Role of C1q in phagocytosis of Salmonella minnesota by pulmonary endothelial cells.

The Re mutant of Salmonella minnesota adheres in much greater numbers than the wild type to endothelial cells derived from the bovine pulmonary artery. Since the Re mutant is distinguished from wild-type S. minnesota by its ability to bind C1q and since endothelial cells possess receptors for C1q, we examined the role of C1q in the phagocytosis of the S. minnesota Re mutant. First, preincubating endothelial cells with C1q-enriched medium resulted in increased adherence of the Re mutant (17.9 x 10(4) versus 6.6 x 10(4]. Second, preincubating the Re mutant with C1q-enriched medium resulted in increased numbers of adherent bacteria (62.1 x 10(4) versus 6.6 x 10(4]. Preincubation of both endothelial cells and bacteria with C1q-enriched medium resulted in increased adherence above control levels but less adherence than when either cells or bacteria were preincubated separately in C1q-enriched medium. If serum depleted of C1q was used for preincubation of endothelial cells or bacteria, adherence was reduced below control levels. Thus, C1q plays an important role in the initial steps (recognition, binding, and ingestion) of phagocytosis. Next, the role of C1q was investigated in the respiratory burst response. Levels of superoxide anion released from endothelial cells 15 min after phagocytosis of the Re mutant (100 bacteria per endothelial cell) were assayed by measurement of the superoxide dismutase-inhibitable reduction of ferricytochrome c. Superoxide anion release was increased during phagocytosis of the Re mutant (35 nmol of O2- per 3 x 10(6) endothelial cells) and was also elevated above control values by incubation with soluble C1q (10 nmol of O2- per 3 x 10(6) endothelial cells). These results indicate a role for C1q in both the ingestion and the response of endothelial cells to the S. minnesota Re mutant.     

Antibody-independent binding of the first component of complement (C1) and its subcomponent C1q to the S and R forms of Salmonella minnesota.

Strong bactericidal effects of normal guinea pig and human sera against the Salmonella minnesota S form and an R form (Re) depend on Ca2+, complement component C4, and subcomponent C1q of complement component C1. Therefore, the interaction of C1 and C1q with these forms was investigated. The bacteria directly bound subcomponent C1q, as demonstrated by fixation and transfer tests and by fluorescent methods. Binding of macromolecular C1 was shown by fixation and transfer tests and by C4 consumption. C1 fixation and transfer tests provide evidence that C1 and C1q were bound more tightly to the Re form than to the S form. At physiological ionic strength, all cell-bound molecules were released from the S form, whereas at least 60% remained on the cell surface of the Re form. The Re form showed another binding behavior for C1: preincubation of bacteria with purified C1q totally prevented C1 uptake by the S form, compared to only 10% inhibition of the uptake by the Re form. Therefore, we conclude that macromolecular C1 is bound differently by the S form than by the Re form. The analysis of five other core-deficient mutants of S. minnesota (Ra, Rb, Rc, Rd1, and Rd2) revealed that the difference could be explained by a deficiency of the O-specific polysaccharide. In contrast, all the C1q bound to Ra, Rb, and Rc mutants was detectable by the transfer test. Therefore, we postulate that binding of macromolecular C1 to these mutants must be due to an additional C1 subcomponent besides C1q.     

Ganglioside modulation of lipopolysaccharide-initiated complement activation

Highly purified preparations of mouse gangliosides have been demonstrated to bind to purified preparations of lipopolysaccharide (LPS). In some instances, the binding has been demonstrated to be dependent upon the presence of sialic acid in the ganglioside preparation. The binding of gangliosides to LPS from the deep rough Salmonella minnesota Re mutant has suggested that the interaction involves the lipid A-2-keto-3-deoxyoctulosanate region of the LPS macromolecule. The interaction between gangliosides and LPS has been demonstrated to result in an abrogation of lipid A dependent activation of the classical pathway of serum complement by Re LPS. Surprisingly, however, the presence of sialic acid containing glycolipids has been shown to enhance significantly the capacity of LPS to initiate activation of the alternative pathway of complement. These data suggest that sialic acid can enhance as well as inhibit the formation of a stable alternative-pathway C3 convertase.     

Monoclonal antibodies specific for Escherichia coli J5 lipopolysaccharide: cross-reaction with other gram-negative bacterial species.

Four monoclonal antibodies against Escherichia coli J5 were studied. Each of these monoclonal antibodies reacted with purified lipopolysaccharides from E. coli J5, the deep rough mutant Salmonella minnesota Re595, Agrobacterium tumefaciens, and Pseudomonas aeruginosa PAO1 as well as with the purified lipid A of P. aeruginosa. Enzyme-linked immunosorbent assays using the outer membranes from a variety of gram-negative bacteria demonstrated that these lipid A-specific monoclonal antibodies interacted with between 84 and 97% of the gram-negative bacterial species tested. One of the monoclonal antibodies, 5E4, was shown to interact with 34 of the 35 outer membrane or lipopolysaccharide antigens tested. Immunoenzymatic staining of Western electrophoretic blots of separated P. aeruginosa outer membrane components was used to demonstrate that antibody 5E4 interacted with a similar fast-migrating band, corresponding to rough lipopolysaccharide, from all 17 serotype strains and all 14 clinical isolates of P. aeruginosa. Similarly, iodinated goat anti-mouse immunoglobulin was used to detect the binding of monoclonal antibody 8A1 to a fast-migrating band on Western electrophoretic blots of purified lipopolysaccharides from Klebsiella pneumoniae and both smooth and rough strains of E. coli, Salmonella typhimurium, and S. minnesota. These results suggest considerable conservation of single antigenic sites in the lipid A of gram-negative bacteria.     

Parameters affecting the enzyme-linked immunosorbent assay of immunoglobulin G antibody to a rough mutant of Salmonella minnesota.

An enzyme-linked immunosorbent assay was developed to detect rabbit immunoglobulin G antibodies against purified lipopolysaccharide of the rough Re mutant of Salmonella minnesota. The time necessary for each step was investigated, and this resulted in a test that could be completed in 1 working day. Serial dilutions of rabbit sera drawn throughout immunization with the Re mutant revealed an elimination of the prozone effect upon prolonged immunization. We interpret this to demonstrate an increase in immunoglobulin G affinity for the lipopolysaccharide on prolonged immunization. We propose that the enzyme-linked immunosorbent assay prozone effect be investigated for use as a measure of antibody affinity.     

Kinetics of the biosynthesis of complement subcomponent C1q by murine macrophages: LPS, immune complexes, and zymosan alone and in combination with interferon-gamma.

We investigated the effects of bacterial lipopolysaccharide (LPS), immune complexes (IC), and C3b opsonized zymosan (AZ) alone and in combination with interferon-gamma (IFN-gamma) priming on macrophage synthesis and secretion of C1q. Our results indicated that LPS, IC, and AZ alone stimulated C1q mRNA and secretion in the absence of IFN-gamma. The increase in mRNA accumulation was detectable after 3 h, peaked at 6 h and was maintained at constitutive levels for 24 h. There was a corresponding early burst of increased secretion of functional C1q after 3 to 6 h which declined rapidly after 9 to 24 h culture of LPS-stimulated macrophages. Priming of macrophages with IFN-gamma and simultaneous triggering with LPS, IC, or AZ produced additive rather than synergistic increases in C1q mRNA accumulation. These same agents inhibited constitutive secretion of C1q in the absence of IFN-gamma priming as determined by autoradiographic analysis of metabolically radiolabeled secretory C1q. Triggering of IFN-gamma primed macrophages with LPS, IC, or AZ also markedly suppressed the increased rate of C1q secretion induced by IFN-gamma in a dose-related fashion. A corresponding dose-dependent increased accumulation of endogenous C1q in cell lysates was detected by Western blot analysis of macrophages which had been stimulated by LPS, IC, or AZ alone or in combination with IFN-gamma. Our findings indicate that LPS as well as FcR and C3bR triggering agents stimulate early and sustained C1q synthesis accompanied by an early and short-lived burst of C1q secretion which rapidly diminished and results in an increased intracellular accumulation of C1q due to ongoing synthesis. IFN-gamma appeared to further amplify the same kinetics of increased C1q mRNA accumulation and decreased extracellular accumulation mediated by LPS, IC, and ZM. Our results suggest that LPS, IC, and AZ alone or in combination with IFN-gamma stimulate early C1q production to modulate macrophage effector functions followed by an inhibition of C1q secretion when the activation process has been culminated.     

Effects of a phagocytosis-stimulating factor derived from polymorphonuclear neutrophils on the functions of macrophages.

The effects of phagocytosis-stimulating factor (PSF) derived from polymorphonuclear neutrophils on macrophage functions were studied. PSF enhanced the initial rate of phagocytosis of serum-opsonized zymosan particles by macrophages, whereas it did not affect the phagocytosis of immunoglobulin G-sensitized and inert zymosan particles. Kinetic studies showed that PSF accelerated the ingestion step, but not the attachment step, of phagocytosis by macrophages. On the other hand, PSF did not affect the other macrophage functions such as O2- generation, chemotaxis, adherence, and enzyme release. These results suggest that PSF may specifically modulate the complement receptor function of macrophages. Immunoblot assay showed the absence of components in macrophage which reacted with purified antibodies against polymorphonuclear neutrophil-derived PSF, and an extract from phagocytosing macrophages had no phagocytosis-stimulating activity, indicating that the macrophages did not produce PSF-like substances.     

Isolation and characterization of macrophage-derived C1q and its similarities to serum C1q

Recently, we have shown that the collagen-like, Fc-recognizing subcomponent C1q of the first complement component is synthesized by human, guinea pig and mouse peritoneal macrophages. To test whether macrophages may contribute to the serum pool of C1q, C1q was purified from guinea pig serum and from guinea pig peritoneal macrophage supernatants and compared for similarities. Both molecules had a similar sedimentation rate (macrophage C1q: 11.3 S, serum C1q: 11.2 S) and showed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions three identical bands with molecular weights of Mr, 29 000, Mr, 27 000 and Mr 23 000 for the A, B and C chains, respectively. Both C1q molecules migrated by immunoelectrophoresis in the gamma region and, in Ouchterlony analysis, showed complete antigenic identity with rabbit anti-serum C1q. These experiments demonstrate the antigenic and protein chemical similarities between serum C1q and C1q secreted by macrophages supporting the idea that macrophages have to be considered as one potential source of serum C1q. Furthermore, macrophage-derived C1q may be of importance in the local microenvironment at an inflammatory site involving macrophages.     

The functions of endogenous C1q, a subcomponent of the first component of complement, as a receptor on the membrane of macrophages

C1q, the Fc-recognizing subcomponent of the first component of complement is synthesized by peritoneal macrophages. During the secretion phase C1q serves as an Fc-binding protein in the membrane of macrophages. The Fc-mediated rosette formation was inhibited in a dose-dependent manner when macrophages were pretreated with anti-C1q F(ab')2, whereas C3b rosette formation was not affected. Furthermore, preincubation of peritoneal macrophages with anti-C1q F(ab')2 abolished, dose- and time-dependently, the polyanion-mediated stimulation of secretion of lysosomal enzymes. Polyanion-induced enzyme release was prevented after incubation of polyanions with highly purified C1q. The inhibition of Fc receptor activity by polyanions (i.e. dextran sulfate, liquoid, polyvinyl sulfate) is completely reversed upon treatment of these macrophages with protamine. These findings are compatible with the hypothesis that C1q produced by macrophages serves in the macrophage membrane as an endogenous receptor for Fc and polyanionic molecules. Thus, C1q mediates cell-bound biological receptor functions before it is released from these cells and is incorporated into the macromolecular C1 complex.     

Inhibition of the Fc receptor-mediated oxidative burst in macrophages by the Yersinia pseudotuberculosis tyrosine phosphatase.

Suppression of host-cell-mediated immunity is a hallmark feature of Yersinia pseudotuberculosis infection. To better understand this process, the interaction of Y. pseudotuberculosis with macrophages and the effect of the virulence plasmid-encoded Yersinia tyrosine phosphatase (YopH) on the oxidative burst was analyzed in a chemiluminescence assay. An oxidative burst was generated upon infection of macrophages with a plasmid-cured strain of Y. pseudotuberculosis opsonized with immunoglobulin G antibody. Infection with plasmid-containing Y. pseudotuberculosis inhibited the oxidative burst triggered by secondary infection with opsonized bacteria. The tyrosine phosphatase activity of YopH was necessary for this inhibition. These results indicate that YopH inhibits Fc receptor-mediated signal transduction in macrophages in a global fashion. In addition, bacterial protein synthesis was not required for macrophage inhibition, suggesting that YopH export and translocation are controlled at the posttranslational level.     

Malaria-induced lymphokines: stimulation of macrophages for enhanced phagocytosis

Culture supernatants from antigen-pulsed spleen cells of mice infected previously with either BCG or Plasmodium chabaudi were used to study macrophage activation as judged by phagocytosis of immunoglobulin G-sensitized erythrocytes and Plasmodium berghei- and P. chabaudi-infected erythrocytes. Resident peritoneal macrophages were incubated in vitro with spleen cell factor and then assayed for ingestion of immunoglobulin G-sensitized or parasitized erythrocytes. Macrophages activated with BCG-induced lymphokine bound and ingested two- to threefold more P. berghei parasitized erythrocytes than macrophages incubated with control spleen cell factor. Similarly, Plasmodium-stimulated spleen cells from mice infected with malaria produced a lymphokine(s) capable of activating macrophages for enhanced Fc receptor-mediated phagocytosis. The stimulation of phagocytosis by the lymphokine is nonspecific in nature, since phagocytosis of parasitized erythrocytes from one species of murine malaria is enhanced by the lymphokine prepared from a heterologous species. Nylon wool-nonadherent, malaria-sensitized spleen cells elaborated a lymphokine which stimulates macrophages for enhanced phagocytosis, whereas anti-0-treated spleen cells failed to produce the phagocytosis-promoting lymphokine. Consequently, this lymphokine appears to be elaborated by sensitized T lymphocytes. Interestingly, enhanced phagocytosis of opsonized trophozoites and schizonts, but not ring stage parasites of P. chabaudi, was displayed by macrophages activated with the lymphokine(s) prepared from P. chabaudi-recovered mice. Preincubation of the malaria parasitized erythrocytes with hyperimmune serum raised against the parasites greatly facilitated both binding and ingestion by the stimulated macrophages.     

Chemical, biological, and immunochemical properties of the Chlamydia psittaci lipopolysaccharide.

The lipopolysaccharide (LPS) of Chlamydia psittaci was extracted from yolk sac-grown elementary bodies, purified, and characterized chemically, immunochemically, and biologically. The LPS contained D-galactosamine, D-glucosamine, phosphorus, long-chain fatty acids, and 3-deoxy-D-manno-2-octulosonic acid in the molar ratio of approximately 1:2:2:6:5. The antigenic properties of the isolated LPS were compared with those of the LPS from Chlamydia trachomatis and Salmonella minnesota Re by the passive hemolysis and passive hemolysis inhibition tests, absorption, hydrolysis kinetics, and Western blot analysis with rabbit polyclonal antisera against chlamydiae and with a mouse monoclonal antibody recognizing a genus-specific epitope of chlamydial LPS. Two antigenic determinants were identified, one of which was chlamydia specific and the other of which was cross-reactive with Re LPS. Both determinants were destroyed during acid hydrolysis, whereby a third antigen specificity was exposed which was indistinguishable from the lipid A antigenicity. In rabbit polyclonal antisera prepared against Formalin-killed elementary bodies or detergent-solubilized membranes, two antibody specificities were differentiated. One of these was chlamydia specific, and the other was cross-reactive with Re LPS. The LPS of C. psittaci was inactive within typical endotoxin parameters (lethal toxicity, pyrogenicity, local Shwartzman reactivity); it was, however, active in some in vitro assays, such as those testing for mouse B-cell mitogenicity and the induction of prostaglandin E2 in mouse peritoneal macrophages.     

Effects of Histoplasma capsulatum on murine macrophage functions: inhibition of macrophage priming, oxidative burst, and antifungal activities.

Histoplasma capsulatum yeast cells fail to trigger an oxidative burst response in normal murine macrophages. The results of this study, in which an in vitro assay of macrophage antifungal effects was used, extend these findings. During 18 h of incubation, unprimed elicited murine macrophages inhibited H. capsulatum growth only when macrophages were present in great excess. Gamma interferon (IFN-gamma)-primed macrophages showed enhanced fungal growth inhibition but a similar requirement for an excess of phagocytes. Macrophages containing heat-killed H. capsulatum exhibited diminished antifungal effects toward viable H. capsulatum and Saccharomyces cerevisiae cells. Parallel experiments showed no comparable effect of ingested latex particles on macrophage antifungal activity. Using chemiluminescence as a measure of the oxidative burst, we found that macrophages primed in vitro with IFN-gamma alone failed to exhibit a significant response to triggering by H. capsulatum yeast cells unless a second priming agent (tumor necrosis factor alpha or bacterial lipopolysaccharide) was added to IFN-gamma. Furthermore, macrophage priming with single agents was blocked by the prior ingestion of heat-killed H. capsulatum. These studies provide evidence that ingestion of H. capsulatum yeast cells can induce a prompt and enduring deactivation of murine macrophages.     

Trypanosoma cruzi but not Trypanosoma brucei fails to induce a chemiluminescent signal in a macrophage hybridoma cell line.

Macrophage-Trypanosoma cruzi interactions were studied by using a newly generated macrophage hybridoma cell line (2C11-12) that was selected for its capacity to produce high levels of reactive oxygen intermediates. This cell line was found to be a suitable host cell for T. cruzi, and intracellular parasitic development could be inhibited by activation with gamma interferon. When exposed to opsonized Trypanosoma brucei, Micrococcus lysodeikticus, or Legionella pneumophila, the activated macrophage cell line produces a high chemiluminescent signal, indicating the release of reactive oxygen intermediates. Alternatively, when opsonized T. cruzi was added to these activated macrophages, this parasite failed to stimulate a chemiluminescent response, suggesting an impairment in the triggering of the respiratory burst.     

Susceptibility of HRS/J mice to listeriosis: macrophage activity.

Macrophage functions, including phagocytosis and bactericidal and oxidative activities, were measured in highly susceptible Listeria monocytogenes-sensitive HRS/J homozygous and heterozygous mice. Phagocytic studies with both caseinate-elicited and L. monocytogenes-immune macrophages revealed comparable engulfment of latex particles, zymosan, and bacteria by mononuclear phagocytes obtained from all experimental mouse strains. Elicited macrophages cultivated from mutant hairless and heterozygous littermates exhibited a reduced capacity to control Listeria infection compared with cells derived from CD-1 mice. However, intracellular killing of the microorganisms by immune macrophages was comparable to that observed with the outbred controls. Studies on oxidative metabolic activities associated with the respiratory burst indicate that while intracellular nitroblue tetrazolium reduction was comparable for macrophages cultivated from all mouse strains, the liberation of superoxide anion and chemiluminescence responses were significantly diminished in caseinate-elicited HRS/J cells. Moreover, immune elicited hr/hr and hr/+ macrophages generated oxidative species at levels comparable to that observed with cells derived from resistant animals. Thus, immunologically elicited HRS/J mice are capable of responding to sublethal Listeria infection with heightened antibacterial and oxidative activities.     

Inhibition of murine macrophage protein kinase C activity by nonviable Histoplasma capsulatum.

Ingestion of Histoplasma capsulatum yeast cells inhibits the oxidative burst response of murine macrophages (M phi's). Since protein kinase C (PKC) is believed to be an integral part of the signal transduction pathway involved in the production of reactive oxygen intermediates, we investigated the relationship between PKC activity and oxidative burst inhibition in H. capsulatum-containing murine peritoneal M phi's. An inhibitory effect on both basal and phorbol myristate acetate-mobilized membrane PKC activities was observed in M phi's which had ingested H. capsulatum but not latex spheres. These results suggest that one way in which H. capsulatum may disrupt the oxidative burst is through a PCK-dependent mechanism.     

On the interactions between macrophages and developmental stages of Schistosoma mansoni: the cytotoxic mechanisms involved in macrophage-mediated killing of schistosomula in vitro

In an in vitro cytotoxicity assay, mouse adherent peritoneal exudate macrophages (APEM), harvested 8-10 weeks post Schistosoma mansoni infection caused sizable (greater than 90%) specific killing of schistosomula. This cidal effect was not diminished by the addition of scavengers of oxidative burst products to the cytotoxicity assay, albeit macrophages from schistosome-infected mice produced more H2O2 than did macrophages from non-infected mice. Of inhibitors of lysosomal enzyme function and release added to the cytotoxicity assay, trypan blue (1 mg/ml) fully abolished the schistosomulicidal effect; hydrocortisone (100 micrograms/ml) was partly effective, and gold salts (1 mg/ml) were ineffective. A cidal effect was not apparent in the absence of L-arginine nor in the presence of excess (greater than 400 micrograms/ml) L-arginine, L-lysine or L-ornithine. Arginase (5 U/ml) totally abrogated the schistosomulicidal effect. The findings suggest that a macrophage protein of a lysosomal origin, dependent on arginine for its reaction and/or production, may be involved in the in vitro killing of schistosomula by macrophages from S. mansoni-infected mice.     

Candida albicans killing by RAW 264.7 mouse macrophage cells: effects of Candida genotype,infection ratios,and gamma interferon treatment

Phagocytic cells such as neutrophils and macrophages are potential components of the immune defense that protects mammals against Candida albicans infection. We have tested the interaction between the mouse macrophage cell line RAW 264.7 and a variety of mutant strains of C. albicans. We used an end point dilution assay to monitor the killing of C. albicans at low multiplicities of infection (MOIs). Several mutants that show reduced virulence in mouse systemic-infection models show reduced colony formation in the presence of macrophage cells. To permit analysis of the macrophage-Candida interaction at higher MOIs, we introduced a luciferase reporter gene into wild-type and mutant Candida cells and used loss of the luminescence signal to quantify proliferation. This assay gave results similar to those for the end point dilution assay. Activation of the macrophages with mouse gamma interferon did not enhance anti-Candida activity. Continued coculture of the Candida and macrophage cells eventually led to death of the macrophages, but for the RAW 264.7 cell line this was not due to apoptotic pathways involving caspase-8 or -9 activation. In general Candida cells defective in the formation of hyphae were both less virulent in animal models and more sensitive to macrophage engulfment and growth inhibition. However the nonvirulent, hypha-defective cla4 mutant line was considerably more resistant to macrophage-mediated inhibition than the wild-type strain. Thus although mutants sensitive to engulfment are typically less virulent in systemic-infection models, sensitivity to phagocytic macrophage cells is not the unique determinant of C. albicans virulence.     

Quantitation of rabbit immunoglobulin G antibodies to Salmonella minnesota Re by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay was developed which allowed the measurement of rabbit immunoglobulin G antibodies directed to Salmonella minnesota Re glycolipid. Efficient adsorption of the antigen to polystyrene could only be effected provided it had been previously dialyzed against 0.2 M EDTA (pH 7.0) and subsequently treated with 0.2% sodium deoxycholate (15 min at 56 degrees C) in 0.05 M diethanolamine buffer (pH 9.6). The method is by far more sensitive than quantitative precipitation in the determination of IgG antibodies. Inhibition by glycolipid of binding S. minnesota Re antibodies to immobilized glycolipid attests to the specificity of the assay and permits the detection of as little as 100 ng of glycolipid.     

A macrophage-suppressing 40-kD protein in a case of pulmonary alveolar proteinosis

Summary Pulmonary alveolar proteinosis (PAP) is a rare disease of unknown etiology. Macrophage dysfunctions are claimed to be involved in the pathogenesis. We investigated phagocytosis and oxidative metabolism of alveolar macrophages in a case of pulmonary alveolar proteinosis. These cells phagocytize normally and phagocytizable stimulants cause a normal oxidative burst. In response to the membrane signals phorbolmyristate acetate and aggregated immunoglobulin, however, no stimulated turnover of the oxidative metabolism can be observed. A 40-kD protein found in the lavage fluid mediates this macrophage-inhibiting effect. This phenomenon may contribute to the frequent opportunistic infections seen in PAP patients. It can be concluded from our data that the high frequency of infections with opportunistic species in these patients can be reduced by therapeutic bronchoalveolar lavage. By this procedure the abnormal macrophage-suppressing protein can be washed out of the lung at an early stage of the disease.Abbreviations AM alveolar macrophage - BM blood monocyte - BAL bronchoalveolar lavage - CL chemiluminescence - Ig immunoglobulin - kD kilodalton - PAP pulmonary alveolar proteinosis - PMA phorbolmyristate acetate - SRBC sheep red blood cells - Z zymosan