero M Facco M Dziejman M Chilosi M Qin S Luster AD Semenzato G Involvement of the IP-10 chemokine in sarcoid granulomatous reactions.J Immunol. 1998; 161: 6413-6420PubMed Google Scholar Cells were scored using a FACScan analyzer (Becton Dickinson, Mountain View, CA), and data were processed using the Macintosh CELLQuest software program (Becton Dickinson). The expression of cytoplasmic cytokine was evaluated after permeabilization of cell membranes using 1:2 diluted PermeaFix (Ortho, Raritan, NJ) for 40 minutes. After permeabilization procedures, anti-IL-4, anti-IFN-γ, and anti-CXCL10 antibodies were added. Because pulmonary cells bore cytoplasmic cytokines in a unimodal expression pattern, indicating that the entire cell population exhibits relatively homogeneous fluorescence, the percentage of positive cells does not represent the most accurate way of enumerating positive cells. The mean fluorescence intensity was used to compare the positivity of these specific antigens on different cell populations. To evaluate whether the shift of the positive cell peak was statistically significant, the Kolmogorov-Smirnov test for analysis of histograms was used according to the Macintosh CELLQuest software user's guide (Becton Dickinson). For immunofluorescence analysis, control IgG1 and IgG2a and IgG2b were obtained from Becton-Dickinson; control rat antiserum consisted of ascites containing an irrelevant rat IgG2b; control rabbit antiserum consisted of rabbit IgG (purified protein) purchased from Serotec (Serotec, UK); goat-anti-rabbit IgG and goat F(ab′)2 anti-rat IgG were obtained from Immunotech (Marseille, France). To verify the ability of AMs to release CXCL10, AMs (1 × 106/ml) were isolated from the BALs of allograft recipients, resuspended in RPMI medium, and cultured for 24 hours in 24-well plates at 37° in 5% CO2. In separate experiments AMs were stimulated with IFN-γ (100 U/ml), phorbol myrisate acetate (10 ng/ml), and lipopolysaccharide (10 μg/ml; Difco Labs., Detroit, MI). After the incubation period, supernatants were harvested, filtered through a 0.45-μm Millipore filter, and immediately stored at −80°C. At the end of the culture time AM viability was always >95%. Chemotactic activity of supernatants was determined as reported below. T-cell migration was measured in a 48-well modified Boyden chamber (AC48 Neuro Probe Inc.). The chamber is made of two sections: different chemotactic stimuli were loaded in the bottom section while cells were added in the top compartment. Polyvinylpyrrolidone-free polycarbonate membranes with 3- to 5-μm pores (for lung T cells obtained from allograft patients and the CXCR3+ and CXCR− T cell lines, respectively; Osmonics, Livermore, CA) and coated with fibronectin were placed between the two chamber parts. Only the bottom face of filters was pretreated with fibronectin; the fibronectin pretreatment maximizes attachment of migrating cells to filters, avoiding the possibility that they may not adhere. Using this procedure in preliminary experiments we demonstrated that only a trivial number of cells may be recovered in the bottoms of the wells. To avoid the shedding of fibronectin, fibronectin-treated filters were extensively washed. In preliminary experiments, fibronectin-treated filters did not induce spontaneous chemotaxis in absence of chemokines. To evaluate the migratory properties of pulmonary T lymphocytes rhIP-10/CXCL10 (200 ng/ml) were used. The CXCR− and CXCR3+ cell lines (300-19, kindly provided by Dr. B. Moser, Theodor-Kocher Institute, University of Bern, Switzerland) were used as negative and positive controls. Thirty μl of chemokines or control medium were added to the bottom wells, and 50 μl of 5.0 × 106 cells/ml T cells or CXCR−/+ cells resuspended in RPMI 1640 were added to the top wells. The chamber was incubated at 37°C with 5% CO2 for 2 hours. The membranes were then removed, washed with PBS on the upper side, fixed, and stained with DiffQuik (Dade AG, Düdingen, Switzerland). Cells were counted in three fields per well at ×800 magnification. All assays were performed in triplicate. In blocking experiments, cell suspensions were preincubated before chemotaxis assay for 30 minutes at 4°C with anti-human CXCR3 mAb at the concentration of 20 μg/ml. The CXCR− and CXCR3+ cell lines were also used to evaluate both the chemotactic activities of macrophage supernatants and the fluid component of BAL samples. Supernatants from cell cultures and the fluid components of BALs were obtained as reported above and used undiluted; different concentrations of CXCL10 were used as a positive control. Chemotactic assays were performed as reported above. In blocking experiments, anti-CXCL10 was added to the cell supernatants before chemotaxis assay at the concentration of 20 μg/ml. Data were analyzed with the assistance of the Statistical Analysis System. Data are expressed as mean ± SD. Mean values were compared using the analysis of variance test. A P value A2>A1). Strongly stained infiltrates of CXCR3+ lymphocytes were also seen in active obliterative bronchiolitis (Figure 1, c and d). Frozen lung samples showed staining intensity and distribution similar to the formalin-fixed lung biopsies used in the study. Flow cytometry analysis of BAL T lymphocytes obtained from allograft recipients who showed histological signs of rejection showed that T cells accumulating into alveolar air spaces were CXRC3+ (Figure 2) and bore IFN-γ but not interleukin (IL)-4, a pattern that is characteristic of Tc1/Th1 cells. BAL T cells of patients with BOS expressed CXCR3 at high density as well as cytoplasmic IFN-γ (Figure 2). CXCR3 was found to be expressed at higher intensity in BAL T cells of patients with higher grade acute rejection than in patients with lower grade rejection, the differences in the mean fluorescence intensity of CXCR3+ peaks being significant in the Kolmogorov-Smirnov analysis (P < 0.01). Although CXCR3 was highly expressed by patients with T cell alveolitis, normal BAL T cells and T cells from patients with normal cellular recovery expressed low or insignificant levels of CXCR3 at the Kolmogorov-Smirnov analysis. To characterize the biological properties of CXCR3, highly purified T cells isolated from the BALs of patients with allograft rejection and T cell alveolitis were assessed for their migratory capabilities in response to different concentrations of CXCL10. The evaluation of the migratory capabilities of pulmonary T cells in normal patients or patients with no signs of rejection was prevented by the low recovery of pulmonary T cells from the BAL. For this reason, the 300-19 T cell lines expressing high levels of CXCR3 or not expressing CXCR3 were used as positive and negative controls for the in vitro chemotaxis assay. As shown in Figure 3, pulmonary T cells purified from the BAL of patients with grade A1 to A3 and BOS exhibited a definite migratory capability in response to CXCL10. The migratory capability was influenced by CXCR expression. In fact, the blocking of the CXCR3 receptor with specific antibodies determined a marked inhibition of CXCL10-induced chemotaxis. These data suggest that pulmonary T cells infiltrating lung allografts express a functional CXCR3 receptor that induces migration in response to CXCL10. In a second set of experiments we evaluated which inflammatory cells infiltrating lung allograft express CXCL10 during rejection episodes. The expression of CXCL10 was observed in macrophage-infiltrating lung biopsies of patients with histological evidence of A1-A3 rejection (Figure 4) or with BOS; interestingly, also a few epithelial cells expressed CXCL10. Flow cytometry analysis confirmed the selective expression of CXCL10. As shown in Figure 5, macrophages isolated from the BAL of recipients with rejection episodes or BOS expressed CXCL10. Cell-free supernatants were obtained from 24-hour cultured AMs and tested for their capabilities of inducing T cell migration. Supernatants obtained from AMs of patients with acute rejection episodes or BOS exerted significant chemotactic activity on the CXCR3+ cell line (Figure 6); the CXCR3-negative cell line did not migrate in the presence of supernatants (data not shown). The addition of an anti-CXCL10 neutralizing antibody (Figure 6), but not of a control antibody, inhibited chemotactic activities of supernatants (data not shown). The inhibitory activity shown by the neutralizing antibody was not complete (range, 46 to 79%). This suggests that other CXCR3 ligands, including CXCL9 and CXL11, are likely to be present in conditioned media and interact with the CXCR3+ T cell clone, mediating its migration. When macrophages were cultured in the presence of IFN-γ the chemotactic activity of cell-free supernatants was higher than the levels obtained from unstimulated AMs (46.7 ± 12.3 versus 58.0 ± 9.1 number of migrating CXCR3+ cells/high-powered field; mean ± SD of pooled results obtained in different lung allograft recipients;P < 0.01). Again, the chemotactic activity of conditioned medium obtained by IFN-γ stimulated AMs was only partially inhibited by the pretreatment with the anti-CXCL10 antibody, suggesting the presence of other CXCR3 ligands in the supernatants. As a confirmation, the addition of an anti-CXCL9 antibody also inhibited chemotactic activities of supernatants (data not shown). Taken together these data suggest the need to evaluate the role of other non-ERL chemokines in the pathogenesis of allograft rejection. To evaluate whether CXCR3 ligands are released in the pulmonary microenvironment in vivo, the fluid component of nine BAL samples was evaluated for chemotactic activity on the CXCR3+ cell line (Figure 7). A definite biological activity was demonstrated in the samples of patients with acute rejection or BOS; this activity was partially inhibited by an anti-CXCL10 neutralizing antibody indicating the presence of other CXCR3 ligands in the fluid component of BAL. This study provides the first evidence that alloimmune T cells trafficking into lung allografts express CXCR3 and migrate in response to CXCL10. In addition, the chemokine is expressed and actively released by graft-infiltrating macrophages during rejection episodes in the lung microenvironment. As recently reviewed,17Tiroke AH Bewig B Haverich A Bronchoalveolar lavage in lung transplantation. State of the art.Clin Transplant. 1999; 13: 131-157Crossref PubMed Scopus (70) Google Scholar BAL associated with transbronchial biopsies may be useful for understanding the natural course of lung transplantation. During the first 3 months after pulmonary transplantation, elevated total cell counts in BAL and neutrophilic alveolitis are common features, representing the cellular response to the graft. Lymphocytic alveolitis with a decreased CD4/CD8 ratio suggests acute rejection, but is also detectable in viral pneumonia and obliterative bronchiolitis. In the case of a combined lymphocytosis and neutrophilia without any evidence of infection, BOS should be taken into account. It is herein shown that graft-infiltrating T cells express CXCR3 whenever a T cell alveolitis occurs. Episodes of grade A2 or greater rejection were associated with a striking expression of this chemokine receptor by pulmonary lymphocytes as well as with cases of patients suffering from BOS and showing BAL lymphocytosis and neutrophilia. We have also shown CXCR3 expression in allograft recipients with clinical signs of viral pneumonia associated with a lymphocytic alveolitis (data not shown). CXCR3 expression is likely to represent a common mechanism that is involved in the recruitment of activated T cells in the pulmonary microenvironment of lung allograft recipients. The continuous recruitment of Th1-type CXCR3+ T cells might play a role not only in the pathogenesis of acute rejection but also in favo
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