Chromosome-mediated resistance of Yersinia enterocolitica serotype O9 to intracellular killing by mouse peritoneal macrophages

Abstract The survival of Yersinia enterocolitica serotype O9 within mouse peritoneal macrophages was investigated. To evaluate the role of the virulence plasmid in the resistance to intracellular killing, an isogenic pair of virulent (plasmid-bearing) and avirulent (plasmid-less) O9 strains was used. The virulent strain was able to express plasmid-encoded outer membrane proteins and to colonize the Peyer's patches of orally infected mice. When mice were infected intraperitoneally, both strains were recovered at similar rates and over the same time from the peritoneal cavity. When in vitro assays were performed, both strains showed similar resistance to intracellular killing by monolayers of resident and inflammatory peritoneal macrophages. Previous opsonization of bacteria did not modify their survival within macrophage monolayers. We concluded that serotype O9 strains display a chromosome-mediated resistance to intracellular killing by mouse peritoneal macrophages. Moreover, macrophage resistance does not seem to be of importance for virulence of serotype O9 strains in mice.     

Phagocytosis is inhibited by autophagic induction in murine macrophages

Recent studies have demonstrated that communication takes place between the autophagic and phagocytic pathways, indicating that the convergence of these two pathways plays an important role in the innate immune response against intracellular microbes. The present study investigated the effect of autophagic induction on the phagocytic capacity of murine macrophages. Autophagy induced by physiological and pharmacological means was shown to reduce the phagocytic capacity of murine macrophages, regardless of cell origin or the nature of the phagocytosed particles themselves. This autophagic inhibitory effect on phagocytosis was shown to be an early and reversible event that results in no loss of cell viability. Furthermore, the data presented herein demonstrate that the induction of autophagy does not affect a macrophage’s capacity to recognize and bind to particles, indicating that autophagy does not inhibit the particle recognition process, even though particle internalization is suppressed. The findings herein support the notion that phagocytosis and autophagy may be interdependent and complementary processes.     

THIS ARTICLE HAS BEEN RETRACTED Antifungal antibiotic hamycin increases susceptibility of Candida albicans to phagocytosis by murine macrophages

Hamycin is an antifungal antibiotic produced by Streptomyces pimprina Thirum. In the present study, the effect of hamycin on (a) the phagocytosis of Candida albicans by murine peritoneal macrophages and (b) the cell surface hydrophobicity (CSH) of C. albicans was investigated. Addition of hamycin to the culture of macrophages and Candida cells increased the susceptibility of Candida cells to the phagocytosis by macrophages. Pretreatment of Candida cells with hamycin increased their vulnerability to killing by macrophages. Examination of physico-chemical properties of Candida cell surface showed a significant decrease in the CSH. These findings suggest that the binding of hamycin to Candida cells induces biochemical/physico-chemical alterations of the surface, so that it becomes more susceptible to phagocytosis by murine macrophages.     

Antibody-independent phagocytosis of tumor cells by human monocyte-derived macrophages cultured in recombinant macrophage colony-stimulating factor

Human monocytes exposed in vitro to recombinant macrophage-colony-stimulating factor (rhMCSF) differentiate into monocyte-derived macrophages (MDM), which mediate efficient antibodydependent cytotoxicity (ADCC) against tumor cells. We and others have shown that this form of ADCC is unusual in that phagocytosis, rather than extracellular lysis, appears to play the major role in target cell killing. In this study, we asked whether the phagocytic form of cytotoxicity seen with ADCC could occur in the absence of an opsonizing antibody. We now report that, whereas cell lines derived from solid tumors are often resistant to antibody-independent cytotoxicity, malignant cells of lymphoid origin appear particularly susceptible to such antibody-independent killing. We found that all of nine lymphocytic leukemia and lymphoma cell lines tested in a total of 35 experiments, plus all four samples of fresh leukemic blasts, were consistently susceptible to antibody-independent MDM cytotoxicity. Antibody-independent cytotoxicity against these cells was efficient (40%–63% killing) at effector: target (E:T) ratios as low as 2:1. Like ADCC, antibody-independent cytotoxicity involved phagocytosis of target cells, as demonstrated by ingestion of fluorescently labeled targets and analysis by flow cytometry. At the time of phagocytosis, the majority of target cells retained membrane integrity, as indicated by the direct transfer of intracellular [⁵¹Cr]chromate from radiolabeled targets to phagocytosing MDM, without release of the label into the medium. However, in contrast to ADCC, we found that the degree of antibody-independent cytotoxicity was not a function of the E:T ratio. Instead, a constant proportion of the available target cells were killed regardless of the E:T ratio, suggesting that target cell recognition, rather than effector cell potency, might be the limiting factor in determining cytotoxicity. In additional experiments, we have also identified a second tumor cell type, nueroblastoma, as being susceptible to antibody-independent phagocytosis (all of five cell lines tested, cytotoxicity 40%–93%, E:T=3:1). Our data thus indicate that the cytotoxicity induced by rhMCSF is not confined to antibody-mediated killing, and that phagocytosis can play a significant role in target cell destruction even in the absence of opsonizing antibody.Supported in part by grants CA-33049 and CA-53624 from the National Institutes of Health, grant IRG-174b from the American Cancer Society, the Friends of Children Toys-R-Us Foundation. Inc., and the Robert Steel Foundation     

Spore diffusate isolated from some strains of Aspergillus fumigatus inhibits phagocytosis by murine alveolar macrophages

Aspergillus fumigatus is a ubiquitous fungus that grows in decaying organic matter. It can cause disease in both immunodeficient and immunocompetent patients by using virulence factors to escape the host defenses. Some of these factors, such as a diffusate, released from the spores of A. fumigatus, have previously been described. This diffusate was demonstrated to inhibit oxidative burst and phagocytosis of coated red blood cells. The present study has shown that this substance can inhibit the phagocytosis of A. fumigatus spores by murine alveolar macrophages (MALU) and evaluated the action of this substance. We quantified phagocytosis by MALU cells with and without diffusate and evaluated the inhibition of phagocytosis by testing diffusates from different strains. We conclude that the spore diffusate of some strains of A. fumigatus can reversibly decrease the ability of alveolar macrophages to ingest A. fumigatus spores.     

Phagocytosis is coupled to the formation of phagosome-associated podosomes and a transient disruption of podosomes in human macrophages

Phagocytosis consists in ingestion and digestion of large particles, a process strictly dependent on actin re-organization. Using synchronized phagocytosis of IgG-coated latex beads (IgG-LB), zymosan or serum opsonized-zymosan, we report the formation of actin structures on both phagocytic cups and closed phagosomes in human macrophages. Their lifespan, size, protein composition and organization are similar to podosomes. Thus, we called these actin structures phagosome-associated podosomes (PAPs). Concomitantly to the formation of PAPs, a transient disruption of podosomes occurred at the ventral face of macrophages. Similarly to podosomes, which are targeted by vesicles containing proteases, the presence of PAPs correlated with the maturation of phagosomes into phagolysosomes. The ingestion of LB without IgG did not trigger PAPs formation, did not lead to podosome disruption and maturation to phagolysosomes, suggesting that these events are linked together. Although similar to podosomes, we found that PAPs differed by being resistant to the Arp2/3 inhibitor CK666. Thus, we describe a podosome subtype which forms on phagosomes where it probably serves several tasks of this multifunctional structure.     

Antibody-mediated platelet phagocytosis by human macrophages is inhibited by siRNA specific for sequences in the SH2 tyrosine kinase, Syk

Immune thrombocytopenia depends upon Fc receptor-mediated phagocytosis that involves signaling through the SH2 tyrosine kinase, Syk. We designed small interfering (siRNA) sequences complementary to Syk coding regions to decrease the expression of Syk in the human macrophage cell line, THP-1. To evaluate the functional effect of siRNA on phagocytosis, we developed a new in vitro assay for antibody-mediated platelet ingestion by THP-1 cells. Incubation of THP-1 cells at 37 °C with fluorescence-labeled platelets and anti-platelet antibody promoted ingestion of platelets that could be quantitated by flow cytometry. Transfection of THP-1 cells with Syk-specific siRNA resulted in a reduction in the amount of FcγRII-associated Syk protein. Coincident with decreased Syk expression, we observed inhibition of antibody-mediated platelet ingestion. These results confirm a key role for Syk in antibody-mediated phagocytosis and suggest Syk-specific siRNA as a possible therapeutic candidate for immune thrombocytopenia.     

Definition of fibronectin-mediated uptake of gelatinized latex by liver slices and macrophages

These studies show that both liver slices and macrophages carried out fibronectin concentration-dependent uptake of ¹²⁵I-labeled gelatin-coated latex (test latex). Lack of phagocytosis of test latex by liver slices was shown directly by electron microscopy and indirectly by trypsin treatment, which caused the release of all test latex taken up in response to fibronectin. Inhibitors of phagocytosis did not alter this uptake. On the other hand, trypsin released only a portion of test latex from macrophages. Inhibitors of phagocytosis did not effect the released radioactive particles from macrophages but greatly reduced the trypsin-resistant radioactivity, taken as representing phagocytized particles. Opsonization of test latex with fibronectin did not require heparin but its association with liver slices occurred only in the presence of heparin. Macrophages, however, readily bound and internalized the opsonized test latex and heparin only potentiated these reactions. Gelatin competed with test latex for fibronectin for opsonization, but did not inhibit binding and phagocytosis of fibronectin-test latex complexes. Finally, soluble fibronectin-gelatin complexes did not compete for binding and phagocytosis of fibronectin-test latex complexes. Thus, fibronectin concentrated on the surface of latex is preferred for interaction with the fibronection receptor of macrophages. Gelatin, however, was not essential for this reaction, because fibronectin directly coupled to latex was also readily taken up.     

Elasticity and adhesion of resting and lipopolysaccharide-stimulated macrophages

Colloidal Force Microscopy was employed to study the viscoelastic and adhesive properties of macrophages upon stimulation with lipopolysaccharide (LPS). Force vs. distance measurements were performed. The adhesion of LPS-stimulated cells (separation force=37+/-3 nN) was almost twice as high as that of resting macrophages (16+/-1 nN). Upon retraction pulling of membrane tethers was observed. Tether lengths and forces at which rupture take place did not depend on stimulation. The reduced Young's modulus K, a measure of cytoskeleton elasticity, was three times lower than that of the control. The data show that LPS has profound effects on cytomechanical and adhesion properties of macrophages.     

Survival of intracellular pathogens within macrophages

Summary The threat caused by intracellular pathogens increases as conventional drag treatments are less and less effective against a wide range of microorganisms. Understanding the molecular mechanisms used by intracellular pathogens to avoid killing and degradation in their host cells is likely to point at new ways to threat infectious diseases. We discuss some of the strategies used by various microorganisms to avoid killing and degradation in phagolysosomes. Interestingly, it appears that microbes have a lot to teach us about the cell biology and molecular mechanisms of organelle sorting in macrophages.     

Specific inhibition of phagosome-lysosome fusion in murine macrophages mediated by Salmonella typhimurium infection

Abstract Phagosome-lysosome fusion in murine macrophages infected with S. typhimurium LT2 or S. typhi 1079 was investigated. Fusion of phagosome containing S. typhimurium LT2 with lysosome was markedly impaired, whereas S. typhi 1079 did not inhibit phagosome-lysosome fusion in murine macrophages. A similar inhibition of fusion was observed with LPS-deficient mutants of S. typhimurium LT2, suggesting that O-antigens do not contribute to the inhibition of fusion. Phagosome-lysosome fusion in macrophages after ingestion of UV-killed S. typhimurium LT2 was much greater than that of live bacteria. Furthermore, treatment of S. typhimurium LT2 with streptomycin, an inhibitor of bacterial protein synthesis, caused an increase in the extent of phagosome-lysosome fusion. Therefore protein synthesis in live bacteria is probably required for the inhibition of phagosome-lysosome fusion. These results suggest that phagosome-lysosome fusion in murine macrophages is impaired by some product(s) of viable S. typhimurium LT2.     

Role of protein kinase C alpha for uptake of unopsonized prey and phagosomal maturation in macrophages

Protein kinase C alpha (PKC alpha) participates in F-actin remodeling during phagocytosis and phagosomal maturation in macrophages. Leishmania donovani promastigotes, which inhibit phagosomal maturation, cause accumulation of periphagosomal F-actin instead of the disassembly observed around other prey [Cell. Microbiol. 7 (2001) 439]. This accumulation is induced by promastigote lipophosphoglycan (LPG), which has several effects on macrophages including inhibition of PKC alpha. To investigate a possible connection between PKC alpha and LPG's effects on actin dynamics, we utilized RAW264.7 macrophages overexpressing dominant-negative PCK alpha (DN PKC alpha). We found increased cortical F-actin and decreased phagocytic capacity, as well as defective periphagosomal F-actin breakdown and inhibited phagosomal maturation in the DN PKC alpha-overexpressing cells, effects similar to those seen in controls subjected to LPG-coated prey. The results indicate that PKC alpha is involved in F-actin turnover in macrophages and that PKC alpha-dependent breakdown of periphagosomal F-actin is required for phagosomal maturation, and endorse the hypothesis that intracellular survival of L. donovani involves inhibition of PKC alpha by LPG.     

Effects of cholesterol in chylomicron remnant models of lipid emulsions on apoE-mediated uptake and cytotoxicity of macrophages

Chylomicron remnants have been suggested to be involved in the development of atherosclerosis. To investigate the mechanisms of chylomicron remnant-induced atherosclerosis, we prepared cholesterol (Chol)-containing emulsion particles as models for chylomicron remnants. Chol markedly increased the apolipoprotein E (apoE) binding maximum of emulsions without changing the binding affinity and thereby promoted emulsion uptake by J774 macrophages. Fluorescence measurements showed that Chol increased acyl chain order and head group hydration of the surface phospholipid (PL) layer of emulsions. The binding maximum of apoE was closely correlated with the hydration and the increase in the PL head group separation at the emulsion surface. From experiments using inhibitors for lipoprotein receptors, heparan sulfate proteoglycans and low density lipoprotein receptor-related protein were found to be the major contributors to the uptake of Chol-containing emulsions. Trypan blue dye exclusion revealed that the uptake of Chol-containing emulsions induced cytotoxicity to J774 macrophages. This study proposes a mechanism of atherosclerosis induced by chylomicron remnants.     

Sphingolipid-mediated inhibition of apoptotic cell clearance by alveolar macrophages

A decreased clearance of apoptotic cells (efferocytosis) by alveolar macrophages (AM) may contribute to inflammation in emphysema. The up-regulation of ceramides in response to cigarette smoking (CS) has been linked to AM accumulation and increased detection of apoptotic alveolar epithelial and endothelial cells in lung parenchyma. We hypothesized that ceramides inhibit the AM phagocytosis of apoptotic cells. Release of endogenous ceramides via sphingomyelinase or exogenous ceramide treatments dose-dependently impaired apoptotic Jurkat cell phagocytosis by primary rat or human AM, irrespective of the molecular species of ceramide. Similarly, in vivo augmentation of lung ceramides via intratracheal instillation in rats significantly decreased the engulfment of instilled target apoptotic thymocytes by resident AM. The mechanism of ceramide-induced efferocytosis impairment was dependent on generation of sphingosine via ceramidase. Sphingosine treatment recapitulated the effects of ceramide, dose-dependently inhibiting apoptotic cell clearance. The effect of ceramide on efferocytosis was associated with decreased membrane ruffle formation and attenuated Rac1 plasma membrane recruitment. Constitutively active Rac1 overexpression rescued AM efferocytosis against the effects of ceramide. CS exposure significantly increased AM ceramides and recapitulated the effect of ceramides on Rac1 membrane recruitment in a sphingosine-dependent manner. Importantly, CS profoundly inhibited AM efferocytosis via ceramide-dependent sphingosine production. These results suggest that excessive lung ceramides may amplify lung injury in emphysema by causing both apoptosis of structural cells and inhibition of their clearance by AM.     

Enhanced phagocytosis of CD47-deficient red blood cells by splenic macrophages requires SHPS-1

The interaction of CD47 on red blood cells (RBCs) with SHPS-1 on macrophages is implicated to prevent the phagocytosis of the former cells by the latter cells. Indeed, the rate of clearance of transfused CD47-deficient (CD47(-/-)) RBCs from the bloodstream of wild-type mice was markedly increased compared with wild-type RBCs. Conversely, the rate of clearance of transfused wild-type RBCs was markedly increased in mice that expressed a mutant form of SHPS-1 lacking most of the cytoplasmic region of the protein. However, we here found that the clearance of CD47(-/-) RBCs in SHPS-1 mutant mice was minimal. In addition, the phagocytosis of CD47(-/-) RBCs by splenic macrophages from SHPS-1 mutant mice was markedly reduced compared with wild-type macrophages. These results thus suggest an additional role for CD47 on RBCs in the negative regulation of phagocytosis by macrophages and in determination of the life span of circulating RBCs.     

Trypanosoma brucei: recognition in vitro of two developmental forms by murine macrophages

In vitro, murine macrophages attach to the midgut form of Trypanosoma brucei in the absence of exogenous proteins. Recognition appears to be specific and saturable. Attachment is mediated by a non-Fc, non-C3 receptor on the macrophage plasma membrane. The attachment mechanism is protease sensitive, temperature sensitive, requires the presence of divalent cations, and is functional in fetal calf serum-free medium. The midgut form is also lysed in normal rabbit serum by activating the alternative complement pathway. The form isolated from the blood is neither lysed in normal serum nor is it spontaneously recognized by the macrophage, in vitro. Partial trypsinization of the bloodstream form, however, results in both the triggering of alternative complement pathway lysis and spontaneous uptake into macrophages. Murine macrophages attach to the midgut form of T. brucei by a receptor on the macrophage plasma membrane which is capable of recognizing particulate activators of the alternative complement pathway.     

思连康对小鼠腹腔巨噬细胞吞噬率和吞噬指数的影响

目的 研究双歧杆菌四联活菌片（商品名：思连康）对小鼠腹腔巨噬细胞吞噬鸡红细胞吞噬率及吞噬指数的影响。方法 将SPF小鼠30只随机分成三组,每组10只,Ⅰ组灌胃生理盐水,Ⅱ组灌胃婴儿双歧杆菌菌悬液,Ⅲ组灌胃双歧杆菌四联活菌片菌悬液,每天给药0.5 mL,菌液浓度为1.0×108 CFU/mL,连续给药10 d后小鼠腹腔注入2%鸡红细胞悬液1 mL（红细胞数量为2×108个/mL）,30 min后处死,取小鼠腹腔洗液,观察并记录吞噬鸡红细胞的巨噬细胞数及被吞噬的鸡红细胞数,计算吞噬率及吞噬指数。结果 与Ⅰ组相比,Ⅱ组和Ⅲ组小鼠腹腔巨噬细胞吞噬鸡红细胞的吞噬率和吞噬指数均显著升高（Ps＜0.05）,其中Ⅲ组高于Ⅱ组（P＜0.05）。结论 双歧杆菌四联活菌片及其婴儿双歧杆菌通过提高小鼠腹腔巨噬细胞的吞噬率和吞噬指数提高机体的免疫力。     

The inhibitory effect of propolis and caffeic acid phenethyl ester on cyclooxygenase activity in J774 macrophages

The effect of an ethanolic extract of propolis, with and without CAPE, and some of its components on cyclooxygenase (COX-1 and COX-2) activity in J774 macrophages has been investigated. COX-1 and COX-2 activity, measaured as prostaglandin E2 (PGE2) production, were concentration-dependently inhibited by propolis (3 x 10(-3) - 3 x 10(2) microgml(-1)) with an IC50 of 2.7 microgml(-1) and 4.8 x 10(-2) microgml(-1), respectively. Among the compounds tested pinocembrin and caffeic, ferulic, cinnamic and chlorogenic acids did not affect the activity of COX isoforms. Conversely, CAPE (2.8 x 10(-4) - 28 microgml(-1); 10(-9) - 10(-4) M) and galangin (2.7 x 10(-4) - 27 microgml(-1); 10(-9) - 10(-4) M) were effective, the last being about ten-twenty times less potent. In fact the IC50 of CAPE for COX-1 and COX-2 were 4.4 x 10(-1) microgml(-1) (1.5 x 10(-6) M) and 2 x 10(-3) microgml(-1) (6.3 x 10(-9) M), respectively. The IC50 of galangin were 3.7 microgml(-1) (15 x 10(-6) M) and 3 x 10(-2) microgml(-1) (120 x 10(-9) M), for COX-1 and COX-2 respectively. To better investigate the role of CAPE, we tested the action of the ethanolic extract of propolis deprived of CAPE, which resulted about ten times less potent than the extract with CAPE in the inhibition of both COX-1 and COX-2, with an IC50 of 30 microgml(-1) and 5.3 x 10(-1) microgml(-1), respectively. Moreover the comparison of the inhibition curves showed a significant difference (p < 0.001).These results suggest that both CAPE and galangin contribute to the overall activity of propolis, CAPE being more effective.     

Attachment of group B streptococci to macrophages is mediated by a 21-kDa protein

Group B Streptococcus (GBS) is able to bind to human macrophages in vitro in the absence of exogenous opsonins. The exact mechanisms that mediate this attachment are unclear. This study was undertaken to determine what protein adhesins are present on the surface of GBS that mediate attachment to macrophages. We have identified a 21-kDa protein from the envelope of GBS type III that directly binds to macrophages as determined by Western blot analysis. Antiserum against this protein was able to inhibit binding of GBS to macrophages by greater than 80% as measured by flow cytometry. Antiserum against the 21-kDa protein cross-reacted with 21-kDa proteins from GBS type Ib, type II, type III (COH31 and MR732) and type IV, as well as Staphylococcus epidermidis , but not GBS type Ia, Listeria monocytogenes or Enterococcus faecalis . This protein may be important in mediating the attachment of GBS to macrophages in an opsonin-poor environment.