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J A Lederer

Publications and source records attributed to J A Lederer.

29 records · Page 2Linked to original sources
PubMed1994

Regulation of cytokine gene expression in T helper cell subsets.

We have examined the transcriptional regulation of the Th1-specific IL-2 gene and the Th2-specific IL-4 gene by transient transfection of promoter-chloramphenicol acetyl transferase (CAT) constructs into T cell clones and by electrophoretic mobility shift assays. Transfection of the Th2 clone D10.G4 with IL-4 promoter-CAT constructs demonstrated anti-CD3 inducible IL-4 promoter activity. In contrast, CAT constructs containing murine IL-2 promoter sequences were not inducible in D10.G4 cells. Transfection analyses in the Th1 clone D1.1 demonstrated inducible IL-2 promoter activity but no IL-4 promoter activity. Electrophoretic mobility shift assays using well-defined regulatory elements in the murine IL-2 gene promoter showed that anti-CD3 stimulation of Th2 clones failed to induce a characteristic increase in the ratio of p65-p50:p50-p50 NF-kappa B binding complexes in the nucleus that did occur in IL-2-producing clones. In addition, other protein complexes with NF-kappa B binding sequences were seen using lysates from Th1 and Th0 cells but not Th2 cells. Thus, expression of the promoter-CAT constructs directly correlates with endogenous IL-2 and IL-4 gene expression in Th1 and Th2 clones, confirming that the differential expression of IL-2 and IL-4 genes in these T cells is transcriptionally controlled. Furthermore, the lack of IL-2 transcription in activated Th2 cells is associated with the failure to generate required IL-2 gene promoter binding proteins, particularly NF-kappa B in the nucleus.

Animals↗
PubMed1994

Species-specific binding of IL-1, but not the IL-1 receptor antagonist, by fibroblasts.

Our laboratory previously reported that bovine thymocytes and fibroblasts proliferated only in response to bovine IL-1, whereas proliferation of murine thymocytes was augmented equally well by murine, human, and bovine IL-1. In this study, we used direct and competitive receptor binding assays to determine whether differential binding of homologous versus heterologous IL-1 accounts for the species-specific response of bovine fibroblasts. Our results demonstrated that bovine and human fibroblasts bound homologous IL-1 with high affinity and heterologous IL-1 with lower affinity. In contrast, murine fibroblasts bound both homologous and heterologous IL-1 with high affinity. Because IL-1 and the human IL-1 receptor antagonist (IL-1ra) both bind to type 1 IL-1 receptors, we also determined whether the IL-1ra demonstrated receptor binding properties similar to human IL-1 for bovine, human, and murine fibroblasts. To our surprise, the human IL-1ra bound equally well to bovine, human and murine fibroblasts. We used this characteristic of the IL-ra to perform affinity cross-linking analysis of the IL-1 receptors on bovine, human and murine fibroblasts. These comparisons demonstrated the IL-1 receptors on bovine and human fibroblasts have similar molecular sizes (M(r) 73 kDa and M(r) kDa, respectively), whereas, IL-1 receptors on murine fibroblasts have an estimated molecular size of M(r) 88 kDa. These data demonstrate that IL-1 receptors on bovine fibroblasts preferentially bind homologous IL-1, but bovine fibroblasts do not discriminate binding by the human IL-1ra. In contrast, murine fibroblast IL-1 receptors bind heterologous IL-1 with high affinity.

3T3 Cells↗
PubMed1993

Costimulator deficient antigen presentation by an endothelial cell line induces a nonproliferative T cell activation response without anergy.

The ability of endothelial cells to activate helper T (Th) cells by antigen presentation was studied using the murine endothelial cell line SVEC4-10 and antigen-specific murine T cell clones. SEVEC4-10 cells constitutively express vascular cell adhesion molecule 1 but not intercellular adhesion molecule 1. Interferon gamma (IFN-gamma) treatment of these cells induced class II major histocompatibility complex (MHC) expression and antigen-presenting capabilities, but did not alter surface integrin expression. IFN-gamma-treated SVEC4-10 cells were competent at mediating antigen-dependent cytokine production and proliferation of a Th2 clone. In contrast, endothelial antigen presentation to Th1 cells did not stimulate T cell proliferation. The addition of MHC mismatched spleen cells as a source of costimulatory molecules resulted in the ability of the endothelial cells to stimulate Th1 cell proliferation in an antigen-specific manner. The failure of the endothelial cell line alone to support Th1 cell proliferation correlated with the failure to stimulate interleukin 2 (IL-2) gene expression. T cell exposure to the endothelial cells plus antigen resulted in upregulation of IL-2 receptors and an enhanced response to subsequent antigen presentation by splenic antigen-presenting cells. Despite the lack of functional costimulators for IL-2 expression, antigen presentation by the endothelial cell line did not induce Th1 cell anergy, indicating that costimulator deficiency for IL-2 expression is not obligatorily linked to anergy induction. Thus, endothelial cells are capable of presenting antigens to helper T lymphocytes, but stimulate only partial T cell responses. These partial responses may serve to selectively stimulate transmigration of antigen-specific T cells and may enhance functional responses upon subsequent, extravascular antigen exposure.

Animals↗
PubMed1993

Transcription of the interleukin 4 gene is regulated by multiple promoter elements.

Activation of T helper cell 1 (Th1) and Th2 results in transcription of the interleukin 2 (IL-2) and IL-4 cytokine genes, respectively. Whereas many of the regulatory elements and factors responsible for IL-2 transcription in T cells are well defined, little is known about parallel mechanisms that drive transcription of the IL-4 gene. Here we have analyzed the murine IL-4 promoter, both in vivo and in a Th2 clone. 3 kb of IL-4 upstream sequence is shown to be sufficient to achieve tissue-specific and inducible expression of a thymidine kinase reporter gene in vivo in a manner that mirrors the expression of endogenous IL-4. Tissue-specific and inducible expression is also demonstrated in a Th2 clone, but not in a B cell line. Deletional and mutational analysis of the IL-4 promoter demonstrated that sequences from -100 to -28 were necessary for a transcriptional response to Concanavalin A or anti-CD3 monoclonal antibody. An overlapping, yet smaller region, spanning the sequences from -60 to -28 bp was shown to be required for the response to ionomycin. Mutation of an 8-bp region from -43 to -35 of the IL-4 promoter completely abrogated IL-4 gene transcription in response to all stimuli tested. In addition, our results show that the effects of the immunosuppressive agent Cyclosporin A map to the same DNA sequences as the positive control elements. These results identify DNA sequences that are functionally important for the control of IL-4 gene transcription both in vivo and in vitro. Although these sequences are highly conserved in the human and murine IL-4 genes, they are largely not present in the IL-2 enhancer complex. Thus, cytokine-specific cis-acting elements may be one mechanism by which these two cytokine genes are differentially regulated.

Animals↗
PubMed1992

Characterization and identification of interleukin 1 receptors on bovine neutrophils.

Interleukin 1 (IL-1) has been shown to modulate various functional activities of neutrophils, presumably by first binding to cell surface IL-1 receptors. In this study, we characterized and identified IL-1 receptors on bovine neutrophils using direct and competitive receptor binding studies and affinity cross-linking analysis. The results of direct binding studies demonstrated that bovine neutrophils bound 125I-labeled bovine IL-1 by a single affinity binding site (Kd = 4.6 nM, 5600 binding sites per cell). Competitive receptor binding studies demonstrated that unlabeled recombinant bovine IL-1 beta, murine IL-1 alpha, and human IL-1 beta competitively blocked neutrophil binding of 125I-labeled bovine IL-1 beta. In contrast, the IL-1 receptor antagonist (IL-1ra) demonstrated no detectable binding to bovine neutrophils as judged by direct and competitive receptor binding assays. Affinity cross-linking of 125I-labeled bovine IL-1 beta to neutrophils was used to identify cell surface IL-1 receptors. Two specific cross-linked products were observed with molecular sizes of 89 kd (a deduced receptor size of 71.5 kd) and more than 200 kd. The latter may indicate a complex of IL-1 receptor-associated signal-transducing components, IL-1 receptors, and IL-1. The presence of 100 nM unlabeled bovine, murine, or human IL-1 during the receptor binding and cross-linking reactions prevented the formation of cross-linked complexes of 125I-labeled bovine IL-1 beta and its receptor. In contrast, the IL-1ra failed to inhibit the formation of cross-linked complexes of 125I-labeled bovine IL-1 beta and its receptor.

Animals↗
PubMed1992

Borrelia burgdorferi stimulates release of interleukin-1 activity from bovine peripheral blood monocytes.

Infection with Borrelia burgdorferi is suspected to be a cause of lameness and arthritis in cattle. Interleukin-1 (IL-1) activity has been detected in joint fluids from human patients affected by various arthritides, including Lyme arthritis. In addition, human monocytes and murine macrophages have been reported to release IL-1 activity when incubated with B. burgdorferi in vitro. To address a possible mechanism by which B. burgdorferi might cause a bovine arthritic syndrome, we determined whether bovine peripheral blood monocytes released IL-1 activity when coincubated with B. burgdorferi in vitro. High-passage and low-passage isolates of B. burgdorferi stimulated release of IL-1 activity from bovine monocytes. The amount of IL-1 activity released was dependent on the number of borreliae added to the monocyte cultures. In addition, live and heat-killed B. burgdorferi cells stimulated release of similar amounts of IL-1. We also obtained no evidence that soluble components released from in vitro-cultured B. burgdorferi stimulated IL-1 release from bovine monocytes. A recombinant IL-1 receptor antagonist blocked the proliferative activity of monocyte-conditioned medium in a thymocyte costimulation assay, thus demonstrating that the costimulatory activity detected was due to IL-1.

Adult↗
PubMed1989

Production and purification of bovine monocyte-derived interleukin 1.

Few studies have addressed the biological and molecular nature of bovine interleukin 1 (IL-1). In an effort to increase our understanding of the role of bovine IL-1 in bovine immunology, we investigated various parameters of its production by LPS-stimulated monocytes in vitro. Bovine monocytes isolated by our methods constitutively released IL-1 activity, as measured by the murine thymocyte IL-1 assay. Monocyte release of IL-1 activity was further augmented when the cells were incubated with 0.005-10 micrograms per ml of Escherichia coli lipopolysaccharide (LPS). The presence of 1, 5, or 10 percent heat-inactivated fetal bovine serum (FBS) enhanced LPS-stimulated bovine monocyte release of IL-1 activity as compared with monocytes cultured under serum-free conditions. We used a combination of size-exclusion and reverse-phase high-performance liquid chromatography (HPLC) to purify bovine IL-1 from serum-free monocyte culture supernatants. Size-exclusion HPLC resulted in a single peak of biological activity with an approximate molecular weight of 18,000 daltons. Further purification by reverse-phase HPLC demonstrated at least three major molecular species with IL-1 activity. Besides providing information about production of IL-1 by bovine monocytes in vitro, this study also describes a protocol to purify bovine IL-1 for future studies addressing its biological functions.

Animals↗
PubMed1989

Species preference of bovine thymocytes and fibroblasts for bovine interleukin 1.

The purpose of this study was to determine whether bovine thymocytes and fibroblasts preferentially respond to bovine IL-1 as compared with human and murine interleukin 1 (IL-1). We compared equivalents units of recombinant murine and human IL-1 alpha, and purified monocyte-derived bovine IL-1, as measured in the murine thymocyte IL-1 assay, for their ability to stimulate the proliferation of bovine thymocytes and fibroblasts. Fetal and adult bovine thymocytes responded to bovine IL-1, but in contrast, did not respond to recombinant human or murine IL-1 alpha at the concentrations tested. Both adult and neonatal murine thymocytes responded similarly to the three IL-1 preparations. Thus, we concluded that the preferential response of bovine thymocytes for bovine IL-1 was not dependent upon the age of the thymus donor. Bovine fibroblasts also preferentially responded to bovine IL-1; fibroblast proliferation was stimulated by bovine but not human or murine IL-1. Our findings, therefore, imply that although IL-1 is highly conserved among mammalian species, its ability to stimulate the proliferation of thymocytes and fibroblasts may vary from species to species.

Animals↗
PubMed1987

"Haemophilus somnus," a facultative intracellular pathogen of bovine mononuclear phagocytes.

We have reported previously that bovine neutrophils are unable to kill the bovine respiratory pathogen "Haemophilus somnus." In the present study we expanded our efforts and examined the interaction of bovine mononuclear phagocytes with this important veterinary pathogen. Bovine alveolar macrophages and blood monocytes ingested but did not kill opsonized "Haemophilus somnus" in vitro, whereas these same cells ingested and killed opsonized Escherichia coli. Because this suggested that "H. somnus" was a facultative intracellular pathogen, we developed an assay to monitor the intracellular fate of ingested "H. somnus" within bovine monocytes. Our results indicated that ingested "H. somnus" multiplied within bovine monocytes (1- to 2-log10 increase in 4 h); equivalent intracellular growth was noted for both a laboratory strain and a recent field isolate of "H. somnus." Bovine monocytes killed ingested E. coli (1- to 2-log10 decrease in 4 h) under the same assay conditions that were used to follow intracellular growth of "H. somnus," thus indicating that the assay conditions did not induce a generalized defect in monocyte antibacterial activity. Light and electron microscopic examination of "H. somnus"-infected monocytes confirmed that intracellular growth had occurred. We did not observe an obvious correlation between the release of superoxide anion from bovine mononuclear phagocytes that had ingested opsonized "H. somnus" and E. coli and the subsequent intracellular survival of the bacteria. The results of this study suggest that infected mononuclear phagocytes sustain "H. somnus" infections in cattle and thus contribute to the subacute to chronic clinical course that has been reported.

Animals↗
PubMed1984

[Sterility caused by protein deficiency: the role of prolactin].

The first piece of research was made on mice in order to try to determine the importance of the ration of protein in the food in the maintenance of pregnancy and the mechanism by which protein lack leads to sterility. Five groups of 100 couples were given diets which contained 23.5%, 12.48%, 10.50%, 7.01% and 0% of protein respectively over a period of 100 days. The drop in the percentage of protein was obtained by adding detoxicated cassava flour and the diet without any protein was made up of starch, maize oil, mineral salts and vitamins. The number of litterings per 100 couples was only lowered when the diet was down to 7.01% of protein and at the same time it was only with this diet that one found a significant reduction in the number of young per litter. When the diet had no protein in it at all no young were born at all. Pregnant rats were fed diets which contained 23.5%, 7.01% or no protein at all. In this last diet we watched to stop coprophagia. There was very little difference in the number of implantations and young born with a diet of 7.01% of protein but a very significant drop with the diet without protein. With this last diet was very little significant difference in the series whether one had watched to stop coprophagia or not. An injection of 10 I.U. of HCG from the 3rd to the 13th day of pregnancy in rats fed a protein-free died did not alter significantly the number of implantations and did lead to complete resorption of the embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗