Phagocytic and candidacidal activities of macrophages from suckling and adult mice pretreated with concanavalin-A.

In this study, we investigated the effect of concanavalin-A (Con-A) on the activation of phagocytosis and killing of Candida albicans by peritoneal macrophages from suckling and adult mice. Pretreatment of adult mice with Con-A dose-dependently increased the percentage of macrophages phagocytosing C. albicans in vitro from 3.8 +/- 0.9 to 24.2 +/- 2.4 in the absence of serum opsonins. Addition of mannan (50 microg) and mannose (50 mM) to the incubation medium reduced phagocytosis from 21.5 +/- 1.3 to 4.7 +/- 1.9, suggesting that treatment with Con-A increased phagocytosis mediated by mannose receptors. Killing of C. albicans was also increased by increasing the dose of Con-A. Pretreatment of suckling mice with Con-A increased the macrophages' phagocytic and candidacidal activities by an amount similar to that observed in adult mice. Furthermore, suckling mice pretreated with Con-A survived an intraperitoneal inoculum of 5 x 10(7) C. albicans, whereas all control mice died within 24-48 h of infection. This suggested that increased phagocytosis and killing of C. albicans stimulated by the action of Con-A conferred early protection upon suckling mice experimentally infected with C. albicans.     

Normal host defense during systemic candidiasis in mannose receptor-deficient mice

Pathogen pattern recognition receptors (PRRs) recognize common structural and molecular motifs present on microbial surfaces and contribute to induction of innate immune responses. The mannose receptor (MR), a carbohydrate-binding receptor expressed on subsets of macrophages, is considered one such PRR. In vitro experiments have implicated the MR in phagocytosis of mannose-bearing microbes, including Candida albicans, and enhancement of antifungal response by macrophages. However, the significance of the MR's contribution to immune response during systemic C. albicans infection has never been directly demonstrated. Using MR-deficient mice in an in vivo infection experiment, we examined the role of the MR in immune response during disseminated candidiasis. MR(-/-) and wild-type control mice were challenged intraperitoneally with C. albicans, and the survival rates, tissue fungal burden, inflammatory cell recruitment, and specific antibody production after infection were evaluated. We found no significant difference in survival between the two mouse strains. MR(-/-) mice had higher average fungal burdens in some of the organs on days 7 and 21 but exhibited competence in inflammatory cell recruitment and antibody production. We also observed in vitro that MR(-/-) peritoneal cavity macrophages were equally capable of C. albicans uptake and that phagocytosis could be blocked with beta-glucan. We conclude that the MR is not required for the normal host defense during disseminated candidiasis or for the phagocytosis of C. albicans and that a beta-glucan receptor may be required for C. albicans phagocytosis.     

Differential cytokine production and Toll-like receptor signaling pathways by Candida albicans blastoconidia and hyphae

Toll-like receptors (TLR) are crucial for an efficient antifungal defense. We investigated the differential recognition of blastoconidia and hyphae of Candida albicans by TLRs. In contrast to Candida blastoconidia, which stimulated large amounts of gamma interferon (IFN-gamma), the tissue-invasive Candida hyphae did not stimulate any IFN-gamma by human peripheral blood mononuclear cells (PBMC) or murine splenic lymphocytes. After stimulation with blastoconidia, the production of IFN-gamma was TLR4 dependent, as shown by the significantly decreased IFN-gamma production in anti-TLR4-treated PBMC and in splenic lymphocytes from TLR4-defective ScCr mice. In addition, peritoneal macrophages from ScCr mice produced less tumor necrosis factor alpha (TNF-alpha) than macrophages of control mice did when stimulated with Candida blastoconidia, but not with hyphae, indicating that TLR4-mediated signals are lost during hyphal germination. In contrast, macrophages from TLR2 knockout mice had a decreased production of TNF-alpha in response to both Candida blastoconidia and hyphae. Candida hyphae stimulated production of interleukin-10 through TLR2-dependent mechanisms. In conclusion, TLR4 mediates proinflammatory cytokine induction after Candida stimulation, whereas Candida recognition by TLR2 leads mainly to anti-inflammatory cytokine release. TLR4-mediated proinflammatory signals are lost during germination of Candida blastoconidia into hyphae. Phenotypic switching during germination may be an important escape mechanism of C. albicans, resulting in counteracting host defense.     

Increased susceptibility of complement factor B/C2 double knockout mice and mannan-binding lectin knockout mice to systemic infection with Candida albicans

Candida albicans is the major cause of systemic fungal infections in immunocompromised patients. We investigated the susceptibility of mice deficient in complement factor B and C2 (Bf/C2(-/-)), C1q (C1qa(-/-)), and mannan-binding lectin (MBL)-A (MBL-A) and MBL-C (MBL-A/C(-/-)) to systemic infection with C. albicans. Animals were infected i.p. with 10(8)C. albicans blastoconidia and monitored for mortality. Bf/C2(-/-) mice showed high mortality (over 90%) within the study period of 3 weeks. In contrast, mortality in C1qa(-/-) mice was below 15% whereas that of MBL-A/C(-/-) mice was 40% (P<0.001). Intravenous infection of mice with 8x10(5) blastoconidia resulted in the same trend with Bf/C2(-/-) mice being highly susceptible compared to the other strains. Histology of kidney sections of infected Bf/C2(-/-) mice showed widespread mycelia confirming the high CFU counts from cultured tissue homogenates. In C1qa(-/-), MBL-A/C(-/-) and wild type C57BL/6 mice hyphal growth was limited. However, massive inflammatory infiltration was apparent, which was not seen in Bf/C2(-/-) mice. The ability of the mouse sera to opsonize C. albicans was determined by quantification of phagocytosis of C. albicans by peritoneal phagocytes. Whilst phagocytosis mediated by Bf/C2(-/-) mouse serum was low (10.6%), more phagocytosis could be seen in MBL-A/C(-/-) (19.9%), C1qa(-/-) mice (23.9%) and wild type mice (29%). Deficiency of classical pathway activation has only a low impact whereas the lectin pathway contributes to the host defence against candidosis. The more pronounced lack of complement activation in Bf/C2(-/-) mice leads to uncontrolled infection due to an opsonophagocytic defect.     

Effect of lentinan and mannan on phagocytosis of fluorescent latex microbeads by mouse peritoneal macrophages: a flow cytometric study

Lentinan, an immunopotentiating beta-1,3-glucan polysaccharide stimulated the in vitro phagocytosis of BSA-coated, C3b- or monoclonal immunoglobulin (IgG2b)-coated fluorescent microspheres by resident or thioglycollate-elicited mouse macrophages in a dose-dependent manner. Analysis of flow cytometric data has shown that microbead phagocytosis of resident macrophages, which exhibit a lower basic phagocytic activity than the thioglycollate elicited ones, has been augmented by up to 900% due to lentinan. The percent ratio of phagocytes among peritoneal exudate cells, however, remained unchanged after short-term lentinan stimulation. Preincubation of the cells with lentinan resulted in increased ingestion of the microbeads. Activation of phagocytosis by lentinan is therefore due in part to the direct stimulation of the cells, however, lentinan also serves as supplementary opsonin for C3b-coated beads. Mannan inhibited the ingestion of C3b-coated microspheres by 75%, which was abolished in part when lentinan was also added to the cells. Mannan did not influence the phagocytosis of BSA-coated or IgG-coated beads. Our data, based solely on in vitro studies, suggest a beta-glucan receptor mediated activation of phagocytes by lentinan. These receptors are different from the C3b, Fc or mannose receptors. It is very likely that stimulation of phagocytic activity of macrophages by lentinan may contribute to the antitumor action of this immunopotentiating polysaccharide.     

Candida albicans stimulates arachidonic acid liberation from alveolar macrophages through alpha-mannan and beta-glucan cell wall components.

Candida albicans is an increasingly important fungal pathogen. Alveolar macrophages respond to fungal components such as zymosan by releasing arachidonic acid (AA) and AA metabolites. However, few studies hypothesized that macrophages respond to C. albicans by releasing AA and generating AA metabolites as a consequence of interaction of mannose and beta-glucan receptors with fungal cell wall components. [14C]AA-labeled rabbit alveolar macrophages released AA following stimulation with either live or heat-killed C. albicans. High-pressure liquid chromatography analysis revealed that 55% of the AA released was metabolized via cyclooxygenase and lipoxygenase pathways. The metabolites consisted of prostaglandin E2, prostaglandin F2 alpha, 6-ketoprostaglandin F1 alpha, thromboxane B2, and leukotrienes B4 and D4. We further examined the roles of alpha-mannan and beta-glucan components of C. albicans in mediating these alterations of eicosanoid metabolism. Prior work in our laboratory has shown that soluble alpha-mannan and beta-glucan inhibit macrophage mannose and beta-glucan receptors, respectively. Incubation of alveolar macrophages with soluble alpha-mannan derived from C. albicans (1 mg/ml) resulted in 49.8% +/- 2.6% inhibition of macrophage AA release during stimulation with intact C. albicans (P = 0.0001 versus control). Macrophage AA release in response to C. albicans was also inhibited to a significant but lesser degree by soluble beta-glucan (36.2% +/- 1.3%; P = 0.008 versus control). These results indicate that C. albicans stimulates macrophage AA metabolism and that these effects are partly mediated by alpha-mannan and beta-glucan constituents of the fungus.     

Effects of a glyconutrient on macrophage functions

Previous studies have shown that mannosylated bovine serum albumin (mBSA) enhances the respiratory burst (RB), phagocytosis, and killing of Candida albicans and Escherichia coli by resident murine peritoneal macrophages (Mphi). Upregulation of the above Mphi functions was associated with the binding of mBSA to the macrophage mannose receptor. The present study was done to determine if certain glyconutrients could stimulate Mphi functions in a similar manner. Resident peritoneal murine Mphi collected from C57BL/6 mice were exposed to the glyconutrients for 10 and 60 min. The RB was measured using chemiluminescence. Both phagocytosis and killing were measured after incubation with each of the following microorganisms: Candida albicans, Escherichia coli and Staphylococcus aureus. The percent phagocytosis and killing were determined using fluorescence microscopy. Results indicated that certain glyconutrients, caused a dose and time dependent effect on Mphi-induced killing of all three microorganisms.     

Interaction of macrophages with "old" red blood cells from syngeneic mice in vitro and the independence of the recognition process on macrophage Fc receptors

The interaction between peritoneal macrophages and "old" red blood cells (RBC) from syngeneic mice was studied in vitro. It seems that this interaction is not mediated directly via Fc receptors on the macrophage. (1) 2-deoxy-D-glucose did not inhibit the phagocytosis of "old" RBC, but did inhibit the phagocytosis of IgG-coated sheep RBC (SRBC). (2) Immobilization of Fc receptors by plating macrophages on coverslips coated with bovine serum albumin: anti-bovine serum albumin (BSA: anti-BSA) complexes had no effect on the phagocytosis of "old" RBC, but inhibited the phagocytosis of IgG-coated SRBC. The interaction between mouse macrophages and "old" RBC is temperature dependent. At 4 degrees C and 22 degrees C "old" RBC attach to macrophages but are not phagocytized; at 37 degrees C phagocytosis occurs. Fetal calf serum (FCS) is required for optimal phagocytosis. Sialic acid residues on the macrophage surface do not play a role in this recognition and phagocytosis process, as neuraminidase treatment of macrophages did not affect the interaction.     

Impairment of Leishmania mexicana phagocytosis in peritoneal macrophages induced by Amaranthus leucocarpus lectin

The lectin from Amaranthus leucocarpus (ALL) is specific for N-acetyl-D-galactosamine and inhibits phagocytosis of Leishmania mexicana promastigotes in Balb/c mice peritoneal macrophages by 38%. The lipophosphoglycan (LPG) purified from L. mexicana inhibits penetration of promastigotes into peritoneal macrophages by 31%; interestingly, treatment of macrophages with both, ALL and LPG, inhibits phagocytosis of promastigotes by 72%, confirming that ALL induces modification of the macrophage's phagocytic activity by a different route than mannose or C3b receptors. The Inhibitory effect of ALL was time-dependent. N-acetyl-D-galactosamine (GalNAc) or O-glycosidically linked glycoproteins modified macrophage phagocytosis of Leishmania. These results suggest that macrophage membrane glycoproteins, possessing constitutive GalNAc, can influence the signaling pathways used by this intracellular parasite to infect.     

In vitro and in vivo effects of Imipenem on phagocytic activity of murine peritoneal macrophages

Imipenem, a new antibiotic beta-lactam, and Tienam (an Imipenem/Cilastatine combination) have been studied in vitro and in vivo respectively, in the phagocytic function of macrophages. In this paper we have seen the variations produced by 50 mg/l of Imipenem and 120 mg/kg of Tienam in the adherence, spontaneous mobility, chemotaxis, opsonization, phagocytosis of Candida albicans and latex beads, candidicid effect and nitroblue tetrazolium (NBT) reduction in peritoneal macrophages from BALB/C mice. This antibiotic significantly increases in vitro and in vivo the adherence, spontaneous mobility and chemotaxis, phagocytosis of latex beads and the digestion of ingested material (nitroblue tetrazolium reduction) in the above-mentioned cells. The number of Candida albicans opsonized and ingested by the macrophages is not modified in the presence of Imipenem, and neither is the candidicid effect.     

Susceptibility of beige mutant mice to candidiasis may be linked to a defect in granulocyte production by bone marrow stem cells.

The beige mutation in mice has a pervasive effect on mechanisms of host resistance to infectious agents. Best characterized are defects in granulocyte chemotaxis and phagocytosis, which are associated with increased susceptibility to bacteria, and a deficiency in the levels of natural killer (NK) cells, which has been linked to decreased resistance to both murine cytomegalovirus and the yeast Cryptococcus neoformans. The objective of the present experiments was to explore the cellular basis of the enhanced susceptibility of beige mice to systemic infection with the yeast Candida albicans. In contrast to murine cytomegalovirus and C. neoformans, infection with C. albicans did not induce any detectable NK cell activity in the spleen of bg/bg or bg/+ mice. Unfractionated bone marrow (BM) displayed some candidacidal activity, mediated by both phagocytic and nonphagocytic cells; however, there was no difference between homozygous and heterozygous mice in the effector function of normal BM cells or mononuclear cells derived from either short- or long-term BM cultures. On the other hand, peritoneal granulocytes from bg/bg mice were significantly more effective than those from bg/+ mice in killing Candida blastoconidia in vitro. A similar comparison of granulocytes from short-term BM cultures showed that the activities of cells from bg/bg and bg/+ mice were equivalent, indicating that the granulocytes derived from the peritoneal cavity of bg/bg mice had probably been exposed to some form of nonspecific stimulation in vivo. Somewhat surprisingly, long-term BM cultures did not support the continual growth of bg/bg granulocytes, and it is possible that the beige mutation may be associated with a lesion in the differentiation pathway that leads to the production of granulocytes. Taken together, the data indicate that, in beige mice, granulocytes rather than NK cells are a major determinant of natural resistance to C. albicans infections.     

Studies of macrophage function in murine systemic lupus erythematosus. 3. The nature, anatomical location, and reversibility of the phagocytic defect

The defect in phagocytosis and binding of antibody-coated sheep erythrocytes (EA) by peritoneal macrophages of (NZB X NZW)F1 or B/W mice is not intrinsic, but is related to the development of the autoimmune disease process. The defect appears to be confined to peritoneal macrophages, since bone marrow (BM)-derived macrophages have normal to elevated activities in vitro. The peritoneal macrophage defect is not due to blockade of Fc receptors in vivo, as shown by long-term culture or recovery of phagocytic and binding activities after removal of Fc receptors by pronase, but represents a reduced number of receptors with slightly delayed turnover. The defect can be reversed by elicitation of activated macrophages with Corynebacterium parvum, thioglycollate, or proteose peptone in vivo. Normal Fc-mediated phagocytosis and binding by BM-derived macrophages cultured from untreated autoimmune mice is enhanced by pretreatment of mice with C. parvum, thioglycollate, or proteose peptone. The cause of the defect in Fc-mediated phagocytosis by resident peritoneal macrophages of autoimmune mice was not ascertained; it may be due to abnormal macrophage kinetics or to the local effects of lymphokines released as a result of other autoimmune changes.     

Antifungal activity of splenic, liver and pulmonary macrophages against Candida albicans and effects of macrophage colony-stimulating factor.

Disseminated infections due to Candida albicans are frequently encountered in immunocompromised patients. We compared the antifungal activities of macrophages residing in spleen, liver and lungs of rabbits against blastoconidia and pseudohyphae of C. albicans. Splenic adherent cells (SAC), Kupffer cells (KC) and pulmonary alveolar macrophages (PAM) all ingested blastoconidia efficiently. SAC caused significantly more damage to unopsonized pseudohyphae compared with KC (P < 0.01) or PAM (P < 0.001). Incubation of SAC with 15 ng ml(-1) of recombinant human macrophage colony-stimulating factor (M-CSF) at 37 degrees C for 2 days significantly enhanced phagocytosis (P = 0.02) and killing (P = 0.05) of blastoconidia. In contrast, M-CSF had no effect on phagocytic activities of KC or PAM against blastoconidia or on damage caused by any of the macrophages to pseudohyphae of C. albicans. Thus, although all three resident macrophage types ingest blastoconidia efficiently, they differ in their capacity to cause damage to pseudohyphae and in their responsiveness to M-CSF for antifungal activation. M-CSF augments the capacity of SAC to ingest and kill blastoconidia and may therefore have a role in the treatment and prevention of hematogenously disseminated candidiasis.     

Effect of physical activity stress on the phagocytic process of peritoneal macrophages from old guinea pigs.

The different stages of the phagocytic function in peritoneal macrophages from old guinea pigs (27 +/- 3-months-old) were studied before, immediately after and 24 h after being subjected to physical activity stress (swimming until exhaustion) which raised the blood levels of corticosterone. The phagocytosis of opsonized Candida albicans was stimulated immediately after physical activity. No modifications in adherence, chemotaxis, ingestion of inert particles, or microbicide capacity, measured by nitroblue tetrazolium (NBT) reduction, were found. At 24 h, when no stress could be shown by corticosterone analysis, the phagocytosis of opsonized C. albicans remained stimulated and chemotaxis was increased while ingestion of inert particles and microbicide capacity remained unchanged. The adherence, however, was at a smaller level. No correlations were found between the corticosterone levels and the status of the phagocytic process of peritoneal macrophages.     

Comparison of Peritoneal Macrophages from Germfree and Conventional Mice

Morphology, lysosomal enzyme activities, and phagocytosis via immunological receptors were tested in peritoneal macrophages from germfree and conventional mice. Nonstimulated macrophages from germfree mice showed less spreading and were more easily detached when seeded on glass than conventional macrophages. The activities of the lysosomal acid phosphatase and cathepsin D were similar in the two cell groups, whereas beta-glucuronidase showed higher activity in macrophages from germfree mice. F(c) receptor-mediated phagocytosis of opsonized sheep erythrocytes was equally effective in germfree and conventional macrophages, and both cell types attached but did not internalize erythrocytes via the C(3)b receptor. Intraperitoneal injections of mineral oil caused a significantly higher influx of macrophages in conventional mice than in germfree mice, whereas the influx of polymorphonuclear cells was enhanced in both animals. Stimulation in vivo with oil or Escherichia coli endotoxin increased cell size, spreading ability, membrane ruffling, and lysosomal enzyme activities in macrophages from both conventional and germfree mice. The Fc-mediated phagocytosis was not influenced by stimulation, whereas the capacity to internalize via C(3)b receptor was triggered in macrophages from conventional mice, but not in corresponding cells from germfree mice. Similar results were obtained after stimulation with endotoxin in vitro. Culture in fetal calf serum for 72 h caused intracellular rises in all three enzyme activities tested in macrophages from conventional mice, whereas only the activity of acid phosphatase was increased in macrophages from germfree mice. Stimulation with zymosan in vitro caused selective release of lysosomal enzyme activity in macrophages from both animal groups. We conclude that peritoneal macrophages from germfree mice share several properties with cells from conventional mice, however, unstimulated beta-glucuronidase activity was increased, whereas spreading on glass, chemotactic response, in vitro induction of lysosomal enzymes, and the capacity to internalize via the C(3)b receptor after stimulation were reduced or absent.     

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.     

Effect of angiotensin II on macrophage functions.

Angiotensin II (At II) has been shown to inhibit in vitro the IgG2a-mediated rosette formation of 51Cr-sheep red blood cells (SRBC) by provoked peritoneal macrophages (PM) at 10(-5)-10(-6) M concentrations. The decreased rosette formation was associated with an increased phagocytosis. It was found that the enhanced rosette formation at 10(-7) M hormone concentration was followed by diminished phagocytosis via Fc gamma receptors (R). Processes mediated through Fc microR were affected only after incubation with 10(-5) M of At II. The attachment and subsequent phagocytosis through C3bR was markedly enhanced by At II in a dose-dependent way. Thus, the relative phagocytosis (RP) through both FcRs was significantly enhanced by 10(-5), i.e. by 10(-5)-10(-6) M of At II, but lowered at 10(-7) M hormone concentration. In addition, there was no RP enhancing effect of At II after preincubation with 10(-5) M of indomethacin (IM), indicating the significance of prostaglandins (PG) in the hormone effect. The medium containing 5 mM of EGTA diminished both the RP enhancing and inhibiting effects of At II. The RP mediated by C3b was not affected by At II, IM or EGTA. The intracellular killing capability, measured by chromium release from Candida albicans, was not altered or even slightly diminished after At II treatment of PMs.     

Serum amyloid P-component-induced colony-stimulating factors production by macrophages

Purified mouse serum amyloid P-component (SAP; 0.5-50 microg/kg), injected intravenously into Swiss mice, induced the production of serum colony-stimulating factors (CSFs); the maximum induction was observed at 10.0 microg/kg. Further, in vitro purified mouse SAP (0.1-50 microg/ml) stimulated the mouse elicited peritoneal macrophages to elaborate CSFs in the conditioned medium (CM); 5.0 microg/ml SAP appeared to be the optimum. Both in vivo and in vitro the maximum production of CSFs occurred 6 h after initiation of stimulation, and returned to the background levels by 48 h. Mannose 6-P, mannose 1-P and mannose, and not other sugars inhibited the SAP-induced production of CSFs by macrophages which suggests that SAP interaction with macrophages was mediated by specific glycoprotein-receptors. A neutralizing (100%) concentration of rabbit antimouse interleukin (IL)-1 polyclonal antibody had no effect on the SAP-induced CSF production, indicating that it would be IL-1-independent. SAP-induced CSFs, both in serum and CM, were functionally similar as they supported the formation of granulocyte (G), macrophage (M) and GM colonies in similar proportions. The production of CSFs appeared to be lipopolysaccharide (LPS)-independent as it was not inhibited by polymyxin B sulfate (25.0 microg/ml), and heat-inactivated (80 degrees C, 1 h, pH 7.0) SAP did not induce the production of CSFs. The CSFs were produced de novo because cycloheximide (50.0 microg/ml) completely inhibited their production. These results demonstrate that purified mouse SAP, in a dose-dependent manner, can induce the production of serum CSFs in mice, and can induce LPS-independent de novo production of CSFs by elicited macrophages in vitro.     

Differential role of MyD88 in macrophage-mediated responses to opportunistic fungal pathogens

Toll-like receptors mediate macrophage recognition of microbial ligands, inducing expression of microbicidal molecules and cytokines via the adapter protein MyD88. We investigated the role of MyD88 in regulating murine macrophage responses to a pathogenic yeast (Candida albicans) and mold (Aspergillus fumigatus). Macrophages derived from bone marrow of MyD88-deficient mice (MyD88(-/-)) demonstrated impaired phagocytosis and intracellular killing of C. albicans compared to wild-type (MyD88(+/+)) macrophages. In contrast, ingestion and killing of A. fumigatus conidia was MyD88 independent. Cytokine production by MyD88(-/-) macrophages in response to C. albicans yeasts and hyphae was substantially decreased, but responses to A. fumigatus hyphae were preserved. These results provide evidence that MyD88 signaling is involved in phagocytosis and killing of live C. albicans, but not A. fumigatus. The differential role of MyD88 may represent one mechanism by which macrophages regulate innate responses specific to different pathogenic fungi.     

Phagocytosis by receptors for C3b (CR1), iC3b (CR3), and IgG (Fc) on human peritoneal macrophages

Human peritoneal macrophages (HPM) obtained via laparoscopy were examined for the presence and functional capacity of complement and Fc receptors. Between 5 and 20 ml of peritoneal fluid containing 1-2 X 10(6) macrophages/ml was available for each study. Macrophages made up 80-95% of the cells in the fluid. Fc and C3 receptors on HPM were characterized by rosette formation with, and phagocytosis of, IgG- and C3-coated sheep erythrocytes (E). ElgG were bound by 82% and ingested by 63% of HPM, with 4-15 E ingested/HPM. The HPM formed rosettes with EC3b (56%) and EC3bi (71%) but not EC3d,g or EC3d. Antibodies to complement receptors type 1 (CR1) and type 3 (CR3) inhibited rosette formation with EC3b and EC3bi, respectively, indicating that HPM possessed separate and distinct receptors for the C3b and iC3b ligands. In 60% of the samples studied, HPM demonstrated the ability to ingest both EC3b and EC3bi, as well as ElgG. Because of the heterogeneous nature of the cells obtained in peritoneal fluid, due to their progressive change from monocytelike cells into mature macrophages, HPM were separated by 1 g velocity sedimentation into fractions of increasing maturity. They were then examined for phagocytosis via Fc and complement receptors. Fc receptor mediated phagocytosis occurred throughout the monocyte-to-macrophage maturation sequence, while the ability of HPM to ingest via CR1 and CR3 was maturation dependent, with ingestion via CR3 occurring before CR1, in a manner analogous to in vitro differentiation of monocyte-derived macrophages.