Pattern of Graft- and Host-Specific MHC Class II Expression in Long-Term Murine Cardiac Allografts
1998; Elsevier BV; Volume: 153; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)65547-2
ISSN1525-2191
AutoresSatoru Hasegawa, Gerold Becker, Hiroaki Nagano, P Libby, Richard Mitchell,
Tópico(s)Mast cells and histamine
ResumoIn solid-tissue allografts, donor vascular cells as well as recipient inflammatory cells can express MHC class II molecules. However, it is uncertain how much residual donor endothelium persists and to what extent donor versus recipient MHC class II expression can contribute to the ongoing immune response, especially in long-term grafts. To establish the origin of class-II-expressing cells in the allograft, we evaluated the expression of donor- or recipient-specific MHC class II molecules in murine cardiac allografts. Donor hearts from BALB/c (H-2d) mice were transplanted into C57BL/6 (B6, H-2b) recipients; B6 isografts served as controls. Untreated allografts ceased functioning at approximately 7 days with severe parenchymal rejection. Allografts from recipients treated with anti-CD4 and anti-CD8 MAbs after transplantation were explanted at 8 to 12 weeks and demonstrated intimal fibroproliferative lesions with a mild parenchymal mononuclear cell infiltrate. Class II expression in isografts was limited to epicardial macrophages. Both acutely rejecting and long-term allografts contained abundant macrophages expressing recipient class II molecules. Occasional cells (passenger leukocytes) in untreated, acutely rejecting allografts bore donor class II molecules; long-term allografts contained few such cells. In contrast, vascular endothelial and medial smooth muscle cells consistently expressed donor class II molecules. These results suggest that ongoing MHC class II expression in donor vascular cells, as well as in recipient macrophages, may contribute to sustained activation of host T cells with consequent release of cytokines that ultimately promote the development of graft arteriosclerosis. In solid-tissue allografts, donor vascular cells as well as recipient inflammatory cells can express MHC class II molecules. However, it is uncertain how much residual donor endothelium persists and to what extent donor versus recipient MHC class II expression can contribute to the ongoing immune response, especially in long-term grafts. To establish the origin of class-II-expressing cells in the allograft, we evaluated the expression of donor- or recipient-specific MHC class II molecules in murine cardiac allografts. Donor hearts from BALB/c (H-2d) mice were transplanted into C57BL/6 (B6, H-2b) recipients; B6 isografts served as controls. Untreated allografts ceased functioning at approximately 7 days with severe parenchymal rejection. Allografts from recipients treated with anti-CD4 and anti-CD8 MAbs after transplantation were explanted at 8 to 12 weeks and demonstrated intimal fibroproliferative lesions with a mild parenchymal mononuclear cell infiltrate. Class II expression in isografts was limited to epicardial macrophages. Both acutely rejecting and long-term allografts contained abundant macrophages expressing recipient class II molecules. Occasional cells (passenger leukocytes) in untreated, acutely rejecting allografts bore donor class II molecules; long-term allografts contained few such cells. In contrast, vascular endothelial and medial smooth muscle cells consistently expressed donor class II molecules. These results suggest that ongoing MHC class II expression in donor vascular cells, as well as in recipient macrophages, may contribute to sustained activation of host T cells with consequent release of cytokines that ultimately promote the development of graft arteriosclerosis. In 1989, we hypothesized that an allogeneic response stimulated by foreign MHC class II molecules promotes the development of graft vascular disease.1Libby P Salomon RN Payne DD Schoen FJ Pober JS Functions of vascular wall cells related to development of transplantation-associated coronary arteriosclerosis.Transplant Proc. 1989; 21: 3677-3684PubMed Google Scholar We subsequently showed that the endothelium of vessels within transplanted human hearts indeed bears MHC class II molecules and that sustained allogeneic class II expression on the graft endothelium may contribute to the development of graft vascular disease.2Salomon RN Hughes CCW Schoen FJ Payne DD Pober JS Libby P Human coronary transplantation-associated arteriosclerosis.Am J Pathol. 1991; 137: 871-882Google Scholar These studies used reagents that recognize framework determinants on the MHC class II molecules rather than discrete polymorphic regions. Therefore, it remained uncertain to what extent donor vascular wall cells capable of instigating an immune response might persist in long-term allografts. Conversely, it was unknown how extensively host-derived vascular cells might insinuate into grafts, in which case host immune recognition would no longer occur. Moreover, the persistence of donor immunogenic passenger leukocytes in long-term allografts remains controversial. For these reasons, defining the origin of these cells in long-term allografts of solid organs has considerable importance. There is older evidence from a rat experimental model that the endothelium of long-term aortic allografts contains some recipient-derived cells.3Williams GM Krajewski CA Dagher FJ Ter Haar AM Roth JA Santos GW Host repopulation of the endothelium.Transplant Proc. 1971; 3: 869-872PubMed Google Scholar In contrast, recent reports using immunohistochemical staining for donor-specific MHC class II molecules have demonstrated that endothelial cells (ECs) lining vessels of acutely rejecting allografts in rodents are of donor origin.4Forbes RDC Gomersall M Darden AG Guttmann RD Multiple patterns of MHC class II antigen expression on cellular constituents of rat heart grafts.Transplantation. 1991; 51: 942-948Crossref PubMed Scopus (17) Google Scholar, 5Milton AD Fabre JW Massive induction of donor-type class I and class II major histocompatibility complex antigens in rejecting cardiac allografts in the rat.J Exp Med. 1985; 161: 98-112Crossref PubMed Scopus (176) Google Scholar, 6Xu R Burdick JF Scott A Beschorner WE Adler W Kittur DS Graft-specific MHC class II gene expression in response to allogeneic stimulus in heterotopic murine cardiac allografts.Immunology. 1992; 75: 361-365PubMed Google Scholar, 7Benson EM Colvin RB Russell PS Induction of IA antigen in murine renal transplants.J Immunology. 1985; 134: 7-9PubMed Google Scholar Nevertheless, it is unclear whether the ECs that line vessels of longer-term allografts originate from the donor and/or recipient. The availability of mouse strains of well defined histocompatibility haplotype, as well as allospecific antisera capable of selectively differentiating MHC molecules, should permit unambiguous determination of the animal of origin of all cell types. We have therefore used long-term total allogeneic-mismatched murine cardiac allografts to examine the pattern of donor versus recipient MHC class II expression in allograft ECs and vascular smooth muscle cells (SMCs) as well as allograft mononuclear inflammatory cells. Male BALB/c (B/c, H-2d) and C57BL/6 (B6, H-2b) mice were obtained from Taconic Farm (Germantown, NY) and were used at 10 to 12 weeks of age; body weight was approximately 25 g. B/c mice were used as allograft donors and B6 mice were used as recipients. These strains are disparate in major histocompatibility complex (MHC) class I, MHC class II, and multiple non-MHC alloantigens. B6 isografts were used as controls. The mice were maintained in the Harvard Medical School animal facilities on acidified water. Sentinel animals surveyed serologically for viral pathogens were negative in the room in which these mice were housed. All experiments conformed to animal care protocols approved by the institutional review group. Heterotopic cardiac transplantation was performed using a modification of the method described by Corry et al.8Corry RJ Winn HJ Russell PS Primary vascularized allografts of hearts in mice. The role of H-2D, H-2K and non H-2 antigens in rejection.Transplantation. 1973; 16: 343-350Crossref PubMed Scopus (791) Google Scholar In brief, donors and recipients were anesthetized with Metofan (Pittman-Moore, Mundelein, IL). Donor hearts were perfused with chilled and heparinized saline via the inferior vena cava and harvested after ligation of the vena cava and pulmonary veins. The aorta and pulmonary artery of donor hearts were anastomosed to the abdominal aorta and inferior vena cava, respectively, of recipients using microsurgical techniques. Ischemic time was routinely approximately 25 minutes, with a success (long-term survival) rate of approximately 90%. The viability of the cardiac allograft was assessed by daily abdominal palpation. Cessation of the graft heartbeat was defined as the day of graft failure, typically indicating severe acute rejection as determined by histopathological examination. Table 1 shows the various experimental groups. Immunosuppression was attained by weekly intraperitoneal injections of 0.2 ml of ascites or comparably concentrated antibody preparations containing anti-CD4 (GK1.5) and anti-CD8 (2.43) monoclonal antibodies (MAbs) beginning 4 days after transplantation.9Nagano H Libby P Taylor MK Hasegawa S Stinn JL Becker G Tilney NL Mitchell RN Coronary arteriosclerosis after T-cell-mediated injury in transplanted mouse hearts: role of interferon-γ.Am J Pathol. 1998; 152: 1187-1197PubMed Google Scholar This protocol permits a single 4-day early acute rejection episode, followed by complete CD4 and CD8 cell ablation and therefore long-term suppression of any further immune-specific response. The model permits long-term (at least 12 weeks) survival of complete allogeneic-mismatched allografts (not possible in our hands with other protocols thus far published) and yields graft arteriosclerosis lesions histologically identical to those seen with conventional pretransplant MAb treatment.9Nagano H Libby P Taylor MK Hasegawa S Stinn JL Becker G Tilney NL Mitchell RN Coronary arteriosclerosis after T-cell-mediated injury in transplanted mouse hearts: role of interferon-γ.Am J Pathol. 1998; 152: 1187-1197PubMed Google Scholar, 10Nagano H Mitchell RN Taylor MK Hasegawa S Tilney NL Libby P Interferon-γ deficiency prevents coronary arteriosclerosis but not myocardial rejection in transplanted mouse hearts.J Clin Invest. 1997; 100: 550-557Crossref PubMed Scopus (235) Google ScholarTable 1Strain Combinations, Treatment, and MorphologyCombinationTreatmentnDurationPRGADIsograft (B6 to B6)None412 weeks0 ± 00 ± 0Isograft (B6 to B6)Anti-CD4 MAb412 weeks0 ± 00 ± 0Anti-CD8 MAbAllograft (B/c to B6)None67.0± 0.2 days*Explanted at time of graft failure.2.8 ± 0.40.3 ± 0.1Allograft (B/c to B6)Anti-CD4 MAb68–12 weeks0.8 ± 0.31.5 ± 0.3Anti-CD8 MAbValues represent the mean ± SD. PR, parenchymal rejection; GAD, graft arterial diseases.* Explanted at time of graft failure. Open table in a new tab Values represent the mean ± SD. PR, parenchymal rejection; GAD, graft arterial diseases. GK1.5 (anti-CD4) and 2.43 (anti-CD8) MAbs for immunosuppression were prepared from hybridoma clones (American Type Culture Collection, Rockville, MD) and used as ascites preparations or from threefold concentrations of serum-free supernatants from an artificial capillary system (Cellmax, Celluco, Rockville, MD); antibodies for CD45, B220, Mac-3, and CD31 (PECAM-1) as well as biotinylated, fluorescein isothiocyanate (FITC)-conjugated or nonconjugated isotype- and species-specific secondary antibodies were purchased from PharMingen (San Diego, CA). Mouse monoclonal anti-smooth muscle α-actin antibody (1A4) was obtained from Sigma Chemical Co. (St. Louis, MO). Horseradish-peroxidase-conjugated anti-FITC antibodies were obtained from Boehringer Mannheim (Indianapolis, IN). Biotinylated, FITC-conjugated and nonconjugated MHC class II haplotype-specific MAbs as well as isotype-matched biotinylated, FITC-conjugated, and nonconjugated IgG2a and IgG2b control MAbs were purchased from PharMingen. The anti-I-Ab antibody was clone AF6–120.1, specific for the Aαb chain, (IgG2a isotype, κ light chain); according to the supplier, this antibody is cross-reactive with cells from H-2k and H-2umice and weakly reactive with cells from H-2p haplotype mice but not with cells from H-2d animals. The anti-I-Ad antibody was clone AMS-32.1 (IgG2bisotype, κ light chain); according to the supplier, this antibody is cross-reactive with cells from H-2f, H-2j, and H-2v mice and weakly reactive with cells from H-2k and H-2q haplotype mice but not with cells from H-2b animals. To verify directly lack of cross-reactivity under our immunostaining conditions, spleens from B6 and B/c mice were stained with both I-A-specific MAbs; as shown in Figure 1, each antibody specifically stains cells from only the appropriate MHC II haplotype animals. Grafts were explanted either at the time of cessation of heartbeat or at 8 or 12 weeks and were transversely sectioned. One-half of each heart was fixed in 10% buffered formalin for routine morphological examination; paraffin-embedded sections of the fixed tissue were stained with hematoxylin and eosin (H&E) or with an elastic fiber stain using Weigert's method. The other half of each heart was frozen in OCT compound (Ames Co., Division of Miles Laboratories, Elkhart, IN) and stored at −80°C. Six-micron-thick sections were fixed in cold acetone for 2 minutes, incubated at room temperature with 5% normal goat serum in PBS for 20 minutes to block nonspecific binding sites, and subsequently immunostained with rat MAbs for CD45, CD4, CD8, Mac-3, or CD31 followed by biotinylated goat anti-rat IgG using an avidin-horseradish peroxidase-biotin complex method11Hsu SM Raine L Fanger H The use of antiavidin antibody and avidin-biotin-peroxidase complex in immunoperoxidase techniques.Am J Clin Pathol. 1981; 75: 816-821Crossref PubMed Scopus (905) Google Scholar(Vector Laboratories, Burlingame, CA) and an aminoethyl carbazole substrate (Vector Laboratories); hematoxylin was used for counterstaining. Controls included comparable concentrations of nonspecific rat IgG in the first step. For immunohistochemical staining for smooth muscle α-actin, frozen sections were stained with mouse 1A4 antibody (IgG2a isotype) and then stained using biotinylated goat anti-mouse IgG2a antibody. Controls included comparable dilutions of nonspecific mouse IgG2aantibody in the first step. Immunohistochemical staining for MHC II was performed using directly biotin-conjugated antibodies to MHC II haplotypes I-Ab or I-Ad and the avidin-horseradish-peroxidase-biotin complex method.11Hsu SM Raine L Fanger H The use of antiavidin antibody and avidin-biotin-peroxidase complex in immunoperoxidase techniques.Am J Clin Pathol. 1981; 75: 816-821Crossref PubMed Scopus (905) Google Scholar Controls included biotin-conjugated isotype-matched non-specific antibody in the first step. Double-staining for MHC II and the EC marker CD31 was performed by sequential incubations with anti-CD31, biotin-conjugated anti-rat IgG, and avidin-alkaline-phosphatase-biotin complex method (Vector Laboratories) using the Vector Blue substrate kit (Vector Laboratories) and levamisole to block endogenous phosphatase activity. This treatment was followed by successive application of FITC-conjugated antibodies to MHC II haplotypes I-Ab or I-Ad and horseradish-peroxidase-conjugated anti-FITC; the slides were developed with the aminoethyl carbazole substrate. By this method, ECs stain blue, and MHC II expression is reflected by a red-brown stain; double-stained cells have a deep purple color. Controls included use of appropriately conjugated, isotype-matched irrelevant antibodies in the first step. For immunofluorescent labeling of MHC class II molecules, frozen sections were incubated with biotin-conjugated antibodies to MHC II haplotypes I-Ab or I-Ad, followed by streptavidin-conjugated Texas Red (Amersham Life Science, Cleveland, OH), using standard techniques. Controls included comparable concentrations of isotype-matched, biotin-conjugated nonspecific antibodies in the first step. Grafts were analyzed by standard H&E and elastin stains, and the severity of parenchymal rejection versus graft arterial disease (GAD) was scored. Parenchymal rejection was graded using a scale modified from the International Society for Heart and Lung Transplantation12Billingham ME Cary NRB Hammond ME Kemnitz J Marboe C McCallister HA Snovar DC Winters GL Zerbe A A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection study group.J Heart Lung Transplant. 1990; 9: 587-593Google Scholar (0, no mononuclear cell infiltrate; 1, mild interstitial or perivascular infiltrate without necrosis; 2, focal infiltrates with necrosis; 3, multifocal infiltrates with necrosis; 4, widespread infiltrates with hemorrhage and/or vasculitis), and a GAD score was calculated from the number and severity of involved vessels (0, 75% occlusion).10Nagano H Mitchell RN Taylor MK Hasegawa S Tilney NL Libby P Interferon-γ deficiency prevents coronary arteriosclerosis but not myocardial rejection in transplanted mouse hearts.J Clin Invest. 1997; 100: 550-557Crossref PubMed Scopus (235) Google Scholar All results are expressed as the mean ± SD. Numerical grades for the intensity and extent of staining for MHC class II expression in both parenchymal inflammatory cells and vessels were averaged from scores determined by three independent, blinded observers (Table 2).Table 2Grading Criteria for MHC Class II ExpressionGradeInflammatory cellsVessel0NoneNo staining1Rare positive cells ( 50%)Uniform and intense Open table in a new tab All isografts, whether recipients were given anti-CD4 and anti-CD8 MAbs or not, continued to beat until harvest at 12 weeks and showed neither mononuclear cell infiltrates nor graft vascular lesions (Table 1). Allografts in nonimmunosuppressed animals ceased functioning at approximately 7 days after transplantation and showed severe parenchymal rejection (Table 1; Figure 2A). Long-term allografts (8 to 12 weeks) in recipients immunosuppressed with weekly anti-CD4 and anti-CD8 MAbs beginning 4 days after transplantation exhibited an ongoing mononuclear cell infiltrate composed predominantly of macrophages.9Nagano H Libby P Taylor MK Hasegawa S Stinn JL Becker G Tilney NL Mitchell RN Coronary arteriosclerosis after T-cell-mediated injury in transplanted mouse hearts: role of interferon-γ.Am J Pathol. 1998; 152: 1187-1197PubMed Google Scholar Arteries in these allografts exhibited intimal fibroproliferative vascular lesions (Table 1; Figure 2B), resembling lesions seen in other murine models of graft arteriosclerosis, as well as typical human graft arteriosclerosis.2Salomon RN Hughes CCW Schoen FJ Payne DD Pober JS Libby P Human coronary transplantation-associated arteriosclerosis.Am J Pathol. 1991; 137: 871-882Google Scholar, 9Nagano H Libby P Taylor MK Hasegawa S Stinn JL Becker G Tilney NL Mitchell RN Coronary arteriosclerosis after T-cell-mediated injury in transplanted mouse hearts: role of interferon-γ.Am J Pathol. 1998; 152: 1187-1197PubMed Google Scholar, 10Nagano H Mitchell RN Taylor MK Hasegawa S Tilney NL Libby P Interferon-γ deficiency prevents coronary arteriosclerosis but not myocardial rejection in transplanted mouse hearts.J Clin Invest. 1997; 100: 550-557Crossref PubMed Scopus (235) Google Scholar Freshly explanted native B6 hearts showed no constitutive class II expression in parenchymal or vascular wall cells (Figure 3A). Class II expression in isografts was confined to infiltrating macrophages in the epicardium, and no vascular or myocardial cells showed staining for class II (Table 3 and Figure 3, B and C). The results were the same regardless of whether isografted animals received weekly anti-CD4 and anti-CD8 MAbs (Figure 3, B and C) or not (not shown).Table 3Summary of Immunohistochemical Staining for Donor- or Recipient-specific MHC IIMHC Class III-Ab (B6-specific)I-Ad (B/c-specific)Inflammatory cellsVesselInflammatory cellsVesselIsograft (B6 to B6)1.1 ± 0.20.1 ± 0.1NANAAllograft (B/c to B6); 7 days, with no treatment3.6 ± 0.30.1 ± 0.11.6 ± 0.53.1 ± 0.2Allograft (B/c to B6); 8 weeks, treated with MAbs3.6 ± 0.30.3 ± 0.20.9 ± 0.52.7 ± 0.7Allograft (B/c to B6); 12 weeks, treated with MAbs2.2 ± 0.80.0 ± 0.00.5 ± 0.22.0 ± 0.9Values represent the mean ± SD; grading is as in Table 2. NA, not applicable. Open table in a new tab Values represent the mean ± SD; grading is as in Table 2. NA, not applicable. Allografts in nonimmunosuppressed recipients failing at approximately 7 days displayed abundant mononuclear inflammatory infiltrates expressing I-Ab (recipient-specific) molecules. These mononuclear I-Ab-positive cells co-localized with cells stained for CD45 and Mac-3 and therefore largely represent activated macrophages (Table 3 and Figure 4, A, B, E, and G) although scattered diffuse CD4- and CD8-positive T cells were also present in the allografts (not shown). In contrast, ECs (CD31-positive) and SMCs (smooth muscle α-actin-positive) in the vessels showed intense I-Ad (donor-specific) staining (Table 3 and Figure 4, C, D, and F). Occasional inflammatory cells around vessels also expressed I-Ad and are interpreted to represent passenger leukocytes (Figure 4F). Allografts in immunosuppressed animals 8 weeks (Figure 5) and 12 weeks (Figure 6) after transplantation demonstrated I-Ab (recipient-specific) molecules on infiltrating macrophages (Mac-3 positive) in the expanded intima and around vessels, but not on ECs or medial SMCs (Table 3; Figure 5, Figure 6). In contrast, I-Ad (donor-specific) molecules were expressed in the vessel wall cells, including ECs and medial SMCs, but not in the perivascular infiltrates (Table 3; Figure 5, Figure 6). Twelve-week allografts in immunosuppressed recipients showed findings comparable to 8-week allografts but displayed overall lower levels of class II expression (Table 3; Figure 6). No myocyte staining for MHC II of either haplotype was ever seen, and in no case did allograft ECs or SMCs express recipient MHC class II molecules.Figure 6Immunostaining for cell type and MHC II expression in allografts 12 weeks after transplantation. Representative serial sections are of B/c allograft hearts transplanted into B6 recipients immunosuppressed with anti-CD4 and anti-CD8 MAbs. Magnification, ×200. A: Staining for CD45, showing leukocyte infiltration in the intima and perivascular area. B: Staining for Mac-3, showing abundant macrophage infiltration in the expanded intima and perivascular area. C:Staining for CD31 (PECAM-1), showing the endothelial layer of the artery and adjacent smaller vessels.D: Staining with 1A4, showing smooth muscle α-actin-positive cells in the media and the expanded intima of the vascular lesion.E to H: Immunofluorescent staining (Texas Red) for MHC class II molecules (E and F) and isotype-matched controls (G andH). E: Staining for I-Ab (recipient MHC class II). Inflammatory cells in the expanded intima and around the vessel express recipient MHC class II molecules; ECs and medial SMCs do not express recipient MHC class II molecules. F:Staining for I-Ad (donor MHC class II). ECs and medial SMCs in the vessel express donor MHC class II molecules intensely, although SMCs in the expanded intima do not express I-Ad molecules. The signal from parenchymal myocytes results from background autofluorescence (see G andH). G: Control for E, using nonspecific biotin-conjugated mouse IgG2a as the first antibody. H: Control for F, using nonspecific biotin-conjugated mouse IgG2b as the first antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To demonstrate unambiguously that vascular wall ECs were of donor origin, double-label immunohistochemistry was performed on allografts 12 weeks after transplantation. In this protocol, ECs are stained blue and MHC II positivity imparts a red-brown color; double-stained cells have a deep purple cast. Samples stained for CD31 and with nonspecific control antibodies isotype matched to the MHC-II-specific antibodies (Figure 7A) show a rim of EC staining on the vascular lumen and only background staining of the vessel wall and surrounding interstitial tissue. Double staining for CD31 and recipient MHC II (I-Ab) shows blue luminal staining of ECs as well as strong I-Ab positivity in intramural and perivascular inflammatory cells, demonstrating the host origin of the inflammatory cells; ECs are not double stained (Figure 7B). In contrast, staining for CD31 and donor MHC II (I-Ad) shows strong double staining in the vessel lumen, demonstrating the donor origin of the ECs (Figure 7C); as in Figure 6, SMCs expressed relatively low levels of donor MHC II. A number of cytokines can regulate MHC class II expression by the various constituent cells in allografts; for example, MHC II on human ECs is induced by interferon (IFN)-γ,13Pober JS Gimbrone Jr, MA Cotran RS Reiss CS Burakoff SJ Fiers W Ault KA Ia expression by vascular endothelium is inducible by activated T cells and by human γ-interferon.J Exp Med. 1983; 157: 1339-1353Crossref PubMed Scopus (502) Google Scholar and MHC II on murine macrophages may be increased by IFN-γ,14Cockfield SM Urmson J Pleasants JR Halloran PF The regulation of expression of MHC products in mice: factors determining the level of expression in kidneys of normal mice.J Immunol. 1990; 144: 2967-2974PubMed Google Scholar tumor necrosis factor-α,15Zimmer T Jones PP Combined effects of TNFα, prostaglandin E2, and corticosterone on induced Ia expression on murine macrophages.J Immunol. 1990; 145: 1167-1175PubMed Google Scholar, 16Freund Y Dedrick RL Jones PP cis-Acting sequences required for class II gene regulation by interferon-γ and tumor necrosis factor-α in a murine macrophage cell line.J Exp Med. 1990; 171: 1283-1299Crossref PubMed Scopus (33) Google Scholar and/or interleukin-4.17Stuart PM Zlotnik A Woodward JG Induction of class I and class II MHC antigen expression on murine bone marrow-derived macrophages by IL-4.J Immunol. 1988; 140: 1542-1547PubMed Google Scholar MHC class II expression on graft endothelium has particular importance as a major determinant of graft immunogenicity.2Salomon RN Hughes CCW Schoen FJ Payne DD Pober JS Libby P Human coronary transplantation-associated arteriosclerosis.Am J Pathol. 1991; 137: 871-882Google Scholar, 13Pober JS Gimbrone Jr, MA Cotran RS Reiss CS Burakoff SJ Fiers W Ault KA Ia expression by vascular endothelium is inducible by activated T cells and by human γ-interferon.J Exp Med. 1983; 157: 1339-1353Crossref PubMed Scopus (502) Google Scholar, 18Pober JS Collins T Gimbrone Jr, MA Libby P Reiss CS Inducible expression of class II major histocompatibility complex antigens and the immunogenicity of vascular endothelium.Transplantation. 1986; 41: 141-146Crossref PubMed Google Scholar, 19Pober JS Collins T Gimbrone Jr, MA Cotran RS Gitlin JD Fiers W Clayberger C Krensky AM Burakoff SJ Reiss CS Lymphocytes recognize human vascular endothelial and dermal fibroblast Ia antigens induced by recombinant immune interferon.Nature. 1983; 305: 726-729Crossref PubMed Scopus (398) Google Scholar Previous reports demonstrated that ECs in acutely rejecting allografts express donor-specific class II molecules.4Forbes RDC Gomersall M Darden AG Guttmann RD Multiple patterns of MHC class II antigen expression on cellular constituents of rat heart grafts.Transplantation. 1991; 51: 942-948Crossref PubMed Scopus (17) Google Scholar, 5Milton AD Fabre JW Massive induction of donor-type class I and class II major histocompatibility complex antigens in rejecting cardiac allografts in the rat.J Exp Med. 1985; 161: 98-112Crossref PubMed Scopus (176) Google Scholar, 6Xu R Burdick JF Scott A Beschorner WE Adler W Kittur DS Graft-specific MHC class II gene expression in response to allogeneic stimulus in heterotopic murine cardiac allografts.Immunology. 1992; 75: 361-365PubMed Google Scholar However, it was unclear whether donor or recipient ECs populated the engrafted vessels in solid organs transplanted for prolonged periods. This study used long-term (8- to 12-week) cardiac allografts in mice with MHC-mismatched donors and recipients to establish the origin of the cells that express class II molecules. Although MHC II expression is a critical component in the stimulation of CD4+ T cells, it should also be emphasized that a number of co-stimulator molecules, beyond the scope of this study (eg, CD40, CD80, and CD86), likewise modulate the local immune response. We have focused on MHC II expression because it plays a central role in T cell stimulation by foreign tissues; persistent donor MHC II would therefore be important in the pathology of long-term allograft failure. The immunosuppressive protocol used in these experiments permits a single, early 4-day episode of untreated rejection, followed by virtually complete CD4 and CD8 cell ablation and therefore long-term suppression of any further immune-specific response.9Nagano H Libby P Taylor MK Hasegawa S Stinn JL Becker G Tilney NL Mitchell RN Coronary arteriosclerosis after T-cell-mediated injury in transplanted mouse hearts: role of interferon-γ.Am J Pathol. 1998; 152: 1187-1197PubMed Google Scholar The model permits long-term (at least 12 weeks) survival of complete allogeneic-mismatched allografts. Moreover, this immunosuppression protocol yields graft arteriosclerosis lesions histologically identical to those seen with conventional pretransplant MAb treatment.9
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