The effects of intracellular signalling pathway inhibitors on phagocytosis by haemocytes of Manduca sexta

The intracellular signalling pathways activated during phagocytosis by larval haemocytes of a lepidopteran, Manduca sexta, were investigated. Using fluorescein-labelled Escherichia coli as bioparticles, a fluorescence-based assay was used to quantify phagocytosis by haemocytes in monolayers in vitro, and the intracellular signalling pathways involved in phagocytosis were examined using inhibitors. Pathways known to be involved in phagocytosis by mammalian cells were selected for the study in haemocytes, and the amino acid sequences of human isoforms of the selected protein targets were used to conduct searches of two completed databases of insect proteins, those of Drosophila melanogaster and Anopheles gambiae and EST databases of moths Bombyx mori and M. sexta. Decreases in phagocytosis produced by pathway inhibitors indicated that tyrosine phosphorlylation phosphatidylinositol 3-kinase (PI3-kinase) and mitogen-activated protein kinase/extracellular-regulated kinase (ERK/MAPK) were required for internalisation of bacteria. Inhibition of Syk, a mammalian kinase, also decreased phagocytosis. JNK/SAPK did not seem to be involved in phagocytosis. The presence of protein phosphatases probably regulates phagocytosis at the intracellular level by dephosphorlyation of serine/threonine residues.     

Lung surfactant protein A (SP-A) enhances serum-independent phagocytosis of bacteria by alveolar macrophages.

Surfactant protein A (SP-A) is the main protein component of lung surfactant. We studied the involvement of SP-A in body defense, i.e., effect of SP-A on the phagocytosis of bacteria by alveolar macrophages. We show here that SP-A enhances the phagocytosis of some non-opsonized bacteria: Escherichia coli growing logarithmically (E. coli/log), Pseudomonas aeruginosa/log as well as from stationary phase (P. aeruginosa/stat) and Staphylococcus aureus/log. Furthermore, not only serum-independent phagocytosis was effected by SP-A but also phagocytosis of serum-opsonized S. aureus/stat. No effect of SP-A on phagocytosis was observed with E. coli/stat neither on serum-independent nor on serum-dependent phagocytosis and on phagocytosis of non-opsonized S. aureus/stat. Thus, effect of SP-A on phagocytosis is dependent on bacterial species and on the growth phase of the microorganisms, and this effect is concentration dependent. We studied two different human recombinant SP-As and SP-A isolated from lung lavage material from proteinosis patients. These SP-A molecules contain different isomeric chains, and they differ in complexity of their structure. Qualitatively, we found the same effect with all three substances. Quantitatively, the proteinosis SP-A that forms the most complex structure was the most effective. Taken together, we demonstrated a stimulating effect of SP-A on serum-independent as well as on serum-dependent phagocytosis of bacteria by alveolar macrophages, both depending on species and growth phase of the bacteria.     

Sec22b Is a Negative Regulator of Phagocytosis in Macrophages

The endoplasmic reticulum (ER) is proposed to be a membrane donor for phagosome formation. In support of this, we have previously shown that the expression level of syntaxin 18, an ER-localized SNARE protein, correlates with phagocytosis activity. To obtain further insights into the involvement of the ER in phagocytosis we focused on Sec22b, another ER-localized SNARE protein that is also found on phagosomal membranes. In marked contrast to the effects of syntaxin 18, we report here that phagocytosis was nearly abolished in J774 macrophages stably expressing mVenus-tagged Sec22b, without affecting the cell surface expression of the Fc receptor or other membrane proteins related to phagocytosis. Conversely, the capacity of the parental J774 cells for phagocytosis was increased when endogenous Sec22b expression was suppressed. Domain analyses of Sec22b revealed that the R-SNARE motif, a selective domain for forming a SNARE complex with syntaxin18 and/or D12, was responsible for the inhibition of phagocytosis. These results strongly support the ER-mediated phagocytosis model and indicate that Sec22b is a negative regulator of phagocytosis in macrophages, most likely by regulating the level of free syntaxin 18 and/or D12 at the site of phagocytosis.     

Undertaker, a Drosophila Junctophilin, links Draper-mediated phagocytosis and calcium homeostasis

Phagocytosis is important during development and in the immune response for the removal of apoptotic cells and pathogens, yet its molecular mechanisms are poorly understood. In Caenorhabditis elegans, the CED2/5/10/12 pathway regulates actin during phagocytosis of apoptotic cells, whereas the role of the CED1/6/7 pathway in phagocytosis is unclear. We report that Undertaker (UTA), a Drosophila Junctophilin protein, is required for Draper (CED-1 homolog)-mediated phagocytosis. Junctophilins couple Ca2+ channels at the plasma membrane to those of the endoplasmic reticulum (ER), the Ryanodine receptors. We place Draper, its adaptor drCed-6, UTA, the Ryanodine receptor Rya-r44F, the ER Ca2+ sensor dSTIM, and the Ca2+-release-activated Ca2+ channel dOrai in the same pathway that promotes calcium homeostasis and phagocytosis. Thus, our results implicate a Junctophilin in phagocytosis and link Draper-mediated phagocytosis to Ca2+ homeostasis, highlighting a previously uncharacterized role for the CED1/6/7 pathway.     

Effects of fluoxetine on the activity of phagocytosis in stressed mice

We studied the effects of 1, 2, 5, 10 and 20 mg/kg of fluoxetine on the activity of phagocytosis in mice subjected to a chronic auditory stressor. Both the in vitro and in vivo activity of phagocytosis, measured using the zymosan-particle uptake method and the carbon clearance test, respectively, were reduced after 2, 4, 8 and 16 days of stress exposure. A partial recovery on the in vivo activity of phagocytosis was found on day 16th. Daily treatment with fluoxetine partially reversed the adverse effects of stress in a dose-dependent manner on both parameters but did not significantly affect the activity of phagocytosis in unstressed mice. Significant differences appeared when fluoxetine was administered at 2 mg/kg. Maximum effect was reached at 5 mg/kg.     

Photo-enhancement of macrophage phagocytic activity via Rac1-mediated signaling pathway: Implications for bacterial infection

Phagocytosis and the subsequent destruction of invading pathogens by macrophages are indispensable steps in host immune responses to microbial infections. Low-power laser irradiation (LPLI) has been found to exert photobiological effects on immune responses, but the signaling mechanisms underlying this photobiomodulation of phagocytosis remains largely unknown. Here, we demonstrated for the first time that LPLI enhanced the phagocytic activity of macrophages by stimulating the activation of Rac1. The overexpression of constitutively activated Rac1 clearly enhanced LPLI-induced phagocytosis, whereas the overexpression of dominant negative Rac1 exerted the opposite effect. The phosphorylation of cofilin was involved in the effects of LPLI on phagocytosis, which was regulated by the membrane translocation and activation of Rac1. Furthermore, the photoactivation of Rac1 was dependent on the Src/PI3K/Vav1 pathway. The inhibition of the Src/PI3K pathway significantly suppressed LPLI-induced actin polymerization and phagocytosis enhancement. Additionally, LPLI-treated mice exhibited increased survival and a decreased organ bacterial load when challenged with Listeria monocytogenes, indicating that LPLI enhanced macrophage phagocytosis in vivo. These findings highlight the important roles of the Src/PI3K/Vav1/Rac1/cofilin pathway in regulating macrophage phagocytosis and provide a potential strategy for treating phagocytic deficiency via LPLI.     

An immobile subset of plasma membrane CD11b/CD18 (Mac-1) is involved in phagocytosis of targets recognized by multiple receptors

CD11b/CD18 is a heterodimeric leukocyte surface receptor which functions in both C3bi-ligand binding and homotypic and heterotypic cell adherence. We have examined the effect of several anti-CD11b/18 mAb on phagocytosis of IgG (EIgG) or complement (EC4b) opsonized erythrocytes by polymorphonuclear leukocytes (PMN) and monocytes. F(ab')2 of two mAb (IB4, an anti-beta-chain mAb and Mo-1 an anti-alpha-chain mAb), inhibited both phagocytosis of EIgG and phorbol ester-stimulated phagocytosis of EC4b by PMN and monocytes. These F(ab')2 inhibited the binding of EIgG to monocytes, but they had no effect on binding of EIgG to PMN, or EC4b to either phagocyte. In addition, IB4 inhibited phorbol-ester stimulated phagocytosis of sheep E opsonized with C component 3bi (EC3bi) without inhibiting rosetting of these same targets. These data separate the anti-phagocytic effect of these mAb from effects on phagocyte-target adherence. When PMN were adherent to an anti-CD11b/CD18 F(ab')2-coated surface, EC3bi binding was abolished, but phagocytosis of EIgG or EC4b was unaffected. Subsequent addition of fluid- phase IB4 or Mo-1 F(ab')2 inhibited phagocytosis of EIgG or EC4b by the adherent cells. This suggested that the CD11b/CD18 involved in C3bi rosetting were mobile in the membrane, whereas those involved in phagocytosis of EIgG or EC4b were not. Cytochalasin treatment of PMN during adherence to F(ab')2-coated plates decreased both apical expression of CD11b/18 and subsequent ingestion of EIgG by 70%, suggesting that microfilaments are important in maintaining immobile CD11b/18 on the apical PMN surface. We conclude that there are functionally distinct populations of CD11b/CD18 on monocytes and PMN: one involved in C3bi rosetting and another involved in the process of phagocytosis mediated via several different receptors. CD11b/18 is not required for optimal target binding in all cases, but is always required for ingestion. As with several other integrins, the CD11b/18 molecules involved in phagocytosis have a functional association with the cell cytoskeleton.     

High prevalence of phagocytic-resistant capsular serotypes of Klebsiella pneumoniae in liver abscess

To better understand the role of capsular polysaccharide (CPS) K1 or K2 in Klebsiella pneumoniae liver abscess as well as the development of metastasis to eye, neutrophil phagocytosis of 70 CPS isolates including K1 (n = 23)/K2 (n = 10), non-K1/K2 (n = 37) was evaluated by flow cytometry, fluorescence imaging, and electron microscopy. K1/K2 isolates were significantly more resistant to phagocytosis (P < 0.0001) than non-K1/K2 isolates and displayed increased resistance to intracellular killing. Although mucoid phenotype (M-type) K1/K2 isolates were significantly more resistant to phagocytosis (P = 0.0029) than M-type non-K1/K2, no significant difference in the phagocytosis rate was observed between K1/K2 isolates with M-type and non-M-type (P = 0.0924). Mucoidy is an associated factor that was predominant in K1/K2 isolates, but which itself is not an independent influence on phagocytic resistance. The K1/K2 CPS proved significantly more resistant to phagocytosis than non-K1/K2 CPS in liver abscess isolates (P < 0.0001) and non-abscess isolates (P = 0.0001), suggesting that K1/K2 isolates were generally more virulent in both liver abscess and in non-liver abscess conditions. These findings indicate that resistance of CPS K1 or K2 K. pneumoniae to phagocytosis and intracellular killing presumably contributes to their high prevalence in liver abscess and uniquely in endophthalmitis.     

Inulin stimulates phagocytosis of PMA-treated THP-1 macrophages by involvement of PI3-kinases and MAP kinases

Inulin is a polysaccharide that enhances various immune responses, mainly to T and B cells, natural killer cells, and macrophages in vivo and in vitro. Previous reports describe that inulin activates macrophages indirectly by affecting the alternative complement pathway. In this study, we examined the direct effect of inulin on PMA-treated THP-1 macrophages. Inulin treatment did not stimulate the proliferation of THP-1 macrophages at all. However, inulin treatment significantly increased phagocytosis of the polystyrene beads without the influence of serum. Doses of around 1 mg/mL had the maximal effect, and significant progression of phagocytosis occurred at times treated over 6 h. Inulin augmented phagocytosis not only with polystyrene beads but also with apoptotic cancer cells. The inulin-induced phagocytosis uptake was suppressed in Toll-like receptor (TLR) 4 mutated C3H/HeJ mice peritoneal macrophages. Moreover, inulin-induced THP-1 macrophage TNF-α secretion was inhibited using a blocking antibody specific to TLR4, suggesting that TLR4 is involved in the binding of inulin to macrophages. Furthermore, we used specific kinase inhibitors to assess the involvement of inulin-induced phagocytosis and revealed that phosphoinositide 3-kinase and mitogen-activated protein kinase, especially p38, participated in phagocytosis. These results suggest that inulin affects macrophages directly by involving the TLR4 signaling pathway and stimulating phagocytosis for enhancing immunomodulation.     

Augmentation of fatality of influenza in mice by inhibition of phagocytosis

Influenza virus-infected cells undergo apoptosis and become susceptible to phagocytosis by macrophages, and this leads to the inhibition of virus propagation in vitro. To assess if this were also true in vivo, mice infected with influenza A/WSN (H1N1) virus were administered with phagocytosis inhibitors and examined for the progress of influenza. Administration of the inhibitors caused a decrease in the level of phagocytosis observed with bronchoalveolar lavage cells. We found that both the lethality in mice and the extent of inflammation in the lung were augmented in those mice. These results suggest that phagocytosis of virus-infected cells helps suppress the progress of influenza in mice.     

Endogenous Gas6 and Ca2+ -channel activation modulate phagocytosis by retinal pigment epithelium

Mutation or loss of MerTK as well as deficiency of vβ5-integrins, gives rise to retinal-degeneration due to inefficient phagocytosis of photoreceptor outer-segment fragments by the retinal pigment epithelium (RPE). This study shows that Gas6 expressed endogenously by human RPE promotes phagocytosis. The RPE expresses Gas6 more highly in vivo and in serum-reduced conditions in vitro than in high-serum conditions, suggesting a negative-feedback control. An antibody-blockage approach revealed that Gas6-expressing RPE phagocytizes photoreceptor outer-segment fragments due to stimulation of MerTK by endogenous Gas6 in vitro. MerTK- and Gas6-antibodies reduced phagocytosis. Blocking L-type Ca²⁺-channels with nifedipine inhibited MerTK dependent phagocytosis in vitro. Application of integrin inhibitory, soluble, RGD-containing peptides or soluble vitronectin reduced L-type Ca²⁺-channel currents in RPE. Herbimycin A, which reduces phosphorylation of integrin receptor-associated proteins and decreases L-type Ca²⁺-channel currents in RPE, eliminates the inhibiting vitronectin effect and abolishes phagocytosis. Thus, Gas6-promoted phagocytosis was inhibited by L-type Ca²⁺-channel blockage, which in turn may be activated by integrin receptor stimulation. These results suggest that L-type Ca²⁺-channels could be regulated downstream of both MerTK and vβ5-integrin, indicating that the binding and uptake mechanisms of phagocytosis are part of a converging pathway.     

Uptake of LPS/E. coli/latex beads via distinct signalling pathways in medfly hemocytes: the role of MAP kinases activation and protein secretion

In response to LPS/E. coli treatment, extracellular signal-regulated kinase (ERK) is activated in medfly hemocytes. To explore the molecular mechanisms underlying LPS/E. coli/latex beads endo- and phagocytosis, we studied the signalling pathways leading to p38 and c-jun N-terminal kinase (JNK) activation. JNK and p38-like proteins were initially identified within medfly hemocytes. Flow cytometry analysis revealed that mitogen-activated protein kinases (MAPK) are required for phagocytosis. Inhibition of specific MAPK signalling pathways, with manumycin A, toxin A, cytochalasin D and latrunculin A, revealed activation of p38 via Ras/Rho/actin remodelling pathway and activation of JNK that was independent of actin cytoskeleton reorganization. ERK and p38 pathways, but not JNK, appeared to be involved in LPS-dependent hemocyte secretion, whereas all MAPK subfamilies seemed to participate in E. coli-dependent secretion. In addition, flow cytometry experiments in hemocytes showed that the LPS/E. coli-induced release was a prerequisite for LPS/E. coli uptake, whereas latex bead phagocytosis did not depend on hemocyte secretion. This is a novel aspect, as in mammalian monocytes/macrophages LPS/E. coli-triggered release has not been yet correlated with phagocytosis. It is of interest that these data suggest distinct mechanisms for the phagocytosis of E. coli and latex beads in medfly hemocytes.     

Yeast mannans inhibit binding and phagocytosis of zymosan by mouse peritoneal macrophages

We have examined the effects of various mannans, glycoproteins, oligosaccharides, monosaccharides, and sugar phosphates on the binding and phagocytosis of yeast cell walls (zymosan) by mouse peritoneal macrophages. A phosphonomannan (PO(4):mannose ratio = 1:8:6) from kloeckera brevis was the most potent inhibitor tested; it inhibited binding and phagocytosis by 50 percent at concentrations of approximately 3-5 μg/ml and 10 μg/ml, respectively. Removal of the phosphate from this mannan by mild acid and alkaline phosphatase treatment did not appreciably reduce its capacity to inhibit zymosan phagocytosis. The mannan from saccharomyces cerevisiae mutant LB301 inhibits phagocytosis by 50 percent at 0.3 mg/ml, and a neutral exocellular glucomannan from pichia pinus inhibited phagocytosis by 50 percent at 1 mg/ml. Cell wall mannans from wild type S. cervisiae X2180, its mnn2 mutant which contains mannan with predominantly 1(arrow)6- linked mannose residues, yeast exocellular mannans and O-phosphonomannans were less efficient inhibitors requiring concentrations of 1-5 mg/ml to achieve 50 percent reduction in phagocytosis. Horseradish peroxidase, which contains high-mannose type oligosaccharides, was also inhibitory. Mannan is a specific inhibitor of zymosan binding and phagocytosis. The binding and ingestion of zymosan but not of IgG- or complement-coated erythrocytes can be obliterated by plating macrophages on substrates coated with poly-L-lysin (PLL)-mannan. Zymosan uptake was completely abolished by trypsin treatment of the macrophages and reduced by 50-60 percent in the presence of 10 mM EGTA. Pretreatment of the macrophages with chloroquine inhibited zymosan binding and ingestion. These results support the proposal that the macrophage mannose/N-acetylglucosamine receptor (P. Stahl, J.S. Rodman, M.J. Miller, and P.H. Schlesinger, 1978, Proc. Natl. Acad. Sci. U.S.A. 75:1399-1403, mediates the phagocytosis of zymosan particles.     

Efficient Measurement of Opsonising Antibodies to Plasmodium falciparum Merozoites

Background

Antibodies targeting merozoites are important in protection from malaria. Therefore, merozoite surface proteins are attractive vaccine candidates. There is a need for robust functional assays to investigate mechanisms of acquired immunity and vaccine efficacy. To date, the study of merozoite phagocytosis has been confounded by the complexity and variability of in vitro assays.

Methodology/Principal findings

We have developed a new flow cytometry-based merozoite phagocytosis assay. An optimized merozoite preparation technique produced high yields of merozoites separated from haemozoin. Phagocytosis by the undifferentiated THP-1 monocytic cell line was mediated only by Fc Receptors, and was therefore ideal for studying opsonising antibody responses. The assay showed robust phagocytosis with highly diluted immune sera and strong inter-assay correlation. The assay effectively measured differences in opsonisation-dependent phagocytosis among individuals.

Conclusions/Significance

This highly reproducible assay has potential applications in assessing the role of opsonic phagocytosis in naturally acquired immunity and vaccine trials.     

RUBCN/rubicon and EGFR regulate lysosomal degradative processes in the retinal pigment epithelium (RPE) of the eye

Macroautophagy/autophagy is an intracellular stress survival and recycling system whereas phagocytosis internalizes material from the extracellular milieu; yet, both pathways utilize lysosomes for cargo degradation. Whereas autophagy occurs in all cells, phagocytosis is performed by cell types such as macrophages and the retinal pigment epithelial (RPE) cells of the eye where it is supported by the noncanonical autophagy process termed LC3-associated phagocytosis (LAP). Autophagy and LAP are distinct pathways that use many of the same mediators and must compete for cellular resources, suggesting that cells may regulate both processes under homeostatic and stress conditions. Our data reveal that RPE cells promote LAP through the expression of RUBCN/Rubicon (RUN domain and cysteine-rich domain containing Beclin 1-interacting protein) and suppress autophagy through the activation of EGFR (epidermal growth factor receptor). In the morning when photoreceptor outer segments (POS) phagocytosis and LAP are highest, RUBCN expression is increased. At the same time, outer segment phagocytosis activates the EGFR resulting in MTOR (mechanistic target of rapamycin [serine/threonine kinase]) stimulation, the accumulation of SQSTM1/p62, and the phosphorylation of BECN1 (Beclin 1, autophagy related) on an inhibitory residue thereby suppressing autophagy. Silencing Rubcn, preventing EGFR activity or directly inducing autophagy in RPE cells by starvation inhibits phagocytic degradation of POS. Thus, RPE cells regulate lysosomal pathways during the critical period of POS phagocytosis to support retinal homeostasis.     

Involvement of FAK/Src complex in the processes of Escherichia coli phagocytosis by insect hemocytes

Recently we demonstrated that lipopolysaccharide promotes activation of the Ras/mitogen-activated protein cascade in hemocytes and that phagocytosis of Escherichia coli by insect hemocytes is mediated by an integrin-dependent process [Foukas et al. (1998) J. Biol. Chem. 273, 14813--14818]. Here we report data concerning the focal adhesion kinase (FAK) tyrosine phosphorylation status in hemocytes in response to E. coli. We demonstrate that E. coli-triggering stimulates a significant increase in tyrosine phosphorylation of FAK in hemocytes. Furthermore, immunoblotting analysis using anti-Y397 demonstrated intense FAK activity at the Y397/SH2-binding site in hemocytes treated with E. coli. In addition, antibody-mediated inhibition of FAK and Src-kinase has been shown to abolish FAK phosphorylation and E. coli phagocytosis, indicating a specific role for the FAK/Src complex in the processes of promoting cell phagocytosis. These findings expand the known signaling functions of FAK and provide insight into signal transduction events associated with hemocyte phagocytosis in response to E. coli.     

The role of beta2 integrins and lipopolysaccharide-binding protein in the phagocytosis of dead Neisseria meningitidis

Phagocytosis of microbial pathogens is essential for the host immune response to infection. Our previous work has shown that lipooligosaccharide (LOS) expression on the surface of Neisseria meningitidis (Nm) is essential for phagocytosis, but the receptor involved remained unclear. In this study, we show that human CR3 (CD11b/CD18) and CR4 (CD11c/CD18) are phagocytic receptors for Nm as illustrated by the capacity of CR3- and CR4-transfected Chinese hamster ovary (CHO) cells to facilitate Nm uptake. A CR3-signalling mutant failed to internalize Nm, showing that the ability of CR3 to signal is essential for phagocytosis. Internalization of Nm by CR3-transfected CHO cells could be inhibited by the presence of CR3-specific antibodies. Furthermore, dendritic cells from leukocyte adhesion deficiency-1 patients, who have diminished expression of β2 integrins, showed markedly reduced phagocytosis of Nm. The CR3-mediated phagocytosis required the presence of lipopolysaccharide-binding protein (LBP). Furthermore, the expression of LOS by Nm was essential for LBP binding and phagocytosis via CR3. These results reveal a critical role of CR3 and LBP in the phagocytosis of Nm and provide important insights into the initial interaction meningococci have with the immune system.     

A chimerical phagocytosis model reveals the recruitment by Sertoli cells of autophagy for the degradation of ingested illegitimate substrates

Phagocytosis and autophagy are typically dedicated to degradation of substrates of extrinsic and intrinsic origins respectively. Although overlaps between phagocytosis and autophagy were reported, the use of autophagy for ingested substrate degradation by nonprofessional phagocytes has not been described. Blood-separated tissues use their tissue-specific nonprofessional phagocytes for homeostatic phagocytosis. In the testis, Sertoli cells phagocytose spermatid residual bodies produced during germ cell differentiation. In the retina, pigmented epithelium phagocytoses shed photoreceptor tips produced during photoreceptor renewal. Spermatid residual bodies and shed photoreceptor tips are phosphatidylserine-exposing substrates. Activation of the tyrosine kinase receptor MERTK, which is implicated in phagocytosis of phosphatidylserine-exposing substrates, is a common feature of Sertoli and retinal pigmented epithelial cell phagocytosis. The major aim of our study was to investigate to what extent phagocytosis by Sertoli cells may be tissue specific. We analyzed in Sertoli cell cultures that were exposed to either spermatid residual bodies (legitimate substrates) or retina photoreceptor outer segments (illegitimate substrates) the course of the main phagocytosis stages. We show that whereas substrate binding and ingestion stages occur similarly for legitimate or illegitimate substrates, the degradation of illegitimate but not of legitimate substrates triggers autophagy as evidenced by the formation of double-membrane wrapping, MAP1LC3A-II/LC3-II clustering, SQSTM1/p62 degradation, and by marked changes in ATG5, ATG9 and BECN1/Beclin 1 protein expression profiles. The recruitment by nonprofessional phagocytes of autophagy for the degradation of ingested cell-derived substrates is a novel feature that may be of major importance for fundamentals of both apoptotic substrate clearance and tissue homeostasis.     

Effector function of leucocytes from susceptible and resistant mice against distinct isolates of Candida albicans

Neutrophils and macrophages were generated in vitro from mice that display either high or low tissue susceptibilities to Candida albicans infection and their ability to phagocytose and kill three isolates of the yeast with different virulence characteristics was evaluated. In the absence of opsonization, phagocytosis by BALB/c and CBA/CaH neutrophils was comparable, but the killing was very poor. Opsonization with normal serum slightly decreased phagocytosis, but it had markedly different effects on killing, either enhancing or inhibiting candidacidal activity, depending on the combination of yeast isolate and mouse strain. In contrast, BALB/c macrophages showed high levels of phagocytosis and killing of both unopsonized yeasts and opsonized yeasts; whereas killing of unopsonized yeasts by CBA/CaH macrophages was poor, it was markedly enhanced by opsonization.     

Characteristics of the beta-glucan receptor of murine macrophages

Phagocytosis of heat-killed yeast (HK-yeast), zymosan, and glucan particles by thioglycollate-elicited mouse peritoneal macrophages (Tg-macrophages) was inhibited by soluble glucan polymers/oligomers. The inhibitory capacity of soluble glucans decreased steeply with the decrease in the degree of polymerization (DPn); i.e., the concentration at which 50% inhibition of phagocytosis was attained was 0.23 microgram/ml for glucan 1 (DPn 24.8), 0.8 microgram/ml for glucan 2 (DPn 21.9), and greater than 40 micrograms/ml for glucan 3 (DPn 13.8). The glucan polymers were obtained by partial hydrolysis of glucan particles with formic acid (90%, 95 degrees C, 20 min) and fractionation according to solubility in ethanol water mixtures. A short preincubation (5 min, 4 or 37 degrees C) of Tg-macrophages with glucan 1 led to a subsequent inhibition of HK-yeast phagocytosis. Recovery of the phagocytic function was slow (27% in 3 h; 68% in 5 h) and required protein synthesis. beta-Glucan receptor expression was also suppressed by dexamethasone treatment. Mannan exerted at high concentrations (5 mg/ml) a partial inhibitory activity which was totally abrogated by beta-glucanase treatment. Treatment of macrophages with glucan together with mannan did not enhance the inhibitory capacity of glucan beyond the component abrogated by enzyme treatment. Contribution of local opsonization of HK-yeast to the phagocytic response (involvement of complement receptors) was indirectly negated; (a) glucan 1 which inhibits HK-yeast phagocytosis by up to 95% is not an activator of complement and therefore could not compete for the opsonizing proteins; (b) cycloheximide treatment in itself inhibited only partially HK-yeast phagocytosis whereas it inhibited the reexpression of the glucan receptors; (c) glucan 1 did not affect the phagocytosis of serum opsonized HK-yeast. Thus under the experimental conditions described, phagocytosis of HK-yeast by murine macrophages is mediated by and large by the beta-glucan receptors, while the mannose receptors and complement receptors do not contribute to the process.