Cell Growth and Antimicrobial Activity of Mouse Peritoneal Macrophages in Response to Glucocorticoids, Choleragen and Lipopolysaccharide

Abstract

Normal, thioglycollate-stimulated and BCG-activated mouse peritoneal macrophages were cultivated in vitro with the conditioned medium of mouse L-929 cells. The thioglycollateand BCG-macrophages rapidly proliferated, whereas normal macrophages grew more slowly. A clear morphological difference between the three types of macrophages in the culture was observed.Glucocorticoids inhibited the growth of the macrophages at pharmacological concentrations. Other steroids, progesterone, diethylstilbesterol and testosterone in that order, had a far lower growth-inhibiting effect. Macrophages cultured with 10-6 M dexamethasone had a reduced antimicrobial effect on Candida parapsilosis compared with that of the untreated cells. Choleragen had the same effect on the macrophages as glucocorticoids. The toxin inhibited growth at a concentration as low as 10 pg/ml and cells treated with 1 ng of choleragen per ml had decreased antifungal activity. Similarly, Escherichia coli lipopolysaccharide at 10 ng/ml inhibited the growth of thioglycollate-macrophages.However, macrophages incubated with the lipopolysaccharide had enhanced anticandida activity. Thus, the immunosuppressors glucocorticoid and choleragen inhibited both the increase in the number of macrophages and the microbicidal activity of the phagocytes. Lipopolysaccharide, an immunostimulant, stimulated macrophage activity, but was toxic for cell growth.The mononuclear phagocytes in blood (monocytes) and tissues (macrophages) play a variety of important roles in host defense responses. One mechanism for controlling the responses due to mononuclear phagocytic (MP) cells is activation of MP cells in the host, for example, by infection with certain bacteria (13). Activated MP cells enhance a variety of cellular functions including killing of intracellular pathogens (12, 21) and cytotoxicity to tumor cells (3,9,20). Another mechanism may be the control of the absolute number of MP cells in vivo. The number of MP cells at a local inflammation site increases not only by the migration of blood monocytes (26, 27) but also by the proliferation of the migrated cells at the inflammation site, probably depending on a growth factor produced by lymphocytes or by fibroblasts, as evidenced in vitro (5,8,11,24).

Suppression of Anti‐Candida Activity of Murine and Human Neutrophils by Glucocorticoids

“…In this experimental system, effects of COR , a major GC existing in murine blood, on anti-Candida activity of murine neutrophils were examined. As shown in Table 1, COR suppressed the anti-Candida activity of murine neutrophils in a dose- sive actions of GC, which make a host vulnerable to Candida infection, were elicited through GCinduced dysfunction of macrophages and/or suppression of cell-mediated immunity because anti-Candida activity of macrophages was weakened by 10-6M of DEX (15) and production of those cytokines involved in cell-mediated immunity, which were related with host defense against Candida infection (18,25), was also suppressed by GC (4). Our finding that GCs even at very low concentration, for example, 10-8 M of DEX, significantly suppressed anti-Candida activity of neutrophils suggests that neutrophils may be one of the crucial targets of GC action which renders a host vulnerable to fungal infections like Candida and probably some other opportunistic fungi (14,22).…”

“…Although GCs are recognized to suppress anti-Candida functions of macrophages (15) and cell-mediated immunity (4), there has been no report on down-regulation of neutrophil antiCandida activity by GC. Here, we studied the effects of major risk-factors for candidiasis such as GC hormones and anti-inflammatory GC agents on Preparation of murine and human neutrophils .…”

Innate immune responses of primary murine macrophage-lineage cells and RAW 264.7 cells to ligands of Toll-like receptors 2, 3, and 4

“…However, the vast majority of these studies either have used primary cells collected from a single anatomical site (e.g., alveolar macrophages), or cells that were elicited by the administration of an inflammatory stimulus (e.g., thioglycollate)[1–3]. In addition, several monocyte-lineage cell lines are available, including the murine macrophage-like RAW 264.7 cell line.…”

“…Activation of monocyte-linage cells via different Toll-like receptors results in recruitment of specific adaptor proteins (e.g., MyD88 and TRIF) to initiate cell-signaling and synthesis of several down-stream products, including cytokines and chemokines [3, 5, 6]. Production of one down-stream product from each of the cell-signaling pathways, was chosen as a tool to characterize activation by the microbial ligands[7–9].…”

Microbicidal Activity of Bone Marrow Cells. Augmentation of Candida Killing Activity of the Cells by Culturing with Fibroblast Conditioned Medium

“…Primary cultures of mouse embryo fibroblasts prepared by the conventional method (cut by scissors and dispersed by trypsin) were seeded in half-gallon glass roller bottles with 100 ml of medium (inoculated cells were equivalent to 2-3 embryos/bottle) and cultured for 48 hr on a roller culture apparatus (Wheaton, Millville, N.J.). Culture media were collected, filtered through a 0.3-,um Millipore membrane (Nippon Millipore, Tokyo) and stored at -20 C. Preparation of conditioned medium of L-929 cells (L-CM) was described elsewhere (14). …”