Some effects of lignocaine on cultured mouse peritoneal macrophages

Macrophages were exposed to lignocaine in perifused and non-perifused cultures using media with or without foetal calf serum. Effects on morphology, viability, phagocytosis and the release of enzymes were assessed. During the period of contact with lignocaine there was a selective release of -glucuronidase. After washing enzyme release continued over a period of 7 hours and, in the absence of foetal calf serum, a decrease in the total -glucuronidase content was found in non-perifused, cultures. Although lignocaine-treated cells phagocytosed particulates the rate of enzyme release was reduced compared with normal cells when subsequently exposed to quartz.     

Immediate cytotoxicity of Chlamydia trachomatis for mouse peritoneal macrophages.

The toxicity of Chlamydia trachomatis was studied with mouse peritoneal macrophage culture. Inoculation of 30 inclusion-forming units of trachoma B/TW-5/OT organisms and 250 inclusion-forming units of lymphogranuloma venereum L2/434/Bu organisms per cell caused immediated toxicity, with the killing of 40 to 90% of the macrophages within 6 h after inoculation. Inhibition of phagocytosis by adsorption at 0 degrees C or by NaF pretreatment of macrophages prevented the toxicity, indicating that chlamydiae must be phagocytized to induce toxicity. Infectivity and toxicity could be dissociated, since ultraviolet-inactivated chlamydiae were still toxic. However, the toxicity was destroyed by heating the organisms at 56 degrees C for 10 min. Tetracycline, and antichlamydial drug, did not prevent toxicity, indicating that multiplication of the organisms was not required to induce toxicity. Toxicity was not prevented by treatment of macrophages with hydrocortisone. The toxicity of trachoma TW-5 was reduced by the rabbit immune serum of trachoma TW-5 but not by the rabbit immune serum of psittacosis meningopneumonitis.     

Suspended mouse peritoneal macrophages. Preparation and properties

Since macrophages (MPH) are able to adhere firmly to solid surfaces, the recovery of viable and functional MPH has proven to be extremely difficult. We have developed a simple method using agarose coating for preparing MPH and culturing the cells in suspension. Their properties were tested over 72 h. The oxidative burst declined with time, but could be restored using the lymphokine rich supernatant of pokeweed-stimulated mouse spleen cells. In contrast, phagocytosis and Candida intra-cellular killing remained unchanged.     

The IgM receptor in mouse peritoneal macrophages.

Purified samples of IgM free of IgG and alpha 2-macroglobulin contaminants were obtained from mice sera. Resident peritoneal mouse macrophages poorly phagocytose sheep red blood cells (E) sensitized with purified mouse IgM anti-E. Conversely 62.5% of thioglycollate-elicited macrophages and 10.11% of BCG stimulated peritoneal cells phagocytose IgM sensitized sheep red blood cells. The monomeric and polymeric forms of IgM blocked almost completely the phagocytosis of erythrocytes opsonized with homologous IgM molecules. These data clarifies the controversy concerning the homologous IgM receptor in mouse macrophages and further suggest that it may be used as a marker for macrophage stimulation or activation.     

Effect of hashish compounds on mouse peritoneal macrophages.

Light microscopy reveals an induction of extensive vacuolation in the macrophage after exposure to either delta 1-tetrahydrocannabinol or cannabidiol. Numerous small vacuoles appear in the cell periphery as early as 15 minutes after exposure (at 37degrees C.) to either of the compounds in 20 per cent newborn calf serum-Dulbecco's modified Eagle medium. The small lucent vacuoles coaleasce and yield enormous vacuoles which dominate the the cytoplasm. At approximately 3 hours, many of the vacuoles seem to burst with a concomitant expulsion of cell interior. The effect of hashish compounds on macrophages is essentially irreversible; exposure for 15 minutes to 10(-5) m of delta 1-THC or cannabidiol and a thorough wash in 20 per cent serum-medium, suffices to trigger the sequence of vacuolation and total cell death in the culture. Two major processes involving early reorganization of cellular membranes have been observed using electron microscopy. One relates to the formation of numerous autophagic vacuoles full of organelles, the other relates to the appearance of cytoplasmic inclusions representing extensive destruction of intracellular constituents. Both types of cytoplasmic change have been observed in alveolar macrophages of hashish smokers. Thus, the conditions in the in vitro studies are similar to conditions in people exposed to hashish smoke.     

Interaction of Chlamydia psittaci with mouse peritoneal macrophages.

L-cell-grown Chlamydia psittaci elementary bodies (EB) were rapidly phagocytized by mouse peritoneal macrophages in vitro. However, the intracellular fate of chlamydiae in macrophages appeared to be dependent on the multiplicity of infection (MOI), i.e., the EB-to-macrophage ratio, and the treatment of the EB. At an MOI of 1:1 or less, survival is maximal, and growth and multiplication of live, untreated chlamydiae did occur. In contrast, at a high MOI (100:1), survival of chlamydiae is reduced, as confirmed by release of 3H-labeled nucleic acid into the supernatant. At the high MOI, macrophage damage occurred that resulted in significant release of the lactic dehydrogenase, beginning 2 h postinfection. This immediated macrophage cytotoxicity as abolished by pretreatment of EB with heat (5 min at 56 degrees C) and was reduced about 50% by coating EB with homologous antibody. Pretreatment of the chlamydia with heat or opsonizing antibody provides increased uptake of EB by macrophages but may contribute to increased destruction of these obligate intracellular pathogens in professional phagocytic cells.     

Cathepsin B activity in stimulated mouse peritoneal macrophages.

The activity of cathepsin B was assayed in murine resident peritoneal macrophages, and after stimulation of the cells in vivo and in vitro. The resident cells showed a very low activity of the enzyme, compared to the activities of three other lysosomal enzymes: cathepsin D, acid phosphatase, and beta-glucuronidase which were tested simultaneously. Endocytosis of carrageenan, latex, or carbon particles in vitro induced a prominent rise in intracellular cathepsin B activity. Addition of endotoxin from Escherichia coli in vivo or in vitro, or cell wall products from streptococci in vitro caused no change in cathepsin B activity. There was a release of enzyme activity to the medium after a 72-hour culture of macrophages. However, the release, calculated as a percentage of total activity, was not influenced by any treatments mentioned. All significant rises in enzyme activity could be inhibited by the addition of cycloheximide, and it was concluded that increased enzyme activity was dependent on new protein synthesis.     

Comparison of cytotoxic and microbicidal function of bronchoalveolar and peritoneal macrophages.

Studies were carried out with mice to explore in vitro the effector function(s) of macrophages from two different anatomical compartments (peritoneal cavity and lungs). The cytotoxic capacity of macrophages was measured by determining their cytostatic and cytocidal effects on EL-4 tumour target cells, and the microbicidal capacity of macrophages was measured by determining their ability to kill or inhibit the intracellular protozoan, Toxoplasma gondii. Neither peritoneal macrophages (PM) nor bronchoalveolar macrophages (BAM) from normal mice were ever microbicidal or cytotoxic. Intravenous treatment with Corynebacterium parvum greatly enhanced (activated) both effector functions of PM but did not activate BAM. Chronic infection with Toxoplasma activated PM throughout the period of observation (greater than 140 days), but the presence of activated BAM was transient and appeared to coincide with the occurrence of an inflammatory response in the lungs.     

Resorption of bone by mouse peritoneal macrophages

An experimental system is described in which mononuclear phagocytes are shown to cause contact-dependent resorption of bone powder in vitro. Endotoxin, which is known to stimulate macrophage functions, and which has also been shown to increase bone resorption in organ culture, increased bone dissolution by mononuclear phagocytes. On the other hand, parathyroid hormone and prostaglandin E2, hormones which increase th number and activity of osteoclasts, had no effect on resorption of bone powder by mononuclear phagocytes. This suggests that the osteoclast, which is probably derived from mononuclear phagocytes, is induced to respond to these hormones indirectly, following a primary hormonal effect on osteoblasts.     

Weakening the bactericidal activity of mouse peritoneal macrophages by a combination of type a staphylococcal enterotoxin and endotoxin

Laboratory of Molecular Bases of Pathogenicity of Bacteria, N. F. Gamaleya Research Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR P. A. Vershilova.) Translated from Byulleten' Éksperimental'noi Biologi i Meditsiny, Vol. 108, No. 11, pp. 591–593, November, 1989.     

Human alveolar macrophages: effects of endotoxin in vitro.

Experiments were performed to evaluate the in vitro effects of Escherichia coli lipopolysaccharide on viability and function of human alveolar macrophages. Alveolar macrophages were obtained by fiberoptic bronchoscopy and saline bronchial lavage from 12 normal, nonsmoking volunteers. Cells were incubated with different concentrations of E. coli endotoxin for 1 and 24 h. Endotoxin (10 microgram/ml and more) was cytotoxic for alveolar macrophages after 24 h of incubation and induced significant inhibition of phagocytosis, adherence, and spreading. The effects of endotoxin on alveolar macrophage viability and function were dose and time dependent and were not influenced by indomethacin. Thus, human alveolar macrophages, like other mononuclear phagocytes, are extremely sensitive to endotoxin effects; these observations may be relevant in conditions in which endotoxin may be in contact with alveolar macrophages in vivo: endobronchial infections with gram-negative organisms, byssinosis, chronic bronchitis of grain handles, and humidifier fever.     

Improved method for the isolation of purified mouse peritoneal macrophages

Mouse peritoneal macrophages were cultured for 45 min in medium supplemented with fetal calf serum (FCS) in petri dishes coated overnight with heat-inactivated FCS. After removal of non-adherent cells by washing, adherent cells were detached by a brief incubation in the presence of sub-toxic levels of ethylenediamine tetraacetate (EDTA). Overall peritoneal macrophage recoveries of 90% can be routinely achieved with this method, and full cell viability is maintained.     

Interaction of Chlamydia psittaci reticulate bodies with mouse peritoneal macrophages.

Noninfectious reticulate bodies of Chlamydia psittaci are readily phagocytized by thioglycolate-elicited mouse peritoneal macrophages in monolayer culture. The internalized reticulate bodies are rapidly destroyed as indicated by a 60 to 70% decrease in trichloroacetic acid-precipitable radioisotopic counts in the macrophage pellet by 10 h and a concomitant increase of the trichloroacetic acid-soluble radiolabeled chlamydial nucleic acid in the cytoplasm. This intracellular destruction of reticulate bodies in macrophages is independent of the multiplicity of infection. Reticulate bodies at a high multiplicity of infection, up to 1,000:1, are also incapable of inducing immediate cytotoxicity in macrophages as evidenced by the lack of early release of the host cell-soluble cytoplasmic enzyme lactic dehydrogenase. Thus, it appears that the virulence factors for (i) initiation or maintenance of intracellular survival via circumvention of phagolysosome formation and (ii) host cell damage are either missing or not expressed by the RB form of this bacterium.     

The production of ceroid by mouse peritoneal macrophages in vitro.

Murine resident peritoneal macrophages accumulated lipid droplets and subsequently insoluble, ceroid-like material when cultured in vitro in a medium containing 33% fetal calf serum. At least some of this insoluble lipid was membrane-bound and by light microscopy it often appeared as ''rings'' with a hollow centre. It is suggested that the production of ceroid may be the consequence of the uptake of lipids from the extracellular medium and the activity of the macrophage''s membrane-bound oxidative microbicidal mechanisms. The results indicate that macrophages are capable of rendering lipids insoluble, supporting the suggestion that this might occur in the atherosclerotic plaque.     

In vitro phagocytosis of Candida albicans by peritoneal mouse macrophages.

The ability of sensitized mouse peritoneal macrophages to phagocytose and inhibit Candida albicans was studied in an in vitro system. Mice were sensitized to C. albicans by intraperitoneal infection with viable organisms or by intracutaneous injection of heat-inactivated cells in Freund complete adjuvant. Development of delayed hypersensitivity to C. albicans was evaluated by footpad tests with cytoplasmic and cell wall antigens as well as by macrophage migration inhibition by these antigens and by whole heat-inactivated cells. Inhibition of macrophage migration by heat-inactivated cells was significantly greater when the mice were sensitized by viable organisms. The macrophages from these mice were also larger and showed a greaer ability to inhibit germ tube production by phagocytosed yeasts. This suggests that macrophages may play a protective role in infection by C. albicans.     

Activation of mouse peritoneal macrophages by maintenance in serum-free medium.

The repetitive epitope of Plasmodium falciparum circumsporozoite protein (Asn-Ala-Asn-Pro)3 [(NANP)3] was coupled to tripalmitoyl-S-glyceryl-cysteine (P3C) and tripalmitoyl-S-glyceryl-cysteinyl-serine (P3CS). The lipopeptide P3CS is a potent B-cell and macrophage activator. The resulting immunogenic lipopeptides were used for immunization of the low responder mouse strain BALB/c. These low molecular weight conjugates induced specific anti-(NANP)3 IgG and IgM levels without any carrier proteins or admixed adjuvants after a single administration.     

Early interactions of herpes simplex virus with mouse peritoneal macrophages.

Adsorption of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) to resident peritoneal macrophages (PM) of 4-week-old Swiss albino (SA) and GR/AFib mice was studied. A significantly (P less than 0.05) higher HSV-2 adsorption rate was found with PM of SA mice than with PM of GR/AFib mice. Of added HSV-2 65% bound to the cells of SA mice over a 120-min period versus 15% to PM of GR/AFib mice. Only 15 to 20% of added HSV-1 bound to PM regardless of the mouse strain. These patterns of adsorption were found with all four HSV-1 and four HSV-2 strains tested. Pretreatment of PM with an HSV-2 mutant blocked the adsorption of added HSV-2. Thus, the receptors for HSV attachment seemed to be virus type selective. To avoid masking of adsorption by phagocytotic activity, the adsorption studies had to be performed at 4 degrees C. Transport of attached HSV-1 and HSV-2 to the nuclei of SA PM was studied with purified virus labeled with 32Pi and [3H]thymidine. In double-isotope experiments, only transport of HSV-2 was detected. The possible importance of differences in density or avidity of virus-binding receptors on the plasma membrane of PM is discussed in relation to macrophage-dependent focal liver necrosis, which was only demonstrable after intraperitoneal inoculation of HSV-2, not HSV-1, only in SA, not GR/AFib, mice.     

Antiserotonin antibodies as stimulators of mouse peritoneal macrophages

Stimulating effect of antiserotonin antibodies on phagocyte activity of peritoneal macrophages from C57B1/6 mice is demonstrated both after systemic administration and in a cell culture.In vitro experiments show that serotonin and antiserotonin antibodies exert similar effects. A possible mechanism of neurotropic effect of antiserotonin antibodies involving immunocompetent cells is discussed. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 12, pp. 663–665, December, 1997     

Role of plasma, lipopolysaccharide-binding protein, and CD14 in response of mouse peritoneal exudate macrophages to endotoxin

Plasma lipopolysaccharide (LPS)-binding protein (LBP) and membrane CD14 function to enhance the responses of monocytes to low concentrations of endotoxin. Surprisingly, recent reports have suggested that LBP or CD14 may be dispensable for macrophage responses to low concentrations of LPS or may even exert an inhibitory effect in the case of LBP. We therefore investigated whether LBP and CD14 participated in the response of mouse peritoneal exudate macrophages (PEM) to LPS stimulation. In the presence of a low amount of plasma (<1%) or of recombinant mouse or human LBP, PEM were found to respond to low concentrations of LPS (<5 to 10 ng/ml) in an LBP- and CD14-dependent manner. However, tumor necrosis factor production (not interleukin-6 production) by LPS-stimulated PEM was reduced when cells were stimulated in the presence of higher concentrations of plasma or serum (5 or 10%). Yet, the inhibitory effect of plasma or serum was not mediated by LBP. Taken together with previous results obtained with LBP and CD14 knockout mice in models of experimental endotoxemia, the present data confirm a critical part for LBP and CD14 in innate immune responses of both blood monocytes and tissue macrophages to endotoxins.