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Biomedical subjects

H R MacDonald

Publications and source records attributed to H R MacDonald.

At least 163 records · Page 9Linked to original sources
PubMed1990

Effect of transforming growth factor beta on the EL4 thymoma variant EL4/6.1: dissociation of inhibition of proliferation from expression of IL-1 and IL-2 receptors.

In order to further characterize the action of transforming growth factor beta (TGF-beta) on lymphoid cells, we investigated the effects of porcine TGF-beta 1 and -2 on the IL-1 sensitive EL4/6.1 thymoma cell line. The proliferation of EL4/6.1 thymoma cells was inhibited by TGF-beta 1 and TGF-beta 2 (1 ng/ml) to a similar degree, the population doubling time was increased by 50-60%, total inhibition was not achieved. This decrease of proliferation was associated with an increase of the number of cells in the G0/G1 compartment of the cell cycle. TGF-beta-mediated inhibition could not be overcome by adding exogenous rIL-1 nor was the binding capacity for IL-1 reduced. In addition, TGF-beta did not interfere with the induction of IL-2 receptors by a combination of Ionomycin+PMA+IL-1. The data suggest that TGF-beta mediated inhibition of thymocyte/lymphocyte proliferation is not associated with an inhibition of the expression or the induction of expression of IL-2 or IL-1 receptors.

Animals↗
PubMed1990

Genes encoding tumor necrosis factor alpha and granzyme A are expressed during development of autoimmune diabetes.

Progressive destruction of the insulin-producing beta cells in nonobese diabetic mice is observed after infiltration of the pancreas with lymphocytes [Makino, S., Kunimoto, K., Muraoka, Y., Mizushima, Y., Katagiri, K. & Tochino, Y. (1980) Exp. Anim. (Tokyo) 29, 1-13]. We show that the genes for tumor necrosis factor alpha and granzyme A, a serine protease associated with cytoplasmic granules of cytotoxic cells, are expressed during the development of spontaneous diabetes mellitus in the nonobese diabetic mouse. Granzyme A-positive cells are found both in and surrounding the islets, implying induction prior to islet infiltration. Tumor necrosis factor alpha expression is exclusively observed in the intra-islet infiltrate, predominantly in lymphocytes adjacent to insulin-producing beta cells, the targets of the autoimmune destruction, implying that tumor necrosis factor alpha expression is induced locally--i.e., in the islet. A considerable portion of cells expressing tumor necrosis factor alpha appear to be CD4+ T cells. This T-cell subset was previously shown to be necessary for development of the disease. Thus, these findings may be important for understanding the pathogenesis of autoimmune diabetes mellitus and potentially also for that of other T-cell-mediated autoimmune diseases.

Animals↗
PubMed1990

Expression of genes encoding cytotoxic cell-associated serine proteases in thymocytes.

A family of homologous serine esterases designated granzyme A-H and the pore-forming protein perforin are present in cytoplasmic granules of mature peripheral cytolytic T lymphocytes and natural killer cells. In vivo, the majority of cytotoxic T cells containing these granule-associated proteins are of the CD4-CD8+ phenotype. It is generally assumed that these cells are derived from immature CD4-CD8- thymocytes. However, the precise intrathymic differentiation steps leading to functionally mature cytotoxic T cells are unclear. Thus we decided to analyze the expression of genes in the thymus which are preferentially expressed in mature cytotoxic cells, i.e. granzyme A, granzyme B, and perforin. In situ hybridization on tissue sections revealed the expression of genes coding for granzyme A and granzyme B in the thymus. No evidence was found, however, for thymocytes expressing the perforin gene. Granzyme A and granzyme B mRNA positive cells in the thymus are almost exclusively CD4-CD8- thymocytes, particularly of the CD3- IL2R- phenotype.

Animals↗
PubMed1989

Positive and negative selection of T cell receptor V beta domains controlled by distinct cell populations in the thymus.

The role of thymic MHC class II-bearing cells in the selection of the TCR repertoire has been investigated in allogeneic radiation bone marrow chimeras. Positive selection of mature CD4+ T lymphocytes expressing the V beta 6+ TCR domain was found to depend upon radioresistant (presumably epithelial) I-E+ thymic cells. On the other hand, negative selection of CD4+ V beta 6+ cells (which was additionally dependent upon expression of the Mls-1a gene product) was controlled by a radiosensitive I-E+ thymic component (most likely dendritic cells). These data argue in favor of a compartmentalization of positive and negative selection events during T cell development.

Animals↗
PubMed1989

Clonal deletion of self-reactive T cells in irradiation bone marrow chimeras and neonatally tolerant mice. Evidence for intercellular transfer of Mlsa.

Tolerance to Mlsa has been shown to be associated with clonal deletion of cells carrying TCR beta chain variable regions V beta 6 or V beta 8.1 in mice possessing I-E antigens. To evaluate the rules of tolerance induction to Mlsa we prepared irradiation bone marrow chimeras expressing Mlsa or Mlsb and I-E by different cell types. Deletion of V beta 6+, Mlsa-reactive T cells required the presence of Mlsa and I-E products either on bone marrow-derived cells or on irradiated recipient cells. Tolerance was induced when Mlsa and I-E were expressed by distinct cells of the chimera. Also neonatally tolerized mice exhibited depletion of V beta 6+ cells after injection of I-E- Mlsa spleen cells (DBA/1) into newborn I-E+ Mlsb mice (BALB/c x B10.G)F1. These results suggest that the product of the Mlsa locus is soluble and/or may be transferred from cell to cell and bound to I-E antigens. The chimera experiments also showed that tolerance to Mlsa is H-2 allele independent, i.e., is apparently unrestricted. Differentiation of chimeric (H-2d/Mlsa x H-2q/Mlsb)F1 stem cells in either an H-2d or an H-2q thymus revealed that tolerance assessed by absence of V beta 6+ T cells is not dependent on the thymically determined restriction specificity of T cells.

Animals↗
PubMed1989

Clonogenic potential of murine CD4+8+ thymocytes. Direct demonstration using a V beta 6-specific proliferative stimulus in Mlsa mice.

Although considerable indirect evidence supports the hypothesis that CD4+8+ thymocytes are developmental intermediates in the generation of mature (CD4+8- or CD4-8+) T cells, the ability of these cells to proliferate in vitro has been highly controversial. We demonstrate here that a fraction of purified murine CD4+8+ thymocytes can be induced to proliferate in response to immobilized anti-TCR mAb. To exclude possible proliferation by trace mature T cell contaminants, we have exploited our recent finding that in Mlsa mice mature V beta 6-bearing thymic T cells are virtually absent (less than or equal to 0.5%) due to clonal deletion, whereas V beta 6 +CD4+8+ thymocytes are present in much higher numbers (approximately 3%). Proliferation of sorted CD4+8+ thymocytes from Mlsa mice was therefore induced at limiting dilution with immobilized anti-V beta 6 mAb to select against any contaminating mature T cells. Under optimal culture conditions, the frequency of CD4+8+ thymocytes proliferating specifically to anti-V beta 6 mAb (1/1000) was higher than those obtained for purified CD4-8+ (1/2000) or CD4+8- (1/5000) subsets, thus demonstrating directly that a proportion (in this case 3%) of CD4+8+ thymocytes are potentially clonable. During culture, V beta 6 +CD4+8+ thymocytes gave rise to a mixture of phenotypically "immature" (CD4-8-) and "mature" (CD4-8+) T cells. This system should be valuable for further analysis of the elusive CD4+8+ thymocyte subset.

Animals↗
PubMed1989

Postnatal disappearance of self-reactive (V beta 6+) cells from the thymus of Mlsa mice. Implications for T cell development and autoimmunity.

The postnatal ontogeny of potentially autoreactive T cells has been studied in a model system where a particular TCR beta chain variable domain (V beta 6) is correlated with reactivity to a minor antigen encoded by the Mlsa locus. Although absent among mature (CD4+ or CD8+) T cells in adult mice expressing Mlsa, brightly staining V beta 6+ cells were readily detectable in the thymus of neonatal animals, reaching a maximum after 4 d and decreasing rapidly thereafter. These V beta 6+ thymocytes were predominantly of the CD4+ phenotype and were localized in the medulla of the developing thymus. Furthermore, the intensity of TCR expression by these CD4+ cells was significantly (twofold) reduced as compared with age-matched Mlsb controls. A rapid disappearance of CD4+V beta 6+ cells (and corresponding decrease in TCR density) could also be observed in the thymus of Mlsb mice that had been injected neonatally with Mlsa spleen cells. Taken together, these results raise the possibility that some autoreactive T cells may persist after birth and that TCR downregulation may occur as a physiological response to tolerogenic signals in vivo.

Animals↗
PubMed1989

Preparation, characterization and application of interleukin-1 beta mutant proteins with surface-accessible cysteine residues.

Two mutants of interleukin-1 beta (K27C and K138C) were produced using site-specific mutagenesis in which lysine residues at positions 27 and 138 of the mature protein sequence were substituted by cysteine residues. The conformations of the mutant proteins were studied by 1H-NMR spectroscopy and shown to be similar to the wild-type protein. The receptor-binding affinity and biological activity of K27C and K138C were also similar to wild-type protein. The substituted cysteines in both mutant proteins were shown to be solvent-accessible as judged by their reactivity towards sulfhydryl reagents. As the wild-type protein contains two cysteines, which are both solvent-inaccessible in the native state, the mutants offer the opportunity to introduce probes in a sequence-specific manner via reaction with sulfhydryl groups. Examples of this are described in which the K138C was disulfide-linked to phycobiliproteins. The highly fluorescent conjugates had similar receptor-binding affinities to that of the wild-type unconjugated protein and were found suitable for flow-cytometric analysis.

Alkylation↗
PubMed1989

Functional responsiveness in vitro and in vivo of alpha/beta T cell receptors expressed by the B2A2 (J11d)- subset of CD4-8- thymocytes.

B2A2-CD4-8- cells represent a rare subpopulation of thymocytes normally comprising 0.5% of the total adult thymus. These cells are known to express CD3-associated T cell receptor (TcR) alpha/beta molecules. In the present study we have examined the functional capacity of alpha/beta heterodimers on B2A2-CD4-8- cells. In the presence of monoclonal antibody (mAb) specific for either murine CD3 or TcR expressing the V beta 8-encoded beta chain (F23.1), B2A2-CD4-8- cells proliferated. Such proliferation was blocked by mAb to interleukin 2 receptor (IL 2R), suggesting an autocrine mechanism involving IL 2 production and subsequent utilization. IL 2 and also IL 3 production by mAb-stimulated B2A2-CD4-8- cells was directly confirmed. Furthermore, a panel of B2A2-CD4-8- clones were derived to assess the role of the TcR in cytolysis. Many clones were isolated which killed Fc receptor-bearing P815 target cells only in the presence of F23.1 mAb. Finally, in vivo treatment of neonatal mice with F23.1 mAb resulted in a marked reduction of V beta 8+ B2A2-CD4-8- thymocytes. Collectively, these results indicate that the TcR alpha/beta complex on CD4-CD8-B2A2- cells is fully capable of transducing signals that lead to proliferation, lymphokine production and cytolysis in vitro, as well as to disappearance of this subset from the thymus in vivo.

Animals↗
PubMed1989

Intradomain H-2Kd/Dd recombinants define the same regions as crucial for recognition by alloreactive or major histocompatibility complex-restricted cytolytic T cells.

To identify residues on class I major histocompatibility complex (MHC) antigens that are important in T cell recognition, we analyzed a series of 11 intradomain recombinant mouse MHC (H-2) molecules in which N-terminal H-2Kd segments of varying lengths are followed by H-2Dd segments. Lysis of L cell transfectant target cells by a series of alloreactive cytolytic T cell (CTL) clones specific for Kd or for Dd revealed several regions that contain residues critical for specific recognition. These residues map within the presumed antigen-binding site of the MHC molecule. Of particular interest was the finding that the two regions identified as important for Kd allorecognition match those that influence the recognition of synthetic peptide antigens by Kd-restricted CTL.

Amino Acid Sequence↗
PubMed1989

Different staphylococcal enterotoxins bind preferentially to distinct major histocompatibility complex class II isotypes.

The stimulation of T cells by staphylococcal enterotoxins (SE) is strictly dependent on major histocompatibility complex (MHC) class II-bearing cells. The interaction between SE and MHC class II molecules was studied on the human B cell lymphoma Raji and its MHC class II-negative variant RJ 2.2.5. Affinity purification with SEA and SEB matrix allowed the isolation of HLA-DR-like molecules from detergent lysates of 125I surface-labeled Raji cells, but not from RJ 2.2.5 cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis also revealed preferences in the binding of other SE such as SED, SEE and toxic shock syndrome toxin 1 to DR-like molecules, SEC2 to HLA-DQ-like molecules and SEC3 to DR- and DQ-like molecules. Preadsorption of the different MHC class II MHC isotypes confirmed the preferential binding of SEA to DR and of SEC2 to DQ. The implications of these findings for the understanding of SE-induced T cell activation and the potency of SE as a tool in the study of MHC class II antigens are discussed.

Chromatography, Affinity↗
PubMed1989

Comparison of phorbol-12-myristate-13-acetate and dioctanoyl-sn-glycerol in the activation of EL4/6.1 thymoma cells.

The present study compared the role of two protein kinase C (PK-C) activating agents, the phorbol ester phorbol-12-acetate-13-myristate (PMA) and the membrane-permeating diacylglycerol dioctanoyl-sn-glycerol (DiC8) in the activation of EL4/6.1 thymoma cells. These cells have been shown to express interleukin-2 receptors (IL-2R) upon stimulation with optimal amounts of PMA (10 ng/ml); also, suboptimal amounts of PMA (1 ng/ml) synergized with the Ca2+ ionophore ionomycin and recombinant interleukin-1 (rIL-1) (Lowenthal et al., 1986). Comparing PMA and DiC8 led to the following results: PMA at 10 ng/ml induced IL-2R; in contrast, DiC8 (30-3 micrograms/ml) alone was unable to induce IL-2R, although it did synergize with ionomycin (0.5 micrograms/ml) and rIL-1. Bihourly additions of DiC8 did not change this pattern. The addition of DiC8 together with rIL-2 also resulted in no IL-2R expression. Furthermore, DiC8 (10 micrograms/ml) effectively translocated PK-C. Therefore, the differences observed between PMA and DiC8 do not seem to be due to differences in metabolism or to an inability to translocate PK-C. Analysis of messenger (m) RNA produced in stimulated EL4/6.1 cells revealed that DiC8 was also unable to induce mRNA for IL-2R. Our data suggest that PMA, especially at "optimal" concentrations, might have effects that cannot be mimicked by diacylglycerol. Furthermore, it seems that the deficient activity of diacylglycerols can be compensated for by a Ca2+ ionophore and, depending on the cellular system, by further signals such as IL-1.

Animals↗
PubMed1989

Transforming growth factors beta slow down cell-cycle progression in a murine interleukin-2 dependent T-cell line.

Transforming growth factors beta (TGF-beta) inhibit the growth of a variety of cell types, including lymphocytes. The immunosuppressive effects of TGF-beta have been attributed to the interference of these molecules with the interleukin-2 (IL-2)-driven component of lymphocyte proliferation. In order to elucidate in more detail the effects of TGF-beta on IL-2-induced proliferation, we investigated the effects of porcine transforming growth factor beta 1 and 2 (pTGF-beta 1 and 2) on the IL-2-driven proliferation of a murine IL-2-dependent T-lymphocyte line (CTLL). The results showed that pTGF-beta 1 and 2 decreased 3H-thymidine incorporation in CTLL cells in a dose-dependent fashion (maximum decrease of 75-85%). Combined-time kinetic analysis of the effects of pTGF-beta on 3H-thymidine incorporation, cell growth, and cell-cycle distribution (monitored as DNA content distribution) revealed that, in the first 48 h of culture, pTGF-beta 1 increased the doubling time from 11.4 to 19.2 h without significantly affecting the cell-cycle distribution of CTLL cells. After 96 h of culture in the presence of pTGF-beta 1, cells started to accumulate in G0/G1, although at this time point 30% of the pTGF-beta 1-treated cells were still in S-G2/M. Furthermore, during the first 48 h, neither the expression of the 55 kd chain of the IL-2 receptor (IL-2R) nor the expression of the transferrin receptor (TfR) was affected by TGF-beta. After 72 h of culture in the presence of pTGF-beta 1, the expression of the IL-2R and TfR was decreased. The data suggest that in CTLL cells TGF-beta initially slows the progression of cells in all phases of cell cycle. In addition, the initial TGF-beta-mediated decrease of IL-2-induced 3H-thymidine incorporation and cell proliferation in CTLL cells is not due primarily to downregulation of the IL-2R and/or TfR.

Animals↗