Paul E. Baker

Prostaglandin inhibition of T-cell proliferation is mediated at two levels

Abstract While numerous investigators have established that E-series prostaglandins inhibit proliferation of lectin- or antigen-activated T cells, the mechanism by which this effect is mediated remained poorly defined. It was recently shown that T-cell replication is mediated by a soluble factor (T-cell growth factor, TCGF), produced by antigen- or lectin-stimulated T cells. Thus, inhibition of T-cell replication by prostaglandins could be controlled either at the level of TCGF production or at the subsequent step of actual proliferation. We have found that differentiated cytotoxic T lymphocytes harvested from TCGF-dependent culture were, indeed, significantly sensitive to as little as 1 n M PGE 1 or PGE 2 ; 1000 n M PGE 1 or PGE 2 reduced TCGF-dependent T-cell proliferation by greater than 43% in all cases. In addition, PGE 1 and PGE 2 suppressed lectin-induced production of TCGF to remarkably similar concentration-dependent levels. Thus, the prostaglandin-E-mediated suppression of lectin-initiated T-cell proliferation could be traced to an inhibition of both the production and action of the T-cell-specific mitogen, TCGF. Since TCGF is produced by one T-cell subpopulation and acts on different T-cell subpopulations including both T helper and T cytotoxic cells, the consequences of prostaglandin-E inhibition of TCGF may have broad effects on cellular and humoral immune responses.

Expression, purification and characterization of recombinant marine granulocyte-macrophage colony-stimulating factor and bovine interleukin-2 from yeast

Expression and secretion of two lymphokines, murine granulocyte-macrophage colony-stimulating factor (MuGM-CSF) and bovine interleukin-2 (BoIL-2), to levels of 50-60 mg per liter were achieved by placing these cDNAs in a Saccharomyces cerevisiae expression vector that utilized the yeast alcohol dehydrogenase-2 promoter and alpha-factor leader peptide. These lymphokines were purified to homogeneity by direct application of the crude yeast medium to reversed-phase high-performance liquid chromatography. Despite the fact that both lymphokines contain at least one N-glycosylation site and have identical N-terminal residues (Ala-Pro-Thr), recombinant (R) GM-CSF was found to be heterogeneously glycosylated by yeast while RBoIL-2 was secreted without glycosylation. Additionally, approximately 40% of the RGM-CSF was found to be proteolytically cleaved after the second amino acid residue, while RBoIL-2 was found to be intact.

Cloning, sequence and expression of bovine interleukin 1α and interleukin 1β complementary DNAs

Abstract Interleukin 1 (IL-1) is a cytokine which mediates a variety of immunoregulatory and inflammatory activities. Using human IL-1α and IL-1β probes, cDNAs for the corresponding bovine genes were isolated from an alveolar macrophage library. The open reading frames of the bovine IL-1α and IL-1β cDNAs encode proteins of 268 and 266 amino acids, respectively, each with a predicted mol. wt of approx. 31,000. Both forms of bovine IL-1 exhibit a high degree of sequence homology with IL-1 gene products from other mammalian species. Based upon comparisons with human IL-1 amino acid sequences, the post-translationally processed, mature forms of bovine IL-1 would occur as 17–18,000 mol. wt proteins. Sequences encoding mature bovine IL-1α and IL-1β were inserted into E. coli expression plasmids and biologically active proteins were synthesized as judged by the ability of the recombinant proteins to induce proliferation of bovine thymocytes. Both IL-1α and IL-1β exist as single genomic copies. In addition, bovine IL-1β mRNA is approx. 10-fold more abundant than IL-1α mRNA in stimulated alveolar macrophages.

Functional and molecular characteristics of t-cell growth factor☆

Abstract T-cell growth factor, a mitogenic protein released from lectin- or antigen-activated mononuclear cells, maintains T-lymphocytes in continuous exponential proliferative culture. Studies which explored the parameters required for TCGF production revealed that both mature thymic-dependent T-cells and adherent cells participated in the release of TCGF. Further experimentation suggested that T-cells released TCGF after receiving two signals; (1) lectin or antigen cell membrane binding, and (2) a soluble product derived from adherent cells. A separate subset of T-cells, which did not require thymic influence, responded to TCGF. TCGF appeared to interact with the TCGF-responsive subset via specific membrane receptors: TCGF could be absorbed only by lectin- or antigen-activated living or glutaraldehyde-fixed T-cells. The T-cell response was initiated by lectin or antigen, but proliferation was mediated solely by purified TCGF. Thus, the T-cell immune response, which culminates in clonal expansion and differentiation of antigenreactive cells, appears to be controlled by an endogenously-derived proliferative hormone, which is obligatory for the realization of a competent effector T-cell.

Bovine GM-CSF: molecular cloning and biological activity of the recombinant protein.

The lymphokine granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates the growth and differentiation of granulocytes and macrophages from bone marrow progenitors, and regulates biological functions expressed by mature cells of these lineages. In order to isolate a bovine GM-CSF cDNA, a cDNA library, generated from the BT2 bovine T cell line, was screened with a human GM-CSF cDNA probe. A cDNA clone was isolated with an insert of 783 bp, that would encode a protein of 143 amino acids, with a predicted mol. wt of 16,160. Bovine GM-CSF exhibits a high degree of sequence homology with mouse and human GM-CSF at both the nucleotide and amino acid levels. Comparison of GM-CSF amino acid sequences from the three species indicates that the bovine GM-CSF precursor contains a putative 17 amino acid signal sequence, cleavage of which would yield a 14,250 mol. wt protein. The cDNA was inserted into a mammalian expression vector and transfected into COS-7 monkey kidney cells. Biologically active bovine GM-CSF was secreted as judged by a bovine bone marrow proliferation assay. Bovine GM-CSF was weakly active in both human and mouse bone marrow proliferation assays. In contrast, human GM-CSF was weakly active on bovine but not murine mouse bone marrow cells and mouse GM-CSF was only active on murine bone marrow cells.