Apoprotein E is synthesized and secreted by resident and thioglycollate-elicited macrophages but not by pyran copolymer- or bacillus Calmette-Guerin-activated macrophages

Macrophages are active secretory cells that display functionally distinct phenotypes that are regulated by inflammation. We have found that apoprotein E (ApoE), a component of plasma lipoproteins, was synthesized and secreted by resident and nonspecifically stimulated macrophages elicited with thioglycollate broth, but not by activated macrophages obtained from mice treated with bacillus Calmette-Guerin, pyran copolymer, whole Corynebacterium parvum, or bacterial endotoxin. ApoE represented approximately 1% of the newly synthesized protein and approximately 10% of secreted protein of resident and thioglycollate- elicited macrophages. ApoE from thioglycollate-elicited macrophages was indistinguishable from ApoE in mouse plasma lipoproteins, as determined by immunoreactivity, peptide mapping, and molecular weight. When specific antibodies were used to localize cell-associated ApoE, strong immunofluorescence was seen in the Golgi region of resident and thioglycollate-elicited macrophages immediately after removal from the peritoneal cavity, as well as after culture for up to 7 d. In contrast, activated macrophages did not synthesize or secrete ApoE to an appreciable extent and had no immunocytochemically detectable intracellular ApoE. When activated macrophages were cultured in medium containing serum, their activated state, as judged by production of H2O2, declined within 48-72 h in parallel with the induction of synthesis and secretion of ApoE and detection of intracellular ApoE by immunofluorescence. During prolonged culture the rate of synthesis and secretion of ApoE increased in both resident and activated macrophages. Therefore, the synthesis and secretion of ApoE may serve as markers for the functional state of macrophages.     

Activation of the guinea pig alternative complement pathway by mouse IgA immune complexes

Activation of the complement system by IgA was investigated with immune complexes containing a mouse IgA myeloma protein with specificity for phosphorylcholine linked to bovine serum albumin (PC-BSA). These IgA anti-PC-BSA immune complexes activated the alternative complement pathway in mouse and guinea pig serum, while human complement was not affected. The activation proceeded with consumption of C3 but little or no consumption of C5. C3 did not bind to the IgA immune complexes during complement activation although it did bind covalently to IgG immune complexes. It is suggested that IgA immune complexes do not supply a suitable surface for C3 binding and effective alternative pathway convertase assembly; therefore, cleavage is limited and occurs primarily in the fluid phase. Without C3 binding, C5 cleavage does not occur nor can the alternative pathway activation proceed to the amplification step.     

Complement 1 inhibitor is a regulator of the alternative complement pathway.

We studied complement 1 inhibitor (C1-INH) as an inhibitor of the alternative complement pathway. C1-INH prevented lysis, induced by the alternative complement pathway, of paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes in human serum. It inhibited the binding of both factors B and C3 to PNH and rabbit erythrocytes and blocked the ability of factor B to restore alternative-pathway function in factor B-depleted serum. C1-INH did not bind to factors B or D but did bind to immobilized C3b and cobra venom factor (CVF), a C3b analogue. C1-INH prevented factor B from binding to CVF-coated beads and dissociated bound factor B from such beads. Factor B and C1-INH showed cross competition in binding to CVF-coated beads. Factor D cleaved factor B into Bb and Ba in the presence of C3b. Cleavage was markedly inhibited when C3b was preincubated with C1-INH. C1-INH inhibited the formation of CVFBb and decreased the C3 cleavage. Removal of C1-INH from serum, in the presence of Mg-EGTA with an anti-C1-INH immunoabsorbant, markedly increased alternative-pathway lysis. C1-INH interacts with C3b to inhibit binding of factor B to C3b. At physiologic concentrations, it is a downregulator of the alternative pathway convertase.     

Activation of the alternative pathway of complement by antiserum to factor B.

Monospecific rabbit and goat antisera to human complement proteins and human immunoglobulins were tested for their ability to activate the alternative complement pathway. This activation was detected by two methods where classical pathway activation was blocked with EGTA and alternative pathway activation was promoted with added magnesium ions. These two methods consisted of lysis of GSHE and conversion of factor B into split products. C1q-depleted serum was used in a third assay system. Only antiserum to human factor B was able to activate the alternative pathway in the various systems used. None of the other anticomplement sera showed such activity. When antiserum to factor B was fractionated by ammonium sulfate and column chromatography, activation of the alternative pathway was found in the IgG fraction, and this activity was completely removed by absorption with purified factor B but not with other purified complement components.     

Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease.

Serum mannose-binding protein (MBP) is a C-type lectin that binds to terminal mannose and N-acetylglucosamine moieties present on surfaces of certain pathogens and activates the classical complement pathway. In the present study, we describe the mechanism underlying the activation triggered by MBP. The human serum MBP fraction was obtained by sequential affinity chromatography on mannan-Sepharose, anti-IgM-Sepharose and anti-MBP-Sepharose in the presence of calcium ions. This fraction contained a C1s-like serine protease as assessed by C4 consumption. The C1s-like serine protease, designated MBP-associated serine protease (MASP), was separated from MBP by rechromatography on anti-MBP-Sepharose in the presence of ethylenediaminetetraacetic acid. MASP exhibited both C4- and C2-consuming activities. The molecular mass of MASP was estimated to be 83 kD with two polypeptides of heavy (66 kD) and light (L) (31 kD) chains linked by disulfide bonds. The serine residue responsible for protease activity is located on the L chain. Reconstitution experiments using MASP and MBP revealed that combination of the two components restores C4- and C2-activating capacity on mannan. Based on analyses of molecular size, antigenicity, and 11 NH2-terminal amino acid sequences of the L chain, we conclude that MASP is a novel protein different from C1r or C1s. Our findings are not in accord with a proposed mechanism by which MBP utilizes the C1r2-C1s2 complex to initiate the classical complement pathway.     

Purification and characterization of a human membrane protein that activates the alternative complement pathway and allows the deposition of homologous complement C3

A human myeloid cell subline, P39+, is found to be a target for human complement (C) via the alternative pathway and to allow the deposition of multiple C3 fragments on its membranes, though expressing the complement regulatory proteins decay-accelerating factor and membrane cofactor protein. The parent cell line, P39-, which is phenotypically similar to the P39+ subline, does not allow the deposition of homologous C3 fragments. In this study, we established a monoclonal antibody, M161 Ab, which reacted with P39+ but not P39- cells. This Ab recognized a 43-kD protein in P39+ cell lysate transblotted onto nitrocellulose. Using this Ab as a probe, we purified the 43-kD protein, namely, M161 antigen (Ag). M161 Ag had a basic isoelectric point (pI), 9.3-9.4 by chromatofocusing, and was precipitated as an insoluble material at the pI point. The purified M161 Ag was a single- chain protein and did not possess N- or O-linked carbohydrates. When the purified M161 Ag was transblotted onto nitrocellulose and incubated with Mg(2+)-EGTA serum, human C3 fragments were efficiently deposited on M161 Ag. The major species of the deposited C3 fragments was C3b. Furthermore, the C3 fragments bound to the M161 Ag were detached by 1 M hydroxylamine, suggesting that a covalent ester linkage sustains M161 Ag-C3b interaction. NH2-terminal amino acid analysis revealed that M161Ag is a novel membrane protein. Hence, it appeared that M161 Ag is a potent activator of human alternative complement pathway on human cells that activates homologous C3 and allows the deposition of C3b on itself. Thus, under some conditions, homeostasis of complement is maintained even on human cells, not only by the complement regulatory proteins, but also by membrane C3-activating molecules on which C3b is deposited.     

Biosynthesis of the complement components and the regulatory proteins of the alternative complement pathway by human peripheral blood monocytes

Short-term cultures of human peripheral blood monocytes were shown to synthesize the alternative pathway complement components C3, factors B (B) and D (D), and properdin, the regulatory proteins C3b inactivator (C3bINA) and beta 1H, in addition to C2, C4, and C5. B, D, properdin, C3bINA, and C2 were detected by functional assays, whereas beta 1H, C4, C3, and C5 could only be detected using immunochemical procedures. Immunoperoxidase localization studies showed that all the cells in each culture contained each component, so it is possible that all monocytes synthesize each component. It is concluded that cells of the monocyte- macrophage series form a mobile source of complement components and regulatory proteins which can be concentrated at sites of inflammation.     

Human mannose-binding protein activates the alternative complement pathway and enhances serum bactericidal activity on a mannose-rich isolate of Salmonella

The human mannose-binding protein (MBP) is a multimeric serum protein that is divided into three domains, a cysteine-rich NH2-terminal domain that stabilizes the collagen alpha helix of the second domain and a third COOH-terminal carbohydrate recognition domain. Previous studies have shown that both native and recombinant human MBP bind to wild-type virulent Salmonella montevideo that expresses a mannose-rich lipopolysaccharide. Interaction with MBP results in opsonization and killing by phagocytes. In this report we show that low concentration of MBP (less than 10 micrograms/ml) markedly enhance complement deposition via the alternative complement pathway on S. montevideo. Despite structural similarities between MBP and the C1q subcomponent of the first complement component, MBP did not restore classical pathway activity to C1q-deficient serum, nor did it activate C1s when added to a mixture of C1r and C1s. In the presence of MBP the C3 bound to S. montevideo during incubation in serum was in the form of C3b and iC3b at a ratio of 1:2. Presensitization of S. montevideo with MBP rendered this normally serum resistant organism susceptible to complement-mediated killing. These results emphasize that MBP and complement cooperate in first line defense of the nonimmune host.     

Lysis of measles virus-infected cells by the purified cytolytic alternative complement pathway and antibody

The dependence of antibody-and-complement-mediated lysis of virus-infected cells on the alternative pathway was examined utilizing the isolated cytolytic alternative pathway--a system consisting of the six purified proteins of the alternative pathway of activation (C3, factors B and D, beta 1H, C3b inactivator and properdin), and the five proteins of the membrane attack pathway (C5--9) of complement. HeLa cells acutely infected with measles virus were lysed by anti-viral IgG and the isolated cytolytic alternative pathway with an efficiency comparable to whole human serum. IgG and its F(ab')2 fragment were equally effective in inducing lysis by the isolated cytolytic alternative pathway, binding of approximately equal to 5 X 10(7) molecules per cell being required for 50% lysis; in contrast, no lysis occurred when equivalen or greater amounts of Fab' were bound to the virus-infected cell. Properdin was required for lysis. No lysis occurred if properdin was deleted from the isolated cytolytic alternative pathway, and lysis was diminished by 80% in properdin-depleted serum. Uptake of [125I]C3b from the isolated alternative pathway onto measles virus-infected cells occurred in the absence of properdin, but was accelerated in the presence of properdin. The 11 proteins of the isolated cytolytic alternative pathway are thus sufficient for lysis of measles virus-infected cells bearing anti-viral IgG or F(ab')2 without any other serum protein.     

Recombinant adenovirus vectors activate the alternative complement pathway, leading to the binding of human complement protein C3 independent of anti-ad antibodies.

Recombinant adenoviruses are one of the most common gene transfer vectors utilized in human clinical trials, but it is also clear that systemic administration of this virus will be met by host innate and adaptive antiviral immune responses. One element of innate immunity is the complement system, a group of proteins that has evolved to rapidly recognize foreign microbes and viruses and to clear them from the circulatory system prior to their gaining entry to vulnerable host cells. Excessive complement activation can initiate or propagate a number of deleterious inflammatory responses, by release of potent cytokines and anaphylatoxins and/or by direct cellular toxicity. These reactions can progress rapidly and are factors important in serious complications, including the systemic inflammatory response syndrome and the adult respiratory distress syndrome.     

Uptake of extracellular enzyme by a novel pathway is a major determinant of cathepsin L levels in human macrophages.

The phorbol myristate acetate (PMA)-differentiated myelomonocytic cell line, THP-1, and human alveolar macrophages contain the cysteine proteinase cathepsin L. This enzyme is synthesized as a 43-kD proenzyme and processed to the active 25-kD form. Differentiation of THP-1 cells in the presence of human serum resulted in an increase in the size of the vacuolar compartment and the accumulation of more 25-kD cathepsin L antigen, as compared with THP-1 cells differentiated in the presence of fetal calf serum. Cells cultured in both types of sera have equivalent levels of cathepsin L mRNA. Metabolic labeling experiments demonstrated equivalent rates of synthesis, processing to the active form, and persistence in both culture conditions. An extracellular source of enzyme was documented by immunoblotting human serum which demonstrated 25-kD cathepsin L antigen; furthermore, we demonstrated that both THP-1 cells, differentiated in human serum, and human alveolar macrophages take up the 43-kD proenzyme and process it to the 25-kD form. Thus, human serum contains a factor(s) that induces both a marked increase in the size of the vacuolar compartment in differentiated THP-1 cells and a novel pathway that is responsible for the uptake and processing of extracellular cathepsin L. The activity of this inducible pathway is a major determinant of levels of intracellular cathepsin L. Cathepsin L is a potent elastase and the regulation of its uptake and processing may play a role in the pathogenesis of disease processes characterized by the destruction of elastin, such as pulmonary emphysema.     

Mechanism of injury of virus-infected cells by antiviral antibody and complement: participation of IgG, F(ab'')2, and the alternative complement pathway

Antibody-mediated C-dependent lysis of cell lines infected with herpes simplex type 1 virus, influenza A degrees virus, measles virus, and mumps virus occurred by the alternative C pathway with the participation of IgG antibodies. Lysis occurred only with immune human sera, Mg++ EGTA immune sera, and immune sera depleted of C4 or treated with Fab anti-C4. Lysis did not occur with nonimmune sera, Mg++ EDTA immune sera, and immune sera heated 50 degrees C for 25 min, depleted of factor B or treated with Fab antifactor B. Lysis was restored to heated and factor B immunodepleted immune sera by addition of factor B, but not by addition of an excess of C2. Further studies showed that lysis of HeLa cells infected with measles virus was induced by both immune IgG and F(ab')2 but not Fab' in the presence of a nonantibody-containing human C source. Lysis of measles virus-infected cells was also indpendent of movement of viral antigens on the surface of the infected cells, as inhibition of viral antigen capping by cytochalasin B or sodium azide was not associated with abrogation of immune lysis.     

Activation of the alternative pathway of complement by grain. I. C3PA conversion and quantification of complement consumption by rye

Ground whole rye and airborne rye dust of comparable size distribution were tested for their ability to activate the complement cascade via the alternative pathway. Precipitin-negative pooled normal human serum was incubated with increasing amounts of the two rye dusts. Electrophoresis of the resultant supernatant fluids demonstrated the conversion of the proactivator of the third component of complement to the gamma-migrating activator of the third component. This activation was completely prevented by pre-treating the serum with the chelator EDTA, while pre-treatment with EGTA allowed suboptimal arc conversion, strongly implying that complement was activated via the alternative pathway. Quantification of the supernanant fluids showed dose-dependent complement consumption as defined by both CH100 immunodiffusion and CH50 tube haemolytic techniques. Airborne rye dust showed a greater quantitative potential than ground whole rye for activating the alternative pathway. These results indicate the possibility of the direct action of airborne organic dusts on the induction of inflammatory sequelae in the lungs of both sensitized and unsensitized individuals.     

LIPOCALIN-2 is a major acute-phase protein in a rat and mouse model of sterile abscess

Lipocalin-2 (LCN-2) is a 25-kDa secretory protein currently used as a biomarker for renal injury and inflammation. Its source and cause of the increased serum levels are unclear. The current study compares LCN-2 gene expression with known major acute-phase proteins in the liver in a rat and mouse model of turpentine oil-induced sterile abscess. Serum LCN-2 concentrations increased dramatically up to 200-fold (20 μg/mL) at 48 h after turpentine oil injection. A strong elevation of LCN-2 mRNA in rat liver was observed starting from 4 h up to 48 h after injection, with a maximum (8,738 ± 2,104-fold) at 24 h, which was further confirmed by Western blot analysis. In contrast, the increases in gene expression of α?-macroglobulin, the major acute-phase protein, and hemoxygenase 1, a positive acute-phase protein, were only 1,025 ± 505-fold and 47 ± 12-fold, respectively, during acute-phase reaction (APR). No considerable change was observed in LCN-2 mRNA in rat kidney and other organs as compared with liver. Using wild-type mice, a massive increase in gene expression of LCN-2, with a maximum of 2,498 ± 84-fold in liver, which is similar to that for serum amyloid A (2,825 ± 233-fold), a major mouse acute-phase protein. However, such an increase was significantly inhibited in interleukin 6 knockout mice during APR. Interleukin 6-treated rat hepatocytes induced a significant time-dependent upregulation of LCN-2.Lipocalin-2 is the major acute-phase protein in rat as compared with α?-macroglobulin and hemoxygenase 1 and comparable with serum amyloid A in mouse whose gene expression is mainly controlled by interleukin 6. The liver is the main source of serum LCN-2 in the case of APR. ABBREVIATIONS-LCN-2-lipocalin-2-α?M-α?-macroglobulin-HO-1-hemoxygenase 1-IL-6-interleukin 6-SAA-serum amyloid A-TO-turpentine oil-APR-acute-phase reaction.     

GMP-140, a platelet alpha-granule membrane protein, is also synthesized by vascular endothelial cells and is localized in Weibel-Palade bodies.

We used an immunoperoxidase procedure to examine the tissue distribution of the platelet alpha-granule membrane protein, GMP-140. In addition to its presence in megakaryocytes and platelets, GMP-140 antigen was found in vascular endothelial cells of diverse human organs, but it was not detected in other types of secretory cells. [35S]Cysteine-labeled human umbilical vein endothelial cells synthesized a GMP-140 molecule containing complex N-linked oligosaccharides similar to those previously demonstrated in platelets and the megakaryocytic HEL cell line. Using an immunogold procedure on frozen thin sections of endothelial cells, we found GMP-140 antigen to be localized to membranes of electron-dense storage granules. In double-label experiments there was colocalization of GMP-140 with vWf, indicating that these granules are Weibel-Palade bodies. When endothelial cells were stimulated with histamine, GMP-140 rapidly redistributed to the plasma membrane. Immunoassays of cell lysates indicated that, relative to total cell protein, less GMP-140 is present in human umbilical vein endothelial cells than in platelets. The restricted expression of GMP-140 in secretory granules of platelets and endothelium suggests that it has a specific function in the vascular system rather than a general role related to inducible secretion.     

The effect of complement on the ingestion of soluble antigen-antibody complexes and IgM aggregates by mouse peritoneal macrophages

Complement was found to stimulate markedly the ingestion of soluble antigen-antibody complexes by mouse peritoneal macrophages. This was shown indirectly by measuring the release of degradation products when the complexes were labeled with 125I, or directly when the antigen, that was human transferrin, was labeled with 59Fe. In this case, the metal which was released from human transferrin inside the cells was not excreted, and its accumulation in the macrophages was a direct index of the uptake of immune complexes. The decay of radioactivity in macrophages after ingestion of 125I-labeled complexes was similar when they were taken up with or without complement, indicating that complement acts primarily on ingestion and not on digestion or excretion. The ingestion of complexes was morphologically confirmed using fluorescein-labeled antigen in the immune complexes. The opsonic effect of complement was also observed with IgM aggregates indicating that soluble complexes can be ingested through complement receptors without involvement of Fc-receptors, as required for particulate antigen-antibody complexes.     

A sensitive method to detect synthesis of the functional classical, alternative and terminal pathway of complement by cells cultured in vitro

A new method used to study in vitro synthesis by human monocytes and alveolar macrophages of the essential complement components for the functional classical, alternative and terminal pathway is presented. The method is based on accumulation of major complements components on activators of the alternative (agarose beads) and classical (lgM-sensitized sheep erythrocytes; ElgM) pathway during co-culture with the phagocytes. There was a time-dependent increase in binding of labelled protein to the co-cultured activators, demonstrating de novo protein synthesis by the phagocytes. Moreover, there was a significant binding to the co-cultured agarose beads and ElgM of monoclonal anti-C3c, anti-C3g, polyclonal anti-C5-C9 and of two monoclonal antibodies (poly C9-MA and MCaEll) to a neoantigen of polymerized C9 present in the terminal complement complex (TCC). In addition, we found a significant binding of polyclonal anti-C4 antibodies to co-cultured ElgM. Incubation of the activators in human serum, subsequently revealed the same pattern of antibody binding. There was no binding of anti-S protein antibodies to the activators after incubation with serum or with the phagocytes. We thus conclude that mononuclear phagocyte-produced complement in the form of C3b, iC3b, and the TCC (C5b-9) was deposited on both activators, whereas C4b was detected on the ElgM. It is our hope that this method can be applied when studying complement biosynthesis by cells other than mononuclear phagocytes.     

Production of leukotrienes and thromboxane by resident and activated rat alveolar macrophages: a possible role of protein kinase C.

Arachidonic acid metabolism in resident rat alveolar macrophages and in those activated with complete Freund's adjuvant (CFA) was studied. Adult Sprague-Dawley rats were injected with 0.05 ml CFA, and macrophages were harvested 10 days later. Macrophages were labeled overnight with carbon 14-labeled arachidonic acid, washed, and then stimulated with calcium ionophore A23187 (IoA), phorbol myristate acetate (PMA), or zymosan for 30 minutes. Prostaglandins, thromboxane, and leukotrienes were extracted from the medium and analyzed by radioimmunoassay or radio high-pressure liquid chromatography. Cell lipids were analyzed by radio thin-layer chromatography. Medium and cell beta-glucuronidase activity and protein kinase C activity of the membrane fraction were also assayed. We found (1) lower leukotriene B4 (LTB4) production in stimulated resident macrophages when compared with resident macrophages after IoA stimulation--the suppressed LTB4 production was reversed by PMA; (2) unchanged or higher LTB4 production in activated macrophages when compared with resident macrophages after zymosan stimulation; (3) inhibition of zymosan-stimulated LTB4 production by staurosporine, a protein kinase C inhibitor, in both groups; and (4) lower diacylglycerol (DAG) production in activated macrophages when compared with resident macrophages after IoA stimulation, but not after zymosan stimulation. These results suggest that the reduced response of activated macrophages to IoA is due to decreased production of an endogenous protein kinase C activator. This hypothesis was further supported by the observation that protein kinase C activation in response to IoA was lower in activated macrophages than in resident macrophages. In contrast, zymosan stimulation resulted in higher protein kinase C activation in activated macrophages when compared with resident cells. We hypothesize that protein kinase activation is necessary for leukotriene production and that the preserved ability of zymosan to activate PKC via DAG accounts for the high leukotriene production in zymosan-activated macrophages. We also found that stimulated thromboxane production was higher in activated than resident cells, regardless of the stimulus, and that thromboxane production was not affected by staurosporine. Thus alterations of eicosanoid metabolism in immunologically activated macrophages depend on the stimulus used and the type of eicosanoid examined. Furthermore, leukotriene biosynthesis in rat alveolar macrophages may be regulated by protein kinase C.     

A quantitative study on the activation of the alternative pathway of complement by mouldy hay dust and thermophilic actinomycetes.

Materials associated with the induction of farmer's lung were incubated with fresh normal human serum in the presence of magnesium ethylene glycol tetra-acetic acid (MgEGTA) or ethylene diamine tetra-acetic acid (EDTA) and results compared with material known to activate the alternative pathway of complement--zymosan. Results show that Micropolyspora faeni organisms are as active as zymosan in reducing complement (C) levels in the presence of MgEGTA, with a 50% reduction in CH50 at approximately 140 mug/ml. Thermoactinomyces vulgaris organisms produced a 50% CH50 reduction at approximately 1-25 mg/ml and two samples of respirable mouldy hay dust (MHD) at approximately 5-6 mg/ml whereas extracts of M. faeni and T. vulgaris reduced the CH50 titre by 17% and 39% respectively at 16 mg/ml in the presence of MgEGTA. Organisms and extracts did not reduce the CH50 titre in the presence of EDTA even at the maximum concentration quoted by more than 3%, thus it is considered that alternative pathway activation was responsible for C utilization in the presence of MgEGTA. Respirable MHD used less than 4% available C at 4 mg/ml in the presence of EDTA but at 8 mg/ml dust 11% and 28% available CH50 were used compared with 79% and 81% respectively in the presence of MgEGTA. The elution of immunoglobulin binding material from MHD may be responsible for apparent CH50 consumption in the presence of EDTA.