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Airway epithelial cell damage mediated by antigen-specific T cells: implications in lung allograft rejection

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Airway epithelial cell damage mediated by antigen-specific T cells: implications in lung allograft rejection
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  Airway Epithelial Cell Damage Mediated byAntigen-Specific T Cells: Implications inLung Allograft Rejection Craig R. Smith, Andre´s Jaramillo, Brian F. Duffy,and T. Mohanakumar ABSTRACT:  The aim of this study is to assess themechanisms associated with airway epithelial cell (AEC)injury, which may have implications in lung allograftrejection. Three AEC lines, KDI-650, Beas-2B and A549were analyzed. Effect of cytokines on the expression of Fas,HLA class I, and HLA class II were assessed by flowcytometry. AEC-specific T cells were generated  in vitro and assessed for lysis by  51 Cr release assay. HLA class I andFas were expressed on all AEC lines. Beas-2B and A549expressed low levels of class II compared with KDI-650,which lack this expression. Expression of HLA class II wasaugmented on KDI-650 and Beas-2B by IFN-   treat-ment. AEC-specific T cells generated  in vitro  were pre-dominantly CD8  and lysed relevant AEC targets. Anti-HLA class I monoclonal antibodies inhibited the lysis of AEC by specific T cells while anti-Fas and anti-HLA classII monoclonal antibodies did not have any effect on the Tcell induced lysis of AECs. AECs cultured with superna-tant derived from T-cell cultures induced the expressionof Fas, HLA class I, as well as HLA class II. These resultssuggest AEC damage is mediated by AEC-specific T cellsprimarily by the conventional HLA class I/peptide com-plex and TCR interaction. Further, the factors released bythese T cells also induce the expression of Fas, as well asHLA class I and class II, which may have implications onthe outcome of the immune response against AECs. Human Immunology 61, 985–992 (2000).  © AmericanSociety for Histocompatibility and Immunogenetics,2000. Published by Elsevier Science Inc. KEYWORDS:  airway epithelial cells; T cells; cytokines;lung allograft ABBREVIATIONS AEC airway epithelial cellBOS bronchiolitis obliterans syndromeCTL cytotoxic T lymphocyte INTRODUCTION Epithelial cell injury and/or antigen expression are im-portant factors implicated in acute and chronic allograftrejection [1]. Airway epithelial cells (AEC), in particular,have been shown to be immunologic targets during lungallograft rejection [2–4]. Recent studies suggest thatimmunologically-mediated injury directed at AECs, en-dothelial cells, and smooth muscle cells of the lungallograft might be the underlying cause of the develop-ment of bronchiolitis obliterans syndrome (BOS) [5].  Invitro  studies describing mechanisms of AEC injury beforethe establishment of clinical symptoms may help ininstituting new therapeutic strategies for the treatmentof rejection.The AEC has many functions as a communicator withthe immune system given its first line of defense againstrespiratory antigens [6]. AEC injury may occur duringthe procurement, preservation, and/or the transplanta-tion phase. During the transplantation phase, in partic-ular, AECs of donor srcin are exposed to host immuneregulators, such as recipient T cells and cytokines. How-ever, the role of T cells responding to donor AEC are notwell characterized.We have previously demonstrated that CD8  cyto-toxic T lymphocytes (CTL) present in the bronchoalveo- From the Department of Surgery (C.R.S., A.J., T.M.), Department of  Pathology and Immunology (T.M.), and the HLA Laboratory (A.J.,B.F.D., T.M.), Washington University School of Medicine, St. Louis, MO,USA. Address reprint requests to: T. Mohanakumar, Ph.D., WashingtonUniversity School of Medicine, Department of Surgery, Box 8109-3328CSRB, 660 S. Euclid Avenue, St. Louis, MO 63110, USA; Tel: (314)362-8463; Fax: (314) 747-1560; E-Mail: kumart@msnotes.wustl.edu. Received March 1, 2000; revised July 20, 2000; accepted July 26, 2000. Human Immunology  61,  985–992 (2000)0198-8859/00/$–see front matter© American Society for Histocompatibility and Immunogenetics, 2000Published by Elsevier Science Inc. PII S0198-8859(00)00175-0  lar lavage fluid during rejection can also lyse HLA-matched AECs [7]. In this communication, we extendthese observations to define the AEC/T-cell interactionsin an allogeneic combination in order to assess the mech-anism of AEC damage. MATERIALS AND METHODSCell Lines The primary AEC line KDI-650 was produced in ourlaboratory from a normal human trachea, the SV40-transformed Beas-2B AEC line and the A549 lung ade-nocarcinoma cell line were obtained from ATCC (Rock-ville, MD, USA). All AEC lines were cultured in LHC-9medium as previously described [8]. Cytokines The effect of cytokines on the expression of Fas, HLAclass I, and HLA class II were determined by incubatingthe confluent AEC cultures with individual cytokines;IL-2 (20 U/ml; Chiron, Emoryville, CA, USA), IL-4(2.5    10 4 U/ml; Genzyme, Cambridge, MA, USA),IFN-   (200 U/ml; Genzyme), 10% T-Stim (Collabora-tive Biomedical Products, Bedford, MA, USA), and su-pernatant from AEC-specific T-cell cultures (1:1, 1:2,and 1:10 dilutions) for 48 h. Immunofluorescence and Flow Cytometry Cell surface expression of Fas, HLA class I, and HLA classII expression on AEC were determined by flow cytomet-ric analysis, using monoclonal antibodies against Fas(APO-1.3; Alexis Corporation, San Diego, CA, USA),HLA class I (W6/32) and HLA class II (KuIA2). AECswere incubated with respective monoclonal antibodiesfor 30 min at 4°C. Mouse IgG was used as a negativeisotype control. AEC were then washed and incubatedfurther with secondary antibody (fluorescein-conjugatedF(ab  ) 2  goat anti-mouse IgG) at a dilution of 1:100 for20 min at 4°C. AEC were washed three times and werefixed in 1% paraformaldehyde. Single-color flow cyto-metric analysis was performed using a FACScan flowcytometer (Becton Dickinson, Mountain View, CA,USA). In stimulation experiments, data is presented asthe mean fluorescence channel shift as compared withuntreated control cultures. Generation of AEC-Specific T Cells AEC-specific T cells were generated  in vitro  by cocultur-ing peripheral blood lymphocytes of normal healthy do-nors (10    10 6 cells) with irradiated AECs (10,000 rad,1    10 6 cells) for 7 days at 37°C in a CO 2  incubatorwith RPMI-1640 media supplemented with L-glu-tamine (2 mM), sodium pyruvate (1 mM), HEPES (2.5mM), penicillin (100 U/ml), streptomycin (100   g/ml), FIGURE 1  Constitutive expression of HLA class I, class II,and Fas by AEC lines. KDI-650 is a primary AEC line estab-lished from a normal human trachea. Beas-2B is a SV40-transformed AEC line and A549 is a human lung adenocarci-noma line. A human lymphoblastoid cell line was used as apositive control. Number of cells analyzed: 3    10 3 . 986  C.R. Smith et al.  and 10% normal human serum (C-six Diagnostics, Ger-mantown, WI, USA). T cells (1  10 6 ) were restimu-lated weekly with irradiated AECs (1  10 6 ) and autol-ogous human peripheral blood mononuclear cells(10  10 6 ) used as feeders in the presence of recombi-nant human IL-2 (30 U/ml). Cytotoxicity assays wereperformed after three stimulations. Cytotoxicity Assay AEC specific T cells were assessed for viability by trypanblue and used as effector cells in a standard CTL activityassay as described previously [9]. Briefly, effector cellsand  51 Cr-labeled AEC targets were added to 96-wellround-bottom culture plates at a ratio of 20:1, in trip-licate wells in 200   l of culture media. After 5 h of incubation in a 5% CO 2  incubator at 37°C, culturesupernatants were harvested and mixed with scintillationfluid at a 1:1 ratio. Counts were read using a MicrobetaPlus, liquid scintillation counter (Wallac Inc., Gaithers-burg, MD, USA). Labeling of target cells by  51 Cr andpercentage specific lysis was defined as specified previ-ously [9]. Blocking experiments were performed usingrespective blocking mAbs to HLA class I, class II, andFas. The targets were incubated with the antibodies (10  g/ml) for 1 h on ice prior to using them in the cyto-toxicity assay. Mouse IgG was used as negative isotypecontrol. RESULTSExpression of Fas, HLA Class I and Class IIby AECs As shown in Figure 1, all the AEC lines expressed highlevels of HLA class I. Beas-2B and A549 expressed lowlevels of HLA class II while KDI-650 lacks the expres-sion of HLA class II. All the AEC lines expressed lowlevels of Fas. High level expression of HLA class I andlow levels of HLA class II and Fas on AEC suggest a dualrole for these cells. First, these cells could be immuno-genic and generate AEC specific T cells and second, they FIGURE 2  Effect of cytokines on the expression of HLAclass II on AEC lines. AEC were cultured in the presence of cytokines for 48 h prior to analyzing the expression of receptormolecules. KDI-650 is a primary AEC line established from anormal human trachea. Beas-2B is a SV40-transformed AECline and A549 is a human lung adenocarcinoma line. Individ-ual values represent the mean    SD of three separate experi-ments. Number of cell analyzed, 3  10 3 ;  p  values for IFN-  and T-stim were  0.05 (Student’s  t  -test). Data is presented asthe mean fluorescence channel shift as compared with un-treated control cultures. 987 Epithelial Cell Damage Mediated by Antigen-Specific T Cells  could also become the targets for those T cells leading tolung epithelium injury. Effect of Cytokines on Expression of Fas, HLAClass I and Class II on Human AEC During rejection it is hypothesized that different cyto-kines may be secreted in the local environment and thatthose factors may play a role in the upregulation of various cell surface proteins and receptors contributing tothe T-cell mediated cell injury and outcome of the allo-graft. To determine the effect of cytokines on the expres-sion of Fas, as well as HLA class I and class II, wecultured AEC in the presence of cytokines as described inthe methods section. As shown in Figure 2, IFN-   andT-stim augmented the expression of HLA class II onKDI-650 and Beas-2B cell lines (  p    0.05) but not onA549 cell line. IL-2 and IL-4 did not increase the HLAclass II expression on the AEC lines (Figure 2). IFN-  ,T-stim, IL-2, and IL-4 cytokines have no significanteffect on the expression of HLA class I and Fas by AECs(data not shown). Lysis of AECs by Specific T Cells is Blocked byAnti-HLA Class I, but not by Anti-Fas Antibodies As AECs expressed high levels of HLA class I and lowlevels of HLA class II, we assessed their immunogenicityby generating AEC-specific T-cell lines  in vitro , by cocul-turing human peripheral blood lymphocytes with irra-diated AECs as stimulators in allogeneic combination.All the bulk lines raised against respective AEC werepredominantly CD8  cells. Antigen specific T-cell linesraised against KDI-650, Beas-2B, and A549 were 75%CD8  /20% CD4  , 80% CD8  /15% CD4  , and 70%CD8  /20% CD4  , respectively. All AEC-specific T-celllines were tested for cytotoxicity against relevant andirrelevant AEC targets using a standard  51 Cr releaseassays. As shown in Figure 3, all of the T-cell lines lysedrelevant targets specifically as compared with irrelevantthird party targets. The lysis was 21% for KDI-650,25% for Beas-2B, and 15% for A549, by respectiveeffectors (Figure 3). As shown in Figure 4, blockingexperiments revealed that the lysis of AEC targets byspecific T cells was inhibited using anti-HLA class ImAb by 52.6% for KDI-650, 70.0% for Beas-2B, and36.9% for A549 (  p  0.05). However, the anti-Fas and FIGURE 3  Lysis of AEC by specific T cells. The antigenspecific T cell bulk lines used as effectors were derived after thethird stimulation. The effector to target ratio was 20:1, withan incubation of 5 h, for all the bulk lines assessed. Individualvalues represent the mean    standard deviation of three sep-arate experiments. 988  C.R. Smith et al.  anti-HLA class II monoclonal antibodies had no inhibi-tory effect on lysis of AEC targets by AEC-specific T celllines analyzed (Figure 4). These results indicate that AECdamage is mediated by specific T cells primarily by theconventional HLA class I/peptide complex and TCRinteraction, but not through the Fas-FasL pathway. Increased Expression of HLA Class I, Class II, andFas on AEC Treated with AEC-Specific T-CellCulture Supernatants As we demonstrated that the T-cell lines generatedagainst AEC can lyse relevant targets, we also addressedwhether the factors released by this activated T cells inthe culture supernatants has any effect on the expressionof Fas, HLA class I, and HLA class II on AECs. Todetermine this, AEC were cultured in the presence of T-cell culture supernatants for 48 h. Subsequently, ex-pression of HLA class I, class II, and Fas were determinedusing flow cytometric analysis. As shown in Figure 5, Tcell culture supernatants augmented the expression of HLA class I, class II, and Fas on all AEC lines. Theseresults suggest that AEC specific activated T cells releasefactors that have a profound influence on the augmenta-tion of cell surface molecule expression, which mighthave important consequences in the immune responsegenerated against AECs. DISCUSSION Epithelial and endothelial cells of donor organs are po-tential targets for an allogeneic immune response in atransplant recipient. It has been suggested that the bron-chial epithelium is capable of eliciting an allogeneicimmune response and expresses a number of growthfactors that could potentially play a role in the develop-ment of BOS [10]. In the present study, we demonstratethat a primary AEC line (KDI-650) was immunogenic  invitro  for the generation of antigen specific T cells. Similarfindings were observed with the Beas-2B and A549 AEClines. Interestingly, expression of HLA antigens on renaltubular cells in culture has been associated with vulner-ability to cell mediated lysis [11]. Regarding the airwayepithelium, it has been demonstrated that HLA class Iand class II antigen expression is induced and correlateswith rejection, following lung transplantation in a rat FIGURE 4  Inhibition of CTL lysis of AEC targets by HLAclass I antibody. The antigen specific T cell bulk lines used aseffectors are derived after third stimulation. The effector totarget ratio was 20:1, with an incubation of 5 h, for all thebulk lines assessed. Mouse IgG was used as negative isotypecontrol. Individual values represent the mean    SD of threeseparate experiments. 989 Epithelial Cell Damage Mediated by Antigen-Specific T Cells
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