Phagocytosis of Candida albicans by concanavalin-A activated peritoneal macrophages.

We evaluated the effect of treatment of mice with concanavalin-A (Con-A) on the phagocytosis of glutaraldehyde-fixed Candida albicans by peritoneal macrophages. The mean number of unopsonized C. albicans blastoconidia phagocytosed in vitro by peritoneal macrophages was doubled (from 1.3+/-0.1 to 2.7+/-0.14) by pre-treatment of the donor mice with Con-A. The percent of peritoneal cells phagocytosing the blastoconidia in vitro was increased about four times (from 22.3+/-8.6 to 80.3+/-3.2) by Con-A. This increase in phagocytosis was about 50% inhibited by addition of mannan (50 microg) plus mannose (50 mM) to the assay medium, suggesting that it was mediated by mannose receptors (MR). Phagocytosis in vitro in the presence of fresh non-immune serum (5%) was also increased, from 84.3+/-5.0 for untreated macrophages to 100% for Con-A activated peritoneal macrophages and the mean number of opsonized C. albicans blastoconidia increased from 2.3+/-0.1 to 4. 6+/-0.1. These results suggest that treatment of mice with Con-A increased both the phagocytosis of C. albicans blastoconidia mediated by mannose receptors and by complement receptors.     

In vitro study of contact-mediated killing of Candida albicans hyphae by activated murine peritoneal macrophages in a serum-free medium.

Activated peritoneal macrophages obtained from Listeria-immune mice were demonstrated to kill nonphagocytosable Candida albicans hyphae by contact-mediated mechanisms in a serum-free synthetic medium. The actual killing of hyphae was confirmed by a microculture technique utilizing the dimorphic nature of the fungus. The most efficient candidacidal activity was demonstrated by the macrophages obtained from mice first immunized with live Listeria monocytogenes and then elicited with heat-killed L. monocytogenes cells. Resident macrophages from control mice showed only low candidacidal activity against C. albicans hyphae and yeast cells. Direct physical contact appeared to be required for macrophages to efficiently kill oversized C. albicans hyphae. Efficient in vitro killing of hyphae also required relatively high effector/target cell ratios (50 or higher). The contact-mediated candidacidal activity of activated macrophages was not significantly abrogated by oxygen-radical scavengers, suggesting the involvement of oxygen-independent mechanisms. These results suggest that the enhanced nonspecific immunity to candidiasis seen in Listeria-immune hosts can be attributed, at least in part, to activated fungicidal macrophages. The ability of macrophages to detect and destroy both yeast and hyphal C. albicans cells is clearly an important element of the host defense against candidiasis.     

Studies on phagocytosis: II. In vitro phagocytosis by macrophages

In an in vitro system containing no neutralizing antibody, `immune' macrophages remove T<sub>1</sub> phage from the system more rapidly than do `non-immune' macrophages. The clearance of T₁ phage from the in vitro system occurs in two phases, similar to those occurring in vivo. The results of the in vitro studies thus mirror the results of the in vivo studies previously described.     

Quantitative measurement of phagocytosis of Neisseria gonorrhoeae by mouse peritoneal macrophages.

The simultaneous labeling of gonococci with [6-3H]uracil and of mouse peritoneal macrophages with L-[U-14C]leucine permits a quantitative assessment of the association of gonococci with macrophages under various experimental conditions. Colony-type T1 (piliated) gonococci associated more than T4 (nonpiliated) organisms at 4 degrees C, but at 37 degrees C the association of T4 gonococci with macrophages exceeded that for the T1 organisms. The association of T1 gonococci with macrophages could be enhanced as much as 70-fold by homologous rabbit antisera prepared against whole, formaldehyde-treated organisms. This immune enhancement represented primarily increased phagocytosis rather than surface attachment, as shown by its inhibition at 4 degrees C or with 2-deoxyglucose. The data further suggested that this enhanced phagocytosis was mediated via the Fc receptor for immunoglobulin G.     

In vitro killing of Ehrlichia risticii by activated and immune mouse peritoneal macrophages.

Normal resident murine peritoneal macrophages inoculated in vitro with Ehrlichia risticii readily phagocytized the organism but were unable to suppress ehrlichial replication as determined by indirect fluorescent-antibody staining of the inoculated cells. In contrast, macrophages from Corynebacterium parvum-inoculated and E. risticii-recovered mice rapidly eliminated the ehrlichiae. Macrophages from E. risticii-recovered mice were as effective as the C. parvum-activated cells in phagocytizing and eliminating the organism. Opsonization of E. risticii with homologous antiserum prior to inoculation of macrophage cultures resulted in enhancement of phagocytosis and greater suppression of E. risticii replication in all macrophage groups. These findings indicate that the pathogenesis of E. risticii infection centers on the ability of the organism to enter and replicate within the macrophage with avoidance of macrophage antimicrobial effects. An immune response results in macrophage activation with enhancement of the macrophage's ability to eliminate E. risticii. Opsonization of E. risticii with anti-E. risticii serum renders E. risticii more susceptible to macrophage destruction.     

Contribution of N-acetyl-beta-D-galactosamine-specific lectin to Fc receptor-mediated phagocytosis by mouse peritoneal macrophages.

The contribution of lectin-like receptors on the cell surface of mouse peritoneal macrophages to the process of phagocytosis of IgG-coated sheep red blood cells (SRBC) through Fc receptors has been investigated. Phagocytosis was activated by conditioned medium containing modified vitamin D3-binding protein (DBP) prepared by the incubation of foetal calf serum (FCS) with lysophosphatidyl-choline-treated splenic non-adherent cells. Fc receptor-mediated phagocytosis of opsonized SRBC was specifically inhibited by the addition of N-acetyl-beta-D-galactosamine. The binding of modified Gc globulin, human DBP, to peritoneal macrophage was only inhibited by the addition of N-acetyl-beta-D-galactosamine, and was dependent on N-acetyl-beta-D-galactosamine concentration. In the presence of cycloheximide, activated phagocytosis was reduced to control levels. By Scatchard plot analysis of binding studies, the number of Fc receptors of macrophages which were activated by conditioned medium increased 3.6-fold in comparison to that of control macrophages. These findings suggest that lectin-like receptors having a specificity to N-acetyl-beta-D-galactosamine are involved in activating the process of Fc receptor-mediated phagocytosis of opsonized SRBC by macrophages, and that modified DBP promotes the synthesis of Fc receptors through the N-acetyl-beta-D-galactosamine-specific lectin on macrophage surface.     

Fungicidal activity of rabbit alveolar and peritoneal macrophages against Candida albicans.

We tested the ability of rabbit macrophages to kill Candida albicans in vitro. Resident (unstimulated) alveolar macrophages killed 28.1 +/- 1.9% of ingested organisms in 4 h, whereas resident peritoneal macrophages killed only 15.2 +/- 1.3% (mean +/- standard error of the mean, P < 0.01). Peritoneal macrophages obtained from rabbits treated 3 weeks earlier with complete Freund adjuvant showed enhanced candidacidal activity relative to normally resident peritoneal cells (28.2 +/- 3.1%, P < 0.01). Candidacidal activity by alveolar macrophages recovered from such treated animals was slightly enhanced relative to untreated alveolar macrophages (32.9 +/- 2.3%). Candidacidal activity by peritoneal and alveolar macrophages was not decreased by several agents (cyanide, azide, sulfadiazine, and phenylbutazone) that inhibit the ability of human blood monocytes to kill C. albicans. In contrast, candidacidal activity by alveolar macrophages was greatly diminished by iodoacetate, an ineffective inhibitor of this function in human monocytes. We conclude that rabbit macrophages kill C. albicans by a fungicidal mechanism distinct from the peroxidase-H2O2 mechanism of human granulocytes and monocytes, and that the fungicidal properties of peritoneal and alveolar macrophage populations are enhanced after nonspecific stimulation with complete Freund adjuvant.     

Mannose receptor contribution to Candida albicans phagocytosis by murine E-clone J774 macrophages

Mannoproteins, as the main constituents of the outer layer of yeast cell walls, are able to interact with phagocytic cells in an opsonin-independent manner through the mannose receptor (MR) and to induce yeast ingestion by the professional phagocytes. Moreover, the MR also mediates endocytosis of soluble ligands through clathrin-coated pits. Here, we studied some aspects of the interaction between the MR and Candida albicans using murine E-clone macrophages and the consequences on MR trafficking. Using a pull-down assay involving mixture E-clone macrophage detergent lysate with mannosylated Sepharose beads and glutaraldehyde-fixed, heat-killed (HK) C. albicans, we found that binding of solubilized MR to mannosylated particles occurred with characteristics similar to the receptor's cell-surface mannose-binding activity. We then demonstrated that MR expressed on E-clone macrophages contributed to phagocytosis of unopsonized, HK C. albicans and that yeast phagocytosis induced a decrease in MR endocytic activity without concomitant degradation of the receptor in the time lapse studied.     

In vitro studies on the phagocytosis of Staphylococcus aureus by peritoneal macrophages of New Zealand mice

The ability of peritoneal macrophages from NZB, NZW and BWF1 mice to phagocytose and subsequently kill Staphylococcus aureus has been studied and compared to peritoneal macrophages from CBA, BALB/c and C₃Hf mice. Phagocytic indices determined in young and old New Zealand mice did not differ from those of the control strains. The bactericidal activity of the peritoneal macrophages did not show any interstrain differences.

Incorporation of serum from old and young NZB mice into the culture medium did not affect phagocytosis or the rate at which phagocytosed bacteria were killed. Studies performed in the absence of serum from the culture medium showed that probably both phagocytosis and bactericidal activity were unimpaired.

     

Effects of interleukin-10 on chemokine KC gene expression by mouse peritoneal macrophages in response to Candida albicans

Chemokine KC has been considered to be a murine homologue of human GRO/MGSA and was identified as chemoattractant for monocytes and neutrophils. This study examined the expression of KC mRNA in thioglycollate-elicited mouse peritoneal macrophages that were stimulated in vitro with Candida albicans (CA). Also examined were the inhibitory effects of IL-10 on the CA-induced expression of KC gene by Northern blot analysis. CA was found to induce chemokine gene expression in a gene-specific manner, CXC chemokine IP-10 mRNA expression was not detected in CA-stimulated macrophages. Maximum KC mRNA expression was observed approximately 2 hr after adding CA. The inhibitory action of IL-10 to CA-induced KC mRNA expression on mouse peritoneal macrophages was independent on concentration and stimulation time of IL-10 and was observed approximately one hour after adding IL-10 and CA simultaneously. IL-10 produced a decrease in the stability of KC mRNA, and CA-stimulated macrophages with cycloheximide blocked the suppressive effect of IL-10. These results suggest that CA also induces chemokine KC from macrophages, and IL-10 acts to destabilize CA-induced KC mRNA and de novo synthesis of an intermediate protein is a part of the IL-10 suppressive mechanism.     

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.     

Ultrastructural and immunological aspects of the phagocytosis of Trypanosoma brucei by mouse peritoneal macrophages.

Trypanosome-activated mouse peritoneal macrophages phagocytized and digested Trypanosoma brucei in vitro and in vivo, but in the absence of specific antiserum and complement the degree of phagocytosis was minimal. Ultrastructurally, the parasites attached to the macrophage by their flagella, and ingestion proceeded flagellum first. Once ingested, T. brucei was degraded, presumably due to fusion of the parasite-containing phagosome with lysosomes. Contrariwise, normal mouse peritoneal macrophages displayed negligible ability to ingest T. brucei, even in the presence of specific antiserum and complement. During trypanosomiasis in deer mice (Peromyscus maniculatus), the development of hypergammaglobulinemia correlated with enhanced phagocytosis of T. brucei by macrophages, but only at early post-inoculation days (PID 5 to 15). Complement lysis of trypanosomes was not identified in these experiments. Between PID 20 to 30, antiserum and complement either had no phagocytosis-promoting ability or depressed the phagocytosis of T. brucei by macrophages. These results indicate that both specific antibody and complement contribute to the ingestion of T. brucei by activated macrophages, but that parasite antigenic variation effectively abrogates the phagocytic defense mechanism.     

Complement and Fc receptor-mediated phagocytosis of normal and stimulated mouse peritoneal macrophages.

Normal and stimulated macrophage populations induced by six different stimuli were tested for their IgG and complement (C)-mediated phagocytic capacity. A similar phagocytic capacity for sheep red cells (E) coated with IgG was exhibited by normal and stimulated macrophage populations with the exception of thioglycollate-stimulated macrophages which exhibited a lower phagocytic potential. The extent of attachment and ingestion of E coated with IgM and C 5-deficient mouse serum (EIgMC) was shown to depend on the concentration of IgM and serum in the opsonizing solutions. A further interaction of macrophage-EIgMC rosettes with C5-deficient mouse serum led to ingestion of a high proportion of the attached ElgMC. Heat-killed yeast cells coated with C5-deficient mouse complement were ingested to a similar extent by normal and stimulated macrophage populations except for thioglycollate-stimulated macrophage populations which exhibited about 30% of the phagocytic response. The data support the notion that complement receptors on macrophage surface are not limited in their function to the attachment phase but that they also are capable of mediating phagocytosis. It is suggested that the prevalent protocols of sequential opsonization of E with IgM and C5-deficient mouse serum do not provide enough bridging moieties between the coated E and macrophages to allow for effective ingestion; the EIgMC complex formed is only capable of attachment to macrophage surfaces.     

Effects of dimethyldioctadecylammonium bromide on phagocytosis and digestion of Listeria monocytogenes by mouse peritoneal macrophages.

Listeria monocytogenes was labelled with [3H]-thymidine and phagocytosis in vivo, measured after the intraperitoneal injection of killed or viable listeria. The loss of intracellular radioactivity after incubation in vitro was used as a measure for digestion. Killing was assessed by counting the numbers of viable listeria before and after in vitro incubation. Peritoneal macrophages from mice immunized with viable listeria showed better phagocytosis and digestion of both killed and viable listeria than macrophages from normal mice. Viable listeria were digested to a lesser degree than killed ones by both normal and immune macrophages. The simultaneous injection of the surfactant dimethyldioctadecylammonium bromide (DDA) with killed or viable listeria greatly depressed the digestion of listeria. Phagocytosis of viable listeria was not affected whereas that of killed listeria was slightly enhanced. The injection of 10(8) killed listeria mixed with DDA greatly impaired the digestion of viable listeria 7 days but not 1 day after injection. The impairment of digestion was accompanied by an increase in the number of intracellular viable listeria.     

In vitro kinetics of phagocytosis and intracellular killing of gonococci by peritoneal macrophages from mice deficient in complement component 5.

Unstimulated resident peritoneal macrophages were harvested from complement-sufficient (C5+) and complement-deficient (C5-) mice by peritoneal lavage and cultured for 14 h. Adherence to cover slips was determined, and the monolayer was infected with transparent T1 gonococci. At various times after infection, the macrophages were observed for both attachment and phagocytosis of the gonococci by scanning and transmission electron microscopy. this analysis indicated that C5+ macrophages were capable of immediate phagocytosis of gonococci, with maximal phagocytosis occurring by 60 to 90 min. In contrast, C5- macrophages had a greater lag time before initiation of phagocytosis; this event was started by 30 min and completed by 90 min. The intracellular gonococci which were phagocytized by either C5+ or C5- mice were completely killed after 30 min of incubation. It appears that C5- mice are at a disadvantage in the early kinetics of the phagocytosis of gonococci, but that this does not affect the ultimate intracellular destruction of gonococci.     

Growth inhibition of Candida albicans by rabbit alveolar macrophages.

Normal rabbit alveolar macrophages were infected in vitro with Candida albicans. Early after infection, germ tube formation of phagocytized C. albicans was inhibited in contrast to extracellular (nonphagocytized) C. albicans. Over and 8-h period, plate counts of C. albicans incubated with alveolar macrophages revealed a decrease in colony-forming units in contrast to C. albicans alone. In addition, an assay was developed which specifically measured C. albicans [3H]leucine incorporation in the presence of alveolar macrophages. Using this assay, we observed a 71 to 93% inhibition of macromolecular synthesis in C. albicans when incubated with alveolar macrophages. Autoradiographic studies showed that the inhibition of leucine incorporation was restricted to the ingested Candida.     

Peroxisomes of rat peritoneal macrophages during phagocytosis.

The peroxisomes of resident macrophages in the rat peritoneal cavity were examined during the phagocytosis of latex microbeads, employing the akaline diaminobenzidine (DAB) technique. Peroxisomes generally were located in close proximity to phagosomes and were often observed in a process of apparent fusion with phagosomes. Cytochemical evidence was also obtained for discharge of catalase from peroxisomes to phagosomes. The profiles indicating fusion were observed after 10 minutes of incubation with microbeads. The number of peroxisomes was increased in macrophage profiles examined 30 minutes after exposure to microbeads. Acid phosphatase was localized in small vesicles that were distinct from peroxisomes, and peroxidase was not demonstrable in peroxisomes. A method for ultrastructural localization of periodate reactive complex carbohydrate demonstrated glycoproteins in numerous small vesicles or granules, some of which possibly represented peroxisomers. The possible function of peroxisomes during phagocytosis in rat peritoneal macrophages is considered.