Beta2-microglobulin-deficient mice are resistant to bullous pemphigoid.

Recent understanding of the mechanism of immunoglobulin G (IgG) catabolism has yielded new insight into antibody-mediated diseases. We proposed that beta2-microglobulin (beta2m)-deficient mice have been protected from systemic lupus erythematosis (SLE)-like syndromes because they lack the beta2m-associated IgG protection receptor (FcRn) and therefore catabolize IgG, including pathogenic IgG autoantibodies, considerably more rapidly than normal mice. Such an hypothesis would predict that beta2m-deficient mice would also be resistant to experimental bullous pemphigoid, a disease with a pathogenesis thought to be much simpler than SLE, being the result of antibody directed toward a pathogenic epitope on the epidermal hemidesmosome that anchors basal keratinocytes to the basement membrane. To test this hypothesis, we administered pathogenic rabbit antibody directed toward the hemidesmosome to beta2m-deficient mice and to normal control mice, both intraperitoneally and intradermally, and assessed the mice clinically, histologically, and immunologically for manifestations of skin disease. We found that the beta2m-deficient mice were protected when the antibody was given intraperitoneally whereas intradermal administration resulted in blisters only slightly less severe than those seen in normal mice. These data would indicate that autoantibody-mediated inflammation might be prevented or controlled by appropriate modulation of FcRn function.

Human cytomegalovirus inhibits major histocompatibility complex class II expression by disruption of the Jak/Stat pathway.

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that is able to persist for decades in its host. HCMV has evolved protean countermeasures for anti-HCMV cellular immunity that facilitate establishment of persistence. Recently it has been shown that HCMV inhibits interferon gamma (IFN-gamma)-stimulated MHC class II expression, but the mechanism for this effect is unknown. IFN-gamma signal transduction (Jak/Stat pathway) and class II transactivator (CIITA) are required components for IFN-gamma-stimulated MHC class II expression. In this study, we demonstrate that both a clinical isolate and a laboratory strain of HCMV inhibit inducible MHC class II expression at the cell surface and at RNA level in human endothelial cells and fibroblasts. Moreover, reverse transcriptase polymerase chain reaction and Northern blot analyses demonstrate that neither CIITA nor interferon regulatory factor 1 are upregulated in infected cells. Electrophoretic mobility shift assays reveal a defect in IFN-gamma signal transduction, which was shown by immunoprecipitation to be associated with a striking decrease in Janus kinase 1 (Jak1) levels. Proteasome inhibitor studies with carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone suggest an HCMV-associated enhancement of Jak1 protein degradation. This is the first report of a mechanism for the HCMV-mediated disruption of inducible MHC class II expression and a direct virus-associated alteration in Janus kinase levels. These findings are yet another example of the diverse mechanisms by which HCMV avoids immunosurveillance and establishes persistence.

Viral effects on antigen processing.

Viruses have evolved numerous mechanisms that modulate MHC-mediated antigen presentation, which in turn protect infected cells from T-lymphocyte-mediated immunosurveillance. Recent studies of previously identified viral immunomodulatory proteins reveal the allelic specificity of these proteins, their ability to function in xenogeneic systems and the difficulty in translating in vitro data to in vivo models; moreover, new mechanisms of viral modulation of MHC expression have emerged.

Tumorigenicity in athymic mice of the human colon carcinoma cell line SW1116 expressing the tumor-associated antigenic determinant CA 19-9.

The human colorectal carcinoma cell line SW1116 under optimal growth conditions synthesized and shed antigens bearing the monoclonal antibody-defined carbohydrate determinant CA 19-9. Antigen expressing CA 19-9 in cell culture supernatant was quantitated by an immunoradiometric assay for CA 19-9. Injection of SW1116 cells s.c. into athymic BALB/c mice resulted in the growth of moderately differentiated tumors possessing a distinct morphological resemblance to a typical adenocarcinoma of the colon. Intervals to tumor appearance were dependent on inoculum dose, but 95% of mice at both 5 X 10(6) and 10(7) cells/mouse developed tumors within 14 to 21 days. CA 19-9 antigen was detected in the sera of all nude mice with SW1116 tumors, and antigen concentration correlated (r = 0.77) with tumor volume throughout the 9-week study. The half-life of this antigen in serum following tumor excision from nude mice was 6.5 +/- 1.5 (S.D.) hr. Carcinoembryonic antigen was also detected in serum from mice bearing SW1116 tumors by an immunoradiometric assay for carcinoembryonic antigen, but its concentration correlated (r = 0.86) with tumor volume for only the first 4 weeks of tumor growth. Significant levels of endogenous immunoglobulin G1 and immunoglobulin G3 antibodies to CA 19-9 antigen were found in the serum of nude mice with SW1116 tumors by radioimmunodiffusion, but no apparent relationship between antibody titer and tumor growth or CA 19-9 antigen level in serum was evident. This tumor model may be useful in devising radioimmunodetection and immunotherapeutic strategies for primary and metastatic human colon carcinomas.

Human cytomegalovirus inhibition of major histocompatibility complex transcription and interferon signal transduction.

Pathogens have evolved diverse mechanisms for escaping host innate and adaptive immunity. Viruses that maintain a persistent infection are particularly effective at disabling key arms of the host immune response. For example, the herpesviruses establish a persistent infection in human and animal hosts, in part through critical immunoevasive strategies. Cytomegalovirus, a beta-herpesvirus, impairs major histocompatibility complex (MHC) class I and class II antigen presentation by decreasing MHC expression on the surface of the infected cell, thus enabling infected cells to escape CD8+ and CD4+ T lymphocyte immunosurveillance. Moreover, cytomegalovirus blocks the interferon signal transduction pathway, thereby limiting the direct and indirect antiviral effects of the interferons. In this review, we focus on an emerging paradigm in which the effectiveness of viruses, particularly human cytomegalovirus, to escape antiviral immune responses is significantly enhanced by their ability to inhibit MHC transcription and interferon (IFN)-stimulated (JAK/STAT) signal transduction.

Human cytomegalovirus does not induce human leukocyte antigen class II expression on arterial endothelial cells.

Human cytomegalovirus (CMV) has been associated with allograft rejection and, in particular, with transplant-associated arteriosclerosis. However, the role CMV plays in the development of transplant-associated arteriosclerosis remains unclear. CMV can infect the endothelium, the interface between allograft tissue and the host immune cells, but the direct induction of endothelial human leukocyte antigen (HLA) class II by CMV remains controversial. Our previous studies with venous endothelial cells (EC) have shown that CMV does not directly induce this antigen on infected EC and, furthermore, renders these cells refractory to interferon (IFN)-gamma induction. However, questions have arisen regarding the relevance of these findings to arterial endothelia. Thus, we have extended these studies to determine whether similar interactions occur in arterial EC. EC derived from human coronary artery, aorta, and umbilical artery were assayed by immunofluorescence flow cytometry and dual immunohistochemical staining following IFN-gamma treatment and/or inoculation with CMV. Data generated by these experiments demonstrate that regardless of vascular origin: (1) CMV does not directly induce endothelial surface or cytoplasmic HLA class II, and (2) although uninfected arterial EC are HLA class II inducible by IFN-gamma, infected cells are completely refractory to this effect. These results suggest that CMV-mediated inhibition of HLA class II expression is a phenomenon shared by human arterial and venous endothelia of both fetal and adult origin.

Progressive histologic injury in kidneys from heart and liver transplant recipients receiving cyclosporine.

Cyclosporine (CsA) causes both acute and chronic nephrotoxicity. The goal of the present studies was to examine the nature of the chronic renal pathologic lesions of CsA nephrotoxicity and to determine whether these lesions progress with prolonged exposure to the drug. With this purpose, we examined large sections of kidneys obtained at autopsy from 15 heart transplant recipients, 7 liver transplant recipients, and 10 nontransplanted controls. Tissue sections were examined blindly by light microscopy, histomorphometry, and color computer image analysis. With increasing time following transplantation, there was a progressive increase in renal arteriolar hyalinosis and in the percentage of glomeruli demonstrating global sclerosis. In addition, there was a statistically significant relationship between arteriolar hyalinosis and global glomerulosclerosis (r=0.61, P<0.003). The percentage of glomeruli with focal glomerulosclerosis was also increased in transplant recipients followed for more than 80 days. The kidneys of heart and liver recipients who died less than 80 days after transplantation, that is, those patients who received no CsA or low doses of CsA, demonstrated significantly more interstitial fibrosis than the kidneys of control individuals. Furthermore, there was no significant increase in the amount of renal interstitial fibrosis in allograft recipients followed for prolonged periods of time. There were no significant relationships between the severity of renal pathologic changes and serum creatinine or systemic blood pressure levels. In conclusion, non-renal transplant recipients demonstrate renal damage that affects primarily the preglomerular arterioles and results in glomerular obliteration. This renal damage increases in relation to the time of exposure to CsA and in relation to the total dose of CsA that the patient receives. These renal structural changes are not reflected initially in changes in glomerular filtration rate, as determined by serum creatinine.

Cytomegalovirus-mediated modulation of adhesion molecule expression by human arterial and microvascular endothelial cells.

BACKGROUND: Cytomegalovirus (CMV), a betaherpesvirus associated with allograft rejection, infects the endothelium, the cellular interface between allograft tissue and the host immune system. Because of recent appreciation of the phenotypic diversity of endothelial cells (EC) from different vascular compartments, controversy now exists on the universality of CMV-mediated adhesion molecule induction previously described on umbilical vein EC. Therefore, we herein extend these previous studies to arterial and microvascular EC, which represent sites of vascular rejection. METHODS: Human coronary artery, aortic, umbilical artery, and microvascular EC were mock or CMV infected and/or treated with tumor necrosis factor-a before flow cytometric and immunohistochemical analysis. RESULTS: CMV directly enhanced intercellular adhesion molecule-1 on all EC isolates but did not induce E-selectin or vascular cell adhesion molecule-1. Furthermore, CMV-infected EC were refractory to tumor necrosis factor-alpha-mediated induction of these molecules. CONCLUSION: CMV-induced modulations of adhesion molecule expression, which may affect allograft immunogenicity, seem common to all EC regardless of vascular origin.

T lymphocyte activation by cytomegalovirus-infected, allogeneic cultured human endothelial cells.

Cytomegalovirus, a source of serious complications among immunosuppressed individuals, infects endothelial cells in vivo, and has been epidemiologically associated with allograft rejection and transplantation-associated accelerated arteriosclerosis (TxAA). As the interface between the host immune system and allograft tissues, the endothelium may be of primary importance in mediating pathogenic immune responses to CMV. We have therefore investigated T lymphocyte responses to CMV-infected allogeneic human umbilical vein endothelial cells (HUVE) in vitro. Proliferation assays demonstrated dramatically enhanced responses by CMV-seropositive donor-derived PBMC or purified T cells cocultured with CMV-infected HUVE, as compared with those elicited by noninfected stimulator cells. As determined by limiting dilution analysis of IL-2-producing cells, the frequency of T cells responding to infected HUVE was generally found to exceed by approximately one order of magnitude those responding to uninfected cells. Similar analyses of isolated T cell subsets revealed that these responses (proliferation and IL-2 production) were nearly entirely accountable to the CD4+ fraction. Responses of CD8+ populations, however, varied among donors. The marked activation of CD4+ cells is particularly intriguing since we have shown that CMV-infected HUVE do not express HLA class II antigens. Responses of lymphocytes derived from CMV-seronegative donors were minimal in all assays. These studies show that purified T cells can respond to CMV in the exclusive context of allogeneic endothelial cells. Furthermore, we demonstrate the heretofore undescribed phenomenon of CD4+ T cell activation in the absence of serologically detectable HLA class II antigen. As vascular rejection and TxAA are characterized by subendothelial T cell infiltration, these findings support a role for CMV/endothelial interaction in their pathogenesis.

In vitro induction of endothelial HLA class II antigen expression by cytomegalovirus-activated CD4+ T cells.

CMV has been associated with allograft rejection and transplantation-associated arteriosclerosis. CMV infects endothelium, the interface between allograft tissue and the host immune system. Although endothelial HLA class II expression is a hallmark of vascular rejection, CMV does not directly induce these antigens on infected endothelial cells (EC) and, indeed, renders them refractory to HLA DR induction by IFN-gamma. Our earlier studies have demonstrated, however, that CMV-infected EC are capable of eliciting vigorous activation responses by allogeneic, CMV-seropositive donor-derived CD4+ T cells. We now show that T cells thus activated can induce HLA DR expression on noninfected EC. Human umbilical vein endothelial cells (HUVEC) were inoculated at low titer with CMV strain VHL/E, cocultured with allogeneic, CMV-seropositive or CMV-seronegative donor-derived CD4+ T cells, then dual immunohistochemically stained for CMV antigen and HLA DR, or assayed for HLA DR expression by fluorescence flow cytometry. Results demonstrated that HLA DR was induced in 20-70% of HUVEC in monolayers containing less than 0.5% CMV-infected EC after coculture with CMV-seropositive donor-derived T cells. No such induction was observed in experiments employing T cells isolated from CMV-seronegative individuals. Expression of this antigen was strictly limited to noninfected cells. Rare HLA DR induction was observed in virus-free cocultures. To determine whether endothelial HLA DR was induced by a soluble factor(s) elaborated by activated T cells, noninfected HUVEC monolayers were treated for 72 hr with cell-free supernatant from CMV-infected or noninfected HUVEC/T cell cocultures and assayed as above. Again, HLA DR expression was induced by supernatant from CMV-positive cocultures (only when cocultured T cells were isolated from CMV-seropositive donors), but not by supernatant from CMV-negative cocultures or from T cells cultured alone. The soluble factor was identified as IFN-gamma by the complete attenuation of HLA DR induction by anti-IFN-gamma mAb. These findings suggest that allograft endothelium harboring low level CMV may be capable of eliciting host CD4+ T cell activation, and that consequent release of IFN-gamma is capable of inducing endothelial HLA class II expression, as observed in vascular rejection and transplantation-associated arteriosclerosis. Results of these studies thus provide insight into mechanisms that may help elucidate the association between CMV and rejection-related immune events in the allograft.

Apoptosis in murine cardiac grafts.

Apoptosis, or the induction of programmed cell death, is a mechanism commonly used by cytotoxic T cells to cause target cell lysis. We evaluated the frequency and distribution of apoptotic cells in DBA/2-->DBA/2 heterotopic cardiac isografts, acutely rejecting DBA/2-->C57BL/6 cardiac allografts, and accepted, 60 day DBA/2-->C57BL/6 allografts from mice treated with anti-CD4 Mab (GK1.5) or gallium nitrate (GN). Apoptosis was identified in histologic sections via TUNEL analysis of nuclear DNA fragmentation. We observed the following. (1) Cardiac isografts display no detectable TUNEL+ cells. (2) Rejecting cardiac allografts display rare (<1% of nucleated cells/field), diffuse TUNEL+ cells, peaking on day 3 and declining to 50% of peak by the day of rejection (approximately day 10), and TUNEL+ cells were localized to regions of cellular infiltrate rather than myocyte regions. (3) Accepted cardiac allografts display relatively high numbers of TUNEL+ cells localized in and around the large cardiac arteries (about 20% of nucleated cells/periarterial field). These arteries often showed evidence of transplant vascular sclerosis characteristic of chronic allograft rejection. While a few TUNEL+ cells were found in the arterial tissue, most were observed in the periarterial cellular infiltrate. Similar frequencies and distributions of TUNEL+ cells were observed in grafts that were accepted due to treatment with the anti-CD4 mAb GK 1.5 or gallium nitrate. In general, apoptosis did not correlate with graft failure or parenchymal cell damage, suggesting that cytotoxic T cell-mediated destruction of graft tissues is rare in cardiac allografts. While apoptosis does not appear to be indicative of acute rejection, the characteristic periarterial clustering of apoptosis in accepted grafts may be indicative of immunoregulatory processes that maintain graft acceptance or repair processes that promote chronic vascular remodeling.

Divergent patterns of ELAM-1, ICAM-1, and VCAM-1 expression on cytomegalovirus-infected endothelial cells.

Endothelial cells, one of several in vivo host cells for cytomegalovirus (CMV), participate in solid organ allograft rejection in part through the expression of leukocyte adhesion molecules. The hypothesis that CMV infection alters the constitutive and induced expression of ELAM-1, ICAM-1, and VCAM-1 on infected human umbilical vein endothelial cells (HUVECs) was examined. HUVECs were infected with an endothelial cell-propagated strain of CMV (VHL/E) for various periods, treated with tumor necrosis factor-alpha (TNF alpha), and examined by flow cytometry or immunohistochemically dual-labeled with monoclonal antibodies to CMV immediate early nuclear protein and ELAM-1, ICAM-1, or VCAM-1. Neither ELAM-1 nor VCAM-1 was induced on CMV-infected HUVECs, and treatment with TNF alpha treatment did not result in their induction. In contrast, ICAM-1 was induced on infected HUVECs by 24 hr postinfection. Endothelial ICAM-1 induction may represent a mechanism by which CMV infection exacerbates the recipient cellular immune response to allografts.

Alloantigenicity of human endothelial cells. III. Quantitated indirect presentation of endothelial alloantigens to human helper T lymphocytes.

We have investigated the contribution of monocytes (Mo) to the activation of purified human T cells by allogeneic human vascular endothelial cells (HUVEC). We have previously demonstrated that allogeneic HUVEC stimulate IL-2 production by CD8+ helper T lymphocytes (HTL), but not CD4+ HTL, in the absence of accessory Mo. We now show that addition of responder-autologous Mo to such cultures stimulates a high frequency of CD4+ HTL (1/6500), but no additional CD8+ HTL (1/35,000), as detected by limiting dilution analysis (LDA). The CD4+ HTL production of IL-2 increased with increasing numbers of Mo. Monoclonal antibodies to MHC class II interfered with HTL responses to allogeneic HUVEC in the presence, but not in the absence of autologous Mo. In contrast, IFN-gamma-treated HUVEC stimulated a high frequency of CD4+ HTL in the absence of autologous Mo. However, deletion experiments revealed that the population of HTL responsive to IFN-gamma-treated HUVEC is distinct from the population that responds to HUVEC in the presence of autologous Mo. These data suggest that Mo promote IL-2 production by presenting HUVEC-derived alloantigens via MHC class II molecules to CD4+ HTL, rather than by providing cytokines that promote more efficient IL-2 production by CD8+ HTL, or by inducing MHC class II expression on the HUVEC. In general, these data demonstrate that autologous Mo can play a significant role in the response of T cells to allogeneic HUVEC. Further, they demonstrate that the activation of human HTL by allogeneic HUVEC is complex, and can occur by at least three pathways: (1) direct stimulation of a small number of CD8+ HTL, but no CD4+ HTL, by quiescent HUVEC, (2) direct stimulation of a large number of CD4+ HTL by IFN-gamma-treated HUVEC, and (3) indirect stimulation of a different subset of CD4+ HTL by HUVEC in the presence of autologous monocytes. These three pathways of alloactivation are not unique to allogeneic HUVEC, but this experimental system provides a convenient and relevant model with which each pathway can be easily and independently investigated.

Morphometric analysis of neointimal formation in murine cardiac grafts: III. Dissociation of intestitial fibrosis from neointimal formation.

BACKGROUND: This study examined the relationship between transplant vascular sclerosis (TVS) and tissue fibrosis, features of chronic rejection that can develop rapidly in accepted heterotopic murine cardiac allografts. METHODS: The rate of development of interstitial fibrosis or TVS development was determined by computerized analysis of tissue sections from DBA/2-->C57BL/6 heterotopic cardiac allografts after immunosuppression with gallium nitrate. RESULTS: In accepted cardiac allografts, neointimal fibrosis developed by 30 days after transplant, whereas TVS was minimal by day 30, and maximal by day 60. Variable levels of fibrosis were found throughout the allografts. DBA/2-->DBA/2 cardiac isografts never displayed TVS in this time period, but displayed allograft-like fibrosis within 60 days of transplantation. CONCLUSIONS: Interstitial fibrosis can be dissociated from the TVS development in this experimental model of chronic cardiac allograft rejection. Apparently, it is caused, at least in part, by alloantigen-independent factors other than TVS-related tissue ischemia.

Morphometric analysis of neointimal formation in murine cardiac allografts: II. Rate and location of lesion development.

BACKGROUND: Transplant vascular sclerosis (TVS) is manifested in transplanted human and murine hearts as a concentric, intimal lesion. The purpose of this study was to characterize the rate, location, and intensity of developing TVS lesions in murine cardiac allografts using quantitative morphometric analysis. METHODS: Murine cardiac allografts, treated with the immunosuppressant gallium nitrate, were explanted at 30, 60, and 90 days after transplant. The grafts were histologically stained and evaluated for intimal thickening by deriving a neointimal index (NI) using a computerized image-analysis system. RESULTS: In cardiac allografts, mild vascular lesions of varying NI were detectable by day 30 and lesion severity increased significantly by day 60. Thereafter, average lesion severity stabilized, although the percentage of affected vessels continued to increase from day 30 to day 90. In contrast, day-90 cardiac isografts showed little to no TVS development. Vascular lesions developed randomly without regard for vessel location or size. TVS developed more regularly in vessels of the interventricular septum than in the right or left ventricular walls. The degree of TVS development fluctuated along the length of individual vessels, even as late as 90 days after transplant. The smaller vessels (<85 microm in diameter) appeared to occlude more quickly than the larger vessels. CONCLUSIONS: TVS developed reproducibly in a random pattern throughout cardiac allografts over a 1-month to 3-month period after transplant. This development can be quantitatively monitored by computerized morphometric analysis. In general, under these experimental conditions, 30-day cardiac allografts seem to provide a useful experimental model for studying early aspects of TVS, whereas 60-day allografts may be better suited for analysis of advanced TVS.

Alloantigenicity of human endothelial cells. IV. Derivation, characterization, and utilization of gonadal vein endothelia to control endothelial alloantigenicity during lymphocyte-endothelial interactions.

Most previous studies to evaluate endothelial cell-T lymphocyte interactions have used human peripheral blood as a source of T lymphocytes and human umbilical vein endothelial cells as a source of endothelia. Implicit in this experimental system are allogeneic lymphocyte-endothelial interactions, which are largely ignored. To overcome this problem, we isolated gonadal vein endothelial cells (GVEC) along with matched splenic macrophages and T lymphocytes from cadaveric donors, thus providing a completely autologous series of cells for experimentation. First, GVEC were analyzed for morphology, surface phenotype, and cytokine mRNA expression, and found to be indistinguishable from human umbilical vein endothelial cells. Using this system, we observed that irradiated GVEC were able to promote a 2- to 3-fold increase in the proliferation of matched autologous splenic T cells after PHA stimulation. This indicates that the costimulator activity of endothelial cells reported by others is an intrinsic property of endothelial cells, and is not a consequence of endothelial alloantigens. We also used this system to assess the relative abilities of GVEC and macrophages obtained from the same donor to stimulate the proliferation of purified allogeneic CD3+ PBL. We found the following hierarchy of alloantigenicity in this experimental system: splenic macrophages > IFN-gamma-treated GVEC >> untreated GVEC = TNF alpha-treated GVEC. These studies demonstrate that allogeneic macrophages are intrinsically more antigenic than endothelial cells derived from the same donor. Furthermore, they illustrate the utility of this experimental system to obtain data regarding lymphocyte-endothelial interactions that are otherwise unobtainable.

Morphometric analysis of neointimal formation in murine cardiac allografts.

BACKGROUND: Transplant vascular sclerosis is expressed in transplanted human and murine hearts as a concentric intimal thickening. The purpose of this study was to characterize the location, distribution, and intensity of transplant vascular sclerosis in murine cardiac allografts using computerized morphometric analysis. METHODS: Murine cardiac allograft recipients were treated with the immunosuppressant gallium nitrate to promote graft survival. The grafts were removed at 60 days after transplantation and histologically stained. The coronary arteries were analyzed for intimal thickening using a neointimal index (NI) derived with a computer imaging system. RESULTS: A cross-section taken from the middle of a cardiac allograft showed four major coronary arteries, each with widely different NI values (65, 0, 92, and 0). The same four vessels in two other grafts also showed highly variable NI values, but different patterns of vessel involvement. Next, NI values were determined along the length of a single vessel from aorta to apex. This revealed variable, fluctuating intimal thickening along the length of the vessel. In general, arteries from the aortic versus apical regions of the grafted hearts expressed similar amounts of intimal thickening (analysis of variance, P=0.4826). Finally, a method was devised to quantitate intimal thickening from a sampling of three tissue cross-sections taken from the middle of each cardiac allograft. This value was statistically indistinguishable from values obtained by analysis of intimal thickening in multiple sections covering the entire heart (P=0.6734, 0.9021, and 0.1474). CONCLUSIONS: Intimal thickening in the coronary arteries of murine cardiac allografts appears to be variable in terms of location, distribution, and intensity. This is true for different regions of the same vessel, different vessels in the same heart region, and the same vessels in different cardiac allografts.

Prolonged murine cardiac allograft acceptance: characteristics of persistent active alloimmunity after treatment with gallium nitrate versus anti-CD4 monoclonal antibody.

We have treated DBA/2-->C57BL/6 murine cardiac allograft recipients with anti-CD4 monoclonal antibody or with gallium nitrate to promote long-term (>60 days) allograft survival. Within this period, all grafts developed histologic evidence of ongoing vascular and parenchymal tissue remodeling, including interstitial fibrosis and neointimal hyperplasia, which are characteristic of chronic allograft rejection. To evaluate residual alloimmunity associated with the pharmacologic avoidance of acute graft rejection and the development of chronic tissue remodeling, we subjected these graft recipients to a battery of histologic and immunologic tests. Similar test results were obtained for graft recipients treated with either of the two immunosuppressive agents. All long-surviving allografts displayed histologic evidence of ongoing microvascular endothelial activation and interstitial leukocytic infiltration. Reverse transcriptase-polymerase chain reaction analyses demonstrated intragraft expression of mRNAs for interleukin (IL)-1, IL-2, IL-4, IL-6, tumor necrosis factor, interferon-gamma, and transforming growth factor-beta. All recipients had limiting dilution analysis-detectable, graft-reactive cytolytic T lymphocytes and helper T lymphocytes in their spleens and grafts, and all produced high titers of graft-reactive alloantibodies. In general, these observations indicate that (1) a similar immune status is achieved in long-surviving allografts and their recipients when either anti-CD4 monoclonal antibody or gallium nitrate was used for antirejection therapy, (2) this immune status is characterized by continuous, long-term inflammatory and immune processes that are qualitatively similar to those observed during acute allograft rejection, and (3) no specific immune responses developed selectively in long-term graft recipients to account for the avoidance of acute graft rejection or the development of chronic tissue remodeling in the graft.

Patterns of inflammatory vascular endothelial changes in murine liver grafts.

We have investigated the vascular endothelial phenotypes found at various times posttransplant in murine B10-->C3H liver grafts. In this model, liver allografts are spontaneously accepted, and survive indefinitely unless the recipient is first allosensitized with a skin allograft, in which case the liver allografts are rejected within five days. In our previous studies, allograft inflammation was associated with the development of vascular endothelial reactivity with the mAbs MECA-32 and M/K-2 (anti-VCAM-1). We observed that vascular endothelia in both liver isografts and allografts develop reactivity with MECA-32 mAb within two days of transplantation, indicating endothelial activation in both situations. In contrast, only the endothelia in liver allografts develop VCAM-1 expression, as detected with M/K-2 mAb. VCAM-1 was expressed in both rejecting and accepting liver allografts, demonstrating that endothelial VCAM-1 expression is indicative of ongoing graft inflammation but not necessarily graft rejection. Liver parenchymal cells did not appear to develop reactivity with either antibody under any of the conditions tested. In contrast, bile duct epithelia developed M/K-2 reactivity (VCAM-1 expression), but not MECA-32 reactivity in liver allografts, but not isografts. These data demonstrate alloantigen-dependent and alloantigen-independent patterns of endothelial behavior in murine liver grafts that are quite similar to those found in murine cardiac grafts. Further, they demonstrate that the expression of VCAM-1 by graft endothelia is not diagnostic for acute rejection of liver allografts.

Analysis of inflammatory endothelial changes, including VCAM-1 expression, in murine cardiac grafts.

We have employed a murine model of cardiac transplantation and two monoclonal antibodies, M/K-2 and MECA-32, to study the responses of graft endothelia during allograft rejection. Using immunohistologic techniques, we demonstrate that the monoclonal antibody M/K-2, which binds to the murine cellular adhesion molecule VCAM-1, reacts with an inducible endothelial epitope found in rejecting cardiac allografts, but not in cardiac isografts, normal cardiac tissues, or extracardiac vasculature from allografted mice. Similar, but focal, M/K-2 reactivity is also found in nontransplanted hearts undergoing virally induced myocarditis. M/K-2 reactivity does not develop in the nonrejecting cardiac allografts from nu/nu mice, and M/K-2 reactivity is found only in grafts that develop CD25+ graft-infiltrating cells--i.e., allografts but not isografts. PCR analyses of grafts during development of VCAM-1 expression indicate that allografts, but not isografts, contain mRNA for the cytokines IL-2 and IFN-gamma, and either of these cytokines may be associated with the expression of M/K-2 reactivity in rejecting allografts. Unlike M/K-2, MECA-32 identifies an inducible epitope that is observed on myocardial endothelia of both isografts and allografts, but not normal cardiac tissues. Further, expression of the MECA-32 epitope can occur in grafts that do not develop CD25+ infiltrating lymphocytes, since it is observed in isografts and the native hearts of transplanted or sham-operated mice. Indeed, MECA-32 reactivity may be T cell independent, since it is also found in nonrejecting allografts of nu/nu mice. PCR analyses of grafts during development of MECA-32 reactivity indicate that cardiac isografts contain mRNA for IL-1, IL-6, TNF, and lymphotoxin. One or more of these might be associated with induction of MECA-32 reactivity.