The bradykinin analog RMP-7 increases intracellular free calcium levels in rat brain microvascular endothelial cells.

The vasoactive peptide bradykinin is believed to cause increased vascular permeability by the activation of B2 receptors on the vascular endothelium. A bradykinin analog, H-Arg-Pro-Hyp-Gly-Thi-Ser-Pro-4-Me-Tyr(psi CH2NH)-Arg-OH (RMP-7), was designed and it was proposed that it might increase cerebrovascular permeability by activating B2 receptors on brain microvasculature. In this report, the effects of RMP-7 and related peptides on bradykinin receptor-induced calcium signaling were examined in rat brain microvascular endothelial (RBME) cultures. RBME cells are responsive to bradykinin and exhibit specific [3H]-bradykinin binding, with Scatchard analysis indicating a major class of sites with a Kd of 3.9 +/- 1.4 nM and a minor class of higher affinity sites (Kd approximately 33 pM). RMP-7 displaces a significant component of specific [3H]-bradykinin binding from RBME cells; RMP-10, and RMP-7 diastereomer with a D-amino acid substitution in the number 9 position, does not. [3H]-bradykinin binding to RBME cells is not displaced by desArg9-bradykinin, which indicates that neither class of sites is a B1 bradykinin receptor. RMP-7 induces an increase in intracellular free calcium levels in RBME cells. The time course, magnitude and concentration dependence of RMP-7-induced calcium signaling is similar to that induced by bradykinin in RBME and other endothelial cells. Compared with RMP-7, RMP-10 is at least 1000 times less potent. However, the level of contamination by RMP-7 in the RMP-12 preparation accounts for its activity, an indication that both the RMP-10 and RMP-12 diastereomers are essentially inactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of erythroid burst-promoting activity (BPA) and granulocyte-monocyte colony-stimulating activity (GM-CSA) by isolated human T-lymphocyte subpopulations.

T-lymphocytes and monocytes are prominent among the classes of normal human cells that have been implicated in the production of the hemopoietic growth factors granulocyte-macrophage colony-stimulating activity (GM-CSA) and erythroid burst-promoting activity (BPA). To investigate the nature of the cooperativity that occurs during the elaboration of these growth factors by activated T-lymphocytes and monocytes in vitro, and to define the subsets of T cells involved in this response, we studied the production of GM-CSA and BPA by populations of T-lymphocytes isolated by fluorescence-activated cell sorting, using the monoclonal antibodies OKT3, OKT4, and OKT8. When OKT3+, OKT4+, or OKT8+ cells were incubated for five days in liquid suspension cultures, their production of GM-CSA and BPA was undetectably low. When 5% autologous monocytes were added to the cultures, no increase in the secretion of either of these classes of growth factors was noted. In the presence of concanavalin A (Con A), measurable quantities of both GM-CSA and BPA were elaborated by all three populations of T cells in the absence of monocytes; however, when autologous monocytes were added to the Con A-stimulated T cells, the secretion of both GM-CSA and BPA was markedly enhanced. In addition, we found that supernates of unfractionated T cells incubated with Con A contained not only GM-CSA and BPA but also a potent inhibitor(s) of BPA that could be demonstrated by dilution of the media and removed by gel filtration. In contrast, no inhibitor of GM-CSA was found. By molecular sieve chromatography of the supernates, GM-CSA and BPA coeluted as a single peak. However, the two biologic activities could be separated on the basis of heat stability, since GM-CSA proved to be heat labile whereas BPA did not. Our data indicate that GM-CSA and BPA derived from human T cells are similar in their apparent molecular weights and in the pattern of their production in suspension cultures in response to lectin stimulation. The secretion of both GM-CSA and BPA by Con A-stimulated T cells is facilitated by the presence of autologous monocytes, and is not restricted to either the OKT4- or the OKT8-defined subset.

The kinetics of the production of granulocyte-monocyte colony stimulating activity (GM-CSA) by isolated human monocytes: response to bacterial endotoxin.

When mononuclear phagocytes are stimulated by bacterial endotoxins, they produce Granulocyte-Monocyte Colony-Stimulating Activity (GM-CSA). In order to study the kinetics of the production of GM-CSA by human monocytes, we prepared suspensions of these cells and studied their response to Salmonella typhi endotoxin. We found that when human monocytes were exposed to this preparation of endotoxin, they synthesized GM-CSA de novo and subsequently secreted it into the extracellular environment; however, within hours, the cells became highly refractory to further stimulation by endotoxin. The resistance to endotoxin which these cells rapidly acquired in vitro could not be accounted for by cell attrition, the accumulation of toxic metabolites in the cultures, negative feedback inhibition by newly synthesized GM-CSA, depolarization of the plasma membrane of the cells, or by degradation of endotoxin. When we studied the binding of tritium-labeled endotoxin to viable monocytes, we found that after monocytes were initially exposed to endotoxin, their subsequent ability to bind lipopolysaccharide molecules onto the surface of the plasma membrane was reduced. When we subjected concentrated supernates from endotoxin-stimulated monocyte cultures to gel filtration and isoelectric focusing, we noted that GM-CSA derived from human monocytes had an apparent molecular weight of approximately 42,000 daltons. Our results indicate that resistance to bacterial endotoxins acquired by mononuclear phagocytes may play a role in the immunologic phenomenon of immediate endotoxin tolerance observed in vivo and may in part be due to down-regulation of endotoxin binding to the outer surface of the cell.

The effects of tumor-promoting phorbol esters on human granulopoiesis in vitro.

In order to determine whether the tumor-promoting phorbol esters are capable of inducing normal human committed granulocytic-monocytic progenitor cells (CFUc) to proliferate and differentiate in the absence of granulocyte-monocyte colony-stimulating activity (CSA), we studied the effects of these compounds on human granulopoiesis in vitro. We found that when light-density human marrow cells or peripheral blood leukocytes were depleted of adherent cells and then incubated in semisolid tissue culture medium under conditions optimal for CFUc growth, phorbol myristate acetate (PMA) and its congeners produced no measurable stimulatory effect on the proliferation of CFUc in the absence of added CSA. Likewise, when light-density marrow cells that had not been depleted of adherent cells were plated in the cultures, no stimulation of CFUc colony growth resulted from the addition of PMA. However, when light-density peripheral blood leukocytes were used as a target source of CFUc without first subjecting them to adherence separation, enhanced proliferation and differentiation of CFUc were noted in cultures that contained PMA. To investigate the possibility that CSA production by monocytes in these cultures in response to activation by PMA might account for the enhanced colony formation that we observed, we incubated isolated peripheral blood monocytes in short-term liquid suspension cultures and found that in the presence of PMA, large quantities of CSA were secreted into the surrounding medium. Finally, we noted that when marrow cell suspensions were suboptimally stimulated by low concentrations of CSA added to the cultures, the effects of PMA on CFUc proliferation were unpredictable, enhancing colony formation in some cases and inhibiting it in others. Our data indicate that although the tumor-promoting phorbol esters do not appear capable of directly stimulating the proliferation or differentiation of human CFUc in the absence of CSA, they may do so indirectly by causing auxiliary cells such as monocytes to secrete CSA.

The production of granulocyte-monocyte colony-stimulating activity by isolated human T lymphocyte subpopulations.

Isolated human T lymphocyte subpopulations were obtained by fluorescence-activated cell sorting using the murine monoclonal antibodies, OKT4 and OKT8. The capabilities of the isolated lymphocytes to produce granulocyte-monocyte colony-stimulating activity (CSA) in response to mitogen challenge were assessed by in vitro assays employing light density nonadherent bone marrow cells. Essentially, no CSA production was noted by any isolated T lymphocyte population [OKT4 positive (+) or OKT8 positive (+)] cultured alone or following the addition of 10(4) autologous monocytes/ml. When phytohemagglutinin (PHA) alone was added, OKT4+ lymphocytes elaborated small amounts of CSA. With the addition of concanavalin A (Con-A) alone, both OKT4+ and OKT8+ cells were able to produce modest amounts of CSA. Significantly enhanced CSA production was observed when either OKT4+ or OKT8+ lymphocytes were coincubated with autologous monocytes in the presence of mitogen. We conclude that highly purified T lymphocyte subpopulations, free of monocytes as assessed by nonspecific esterase staining, can elaborate small amounts of CSA in response to PHA or Con-A challenge. A synergistic augmentation of CSA production was noted with coincubation of sorted lymphocytes and autologous monocytes in the presence of mitogen. Finally, our results suggest that the ability of T lymphocytes to make CSA is not exclusively limited to either the OKT4+ or OKT8+ defined subsets.

The synthesis and secretion of granulocyte-monocyte colony-stimulating activity (CSA) by isolated human monocytes: kinetics of the response to bacterial endotoxin.

We studied the kinetics of the synthesis and secretion of granulocyte-monocyte colony-stimulating activity (CSA) by human monocytes stimulated by S. typhi endotoxin. We found that these cells initially secrete copious quantities of CSA when exposed to endotoxin but rapidly become refractory to its stimulatory effect. When monocytes were incubated in liquid suspension cultures, large amounts of CSA were generated during the first 24 hr of culture after the addition of as little as 10 ng/ml of endotoxin to previously unstimulated monocytes. After the addition of endotoxin, CSA secretion abruptly took place after an initial 1 to 2 hr lag phase, and occurred primarily within the first 6 to 12 hr of culture. The addition of puromycin or cycloheximide to the cultures significantly inhibited CSA secretion in response to endotoxin, suggesting that CSA production by stimulated monocytes requires de novo protein synthesis and does not solely result from the release of preformed active CSA. After initial exposure to endotoxin, CSA production by monocytes steadily decreased after 24 hr and ceased after 72 hr. Subsequent reexposure to the same concentration of endotoxin resulted in little CSA production, whereas the refractory state could be overcome by increasing the quantity of endotoxin added. Additional studies demonstrated that refractoriness of monocytes to further CSA production after initial response to endotoxin was not due to a loss of cell viability, degradation of endotoxin, or inhibition of CSA synthesis by soluble metabolites generated by stimulated monocytes. Our data suggest that the capacity of human monocytes to synthesize CSA in response to endotoxin rapidly becomes blunted after initial exposure of the cells to the lipopolysaccharide, but that the refractory state of these cells can be overcome by increasing the concentration of endotoxin. Acquired hyporesponsiveness of cells that produce CSA may in part account for the phenomenon of immediate endotoxin tolerance observed in vivo.