Yacob Weinstein

Specific Leukocyte Receptors for Small Endogenous Hormones. DETECTION BY CELL BINDING TO INSOLUBILIZED HORMONE PREPARATIONS

Receptors for small endogenous hormones on human leukocytes were studied by insolubilizing the hormones and incubating them with the cells. Histamine, norepinephrine, and prostaglandin E(2) (PGE(2)) were conjugated to either of two types of carrier: (bovine or rabbit) serum albumin or a random copolymer of DL-alanine and L-tyrosine. The conjugates were linked to agarose beads (Sepharose) and the resultant drug-conjugate-beads were incubated with leukocytes. Norepinephrine (when linked to its carrier via glutaraldehyde) and histamine preparations bound the majority of leukocytes. The binding appeared to be specific for the hormones tested. For example, the binding by histamine-rabbit serum albumin-Sepharose was prevented or reversed by high concentrations of histamine and histamine antagonists, but not by catecholamines or their pharmacologic antagonists. Similarly, binding of cells to the norepinephrine conjugate was inhibited by some catecholamines and propranolol, but not by histamine or histamine antagonists. Conjugates of norepinephrine linked via carbodiimide did not bind cells. The protein or copolymer carriers did not contribute to binding per se. The hormone-protein-conjugates bound more cells than the hormone-polymer conjugates. The former (unlike the free amines) failed to stimulate accumulation of cyclic AMP in leukocytes. The norepinephrine linked to polymer via glutaraldehyde, however, did stimulate leukocyte cyclic AMP accumulation, possibly because of the flexibility of the polymer. Columns of the various Sepharoses were used to determine the distribution of receptors to each hormone in mixed leukocyte populations. The majority of cells appeared to have receptors for both histamine and norepinephrine (bound through glutaraldehyde). Receptors to prostaglandins may have been detected by the column procedure, but their distribution could not be quantitated. The approach described provides a means to separate leukocytes on the basis of what are likely to be preformed receptors to small endogenous hormones, and to study the physiologic importance and function of the receptors.

The effect of cyclic nucleotides on purine biosynthesis and the induction of PRPP synthetase during lymphocyte activation

Abstract The activation of lymphocytes has been used to study the regulation of mammalian gene expression. Concanavalin A (Con A) added to mouse spleen lymphocytes in serum-free medium leads to an increase in the rate of DNA synthesis as great as 1000 fold, commencing 20 hr after its addition. Prior to 20 hr, the rate of purine synthesis increases 10–100 fold as measured by accumulation of the purine intermediate, formyl glycineamide ribonucleotide (FGAR). Addition of dibutyryl cyclic GMP to the lymphocyte suspensions results in a 10 fold increase in the rate of DNA synthesis in the absence of Con A and enhances both purine synthesis and DNA synthesis in its presence. The activity of phosphoribosyl pyrophosphate synthetase (PRPP synthetase), an enzyme central to purine and pyrimidine biosynthesis, is increased 2–10 fold during the activation. The increase begins to appear 8 hr after Con A addition and requires concomitant protein synthesis. The induced PRPP synthetase activity is stimulated by the presence of cyclic GMP in the enzyme assay. Addition of dibutyryl cyclic AMP to Con A-stimulated lymphocytes inhibits FGAR production, the stimulation of DNA synthesis, and the appearance of cyclic GMP-sensitive PRPP synthetase. These studies suggest that cyclic nucleotides play a significant role in the molecular mechanism of lymphocyte activation, the regulation of purine biosynthesis, and of eucaryotic genetic expression.

Separation of Specific Antibody-Forming Mouse Cells by their Adherence to Insolubilized Endogenous Hormones

Spleen cells from mice immunized with sheep red cells were separated by differential adherence to insolubilized histamine, catecholamines, and prostaglandins. The hormones were insolubilized by linking them to Sepharose beads through a protein carrier. We measured hemolytic plaque formation (per million splenic leukocytes) of cells which passed through columns of hormone-carrier-Sepharose beads (i.e., those cells that failed to bind). As compared with control (no column) cells, the number of plaque-forming cells was substantially reduced by passage through histamine, epinephrine, isoproterenol, and prostaglandin-E(2) columns. Plaque-forming cells were not significantly reduced by passage through carrier Sepharose (another control) or norepinephrine- and prostaglandin-F(2alpha)-carrier Sepharose columns. Thus, the ability of an insolubilized hormone preparation to subtract plaque-forming cells roughly correlated with the presence of pharmacologic receptors for the corresponding free hormones, as judged by stimulation of cyclic AMP accumulation in the same cells, reported previously. Both 19S and 7S plaque-forming cells were subtracted by columns prepared from pharmacologically active hormones, but none of the insolubilized hormones stimulated accumulation of intracellular cyclic AMP. The cell membrane phenomenon that allows adherence to a given hormone-carrier-bead column may be identical with the cell receptor.

In vitro correction of antigen-induced immune suppression: effects of histamine, dibutyryl cyclic AMP and cholera enterotoxin.

Abstract Reduced immune response potential of SJL/J mouse spleen cells to the synthetic polypeptide poly ( l Tyr, l Glu)-poly l Pro—poly l Lys [(T,G)-Pro—L]was observed when the spleen cells were incubated in vitro with (T,G)-Pro—L prior to in vivo challenge of the cells with the same antigen in syngeneic, irradiated recipients. Incubation of the cell-antigen mixture in the presence of histamine, prostaglandins E1 and F2α, dibutyryl cyclic AMP, or cholera enterotoxin abrogated the antigen-induced loss of responsiveness. The immunological effects of histamine, prostaglandins, and cholera enterotoxin were associated with increased accumulation of cyclic AMP in the spleen cell suspension. Addition of the histamine antagonist diphenhydramine or anti-cholera toxin to the respective in vitro incubation mixtures abolished the effects of histamine and cholera enterotoxin on both the immune response and increased cyclic AMP levels. These results suggest that this type of antigen-induced immunosuppression can be reversed by pharmacological agents which elevate levels of cyclic AMP.

Reciprocal changes in interferon production and immune responses of mouse spleen cells fractionated over columns of insolubilized conjugates of histamine

Abstract Cultures of spleen cells from unimmunized and immunized BALB/c mice can support both direct and indirect anti-sheep erythrocyte plaque-forming responses. The responsiveness of spleen cells to this thymus-dependent sheep cell antigen can be altered by fractionation of the cells over insolubilized conjugates of histamines (H-R-S). Cells that do not adhere to H-R-S (i.e., those that pass through the columns) produce a significantly greater plaque-forming cell response than do non-chromatographed cells or cells that have passed through a control column of rabbit serum albumin-Sepharose (R-S). In contrast, the direct plaque-forming cell response of the same culture to Salmonella typhimurium lipopolysaccharide (a T-lymphocyte-independent antigen) was not significantly different in any of the groups of cells tested. In addition, spleen cell filtration over H-R-S also resulted in a significant increase in the blastogenic response to phytohemagglutinin as well as a significantly lower production of interferon in response to phytohemagglutinin. The possibility that suppressor cells (which were shown to adhere to H-R-S) produce their inhibitory effect via interferon is discussed.