(LEW X BN)F1 cardiac allografts are rejected acutely (7 days) in unmodified LEW rats, yet survive indefinitely following cyclosporine (CsA) treatment (15 mg/kg im for 7 days) or in T-cell-deprived (B) recipients. Using these models, the function of T helper cells (Th) in the maintenance phase of CsA-mediated long-term graft survival was examined. With monoclonal antibody immunoaffinity fractionation techniques, Th (W3/25+OX8-) were separated from spleens of CsA-treated hosts 3-4 weeks after grafting (CsA-Th), from specifically sensitized (s-Th), or from normal ungrafted (n-Th) rats. Adoptive transfer of 60 X 10(6) CsA-Th into B recipients produced rejection of donor-specific, but not third-party grafts in 21 +/- 7 days, comparable to s-Th (17 +/- 4 days), but faster than n-Th (4-5 weeks, P less than 0.025). CsA-Th recombined with T cytotoxic/suppressor phenotype (CsA-Tc/s, OX8+W3/25-) in numbers contained in 100 X 10(6) CsA-T cells were ineffectual, even when supplemented with exogenous interleukin 2-rich conditioned medium (IL-2CM); in contrast 100 X 10(6) s-T cells + IL-2CM inevitably caused acute rejection in B hosts (11 +/- 3 days). Increasing numbers of Th incrementally to 100 X 10(6) augmented the effectiveness of s-Th (rejection in 13 +/- 2 days), but did not improve potency of CsA-Th (20 +/- 2 days). Suppressor activity produced by small numbers of contaminating CsA-Tc/s (c. 0.4%, 4-5 X 10(5) cells in 100 X 10(6) CsA-Th) accounted for extended graft survival in B recipients, as this small number of CsA-Tc/s transferred into untreated syngeneic rats increased test graft survival to c. 16 days (P less than 0.001). IL-2 production by spleen cells, depressed during CsA treatment, returned to normal levels 2-3 weeks following drug withdrawal, whereas transfer of CsA-Th into B recipients induced a shift of IL-2 levels from dramatically depressed to normal, findings suggesting normal IL-2 production by CsA-Th. This report demonstrates that an unresponsive state in CsA-treated animals is achieved despite the presence of fully potent donor-specific Th. Active suppressor activity plays a critical role in the maintenance phase of graft survival in rats treated transiently with CsA.

Animals↗

PubMed1985

Phenotype, activation status, and suppressor activity of host lymphocytes during acute rejection and after cyclosporine-induced unresponsiveness of rat cardiac allografts.

Serial changes in phenotype, cell cycle, and functional behavior of lymphocyte subpopulations occurring both during acute rejection in unmodified hosts and in long-surviving heterotopic cardiac allografts in rats treated with cyclosporine (CsA) were studied. Using flow cytometry, RNA and DNA content of cells was examined during various phases of cell activation. In animals acutely rejecting their grafts, numbers of cells infiltrating the grafts and in host spleen in G1 phase (higher RNA content) increased, starting from day 3, and peaked by 5-6 days posttransplantation, and numbers of cells in S/G2/M phase (higher DNA content) remained stable. Similar, although slightly delayed changes were noted in CsA-treated recipients. The ratio of T helper (Th) to T suppressor/cytotoxic (Ts/c) phenotype cells infiltrating acutely rejecting grafts by day 3, was 1.6; it inverted abruptly to 0.7 by days 5-6, suggesting a preponderance of Ts/c during the later stages of allograft rejection. Ratio inversion occurred slightly later in host spleen and later in peripheral blood. Similarly, treatment with CsA produced a transient depression of Th, with recovery of the Th: Ts/c ratio during weeks 3-4 following transplantation. Adoptive transfer studies were then performed to investigate the functional significance of the T cell subsets. Survival of test grafts was prolonged significantly (ca. 14 days, P less than 0.001) when cells infiltrating grafts and spleen were transferred during inversion of Th:Ts/c; before that period, test graft survival was shortened in a second-set manner. These experiments suggest that suppressor cells may be responsible for resolution of acute rejection, as well as for host unresponsiveness seen after CsA treatment, and they represent an important homeostatic host mechanism following immunological stimulation.

Animals↗

PubMed1985

Lack of evidence for an active role for natural killer cells in acute rejection of organ allografts.

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Animals↗

PubMed1984

Apparent superiority of H2-receptor stimulation and simultaneous beta-blockade over conventional treatment with beta-sympathomimetic drugs in post-acute myocardial infarction: cardiac effects of impromidine--a new specific H2-receptor agonist-in the surviving catecholamine-insensitive myocardium.

Left ventricular infarction (AMI) was produced in experimental animals and the contractile response to beta-adrenergic and H2-histaminergic stimulation by isoproterenol and impromidine tested in the isolated perfused heart preparation. Adenylate cyclase activity as well as binding characteristics of [3H]-dihydroalprenolol ([3H]-DHA), [3H]-methyl-tiotidine ([3H]-TIOT) and [3H]-quinuclidinyl benzilate ([3H]-QNB) to cardiac beta 1-, H2- and cholinergic muscarinic receptors were determined in sarcolemmal membrane preparations of the right ventricle of the same hearts. In addition, an attempt was made to elucidate the therapeutic value of post-AMI treatment with impromidine in the presence and absence of beta-blockade, in contrast to administration of prenalterol and the conventional therapy with beta-sympathomimetic drugs, e.g. dobutamine. Three days post-AMI the dose-response curve for isoproterenol of right ventricular dP/dtmax was significantly depressed, while the inotropic effect of impromidine was not impaired. Stimulation of adenylate cyclase activity by isoproterenol was reduced by 80% whereas impromidine and NaF stimulation rates were unaltered. Receptor-binding studies indicated a 90% loss and 10-times lowered affinity (KD) of the remaining beta-receptors while specific [3H]-TIOT- and [3H]-QNB-binding was unchanged. Administration of dobutamine increased mortality rates and extension of infarct size, led to a further decrease in contractile response to isoproterenol, induced complete insensitivity of adenylate cyclase to isoproterenol stimulation and caused pronounced additional reduction of number and affinity of [3H]-DHA-binding sites. In contrast, all above alterations were prevented by treatment with either prenalterol or combined administration of impromidine plus metoprolol. It is concluded, that these alterations in the non-ischemic, uninvolved myocardium post-AMI are the result of catecholamine-induced specific damage of sarcolemmal beta-receptors. Furthermore, treatment with H2-agonists in combination with beta-blocking agents may have beneficial effects, whereas conventional therapy with beta-sympathomimetic drugs tends to worsen the already depressed function of the beta-adrenergic stimulation mechanism.

Adenylyl Cyclases↗

PubMed1984

Restoration of allograft responsiveness in B rats. IV. The divergent migratory behavior of lymphocyte populations mediating cardiac allograft rejection.

The T lymphocyte-deprived (B) rat, produced by X-radiation and bone marrow reconstitution of adolescent thymectomized animals, exhibits a true immunological deficit and are unable to reject histoincompatible heterotopic cardiac allografts. A comprehensive survey of lymphocyte traffic in B recipients was performed to correlate the differential potency of specifically sensitized lymphocyte populations mediating re-establishment of immune responsiveness toward the graft, with their migratory and recirculatory behavior. 111In-oxine-labeled thoracic duct lymphocytes (TDL) were retained in the peripheral blood and migrated from nonlymphoid organs to lymph nodes of B recipients in higher proportion than any other lymphoid population, particularly splenic lymphocytes (SL). Although all cell groups but TDL were sequestered in the spleen in equal and relatively large numbers, no differences were found between the lymphocyte populations tested in their capacity to accumulate in the grafts. In contrast, an increased avidity in the allograft of 125IUdR-labeled TDL and lymph node (LNL) lymphoblasts, as compared to 125IUdR-labeled SL, resembles closely the results of functional studies of the differential potency of adoptively transferred cells. We assume that specific cellular interactions induced by the accumulated 125IUdR-labeled cells invoke nonspecific mechanisms for the recruitment of other uncommitted 111In-labeled lymphocytes which recirculate between blood and lymph and localize indiscriminately in the allograft amplifying its rejection. The latter lymphocytes can be "armed" by adherent cells residing in the lymphoid organs of graft recipients, particularly spleen, and subsequently increase the penetration of the foreign tissue. When radiolabeled lymphocytes were traced in B recipients experiencing rejection of their allografts following transfer of sensitized cells plus lymphokine, their migration patterns as well as blastogenic response in B hosts were similar to those observed during acute rejection of cardiac allografts in unmodified hosts. Thus the similarities between the rejection network brought by alloimmune cells into otherwise unresponsive animals and immunocompetent animals able to reject their grafts are stressed.

Animals↗

PubMed1984

Mechanisms of rejection and prolongation of vascularized organ allografts.

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Animals↗

PubMed1984

Interactions between T lymphocyte subsets supported by interleukin 2-rich lymphokines produce acute rejection of vascularized cardiac allografts in T cell deprived rats.

A series of adoptive transfer studies were performed to define both the role of lymphocyte subpopulations and of lymphokines in the reestablishment of immunologic responsiveness towards vascularized organ allografts in T cell-deprived B rats. Acute rejection of otherwise indefinitely surviving cardiac allografts (10.9 +/- 2.9 days) occurred when sensitized T cells were adoptively transferred with a course of partially purified, lectin-free IL 2-conditioned medium (IL 2CM), whereas rats receiving T cells alone rejected their grafts at 19.8 +/- 4.6 days (p = 0.025). Rejection is mediated primarily by alloactivated T helper cells highly enriched for W3/25 phenotype. The relationship between the mean allograft survival time and the cell number transferred was of exponential order. In contrast to rejection occurring after the transfer of intact T cells, that produced by W3/25+ cells was independent of the administration of exogenous IL 2CM. W3/25+ T helper cells were also found to be less potent in mediating graft rejection than were whole T cells when limited cell numbers were transferred. The OX8+ T cytotoxic/suppressor cell subpopulation, even when supplied with a course of IL 2CM, was unable to bring about rejection, but induced transient graft enlargement in about 50% of the animals. Recombination of individual subsets caused acute rejection at a rate comparable to that of unseparated T cells when IL 2CM was supplied (10.8 +/- 1.0 days), in contrast to those rats which received the recombined inoculum in the absence of IL 2CM (16.8 +/- 2.2 days, p = 0.025). Lymphocyte migration studies revealed a more vigorous homing of OX8+ cells to the allograft as compared to W3/25+ cells. However, to produce maximal accumulation of transferred cells in the graft, both T cell subsets supplemented by lymphokines must be introduced together into T cell-deprived hosts.

Animals↗

PubMed1984

Cyclosporine therapy of rat heart allograft recipients and release of interleukins (IL 1, IL 2, IL 3): a role for IL 3 in graft tolerance?

LEW rat recipients of (LEW X BN)F1 strain heterotopic cardiac transplants treated with cyclosporine A (CsA) (15 mg/kg/day intramuscularly, 7 days) retain grafts indefinitely despite drug withdrawal. Donor-specific suppressor T cells that are active in passive transfer experiments have been harvested from long-term CsA-treated hosts. Although CsA is known to inhibit in vitro cytokine release, the in vivo effects of the CsA on the lymphokine cascade are not known. We investigated the action of the drug upon spontaneous and mitogen-induced interleukin 1 (IL 1), interleukin 2 (IL 2), and interleukin 3 (IL 3) release by spleen cells obtained from the following groups of rats: 1) normal, i.e., untreated and ungrafted; 2) grafted, acutely rejecting; 3) grafted, actively treated; and 4) under CsA-induced state of "tolerance." The results demonstrate that in vivo CsA therapy inhibits monocyte (IL 1 release) as well as lymphocyte function (IL 2 and IL 3 release) only during the inductive phase (the 7 days of treatment). During the "tolerant" phase, mitogen (Con A and LPS)-induced release of interleukins was quantitatively similar to that noted in normal animals. In contrast, a remarkable increase in the spontaneous production of IL 3 was observed in the "tolerant" group. Because cytokine release is not inhibited in the "tolerant" state, our data strongly support the concept that maintenance of the state of unresponsiveness is governed by the emergence of suppressor cells. The correlation of increased spontaneous production of IL 3 during this period leads us to postulate that this interleukin may be implicated in the activation or clonal expansion of suppressor cells, and hence may play a role in graft tolerance.

Animals↗

PubMed1984

Modification of function and migration patterns of thymocyte populations by cyclosporine after organ transplantation in rats.

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Animals↗

PubMed1983

Restoration of allograft responsiveness in B rats. II. Requirements for lymphoid populations and lymphokine.

B rats, produced by lethal x-irradiation of thymectomized animals that are then reconstituted with bone marrow cells from syngeneic, thymectomized, and thoracic-duct-drained donors, are unable to reject histoin-compatible heterotopic cardiac allografts. In these studies, we have continued to investigate the role of immune competent cells and lymphokine in restoring acute allograft rejection in these animals, noting that adoptive transfer of 10(8) sensitized splenocytes (sSL) plus Interleukin-2-rich conditioned medium, IL-2 (CM) will reproducibly produce acute responsiveness toward long-standing, well-functioning, heart grafts. We have now noted that sensitized lymph node cells or thoracic duct lymphocytes can also reverse the unresponsive state, with IL-2 rich conditioned supernatants increasing the effectiveness of small numbers of cells from these populations in inducing allograft rejection. Furthermore, the major cellular components within the spleen have been separated using antibody techniques, and their individual role in the rejection reaction has been assessed. Although 10(8) sSL plus IL-2 (CM) can produce acute rejection in a time comparable to that occurring in unmodified recipients (n = 20, MST +/- SD = 8.4 +/- 1.3 days), neither the T cell fraction (6 x 10(7] of 10(8) SL + IL-2 (CM) (n = 10, MST +/- SD = 17.4 +/- 7.3 days), nor addition of the adherent cell fraction to the T cell component (MST +/- SD = 18 +/- 2 days) or addition of the B cell fraction (MST +/- SD = 15.5 +/- 2.1 days) could restore acute responsiveness. However, increasing the numbers of T cells to 10(8) and transferring concomitantly with IL-2 (CM) again produced acute rejection (n = 4, MST +/- SD = 9 +/- 1.1 days). These data suggest that T cells form the essential element of transfer in the B rat, although other cells present in SL appear necessary to provide an optimal milieu. Our experiments also suggest that this response is independent of the route of administration or of higher doses of IL-2. Finally, using a dual allograft model, it appears that graft destruction is determined by the specific sensitivity of transferred SL; IL-2 (CM) being a nonspecific factor in the response.

Animals↗

PubMed1982

Deleterious effects of cimetidine in the presence of histamine on coronary circulation. Possible clinical implications in anaphylactic states in individuals with coronary heart disease.

The present study characterizes effects of histamine in the presence of the H2-antagonist cimetidine on the coronary circulation of the isolated perfused spontaneously beating guinea pig heart. Infusion of histamine (2 x 10(-8) mol/1-5 x 10(-6) mol/l) induced a dose-dependent coronary dilation, comparable to the effect of isoproterenol and two highly-selective H2-agonistic compounds, impromidine and dimaprit. In the presence of cimetidine (5 x 10(-6) mol/l), however, coronary response to histamine was reversed in a manner that a dose-dependent coronary constriction occurred with coronary spasm and a flow rate approaching zero at histamine concentrations above 8 x 10(-7) mol/l, whereas the dilatory effect of impromidine and dimaprit was completely antagonized. In contrast, the histamine-induced constriction in the presence of cimetidine could be nearly abolished by additional infusion of the H1-antagonistic compound mepyramine (5 x 10(-5) mol/l). Is is concluded that H1- and H2-receptors are present in the coronary smooth muscle, H1-receptors mediating constriction and H2-receptors mediating coronary dilation. Speculation is provided that histamine may have hazardous effects in anaphylactic states if cimetidine is administered simultaneously, e.g., to prevent or cure peptic ulcer. Other possible clinical implications will be discussed.

Anaphylaxis↗

PubMed1981

Cardiac contractile and metabolic effects mediated via the myocardial H2-receptor adenylate cyclase system. Characterization of two new specific H2-receptor agonists, impromidine and dimaprit, in the guinea pig and human myocardium.

The present study chartacterizes myocardial effects of two new histaminergic H2-receptor specific compounds, impromidine, and dimaprit, on cardiac contractile and metabolic parameters of the guinea pig heart and human papillary muscle in comparison to the well-known effects of catecholamines. Impromidine and dimaprit produced a dose-dependent stimulation of the right and left ventricular contractile force in the isolated perfused biventricular catheterized guinea pig heart with maximal stimulation rates equal to those of isoproterenol. Hemodynamic equieffective doses of isoproterenol (2.8X10(-9) mol/l), histamine (1.1X10(-5) mol/l), impromidine (4.6X10(-7) mol/l, and dimaprit (8.5X10(-6) mol/l) induced nearly identical increases in tissue concentrations of c-AMP. All compounds dose-dependently enhanced the activity of the myocardial adenylate cyclase with very similar KA-values in a particulate sarcolemmal membrane preparation of both guinea pig ventricles and human papillary muscles. No effect of either compound was seen on cardiac phosphodiesterase activity. Selective administration of the beta1-blocking agent metoprolol and the H2-receptor antagonist cimetidine clearly discriminates two independent receptors linked to the sarcolemmal adenylate cyclase system in the guinea pig and human myocardium. This is further supported by results obtained from beta-receptor-binding studies in which an interference of impromidine and dimaprit with the stereospecific binding of (-)[3H]-dihydroalprenolol to cardiac beta-receptors could be definitely excluded. The possible therapeutic role of both H2-agonists on the non-ischemic, surviving myocardium, which is transiently refractory to beta-adrenergic stimulation by catecholamines after myocardial infarction, will be discussed.

Adenylyl Cyclases↗

PubMed1980

[Experimental investigations of rat ileum enterotomies after application of physiological plasma fractions (author's transl)].

Standardised enterotomies of the rat ileum were performed by conventional enterorrhaphy and in combination with the fibrin adhesion technique. When enterotomy is also sealed with fibrin adhesive, the initial bursting strength is significantly higher. In the postoperative course this is only valid if a successful inhibition of fibrinolysis can be achieved by adding fibrinolytic inhibitors. Histochemical investigations of tissue enzyme activities confirm these findings.

Animals↗