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M H Sayegh

Publications and source records attributed to M H Sayegh.

At least 163 records · Page 9Linked to original sources
PubMed1994

Differential effects of oral versus intrathymic administration of polymorphic major histocompatibility complex class II peptides on mononuclear and endothelial cell activation and cytokine expression during a delayed-type hypersensitivity response.

Oral and intrathymic exposure to antigen can each induce systemic antigen-specific immune tolerance, but the mechanisms have not been well defined. We studied the effects that the route of exposure to antigen has on the mechanisms of tolerance in vivo using synthetic polymorphic class II major histocompatibility complex (MHC) peptides in a skin delayed-type hypersensitivity (DTH) response model. Lewis rats were immunized by injection in the footpad with synthetic peptides (RT1.Bu and/or RT1.Du) derived from the hypervariable domain of MHC class II beta chain of the Wistar-Furth rat in complete Freund's adjuvant and challenged 2 weeks later by injection in the ear with the MHC peptides. An "oral" group received the peptide mixture by gavage (100 micrograms/day for 5 days) 3 days before immunization, and an "intrathymic" group received a single intrathymic injection of 100 micrograms of peptides 48 hours before immunization. Oral therapy reduced the DTH response to 23 +/- 7%, and intrathymic exposure reduced the DTH response to 26 +/- 6% (P < 0.001) as compared with control DTH responses of unmodified Lewis animals. Immunohistological evaluation of DTH skin lesions showed that oral and intrathymic therapy each decreased mononuclear cell infiltration, fibrin deposition, and endothelial activation when compared with that seen in control rats. In contrast, while both protocols markedly reduced interleukin (IL-2) and interferon-gamma expression, they had differing effects on local expression of IL-4, transforming growth factor-beta, IL-2R, and CD45RC (a possible discriminant between Th1 and Th2 cells in rats). Oral therapy was associated with increased expression of IL-4 and preservation of transforming growth factor-beta expression by residual IL-2R+, CD45RC- mononuclear and endothelial cells, whereas thymic exposure suppressed essentially all features of immune activation including IL-2R induction and cytokine expression. Our data a) document the detailed pattern of cytokine expression and mononuclear and endothelial cell activation markers during DTH responses and b) confirm that oral tolerance is associated with immune deviation to a predominance of Th2 cell function, whereas intrathymic tolerance may be mediated by T-cell anergy or clonal deletion.

Administration, Oral↗
PubMed1993

CD4 mAb therapy modulates alloantibody production and intracardiac graft deposition in association with selective inhibition of Th1 lymphokines.

The accelerated (24 h) rejection of (LEWxBN)F1 cardiac allografts (Tx) in LEW rats sensitized with BN skin grafts, is abrogated with CD4 mAb (BWH-4) administration between skin (day -7) and heart (day 0) transplantation (Tx survival ca. 11 days, p < 0.0001). This study analyzed the effects of CD4-targeted therapy upon host IgG and IgM alloantibody (allo-Ab) within the serum by two-color flow cytometry, and within the Tx, by immunohistology. These data were correlated with mRNA and protein production profiles of Th1 (IL-2, IFN-gamma) vs Th2 (IL-4) specific cytokines (polymerase chain reaction and/or immunohistology). Skin grafts elicited a strong systemic IgM allo-Ab response, which peaked at the time of cardiac Tx rejection at 24 h. It was associated with extensive deposits of IgM on Tx endothelium. Treatment with BWH-4 mAb diminished circulating IgM allo-Ab levels, and only low levels of IgM could be detected at the Tx site. Conversely, the low circulating IgG allo-Ab levels during rejection at 24 h in untreated recipients were accompanied by a strong labeling for intra-Tx IgG. BWH-4 mAb therapy did not prevent totally the switch of the IgM to IgG, but the IgG allo-Ab response was earlier, less intense and more transient than in untreated recipients. Accelerated rejection triggered sequential lymphokine mRNA expression in cardiac Tx, with the peak of transcription for IL-2 (6-12 h) preceding that for IL-4 (24 h). Interestingly, although CD4 targeted therapy virtually ablated the induction of IL-2 mRNA, it preserved transcription of the IL-4 gene. BWH-4 mAb therapy decreased otherwise abundant intra-Tx IL-2 and IFN-gamma, but allowed a vigorous elaboration of IL-4, confirming the translation of mRNA to the protein in vivo. Thus, CD4 mAb-mediated abrogation of accelerated cardiac Tx injury correlates with suppression of Th1 responses (depressed IL-2 and IFN-gamma production), but sparing of the Th2 function (enhanced IL-4 elaboration). Indeed, CD4 mAb-induced allo-Ab depression and immunosuppressive effects may reflect selective targeting of proinflammatory Th1-like cells and the multifaceted effects of IL-4 produced by unopposed Th2-like cells.

Animals↗
PubMed1993

Acquired tolerance to experimental autoimmune encephalomyelitis by intrathymic injection of myelin basic protein or its major encephalitogenic peptide.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system that can be induced in a number of species by immunization with myelin basic protein (MBP) in adjuvant, and serves as an experimental model for the study of multiple sclerosis. The role of the thymus in acquired tolerance in autoimmune models has not been thoroughly investigated. In this study, we examined the effects of intrathymic injection of MBP or its major encephalitogenic peptide on the course of EAE in Lewis rats. A single intrathymic injection of MBP 48 h pre- but not postimmunization protects animals from actively induced EAE. An intact MBP-primed thymus was required up to 10 d postimmunization, as thymectomy on days 1, 2, and 7 postimmunization abrogated the protective effect, whereas thymectomy on day 10 did not. The proliferative response of primed lymphocytes was significantly reduced in animals that were intrathymically injected with MBP. Protection against clinical EAE was induced by thymic injection of the major encephalitogenic region (residues 71-90) but not a nonencephalitogenic (21-40) MBP epitope. Immunohistologic examination of the brain from rats intrathymically injected with encephalitogenic peptide showed markedly reduced cellular infiltrate and virtual absence of activation and inflammatory cytokines as compared with rats intrathymically injected with the nonencephalitogenic peptide. These results indicate that the thymus may play an active role in acquired systemic immunologic tolerance in T cell-mediated experimental autoimmune diseases. This effect may be mediated by a process of clonal inactivation of autoreactive T cell clones circulating through the thymus.

Animals↗
PubMed1993

Oral, but not intravenous, alloantigen prevents accelerated allograft rejection by selective intragraft Th2 cell activation.

We studied the mechanisms by which oral or intravenous administration of allogeneic splenocytes prevents sensitization by skin allografts and development of accelerated rejection of subsequent cardiac allografts. LEW rats were sensitized with BN skin allografts 7 days prior to receiving heterotopic (LEW x BN)F1 vascularized cardiac allografts. While unsensitized cardiac allografts are rejected on days 6-8, control sensitized grafts were rejected within 24 to 48 hr. Oral administration of BN splenocytes during the sensitization phase (between skin and heart grafting) has been found to prevent accelerated allograft rejection and prolong cardiac allograft survival to 7 days. An alternative route of antigen exposure, specifically intravenous administration of BN splenocytes (50 x 10(6) daily for 5 days starting on the day of skin grafting), also prevented accelerated cardiac allograft rejection and prolonged allograft survival to 9 +/- 1 days (n = 5). Immunoperoxidase studies of cardiac allografts harvested 24-48 hr posttransplant showed that, when compared with sensitized controls, animals that received oral splenocytes had reduced deposition of IgG (end-point titer of 1/1000 vs. 1/4000), IgM (1/1000 vs. 1/16000), C3 (1/4000 vs. 1/16000), and fibrin (1/4000 vs. 1/16000). There was also decreased infiltration by macrophages (18 +/- 8 vs. 37 +/- 8 cells/HPF, P < 0.01), T cells (5 +/- 3 vs. 19 +/- 7, P < 0.01), and IL-2R+ T cells (5 +/- 3 vs. 15 +/- 4, P < 0.01), and a significant reduction in the numbers and extent of intragraft mononuclear cells stained with antibodies to IL-1, IL-2, IL-6, IL-8, IFN-gamma, and TNF-alpha. In contrast, these grafts showed markedly increased IL-4 staining (including most mononuclear and all endothelial cells), as compared with control grafts (< 20% of mononuclear cells and only focal endothelial staining). Immunoperoxidase studies of cardiac allografts harvested from rats receiving intravenous splenocytes also showed markedly reduced humoral deposits and cellular infiltrates, comparable to that found in the oral splenocytes-treated group, but showed significantly different cytokine expression. In particular, some intragraft mononuclear cell labeling for IFN-gamma remained, and IL-4 staining was not increased relative to control grafts. Attempts were then made to abrogate spleen cell-induced prolongation of cardiac allograft survival by daily injections of CD4 monoclonal antibody (BWH-4 mAb, 700 micrograms) from the time of cardiac transplantation, therapy previously shown unable to prolong cardiac survival in this model when commenced after skin graft-induced sensitization has occurred.(ABSTRACT TRUNCATED AT 400 WORDS)

Administration, Oral↗
PubMed1993

Thymic recognition of class II major histocompatibility complex allopeptides induces donor-specific unresponsiveness to renal allografts.

Recent data show that intrathymic injection of allogeneic cells induces donor-specific unresponsiveness to allografts. There is also evidence to suggest that, in addition to recognizing intact MHC molecules, T cells can recognize processed MHC peptides, although the role of this indirect mode of allo-recognition in allograft rejection is unknown. We report that a single intrathymic injection of 100 micrograms of a mixture of eight 25-mer synthetic polymorphic class II MHC allopeptides, representing the full-length sequence of RT1.Bu beta and RT1.Du beta (WF) into incompatible (RT1l) LEW recipients, induced a state of long-term unresponsiveness to subsequent engraftment 2 days later of WF, but not third party (RT1n) BN renal allografts. Intrathymic injection of 100 micrograms of either RT1.Bu beta or RT1.Du beta peptide mixtures alone were insufficient to prolong renal allograft survival. Intravenous or intrasplenic injection of the allopeptide mixture did not affect renal allograft survival, establishing the role of thymic recognition of class II MHC allopeptides in inducing systemic unresponsiveness. The induction of intrathymic donor-specific unresponsiveness was abrogated if thymectomy was performed on the day of renal transplantation or 5 days later. PBLs from long-term surviving animals exhibited marked reduction of proliferation to WF, but not third party BN stimulator lymphocytes in the standard mixed lymphocyte response assay in vitro. These observations emphasize the role of recognition of processed MHC molecules in vascularized allograft rejection and confirm the role of the thymus in acquired systemic tolerance to alloantigens.

Animals↗
PubMed1993

Chronic rejection in experimental cardiac transplantation: studies in the Lewis-F344 model.

Despite recognition of chronic vascular injection by numerous investigators since the beginning of experimental and solid organ transplantation, the pathogenesis of graft arteriosclerosis remains poorly understood. We have defined a reproducible model of this disease by transplanting heterotopic cardiac grafts across minor histocompatibility barriers using inbred strains of rat. We found that long-term surviving Lewis grafts in untreated F344 recipients are subjected to a chronic rejection process which results in the development of diffuse graft arteriosclerotic lesions, indistinguishable in appearance from those seen in human cardiac grafts. Immunohistochemical studies confirm that end-stage lesions are similar in composition to human lesions, made up predominantly of vascular smooth muscle cells with occasional monocytes and T cells. Analysis of serial rejecting allografts demonstrates that a distinct inflammatory stage precedes smooth muscle cell accumulation in areas of intimal thickening, suggesting that mononuclear cells play a role in the developing lesion. Endothelial expression of class II and ICAM-1 appears to lead to early mononuclear cell adherence to the endothelium. Analysis using quantitative RT-PCR confirms that MCP-1 is expressed by ED1-positive monocyte/macrophages in rejecting cardiac grafts, suggesting that this chemoattractant may help drive mononuclear cell accumulation in the expanding intima. Immunohistochemical labelling of PDGF, TNF, and IL-1 in vascular lesions suggests that these growth factors may trigger intimal vascular smooth muscle cell proliferation in chronically rejecting allografts. Hypercholesterolemia did not enhance the severity of lesion development in long-term surviving allografts, suggesting that lipid levels are not a major etiologic factor in graft arteriosclerotic lesion formation in the Lewis-F344 model. Finally, the dietary manipulation of EFAD reduced graft infiltration by mononuclear cells and markedly diminished arterial lesion development in chronically rejecting grafts. Heparin anologues have previously been shown to inhibit proliferative vascular smooth muscle cell lesions in rats following endothelial injury (Clowes & Karnovsky 1977), and we are currently assessing the role of heparin in the therapy of graft arteriosclerosis in the Lewis-F344 model. We are also investigating the role of CD4-positive mononuclear cells in the pathogenesis of lesion development, using the anti-CD4 monoclonal antibody BWH-4 to deplete recipients of CD4-positive cells (Sayegh et al. 1991). In summary, our studies in the Lewis-F344 model suggest that monocyte/macrophages play an important role in the pathogenesis of cardiac graft arteriosclerosis. Future studies utilizing this model should help further elucidate the mechanisms resulting in--and help define potential therapies for--chronic rejection in cardiac transplantation.

Animals↗
PubMed1992

Induction of immunity and oral tolerance with polymorphic class II major histocompatibility complex allopeptides in the rat.

We studied the immunogenicity and tolerogenicity of class II major histocompatibility complex (MHC) allopeptides in the rat. Inbred LEW (RT1l) rats, used as responders, were immunized in the foot pad with a mixture of eight synthetic class II MHC allopeptides emulsified in complete Freund's adjuvant. These sequences represent the full-length second domain of RT1.Bu and RT1.Du (WF) beta chains. In vitro, responder lymphocytes harvested from popliteal and inguinal lymph nodes of immunized animals exhibited significant proliferation to the MHC allopeptide mixture. In addition, these responder lymphocytes had significantly increased proliferation to allogeneic WF (RT1u) stimulator cells, when compared to naive controls in the standard one-way mixed lymphocyte response. In vivo, peptide-immunized LEW animals were challenged in the ear 2 weeks after immunization with the allopeptide mixture, the individual allopeptide sequences, or allogeneic WF splenocytes. When compared to controls, these animals had significant delayed-type hypersensitivity responses to the allopeptide mixture, to the beta-pleated sheet allopeptide sequences, and to allogeneic WF splenocytes but not to the alpha-helix allopeptide sequences, to syngeneic LEW splenocytes, or to third party allogeneic BN splenocytes. Oral administration of the allopeptide mixture to LEW responder rats daily for 5 days before immunization effected significant reduction of delayed-type hypersensitivity responses both to the allopeptide mixture and to allogeneic splenocytes. This reduction was antigen-specific, since there was no reduction of delayed-type hypersensitivity responses to mycobacterium tuberculosis. These data demonstrate that lymphocytes from animals immunized with polymorphic class II MHC allopeptides can recognize and proliferate to the same amino acid sequences on allogeneic cell surface MHC molecules. In addition, oral administration of these peptides down-regulates the systemic cell-mediated immune response in a specific fashion. Synthetic MHC allopeptides should allow the study of alloimmunity in vivo, including induction of immune tolerance.

Amino Acid Sequence↗
PubMed1992

Down-regulation of the immune response to histocompatibility antigens and prevention of sensitization by skin allografts by orally administered alloantigen.

The effects of oral administration of major histocompatibility antigens on the alloimmune response have not been investigated. Lymphocytes from inbred LEW (RT1u) rats that were pre-fed allogeneic WF (RT1l) splenocytes exhibited significant antigen specific reduction of the mixed lymphocyte response in vitro and delayed-type hypersensitivity response in vivo, when compared with unfed controls. In an accelerated allograft rejection model, LEW rats were presensitized with BN (RT1n) skin allografts 7 days before challenging them with (LEW x BN)F1 or BN vascularized cardiac allografts. While sensitized control animals hyperacutely reject their cardiac allografts within 2 days, animals prefed with BN splenocytes maintained cardiac allograft survival to 7 days, a time similar to that observed in unsensitized control recipients. This phenomenon was antigen-specific, as third-party WF grafts were rejected within 2 days. Immunohistologic examination of cardiac allografts harvested on day 2 from the fed animals had markedly reduced deposition of IgG, IgM, C3, and fibrin. In addition, there were significantly fewer cellular infiltrates of total white blood cells, neutrophils, macrophages, T cells, IL-2 receptor-positive T cells, and mononuclear cells with positive staining for the activation cytokines IL-2 and IFN-g. On day 6 posttransplant, the grafts from fed animals showed immunohistologic changes typical of acute cellular rejection usually seen in unsensitized rejecting controls. Feeding allogeneic splenocytes prevents sensitization by skin grafts and transforms accelerated rejection of vascularized cardiac allografts to an acute form typical of unsensitized recipients. Oral administration of alloantigen provides a novel approach to down-regulate the specific systemic alloimmune response against histocompatibility antigens.

Administration, Oral↗
PubMed1992

Blocking of mononuclear cell accumulation, cytokine production, and endothelial activation within rat cardiac allografts by CD4 monoclonal antibody therapy.

CD4 monoclonal antibody therapy prolongs allograft survival in a variety of experimental models and is currently undergoing clinical trials, though surprisingly little is known about the effects of CD4 mAb therapy on intragraft effector mechanisms that mediate rejection. We previously reported the significantly improved survival of (LEWxBN)F1 cardiac allografts in LEW rats treated for 10 days with the new CD4 mAb, BWH-4, at a dose of 700 micrograms/day, i.v., starting at the time of engraftment. Thus, CD4-treated rats showed prolongation of allograft survival to a median of 37 days (range 22 to greater than 100 days) post-Tx, compared with rejection at 7 days in untreated controls. We now report the results of detailed immunohistologic studies of allografts collected from these rats. Comparison of acutely rejecting allografts in untreated rats with well-functioning allografts collected at day 7 post-Tx from CD4-treated rats showed that CD4 mAb: (1) significantly reduced mononuclear cell infiltration, interstitial edema, hemorrhage formation and vascular and extravascular thrombosis; (2) inhibited mononuclear cell induction of receptors for IL-2 and transferrin, and upregulation of class II antigens and ICAM-1 on leukocytes and endothelial cells; (3) suppressed intragraft mononuclear cell and/or endothelial production of the cytokines IL-1, IL-2, IL-6, IFN-gamma, and TNF; and (4) blocked upregulation of endothelial tissue factor and downregulation of thrombomodulin, and consequently inhibited fibrin deposition. Studies of allografts from CD4-treated rats collected at day 30 post-Tx, prior to clinical rejection, showed a resurgence of CD4+ cells within allografts and a dense cellular immune response. We conclude that short-term CD4 mAb therapy has potent and extensive inhibitory effects on cytokine-related mononuclear cell and endothelial activation in vivo, blocking multiple afferent and efferent steps of the alloresponse.

Animals↗
PubMed1992

Evidence that therapeutic strategies targeted at CD4+ cells modulate accelerated rejection of cardiac allografts in sensitized rats by different mechanisms.

(LEW x BN)F1 cardiac allografts are rejected within 36 hr in LEW rats presensitized with BN skin grafts 7 days earlier (acute rejection occurs within 8 days). We have previously described the effects of individual CD4 (BWH-4), CD25 (IL-2R, ART-18) mAbs, and CsA therapeutic regimens upon cardiac allograft survival in sensitized hosts. The present studies were designed to probe an adjunctive use of ART-18 or CsA upon BWH-4-mediated suppression of accelerated graft injury. Sequential therapy with BWH-4 and ART-18 in the sensitization phase (days -7 to -1) and effector phase (from day 0, the day of cardiac transplant), respectively, prolonged graft survival additively to c. 22 days. Treatment with BWH-4 markedly diminished host humoral response against ART-18 preparation. BWH-4 given in concert with subtherapeutic dose of CsA produced graft survival comparable to that induced by mAb alone (c. 13 days) with concomitant decreased host anti-BWH-4 response. None of the combined regimens affected the frequency of circulating CD4+ cells, as compared with that exerted by BWH-4 monotherapy. Thus this study defines principles and some mechanistic aspects of optimal immunosuppressive strategies potentiating the effects of CD4-targeted therapy.

Animals↗
PubMed1991

Differential role of CD4+ cells in the sensitization and effector phases of accelerated graft rejection.

Although CD4-targeted therapy markedly prolongs survival of organ allografts in naive rodents, its effects in primed hosts have not been studied. In our model of accelerated rejection (ACCR) of cardiac Tx in rats, treatment with BWH-4, a CD4 mAb (IgG2a), in the sensitization (between skin and heart Tx) but not in the effector (after cardiac Tx) phase, abrogated fulminant less than 36 hr rejection response and prolonged Tx survival to ca. 11 days. This effect correlated with decreased frequency of circulating CD4+ cells, but it did not depend upon their total depletion. It was also related to BWH-4 mAb-mediated elimination/depression of strong anti-donor humoral responses and cellular responses as determined by lymphocyte-mediated cytotoxicity and mixed lymphocyte reaction and mounted otherwise at the time of engraftment by untreated sensitized hosts. Immunoperoxidase studies of cardiac Tx from BWH-4-conditioned recipients revealed reduced T and B cell activities, reflected in abolition/reduction in deposition of humoral mediators, infiltrating cells, intra-Tx elaboration of interleukin-2 and interferon-gamma, and cell activation. This first report of the successful use of CD4 mAb in sensitized recipients of vascularized organ Tx, stresses the role of CD4+ cells as potential targets for immunosuppression in the sensitization phase of accelerated Tx injury. The beneficial therapeutic effect, probably due to both depletion and functional inhibition of CD4+ T cells, has been achieved by using relatively low doses of BWH-4 mAb.

Animals↗
PubMed1991

Effects of BWH-4 anti-CD4 monoclonal antibody on rat vascularized cardiac allografts before and after engraftment.

We studied the effects and mechanism of action of BWH-4, an IgG2a mouse antirat CD4 monoclonal antibody that recognizes a distinct epitope on the CD4 molecule, in LEW recipients of (LEW x BN)F1 vascularized heterotopic cardiac allografts. Ten animals received daily injections of 700 micrograms BWH-4 for ten days after engraftment. Median actuarial allograft survival was 37 days for the treated animals compared with 7.5 days for controls (n = 6). There was depletion of peripheral blood CD4+ lymphocytes to 4 +/- 1% one week posttransplant (normal = 48 +/- 4%, n = 6). Six additional animals were treated with a lower dose (100 micrograms) of BWH-4 daily for ten days. Median actuarial allograft survival was 10 days and the circulating CD4+ cells decreased to 30 +/- 6% at one week posttransplant. All six low-dose-treated animals mounted an anti-BN alloantibody response by 3 weeks, while only one of the ten high-dose-treated animals had positive alloantibodies two weeks posttransplant. Lymphocyte-mediated cytotoxicity against donor lymphoblasts was completely abolished in the high-dose-treated animals when compared with acutely rejecting controls. We also used low-dose (100 micrograms) BWH-4 to pretreat eleven experimental animals for 7 days prior to engraftment. The circulating CD4+ cells decreased to only 12 +/- 2% on the day of transplantation and 26 +/- 9% one week posttransplant. However, six (55%) pretreated animals survived more than 55 days. There was a 66% decrease in lymphocyte-mediated cytotoxicity against donor lymphoblasts when compared with acutely rejecting controls. We conclude that the anti-CD4 mAb, BWH-4, prevents acute rejection of vascularized heterotopic rat cardiac allografts; this effect is mediated by depletion of the CD4+ T cell subset, suppression of alloantibody production, and inhibition of lymphocyte-mediated cytotoxicity.

Animals↗