Suppression of fibroblast proliferation by activated macrophages: involvement of H2O2 and a non-prostaglandin E product of the cyclooxygenase pathway.

Macrophages are considered promoters of fibroblast proliferation; however, suppression by activated macrophages may outweigh this effect. Activated murine peritoneal macrophages obtained by in vivo exposure to C. parvum or by in vitro LPS-activation of thioglycollate-induced macrophages, were tested for their effect on normal syngeneic dermal fibroblasts. C. parvum-activated macrophages, but not resident peritoneal macrophages suppressed fibroblast proliferation. Similarly, macrophages activated in vitro by LPS, but not those unexposed to LPS, suppressed fibroblast proliferation. Catalase partially protected fibroblasts from suppression by either activated macrophage population, suggesting involvement of H2O2 in the suppression. The effect of cyclooxygenase inhibitors on the suppression was also tested. Indomethacin, acetylsalicyclic acid, or eicosatetraynoic acid, all partially protected the fibroblasts from macrophage-mediated suppression. Prostaglandins E2, E1, and F2 alpha, added exogenously at concentrations as high as 10(-6) M, failed to suppress the proliferation of the fibroblasts. These findings suggest that a non-prostaglandin product of the cyclooxygenase pathway is involved in macrophage-mediated suppression of fibroblast proliferation.

Genetic resistance of mice to persistent infection with Mycobacterium lepraemurium in vitro: association with macrophage bactericidal responsiveness to lymphokines and dissociation from production of hydrogen peroxide by macrophages.

Peritoneal and splenic adherent macrophages (SAC) from M. lepraemurium susceptible (C3H/HeJ) and resistant (C57B1/6J) mice were studied for their abilities to generate H2O2 in vitro. Unexpectedly, SAC from the susceptible C3H/HeJ strain produced more H2O2 than those of the resistant C57BL/6J. In vivo sensitization with M. bovis (BCG), or C. parvum increased production of H2O2 by SAC from both strains, whereas in vivo sensitization with M. lepraemurium enhanced H2O2 production only in the C3H/HeJ susceptible strains. In vitro addition of a crude lymphokine enhanced H2O2 production by C3H/HeJ SAC more than by C57BL/6J SAC. In vitro addition of M. lepraemurium caused an inhibition of H2O2 production by SAC from both strains but the inhibition was greater for the resistant C57BL/6J strain. M. lepraemurium phagocytosed in vitro by untreated peritoneal macrophages of both mouse strains were morphologically altered to the same extent. However, the addition of lymphokine dramatically increased the degree of bacterial lysis in only the C57BL/6J strain. These results, support the view that H2O2 plays a limited, if any, role in the protection of the host from M. lepraemurium and may even contribute to susceptibility by inhibiting the host's immune response.

Inhibition of lymphocyte proliferation and antibody production in vitro by silica, talc, bentonite or Corynebacterium parvum: involvement of peroxidative processes.

This study was undertaken to determine whether and by what means particles which induce granulomata in vivo can affect murine spleen lymphoproliferative and antibody responses in vitro. Particles of silica, talc, Bentonite or C. parvum cells inhibited lipopolysaccharide- or concanavalin A-stimulated proliferation and sheep red blood cell-induced antibody response in vitro. The inhibition required at least 48 hours exposure of the cells to the particles. The late onset of inhibition and its reproducibility at different cell or mitogen concentrations implicated particle-induced injury to both phagocytes and lymphocytes. Either alpha-tocopherol or 2-mercaptoethanol prevented the particle-induced inhibition of spleen cell responses. alpha-Tocopherol and 2-mercaptoethanol have in common the capacity to protect cells against membrane lipid peroxidation. The inhibitory peroxidative process(es) implicated by these studies are most likely attributable to: (a) stimulation of oxidative metabolism of phagocytic cells by particles; and (b) iron-catalyzed peroxidation directly by the particles. These data may be relevant in understanding the pathogenesis of and devising therapeutic approaches toward various granulomatous conditions.

Evidence for the involvement of monocyte-derived toxic oxygen metabolites in the lymphocyte dysfunction of Hodgkin's disease.

This study was performed to see if adherent cell-derived toxic oxygen metabolites contribute to the suppression of mononuclear cell blastogenic responses in Hodgkin's disease. Peripheral blood mononuclear cells from 10 patients with Hodgkin's disease were stimulated in culture with the mitogen PHA in the presence of the prostaglandin inhibitor indomethacin and the antioxidants catalase or vitamin E. Patient lymphocytes showed significant increases in PHA-induced proliferation at all PHA doses when cultured with indomethacin. Further augmentation of lymphocyte proliferation was achieved with the addition of catalase or vitamin E to indomethacin in the culture system. The increases in proliferation seen on culture with these agents were greatest in patients with more depressed initial PHA responses. When adherent cells were removed before culture, the agents no longer facilitated increases in proliferation. These data suggest that abnormal lymphocyte proliferative responses seen in Hodgkin's disease may result in part from the excessive production of toxic oxygen metabolites as well as prostaglandins by adherent cell populations.

Regulation by PGE2 of the production of oxygen intermediates by LPS-activated macrophages.

The regulation by prostaglandin E2 (PGE2) of production of oxygen radicals by bacterial lipopolysaccharide-(LPS) activated macrophages was studied in vitro. A 48-hr incubation of murine thioglycollate-elicited macrophages with LPS (0.1 micrograms/ml) resulted in an enhanced ability of these cells to produce oxygen radicals when challenged with phorbol myristate acetate (PMA). Macrophages incubated for 48 hr without LPS did not produce measurable amounts of oxygen radicals when exposed to this triggering stimulus. Thus, PMA-triggered production of oxygen radicals was the result of macrophage activation by LPS. The PMA-triggered production of oxygen radicals by the LPS-activated macrophages was inhibited when PGE2 (10(-5) to 10(-9) M) was present during the incubation with LPS. Inhibition by PGE2 occurred during the early stages of macrophage activation, since the addition of PGE2 24 hr after LPS no longer inhibited the production of oxygen radicals by the macrophages. This inhibitory effect of PGE2 on the LPS-induced activation of macrophages could be reproduced by cyclic-adenosine-monophosphate (cAMP) agonists, such as isoproterenol and cholera toxin as well as by the cAMP analog dibutyryl-cAMP, suggesting a cAMP-mediated mechanism for the inhibitory effect of PGE2 on macrophage activation by LPS. Previous reports have implicated prostaglandins as mediators of destructive processes associated with chronic inflammation. Our findings suggest that PGE2 may, on the other hand, reduce tissue damage in a chronic inflammatory site by inhibiting the production of oxygen radicals by macrophages activated in the sera.

Agents which block membrane lipid peroxidation enhance mouse spleen cell immune activities in vitro: relationship to the enhancing activity of 2-mercaptoethanol.

A broad spectrum of agents known to block various steps in the lipid peroxidation process were tested for their ability to protect mouse spleen cells and thereby enhance their activities, in vitro, in either the primary antibody response or the lipopolysaccharide-stimulated proliferation response. Each agent (superoxide dismutase, butylated hydroxyanisole/butylated hydroxytoluene/n-propyl gallate, lucigenin, and alpha-tocopherol) was able to enhance the cellular response in both assay systems. The degree of enhancement of these immune functions was in proportion to the efficacy of each agent in blocking the overall process in lipid peroxidation. Previous work in this laboratory has shown that the enhancement of the primary antibody response by 2-mercaptoethanol (2-ME) is mediated by the enhanced availability of reduced glutathione in the culture medium. Suboptimal doses of each lipid antioxidant agent were able to enhance the antibody response in the additive manner with a suboptimal dose of 2-ME up to a maximum response equal to that achieved with an optimal dose of 2-ME alone. These data support the hypothesis that the enhancement of cellular responses in the presence of 2-ME is mediated by the lipid antioxidant activity of reduced glutathione.

Enhancement of the primary antibody response by 2-mercaptoethanol is mediated by its action on glutathione in the serum.

2-Mercaptoethanol (2-ME) is widely used in rodent lymphoid cell cultures as an enhancer of multiple cellular functions. We have confirmed that the action of 2-ME must be on a serum component(s), rather than a direct action on the cells. The serum component(s) is contained within the dialyzable fraction of fetal calf serum (FCS) since: (a) dialysis of FCS diminished the ability of FCS to support an antibody response even in the presence of 2-ME; and (b) FCS dialysate, pulsed with 2-ME, restored the ability of dialyzed FCS to support an antibody response. Diminution of the reduced glutathione content of FCS by heating reduced the capacity of FCS to support an antibody response, whereas addition of 2-ME-pulsed glutathione restored the supportive capacity of heated FCS. Conversely, oxidized glutathione inhibited the antibody response in the absence of 2-ME, but that inhibition was not seen in the presence of 2-ME. We have concluded that reduced glutathione is an essential component in FCS in order for 2-ME to produce its enhancing effect. The most plausible explanation for the enhancement of antibody responses, in vitro, by 2-ME, is the concomitant reversal of the inhibitory effect of oxidized glutathione and the increased availability of reduced glutathione which can scavenge oxygen-derived radicals, thus protecting macrophages and lymphocytes from the deleterious effects of oxygen-derived free radicals.

Macrophage-mediated suppression. I. Evidence for participation of both hdyrogen peroxide and prostaglandins in suppression of murine lymphocyte proliferation.

Macrophage-mediated suppression of Con A induced proliferation of murine splenic lymphocytes was studied in vitro. Either Corynebacterium parvrum-induced peritoneal exudate cells (PEC) or thioglycollate-induced PEC could totally suppress lymphocyte proliferation at a PEC:lymphocyte ratio of 2:10, whereas a ratio of 1 to 1.5: 10 caused a partial (60 to 68%) suppression. Exogenous PGE1 and PGE2 at concentrations of 10(-8) to 10(-6) M could not totally suppress lymphocyte proliferation. Conversely, indomethacin reversed the partial suppression by macrophages but only partially protected the totally suppressed lymphocyte cultures. Macrophage-mediated cytotoxicity and cytostasis have been proposed to be mediated by hydrogen peroxide. Therefore, hydrogen peroxide was investigated as a possible additional cause for macrophage-mediated suppression, by testing the anti-inhibitory effects of catalase. Partially suppressed cultures were effectively protected from suppression by catalase. In totally suppressed cultures, catalase alone was only minimally effective, but a synergistic effect of catalase and indomethacin was obtained, which provided complete protection from maximal macrophage-mediated suppression. Catalase presumably contributes to the reversal of macrophage suppressive effects both by reducing the direct toxic effect of H2O2 and by preventing the H2O2 from generating additional prostaglandins.

Antigen-specific and nonspecific mediators of T cell/B cell cooperation. IV. Development of a model system demonstrating responsiveness of two T cell functions to HGG in vitro.

Previous work in this laboratory has defined two functionally distinct T cell subpopulations (viz., antigen-specific helper) and characerized some of the parameters of these subpopulations derived from the keyhole limpet hemocyanin (KLH)-primed mouse spleen. Characerization of further parameters such as relative susceptibility to tolerance induction and relative degree of specificity was not possible with the use of KLH as the antigen. In order to overcome this impediment, an experimental protocol was developed, as herewith described, to permit the study of these two T cell subpopulations in response to the antigen human gamma-globulin (HCG). This protocol qualitatively duplicates the parameters defined for the KLH system and provides an extremely useful model for the study of the response to serum proteins in the Mishell-Dutton culture system.

Antigen-specific and nonspecific mediators of T cell/B cell cooperation. V. Unresponsiveness to HGG in vitro of these two T cell subpopulations.

In previously published experiments we have distinguished two subpopulations of helper T cells (specific helper and nonspecific helper) in the spleens of antigen-primed mice. These subpopulations can be differentiated by their modes of action, and their activities and dose-response characteristics in mouse spleens primed with human gamma-globulin (HGG). In experiments designed to test shether the two subpopulations were derived from the same pool of precursors, or different pools, mice were injected with limiting doses of a tolerogenic form of HGG. They were subsequently challenged with an immunogeneic dose of HGG and their spleens assayed for specific helper and nonspecific helper activities in response to HGG in vitro. Over the dose range of tolerogenic HGG studied, both the helper activities were found to be equally tolerized. The question of the possible involvement of suppressor T cells in the maintenance of the state of unresponsiveness was also addressed quantitatively by the use of this model system. No active suppression was demonstrable in the maintenance of tolerance in either T cell subpopulation. These results suggest that the two types of helper T cells are derived from a common precursor pool, or from different pools with identical susceptibilities to tolerogen. Suppressor cells do not appear to play a role in this form of tolerance.