Biomedical subjects
Publications and source records attributed to R R Rich.
Transureteroureterostomy in 6 dogs with the ureter transposed to cross the arterial notch between the caudal (inferior) mesenteric artery and aorta did not result in vascular induced ureteral obstruction.
Four days after injection of allogeneic lymphocytes BALB/c splenic T cells suppress proliferation of syngeneic cells in mixed lymphocyte reactions (MLR). Conversely, lymph node cells from the same mice amplify MLR responses. To further characterize these functional subpopulations, alloantigen-primed lymphocyte suspensions from both organs were fractionated by velocity sedimentation at unit-gravity. After fractionation MLR suppressor cells from spleens localized exclusively in rapidlly sedimenting fractions of large cells. MLR suppressor activity of cells from these fractions, as well as that of unfractionated spleen cell suspensions, was abolished by treatment with anti-Thy-1.2 serum and complement. Spleen cell fractions of similar sedimentation velocity also secreted a soluble MLR suppressor into culture supernatants. Although inhibitory of MLR, spleen cells of rapid sedimentation velocity did not suppress responses to T cell mitogens. In marked contrast with the effects of spleen cells, large 4-day-alloantigen-primed lymph node cells had no suppressive activity in MLR. MLR amplifier cells of uncertain derivation were found in fractions of medium sedimentation velocity from both spleens and lymph nodes. Fractionation of alloantigen-primed lymph node cell suspensions did reveal, however, a subpopulation of small cells with MLR suppressor acitivty which was unaffected by treatment with anti-Thy-1 serum and complement. The data thus indicate that large alloantigen-activated lymphocytes are not intrinsically suppressive nor are cells which suppress MLR necessarily large. We consequently conclude that regulation of MLR responses by alloantigen-primed lymphocytes involves a complex interaction between distinct functional subpopulations of cells which are separable both by physical and biologic properties.
Spleen cells obtained from BALB/c mice 4 days after allosensitization to C57BL/6 spleen cells via footpad injection suppressed the in vitro generation of BALB/c cytotoxic lymphocytes (CL) against C57BL/6 spleen cells in mixed leukocyte cultures (MLC). Suppressor activity was demonstrated by spleen cells at 4 and 7 days but not at 2, 10, or 14 days after allosensitization and was abolished by treatment with anti-Thy-1,2 serum and complement. A weak and transient cytotoxic response directed against the sensitizing alloantigen was associated with suppressor spleen cell populations, but was dissociated from suppressor function by two experimental approaches. First, increasing stimulatory cell concentration in MLC did not competitively diminish the suppressor activity; rather, the magnitude of suppression increased as the stimulatory cell concentration was increased. Second. BALB/c suppressor cells generated in vivo by either H-2b or H-2k alloantigens suppressed CL responses generated simultaneously against both alloantigens in vitro. CL responses generated against one or the other H-2 haplotype in vitro were suppressed only by suppressor cells activated by that haplotype. Therefore, splenic suppressor cells activated by alloantigen in vivo required antigen-specific restimulation in vitro; thereafter, responder cells syngeneic with the suppressor cell were rendered hyporesponsive to alloantigens by an antigen-nonspecific mechanism.
Suppression of the mixed lymphocyte reaction (MLR) by a soluble factor produced by alloantigen-activated spleen cells requires genetic homology between the factor-producing cells and responder cells in MLR. The ability of lymphocytes used as MLR responder cells to adsorb MLR suppressor factor was tested to investigate the expression of a receptor structure for suppressor molecules. Normal spleen or thymus cells had no effect on suppressor activity. Concanavalin A (Con A)-activated thymocytes, however, effectively removed suppressor activity, suggesting that the receptor is expressed only after activation and is not present or not functional on resting cells. Significantly neither phytohemagglutinin- nor lipopolysaccharide-activated lymphoid cells absorbed the factor. Furthermore, only Con A-activated thymocytes demonstrating genetic homology with the cell producing suppressor factor for H-2 regions to the right of I-E were effective absorbants. Alloantigen-stimulated spleen cells syngeneic to the suppressor cell also removed suppressor activity. These data support an hypothesis that subsequent to stimulation in MLR, T lymphocytes express a receptor, either through synthesis or alteration of an existing molecular structure, which then provides the appropriate site for interaction with suppressor molecules.
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Active suppression of mixed lymphocyte reaction (MLR) response is mediated by a soluble factor released by alloantigen-activated murine suppressor cells. Genetic restrictions controlling suppressor factor interaction with MLR responder cells were elucidated in this study. Non-H-2 genetic background was irrelevant to effective interaction. Using congenic strains and strains with intra-H-2 recombinants the genetic locus controlling suppressor T-cell-responder cell interaction was mapped in the I-C or S regions of the H-2 complex. Similarly, recombinant strains were used to exclude the presence of another suppressor cell-responder cell interaction locus in K,I-A, and I-B regions. It thus appears that the I-C subregion of the H-2 complex controls suppressive cell interactions in this T-cell-mediated immune response.
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Supernatant fluids from murine spleen cell cultures incubated with concanavalin A for 48 hr contain a factor(s), soluble immune response suppressor (SIRS), which suppresses plaque-forming cell responses to sheep erythrocytes by murine spleen cells in vitro. In the present studies, some of the biochemical and biophysical properties of SIRS were investigated. SIRS was non-dialysable; the suppressive activity was stable at 56 degrees C for 30 min, but was destroyed by treatment at 70 degrees C for 30 min, 80 degrees C for 10 min, or at pH 2. The suppressive activity was not absorbed by the stimulating antigen, SRBC, or antisera against murine IgG or mu-chain, suggesting that SIRS does not contain immunoglobulin determinants. Murine spleen and thymus, but not kidney cells, however, absorbed SIRS activity. Enzyme treatments revealed that SIRS was resistant to DNase and RNase, but was destroyed by trypsin and chymotrypsin. In gel filtration with Sephadex G-100, SIRS activity eluted in the fraction corresponding to m.w. in the range between 48,000 and 67,000. With polyacrylamide gel electrophoresis, SIRS activity migrated in the region cathodal to albumin. Isopycnic centrifugation in a cesium chloride gradient suggested that SIRS is a glycoprotein. These supernatant fluids with SIRS activity were also found to contain macrophage migration inhibitory factor (MIF). In the experiments using gel filtration, polyacrylamide gel electrophoresis, and isopycnic centrifugation to fractionate supernatant fluids, SIRS and MIF activity were found in the same fractions, and to date we have been unable to dissociate definitively SIRS activity from MIF activity.
The mechanism of alloantigen-activated spleen cell suppression of mixed lymphocyte reaction (MLR) is explored in this report. Activated murine suppressor spleen cells elaborated a soluble noncytotoxic factor which suppressed MLR responses by 55-95%. Generation of suppressor factor required both in vivo alloantigen sensitization and specific in vitro restimulation. Suppressor factor was not produced by activated spleen cells which had been treated with anti-Thy-1.2 serum and complement. Antigenic specificity toward alloantigens of the stimulator cells was not demonstrable. In contrast, suppressor factor effectively inhibited MLR response only of responder cells of those strains that shared the D-end and the I-C subregion of the H-2 complex with the cells producing suppressor factor. Therefore, active suppression appears to require an MHC-directed homology relationship between regulating and responder cells in MLR.
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Thymus-derived lymphocytes (T cells) from mouse spleen, activated in vitro or in vivo with concanavalin A (Con A), suppress proliferative responses of syngenic lymphocytes in mixed lymphocyte reactions (MLR). Replication in vitro was not required for expression of suppressor activity by Con A-activated cells and was blocked in MLR by treating suppressor cells with mitomycin C or irradiation. Kinetics of MLR responses and viability of cultures were not altered by addition of activated suppressor cells. The data are consistent with a direct inhibitory effect of suppressor T cells on antigen-induced DNA replication. These observations extend a model previously described for regulation of antibody synthesis by Con A-activated T cells to control of cell-mediated immune responses. This model should be particularly useful in further definition of regulatory T cell subpopulations, and in investigation of interactions and relationships between such populations.
The effects of Con A on homing patterns of mouse spleen cells were investigated. After 1 to 18 hr incubation with or without the mitogen, cells were labeled with 51Cr and injected i.v. into syngeneic hosts. Treatment of spleen cells with Con A decreased localization in lymph nodes 75 to 95% and inhibited homing to recipient spleens 17 to 50%. Conversely, localization in liver and lungs was increased. Migration of cells labeled with both 125I-Con A and 51Cr revealed that cells binding the largest amount of Con A were trapped in lungs whereas cells migrating to lymph nodes and spleen had a much lower Con A content. Elution of Con A from lymphocyte surfaces with methyl-alpha-D-mannoside (MAM) reduced the percentage of cells localizing in lungs, with a concomitant increase in the percentage of cells migrating to lymph nodes. However, even after MAM treatment, a cell population with a relatively large Con A content localized in the lungs. These data indicate that both surface-bound Con A and Con A-induced changes in lymphocyte membranes inhibit migration of Con A-activated cells to recipient lymph nodes. They suggest that after Con A treatment lymph node-seeking cells do not migrate normally because of preferential sequestration in lungs of lymphocytes with either Con A-binding sites of increased density or avidity or with an increased propensity for Con A internalization.
Regulatory effects of alloantigen-activated thymus-derived lymphocytes in mixed lymphocyte reactions have been demonstrated. Mice were injected into foot pads with allogeneic spleen cells; 4 days following sensitization spleen or regional lymph node cells from these animals were treated with mitomycin C and incorporated into MLR as regulator populations syngeneic to the responder cell type. Activated spleen cells suppressed MLR responses 60-90% whereas activated lymph node cells from the same animals enhanced MLR responses. Suppression by activated spleen cells was not due to cytotoxic effects nor to altered kinetics of the proliferative response. Studies of splenic suppressor cell generation in vivo revealed peak activity four days after alloantigen stimulation with no activity demonstrable at 7 days or at later times. Suppressor cell activity was abrogated by treatment with anti-thetaC3H serum and complement, and was not alloantigen specific.
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A population of thymus-derived lymphocytes has been identified that, upon activation by the nonspecific plant mitogen concanavalin A, suppresses the development of plaque-forming cell responses in fresh or 48-h antigen-stimulated cultures of mouse spleen cells. Suppressor cells can inhibit both primary and secondary IgM and IgG responses in vitro. X-irradiation before activation of peripheral thymus-derived cells by concanavalin A abrogates generation of suppressor cells. After a 48 h activation period, however, the function of concanavalin A-activated suppressor cells is radioresistant. As yet uncertain is whether these suppressor cells are a population of cells distinct from thymus-derived "helper" cells. In certain important regards, the cells mediating these two opposing functions share similar characteristics; the effect observed may be determined by the circumstances of activation or the numbers of activated cells, and may consequently represent different functions of a single thymus-derived regulator cell population.
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