Endothelial HLA class II induction mediated by allogeneic T cells activated by cytomegalovirus-infected cultured endothelial cells.
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Cells, Cultured↗
Biomedical subjects
D D Sedmak
Publications and source records attributed to D D Sedmak.
Importance of endothelial VCAM-1 for inflammatory leukocytic infiltration in vivo.
The microvascular endothelia of rejecting DBA/2----C57B1/6 murine cardiac allografts develop reactivity with the mAb M/K-2, which recognizes the murine homologue of the human leukocyte adhesion molecule VCAM-1. This reactivity does not develop in DBA/2----DBA/2 cardiac isografts or normal DBA/2 cardiac tissues. To determine whether endothelial VCAM-1 plays a role in allograft inflammation, cardiac allograft recipients were treated with M/K-2 antibody and monitored for leukocytic graft infiltration and graft survival. Treatment of the graft recipients with 200 micrograms/day M/K-2 Ig prolonged graft survival by 5 to 6 days (statistically significant); whereas treatment with 100 micrograms M/K-2 Ig every other day after transplant did not influence graft survival. Neither treatment interfered with leukocytic infiltration, as detected histologically or by limiting dilution analysis. The high dose treatment, but not the low dose treatment, resulted in high circulating levels of M/K-2, as detected by serum ELISA, and prominent antibody deposition on the graft vascular endothelia, as demonstrated by immunohistologic analysis. These data demonstrate that VCAM-1 is uniquely expressed on the vascular endothelia of rejecting murine cardiac allografts, and that a mAb to VCAM-1 can interfere with the allograft rejection process. Although this antibody can interfere with lymphocyte-endothelial adhesion in vitro, it has little effect on leukocytic infiltration in rejecting allografts suggesting that this process does not depend exclusively on VCAM-1 expression.
Animals↗
Alloantigenicity of human endothelial cells. 1. Frequency and phenotype of human T helper lymphocytes that can react to allogeneic endothelial cells.
To determine the relative ability of allogeneic endothelial cells to stimulate helper T lymphocytes (HTL), human PBMC or purified T cells were incubated in conventional lymphocyte microcultures or in limiting dilution microcultures with allogeneic human umbilical vein endothelia (HUVE), with cytokine-treated allogeneic HUVE, or with allogeneic peripheral blood monocytes. These cultures were tested for IL-2 production as an index of HTL stimulation. Dose-response studies in conventional lymphocyte cultures indicated that allogeneic monocytes were better than allogeneic HUVE at stimulating IL-2 production. Limiting dilution analyses revealed that untreated HUVE and TNF-treated HUVE stimulated small numbers of HTL (approximately 1 HTL/30,000 PBMC), whereas 5 to 10 times more HTL were stimulated by IFN-gamma-treated HUVE and 10 to 20 times more HTL were stimulated by allogeneic monocytes. Serologic deletion studies revealed that most of the high frequency HTL responding to IFN-gamma-treated HUVE were CD4+, whereas most of the low frequency HTL responding to nontreated HUVE or to TNF-treated HUVE were CD8+. Interestingly, mAb to MHC class I and class II molecules, which significantly impaired HUVE-induced proliferation, caused little interference with HUVE-induced IL-2 production. Finally, polymerase chain reaction analysis demonstrated that untreated allogeneic HUVE cells could stimulate PBMC to produce mRNA for IFN-gamma, as well as for IL-2. These data demonstrate the following hierarchy of allogeneic stimulatory capacity for human HTL: monocytes greater than IFN-gamma-treated HUVE much greater than TNF-treated HUVE = nontreated HUVE. Further, these data suggest that non-activated allogeneic endothelial cells can initiate immune responses by inducing IL-2 and IFN-gamma. Because IFN-gamma can induce MHC class II expression by the endothelial cells, this could recruit large numbers of CD4+ T cells for IL-2 production.
Antigens, Differentiation, T-Lymphocyte↗
Role of the endothelial adhesion molecule VCAM in murine cardiac allograft rejection.
Murine heterotopic cardiac isografts (C57B1/6----C57B1/6) undergo transient, non-destructive inflammation that is characterized by the acquisition of microvascular endothelial reactivity with the antibody MECA 32. Cardiac allografts (C57B1/6----DBA/2) undergo destructive inflammation that is characterized by the acquisition of reactivity with the antibody M/K-2, in addition to MECA 32. M/K-2 recognizes the murine endothelial adhesion molecule, VCAM-1. Hence, there appear to be antigen-dependent and antigen-independent forms of graft inflammation. Treatment of cardiac allograft recipients with 200 micrograms/day M/K-2 antibody retarded graft loss by only a few days, and did not interfere significantly with leukocytic infiltration, as detected by limiting dilution analysis of graft-reactive CTL, despite the fact that large amounts of M/K-2 could be detected on graft microvascular endothelia and in the peripheral blood as rejection progressed. These data indicate that VCAM is apparently not essential for the leukocytic infiltration and subsequent rejection of cardiac allografts, and is not involved in leukocytic infiltration of murine cardiac isografts.
Animals↗
Evaluation of a rapid peptide-based anti-human immunodeficiency virus-1 antibody immunoassay.
A qualitative, visually interpreted, rapid, and synthetic peptide-based anti-human immunodeficiency virus-1 (HIV) antibody immunoassay has been developed that may be of value in situations in which rapid determination of HIV-1 status is important. Because questions have been raised about the accuracy of rapid anti-HIV-1 assays, the sensitivity, specificity, interobserver and intraobserver variability of the Genie HIV-1 assay (Genetics Systems, Seattle, WA) were determined. Sera from 56 patients with HIV-1 infections documented by enzyme immunoassay and western blot tested positive by this assay. Enzyme immunoassay- and western blot-negative sera from 30 visceral organ transplant donors were negative using the Genie assay. Specificity was examined further by testing sera from 29 patients hospitalized with a variety of medical disorders, including acute bacterial pneumonia, acute myocardial infarction, monoclonal gammopathy, and high titer antinuclear or antimitochondrial antibodies. Two of these patients were reactive with the enzyme immunoassay, both of which tested negative by western blot. All 29 tested negative using the Genie assay. In addition, sera from five patients with repeatedly reactive enzyme immunoassays and negative western blots tested negative by the Genie system. There was 100% agreement in interobserver and intraobserver studies. With the western blot as the reference method, the Genie assay exhibited 100% sensitivity and specificity and there was no observer variability.
AIDS Serodiagnosis↗
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.
CD4 Antigens↗
Heterogeneity of peripheral blood reticulocytes: a flow cytometric analysis with monoclonal antibody HAE9 and thiazole orange.
The expression of a human erythroid cell surface antigen recognized by monoclonal antibody (mAB) HAE9 has been studied on peripheral blood reticulocytes by one- and two-color flow cytometry. Total reticulocyte count was determined using Thiazole Orange (TO) and flow cytometry. In normal individuals, 4.56% of reticulocytes were stained by FITC-labeled mAB HAE9. The correlation between reticulocyte percentage by TO and HAE9 staining was 0.828 (P less than 0.0001) in patients with hematocrits less than 0.25. A HAE9-positive reticulocyte percentage of 6-44% was observed when analyzed by two-color flow cytometry with TO and mAB HAE9. These findings, in conjunction with previous studies, suggest that mAB HAE9 recognizes an early, less differentiated population of peripheral blood reticulocytes. Enumeration of immature reticulocytes may be of clinical utility.
Antibodies, Monoclonal↗
Preservation of natural endothelial cytopathogenicity of cytomegalovirus by propagation in endothelial cells.
Cytomegalovirus (CMV) is a source of major complications in immunosuppressed individuals, and endothelial involvement in CMV infection is well documented. Traditionally the virus has been propagated in fibroblasts, however this process may alter CMV's characteristics, thereby limiting the fibroblast model's utility as a research tool. In our efforts to develop a more accurate in vitro model of CMV/endothelial cell interaction, we have propagated a recent isolate (CMV VHL) through multiple passages in human umbilical vein endothelial cells (HUVE) and, collaterally in neonatal human dermal fibroblasts (NHDF). Infection of HUVE inoculated with either sub-strain of the virus was confirmed by CMV-specific in situ hybridization and by immunocytochemical staining for CMV antigens. Whereas infection of HUVE by substrain VHL/E (endothelial-raised) was accompanied by dramatic cytopathology resembling that observed clinically, the endothelial cytopathic potential of VHL/F (fibroblast-raised) was lost by its 20th passage in NHDF. Similarly, the ability of VHL/F to initiate sustained productive infection in HUVE was severely attenuated; plaque assay of culture supernatants and infected cell fractions, as well as virus-specific DNA polymerase assay of cell lysates, demonstrated progressive viral reproductive activity in VHL/E-inoculated HUVE, whereas VHL/F reproduction was barely detectable. Since properties of VHL/F bear strong resemblance to those of the fibroblast-raised AD169, these studies suggest that while the fibroblast adaptation process commonly employed in the propagation of CMV restricts the host range of the virus and attenuates its spectrum of cytopathic potential, endothelial-based propagation preserves the natural endothelial cytopathogenicity of the original isolate.
Antigens, Viral↗
Serum C3 levels are diagnostically more sensitive and specific for systemic lupus erythematosus activity than are serum C4 levels. The Lupus Nephritis Collaborative Study Group.
To determine whether serum C3 or C4 is more likely to be normal during systemic lupus erythematosus (SLE) remission and abnormal during SLE relapse we studied twelve SLE patients who presented with severe nephritis. The patients were followed long term (12 to 77 months) through multiple relapses (N = 41) and remissions (N = 13) defined by protocol. A total of 471 serum samples were obtained at defined intervals during these relapses and remissions and were analyzed for C3 and C4 levels by two different methods: nephelometry (N) and radial immunodiffusion (R). During SLE remission (defined by protocol and without reference to serum complement levels), C3 measured by N-assay (C3N) and by R-assay (C3R) tended to be normal (specificity of 93% and 71%, respectively). By contrast, C4 measured by N-assay (C4N) and by R-assay (C4R) showed no such tendency (specificity of 50% for both C4N and C4R). During SLE relapse (defined by protocol and without reference to serum complement levels), C3N and C3R were more likely to be abnormal (sensitivity 95% and 85%, respectively) compared with C4N and C4R (sensitivity 56% and 54%, respectively, P less than 0.001 compared with corresponding values for the C3 assay). Analysis by receiver-operator characteristic (ROC) curves demonstrated that the reduced diagnostic sensitivity of C4 versus C3 is not explained by use of an inappropriate lower limits of normal (LLN) for C4.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
The role of vascular endothelial cells in transplantation.
The interface between an allograft and the recipient's immune system is the endothelium of the allograft vasculature. In this boundary position, endothelial cells may play important roles in the afferent and efferent phases of allograft rejection, in the response of the allograft to pretransplant perfusion and to drug therapy, and in the response to viral infection of the host. The expression by endothelial cells of granule membrane protein-140 (GMP-140) and endothelial leukocyte adhesion molecule-1 (ELAM-1), increased tissue factor activity, increased secretion of plasminogen activator inhibitor, and decreased thrombomodulin may all contribute to hyperacute rejection. Similarly, endothelial cells may actively participate in acute cellular rejection and in the development of transplant-associated arteriopathy as a result of induction of antigen-presenting function (ie, HLA class II expression), upregulation of adhesion molecules for lymphocytes and monocytes, and release of platelet-derived growth factors. Endothelial cell functions, which are important for normal inflammatory responses and vessel behavior, may be pathogenic in the allograft.
Acute Disease↗
Expression of IgG Fc receptor antigens in placenta and on endothelial cells in humans. An immunohistochemical study.
Expression of leukocyte IgG Fc receptor (Fc gamma R) antigens by placenta, endothelial cells (EC) of normal tissues, and ECs of kidney and skin from subjects with immune complex diseases was studied immunohistochemically using anti-Fc gamma R monoclonal antibodies (MAb). Monoclonal antibodies against all three leukocyte Fc gamma R classes stained placental villous macrophages. Placental villous trophoblasts were stained intensely by anti-Fc gamma RIII MAb 3G8, while both anti-Fc gamma RI (MAb 32) and anti-Fc gamma RII MAbs IV3, KU79, CIKM5, 2E1, KB61, and 41H16) antibodies did not react with these cells. Anti-Fc gamma RII MAbs IV3, KU79, CIKM5, 2E1, KB61, and 41H16 immunostained placental villous capillary EC, in contrast to anti-Fc gamma RI MAb 32 and anti-Fc gamma RIII MAb 3G8, CLB-Granl, and B73.1, which did not bind. Anti-Fc gamma RI MAb 32, anti-Fc gamma RII MAb IV3 and CIKM5, and anti-Fc gamma RIII MAb 3G8 did not react with the ECs of tonsil, liver, kidney, spleen, intestine, lung, or uterus. Similarly no EC staining was seen with these four MAbs in 14 skin and 14 kidney biopsies from subjects with immune-complex diseases. Fc gamma R antigens are expressed constitutively only by placental villous ECs and are not induced on nonplacental ECs by immune-complex-mediated diseases.
Antibodies, Monoclonal↗
Immunoglobulin G subclass deficiency and pneumococcal infection after allogeneic bone marrow transplantation.
Serum immunoglobulin (Ig) G subclass levels were measured in a radial immunodiffusion assay in 25 leukemic patients before and after allogenic bone marrow transplantation. All patients received a conditioning regimen of busulfan and cyclophosphamide followed by infusion of marrow from an HLA-identical sibling. Intravenous infusions of a commercial Ig preparation were administered every 2 weeks until day 120 posttransplant. Nine patients developed pneumococcal infections at 6 months or greater posttransplant. Infection was associated with low levels or the absence of detectable serum IgG2 and IgG4. At the time of infection, 4 of 7 patients evaluated had undetectable IgG2, while 5 of 7 had undetectable levels of IgG4. After infection, none of the 8 patients evaluated had detectable levels of IgG2, and only 2 of 8 had detectable levels of IgG4. In contrast, all 16 patients without pneumococcal infection had IgG2 levels of 102 mg/dL or greater, and IgG4 levels of 20 mg/dL or greater. It appears that IgG2 and IgG4 subclass deficiencies after allogenic bone marrow transplantation contribute to susceptibility to pneumococcal infection. After pneumococcal infection, IgG2 and IgG4 levels remain low for a prolonged period and patients remain susceptible to infection by encapsulated organisms.
Adult↗
Vascular endothelial differentiation in sponge matrix allografts.
These studies test the hypothesis that vascular endothelia in sponge allografts may develop a function and phenotype resembling the high endothelial venules (HEV) in lymph nodes, thus facilitating the lymphocytic infiltration that is characteristic of allograft rejection. Using limiting dilution analysis to quantitate helper-T-cell accumulation at graft sites, immunohistologic analysis of graft reactivity with the HEV-specific monoclonal antibody MECA 325, and ex vivo lymphocyte-endothelial adhesion assays with graft tissues, we obtained evidence to suggest that HEV-like endothelia may develop at a graft site but that the process whereby lymphocytes accumulate at a graft site is more complex than was initially expected.
Animals↗
Relationship between leukocytic infiltration and endothelial specialization in murine cardiac allografts.
We have used immunologic and histologic techniques to analyze the rejection of heterotopic murine cardiac allografts. These studies were designed to test the hypothesis that lymph-node-like mechanisms of lymphocyte extravasation develop in immediately vascularized organ allografts during the process of rejection. The experimental results suggest that lymph-node-like mechanisms are not involved, and that an unexpected pattern of lymphocyte infiltration develops in these allografts.
Animals↗
Experimental glomerulonephritis induced by antigen that binds to glomerular fibronectin.
In previous studies we demonstrated that antigens with the capacity to bind to the glycoprotein fibronectin (FN), localized in the glomerular mesangium after intravenous (IV) injection. In the present study we sought to determine if the localization of FN-binding antigens in the mesangium is capable of inducing glomerulonephritis. A group of rats were injected IV with the FN-binding antigen phenylated gelatin (DNP-GL) followed 2 hours later by rabbit anti-DNP antibodies. This experimental protocol resulted in the development of a glomerulonephritis characterized by an initial heterologous phase and a late autologous phase. Both phases of the disease were characterized by significant histologic changes, including focal segmental mesangial matrix expansion, inflammatory cell infiltration, and an increase in endothelial and mesangial cells. By immunoperoxidase we demonstrated mesangial deposition of rabbit IgG and rat C3 during the initial heterologous phase and mesangial deposition of rat IgG and C3 during the autologous phase. Significant proteinuria developed during the heterologous phase, but not during the autologous phase of the disease. By contrast, rats injected with DNP-GL alone or anti-DNP antibodies alone did not develop significant histologic glomerular changes or proteinuria. In conclusion, antigens that bind to mesangial FN are capable of inducing glomerulonephritis. This mechanism of localization of antigens in the glomeruli may be relevant to human immune complex (IC)-mediated diseases involving antigens that have the capacity to bind to FN.
Animals↗
Cellular receptors for matrix proteins in normal human kidney and human mesangial cells.
In the present study we evaluated the distribution of cell adhesion molecules, referred as very late antigens (VLA), in the normal human kidney and in mesangial cells in culture (MC). In addition, we assessed the functional properties of VLA proteins on MC. Normal human kidney and MC were stained by immunoperoxidase with mouse monoclonal antibodies to VLA proteins. We demonstrated that VLA-3, a protein that binds FN, laminin and collagen, is the predominant VLA protein in the human glomerulus and on MC. VLA-3 is located in the mesangium and on the glomerular visceral epithelial cell and endothelial cell surfaces in contact with the glomerular basement membrane. VLA-1 was demonstrated in the glomerular mesangium and VLA-5, an FN specific receptor, was present in the mesangium on glomerular endothelial cells and on MC. VLA-2 and VLA-4 were not present in the normal glomerulus nor on MC. In functional studies we evaluated the binding of MC to FN coated surfaces and the binding and phagocytosis of FN coated fluorescent beads by MC. We showed that MC bind to FN coated surfaces and that the binding is inhibited by anti-FN antibodies, EDTA and peptides containing the amino acid sequence Arg-Gly-Asp (RGD). In addition, anti-VLA-5 but not anti-VLA-3 antibodies inhibited significantly the binding of MC to FN, MC demonstrated binding and phagocytosis of FN coated beads and, purified FN inhibited both phenomena. By affinity chromatography and immunoprecipitation we demonstrated that MC FN binding proteins and MC VLA proteins are composed of two distinct protein chains that have Mr characteristics similar to those of normal human fibroblasts VLA proteins. In conclusion, the glomerular distribution of VLA-3 suggests that this protein is primarily involved on the adhesion of glomerular cells to basement membranes and matrix. MC FN receptors (VLA-5) mediate the binding of MC to FN and could mediate the phagocytosis of FN coated antigen or immune complexes by mesangial cells.
Binding Sites↗
Sensitivity and interobserver variability of the Recombigen-HIV-1 LA test.
The Recombigen-HIV-1 LA Test (Cambridge BioScience Corporation, Worcester, MA) uses recombinant peptides derived from the env gene product in a latex agglutination assay for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1) and was recently approved by the Food and Drug Administration for marketing in the United States. It is intended for use as a screening test in physicians' offices, emergency rooms, and other settings where enzyme immunoassays are not practical or available. Concern has been raised over the sensitivity, specificity, and difficulty in interpretation of the agglutination pattern. The authors report on the sensitivity and interobserver variability of the assay as performed in a blinded fashion in a hospital laboratory by technologists experienced with other latex agglutination assays. In the first study, sera from 50 patients positive by enzyme immunoassay (EIA) (Abbott HIV EIA) and western blot (WB), performed with EPITOPE HIV western blot strips were assayed by one technologist using the latex agglutination technique. Forty-six samples were positive and four were negative, yielding a sensitivity of 92%. In the second study, 30 samples consisting of 10 negative by EIA and WB, 10 borderline by EIA and/or indeterminate by WB, and 10 positive by EIA and WB were evaluated by three technologists with the latex agglutination technique. There was agreement among all three technologists in 24 of 30 samples (80%). There was disagreement over one sample from the negative group (one technologist obtained a single false positive result), three from the borderline/indeterminate group, and two from the positive group (three technologists obtained false negative results on two samples). In summary, the authors report interobserver variation in interpreting 20% of tests, reflecting difficulty in assessing weak agglutination. Sensitivity of 92% is below that achievable with the EIA or WB techniques and limits the usefulness of the latex agglutination assay as a screening test.
AIDS Serodiagnosis↗
Inability of cytomegalovirus infection of cultured endothelial cells to induce HLA class II antigen expression.
Cytomegalovirus infection in the renal allograft recipient has been associated with the initiation of acute rejection. The mechanism of this induction is unknown. It may be related to renal tubular epithelial and endothelial expression of HLA class II antigens or a CMV immediate-early antigen that exhibits immunologic crossreactivity with HLA DR. In this study the ability of CMV to both infect and subsequently induce class II antigen expression on cultured human umbilical-vein endothelial cells (HUVEs), in the absence of cytokines, particularly gamma interferon, was tested. Individual HUVE cell lines were first proven to express HLA class II antigens in the presence of 10, 100, and 200 units of recombinant IFN-gamma as early as 24 hr postincubation by an immunohistochemical technique and by flow cytometry. These cell lines were successfully infected with CMV strains AD169 and CMV3 as determined by the presence of early and late viral antigens and CMV DNA. The degree of infection was dose and incubation-time dependent. Infection of HUVEs with these CMV strains and a nonattenuated clinical isolate failed to induce HLA DR, DP, or DQw1 expression in the absence of IFN-gamma. These findings support the hypothesis that endothelial cells in vivo may serve as reservoirs of CMV infection. They do not support the hypothesis that CMV produces an immediate-early antigen that has immunologic cross-reactivity with HLA DR. Furthermore, there is no support for the hypothesis that CMV independently induces HLA class II antigens in the absence of IFN-gamma.
Cells, Cultured↗