Human Alveolar Macrophages Synthesize the Functional Alternative Pathway of Complement and Active C5 and C9 in Vitro

Attachment of protein to agarose beads cultured with macrophages in protein-free medium containing 3H-leucine, shows that de novo synthesis of protein with affinity to the beads takes place. We also found that monoclonal antibodies against human C3c, C3g, and a C9-neoantigen as well as polyclonal antibodies against human C5 and C9, bound to agarose beads that had been kept with the macrophage cultures. Demonstration of C3 derivatives on the agarose beads shows that the essential complement factors of the alternative pathway are synthesized and have been activated by the beads. Deposition of C5 and the detection of a neoantigen of C9 on the beads, indicates that the whole terminal complement pathway has been formed and activated. We conclude that human alveolar macrophages form in vitro the functional alternative pathway of complement, C5 and C9, and we have indirect evidence for synthesis of C6, C7, and C8.     

Synthesis of C3, C5, C6, C7, C8, and C9 by Human Fibroblasts

We investigated the ability of human fibroblasts to produce the components of the final common pathway (C3-C9) of complement in vitro by co-culturing an alternative complement activator (agarose beads) with the cells. The test system involved incubation of beads with anti-complement antibodies followed by radioactive-labelled anti-Ig detection antibodies. Subsequently, the beads were examined in a radioimmunoassay. Our results indicate that human fibroblasts produce C3, C5, C6, C7, C8, and C9. A neoepitope selectively expressed on activated C9 was detected, indicating assembly of the terminal complement complex and thus formation of a functional terminal complement pathway by the fibroblasts.     

Human Alveolar Macrophages Synthesize Active Complement Components C6, C7, and C8 in Vitro

We investigated whether serum-free human alveolar macrophage cultures synthesize active C6, C7, and C8. There was a significant binding of polyclonal anti-human C6 antibodies to agarose beads incubated with unstimulated macrophages for 24 or 48 h. Endotoxin stimulation of the macrophages was necessary for significant binding of polyclonal anti-C7 and anti-C8 antibodies to agarose beads co-cultured for 48 or 96 h. Two monoclonal antibodies (poly C9-MA and MCaE11) specific for a neoantigen of polymerized C9 in the terminal complement complex (TCC), bound to beads mainly incubated with endotoxin stimulated macrophages. The MCaE11 was more sensitive than the poly C9-MA in detecting the C9 neoantigen on beads incubated with the macrophages or human serum diluted 1:16. We thus conclude that human alveolar macrophages synthesize active C6, C7, and C9 that together with C5 and C9, assemble as the TCC on co-cultured agarose beads. Activation of the alternative pathway on the agarose with generation of fixed C3 and C5 convertases is a prerequisite for the subsequent generation of the TCC.     

Human Umbilical Vein Endothelial Cells Synthesize Functional C3, C5, C6, C8 and C9 In Vitro

Human endothelial cells (EC), cultured serum-free, synthesize de novo protein which increasingly bind to agarose beads (an alternative pathway activator), until a plateau phase is reached after 24-48 h. EC synthesize functional C3, C5, C6, C8 and C9, which were detected on co-cultured agarose beads, using relevant polyclonal anti-complement antibodies. Two monoclonal anti-C9 neoepitope antibodies (aE11, poly C9-MA) bound to the co-cultured beads, showing that the terminal complement complex (TCC) (C5b-9) was assembled on the beads. This also suggests that C7 is synthesized. There seems to be a positive correlation between the amount of agarose-bound labelled protein and agarose-bound complement. The results indicate that EC produce and secrete the components for the functional alternative and terminal pathways of complement.     

Synthesis of Complement Components C5, C6, C7, C8 and C9 in Vitro by Human Monocytes and Assembly of the Terminal Complement Complex

Monocytes cultured under serum-free conditions secreted protein which bound covalently and non-covalently to agarose beads, an activator of the alternative pathway of complement. There was a significantly binding of monoclonal anti-C3c antibodies, polyclonal anti-C5, anti-C6, anti-C7, anti-C8, and anti-C9 antibodies, and of a monoclonal antibody against a neoantigen of polymerized C9 to agarose beads incubated with the monocytes for 24, 48, 72 or 96 h. From these results, we conclude that monocytes produce C5, C6, C7, C8 and C9 that assemble as the terminal complement complex on the surface of the agarose beads. Activation by agarose of the alternative pathway with generation of particle bound C3 and C5 convertases is a prerequisite for the subsequent formation of the terminal complement complex. Whether SC5b-9 or the membrane attack of complement (C5b-9) is formed on the beads will be examined.     

Attachment and phagocytosis by salmon macrophages of agarose beads coated with human C3b and C3bi

Agarose beads (diameter 5-10 micron) preincubated in human serum became associated (attached and ingested) to 50-60% of the salmon macrophages within 60 minutes. However, Beads preincubated in serum treated with heating (50 degrees C, 20 min) or with EDTA (10 mM) to inhibit the activation of alternative complement pathway, were not associated to the phagocytes. Furthermore, agarose beads coated with human C3b and C3bi after incubation with isolated complement factors (C3, D, B), were associated to 30-40% of the phagocytes. About 80% of the cell-associated agarose beads was intracellularly located. Conversion by trypsin treatment (0.01%) of agarose bound C3bi to C3d, abolished the association of such beads to the macrophages. The results demonstrate that salmon macrophages possess complement receptors that bind human C3b and C3bi. Agarose beads coated with these ligands (C3b and C3bi) are attached and ingested by the phagocytes.     

Complement (C3) Receptor-Mediated Attachment of Agarose Beads to Mouse Peritoneal Macrophages and Human Monocytes

We have determined the receptors on human monocytes and mouse peritoneal macrophages producing agarose binding. By using isolated human complement factors C3, B and D, agarose beads were coated with C3b. In some experiments C3b was converted to C3bi by using human serum diluted 1:20. Agarose beads coated with C3b or C3bi bound strongly to monocytes. Only agarose beads coated with C3bi were attached to mouse macrophages. Trypsinization of agarose beads coated with C3bi abolished the attachment of the beads to macrophages and monocytes, probably because of conversion of C3bi to C3d. Endocytosis by macrophages of agarose preincubated in human serum or in C5-deficient AKR mouse serum reached the same levels, indicating that the amount of C5 present in serum during preincubation is not important for the degree of endocytosis. It is concluded that internalization of agarose by macrophages is mediated via the C3bi receptor.     

Synthesis of Soluble C3 and C9 Neoepitopes by Human Alveolar Macrophages in Vitro

The aim of this study was to examine whether soluble neoepitopes of activated C3 (C3b, iC3b, C3c) and C9 are produced by human alveolar macrophages cultured in serum-free medium. There was a significant and inhibitable production of C3 and C9 neoepitopes and C9 by the macrophages from all donors, as detected by enzyme-linked immunosorbent assays based on monoclonal (bH6, aE11) and polyclonal (anti-C9) antibodies. A strong donor-dependent variation in the levels of the C3 neoepitope and C9 (five- to sevenfold) and the C9 neoepitope (twofold) was found. After 1 day (24 h) of incubation, the complement levels were largely unaltered. The presence of an exogenous alternative pathway activator (agarose beads) reduced the amount of soluble complement because of binding to the agarose. However, the relative fraction of C9 neoepitope versus C9 increased (two- to threefold), due to agarose-mediated activation of C9. The results demonstrate activation of the complement system in serum-free alveolar macrophage cultures, irrespective of the presence of a known complement activator.     

S protein binds to serum-treated agarose beads independently of complement activation and the formation of the terminal complement complex on the beads.

Comparison of initial (early-phase) and terminal (late-phase) sequence activation of complement by agarose beads and endotoxin was evaluated in an enzyme immunoassay (EIA) of serum levels of C3c and C9 neoepitopes, respectively. EIA and Western blotting with anti-S protein monoclonal antibody revealed lower S protein values and weaker S protein bands in serum activated by agarose beads than by endotoxin, implying that S protein was removed from serum by binding to agarose. The binding of S protein to the beads was confirmed by radioimmunoassay and was found to be equal in normal and heat-inactivated serum. In contrast, the terminal complement complex was formed only on agarose beads incubated with normal serum and not with inactivated serum.     

Formation of the Terminal Complement Complex on Agarose Beads: Further Evidence that Vitronectin (Complement S-Protein) Inhibits C9 Polymerization

Vitronectin occupies the metastable binding site of C5b-7, which is unable to insert membranes as part of the complement lytic attack. Some evidence has been presented that vitronectin inhibits also membrane-associated pore formation by inhibiting C9 polymerization in the terminal complement complex (TCC). The authors wished to add to this background by studying the effect of vitronectin on formation of TCC on a carbohydrate surface like agarose beads, an alternative complement pathway activator. Bound TCC was detected by monoclonal and polyclonal antibodies to C9-neoepitopes. Soluble SC5b-7 and TCC (SC5b-9) did not bind to the agarose beads. Using serum or isolated complement factors for the alternative and terminal pathways, the authors found that vitronectin reduced the density of C9-neoepitopes on the beads. As there was no convincing evidence for association of vitronectin with the factors C5b-8 of the agarose-bound TCC, it was concluded that vitronectin bound directly to C9 in TCC and inhibited C9 polymerization within the complex. The authors have shown that TCC can bind to a carbohydrate surface like agarose (an alternating polymer of galactose moieties) in the absence of lipid. These results suggest that vitronectin can limit the lytic effect of membrane-bound TCC by inhibiting C9 polymerization.     

Synthesis of Complement by Alveolar Macrophages from Patients with Sarcoidosis

Sarcoidosis is a granulomatous disorder of unknown aetiology. Alveolar macrophages (AM) in sarcoidosis release a variety of mediators important to the pathogenesis of the disease. Complement is essential for the inflammatory response and we investigated whether there were any major defects in the potential for sarcoidosis AM to synthesize complement in vitro. AM from 11 patients with active sarcoidosis and three healthy controls were cultured under serum-free conditions. There was a significant binding of polyclonal (anti-C5, -C6, -C7, -C8) and monoclonal anti-complement antibodies (anti-C3c and anti-C9 neoepitope (aE11] to agarose beads incubated with unstimulated AM for 24, 48, or 72 h. A significant and inhibitable production of soluble C3c, C5, C9, and S-protein was found in the harvested medium as detected by enzyme immunoassays. Activated C3 and C9 were also detected based on neoepitope expression. Presence of co-cultured agarose beads reduced the amount of soluble S-protein due to deposition on the agarose. We argue that the C9 neoepitope is an integral part of the terminal complement complex (TCC), both in the fluid and solid phase when bound to the agarose. In the fluid phase, SC5b-9 was generated, whereas the agarose-bound S-protein is assumed not to be associated with TCC on the beads. The results demonstrate for the first time that AM from sarcoidosis patients synthesize the functional alternative and terminal pathway of complement.     

Co-localization of terminal C5b-9 complement complexes and macrophages in human atherosclerotic arterial walls

The co-localization of terminal C5b-9 complement complexes and macrophages was investigated in human arteries with atherosclerosis using a double-labeling immunohistochemical technique. Macrophages were found in all the atherosclerotic arteries, with the accumulation correlating positively with the degree of atherosclerosis. This accumulation was associated with an increase of C5b-9 deposits, as well as with an increase in the number of deposits containing both complement components and macrophages ('co-localization'). This co-localization was found to pertain both to intact macrophages and to macrophage remnants. These data suggest that C5b-9 complement complex might be formed on macrophages with subsequent promotion of inflammatory events and progression of the atherosclerotic lesions.     

Fibronectin Promotes Binding But Not Ingestion of Agarose Beads by Mouse Macrophages and Human Monocytes

We have examined to what extent human fibronectin associated with agarose beads with a 5- to 10-μm diameter mediates binding and uptake of the heads by mouse macrophages and human monocytes. Native agarose beads preincubated with ¹²⁵I-fibronectin were neither associated with nor taken up by mouse macrophages after 30 min of incubation under serum-free conditions. When fibronectin was cross-linked to cyanogen bromide-activated agarose heads or incubated with gelatinized heads, this resulted in a significant increase in particle binding by macrophages and monocytes as compared with gelatinized beads, whereas the fraction of cells with ingested particles remained unaltered. Native agarose heads activated by cyanogen bromide and treated with ethanolamine were to a greater extent associated with and taken up by phagocytes than fibronectin- or gelatin-coated heads. Our results indicate thai fibronectin acts as an adhesive glycoprotein and not as an opsonin. Since agarose beads are activators of the alternative pathway of complement, and fibronectin is reported to bind to factor C3, we speculate that cell-derived C3b is bound to the beads and fibroneetin-coaled beads arc ingested by the phagocytes via complement C3b receptors on the cells.     

Phagocytosis of Agarose Beads by Receptors for C3b (CR1) and iC3b (CR3) on Alveolar Macrophages from Patients with Sarcoidosis

Alveolar macrophages (AM) from sarcoidosis patients exhibit no detectable defect in their potential to synthesize the functional alternative and terminal pathway of complement. They also synthesize more C9 than AM from healthy controls. Various authors [4, 6] have suggested that sarcoid AM have decreased phagocytic ability. In the present work we studied whether there was any difference in C3 receptor-mediated phagocytosis of serum-treated and native agarose beads by AM recovered from patients with active sarcoidosis compared with controls, AM from seven patients with active sarcoidosis and seven healthy controls were cultured under serum-free conditions for 2, 12, 24. and 48 h. We found a significantly increased CR1 and CR3 receptor-mediated phagocytosis of native agarose heads by AM from the seven patients. CR1 and CR3 were also detected on AM directly recovered from bronchoalveolar lavage fluid using fluorescein-conjugated monoclonal anti-receptor antibodies. The percentage of AM expressing CR appeared to be increased in sarcoidosis. The reason for the enhanced phagocytosis of agarose beads by the sarcoid AM is probably the result of both increased synthesis and receptors of complement. Altered complement production and complement receptors may be important for the pathogenesis of this granulomatous disorder.     

Regulation of complement activation at the C3-level by serum resistant leptospires

Leptospires are spirochetes that are transmitted to humans through contacts with wild or domestic animals or via an exposure to contaminated soil or water. In this study we have compared the serum-sensitivity of five pathogenic strains of leptospires (L. interrogans) to an environmental isolate (strain Patoc). Different levels of sensitivities to human serum were seen. Interestingly, the most sensitive strain was the non-pathogenic Patoc strain. The fully and intermediately resistant strains have been isolated from human patients. Testing was performed in the absence of specific antibodies, and killing was found to be dependent on the complement system. The serum sensitive Patoc strain was killed in human serum within minutes, whereas the most resistant strains tolerated serum up to 4h. We also tested the deposition of the complement components C3, C5, C6, C8 and C5b-9 to the surfaces of the sensitive and resistant strains of Leptospira by immunofluorescence microscopy and ELISA. C3 was deposited on both the sensitive and resistant strains, but the terminal complement components were detected only on the surface of the complement-sensitive strain. The complement resistant and intermediate strains were found to bind more factor H from human serum than the complement sensitive strain. Thus, binding of this major alternative complement pathway inhibitor is related to serum resistance in Leptospira spirochetes.     

Formation of the Functional Alternative Pathway of Complement by Human Monocytes in Vitro as Demonstrated by Phagocytosis of Agarose Beads

Native agarose beads (diameter 5-10 micron), activators of the alternative complement pathway, are slowly phagocytosed when incubated with human monocytes cultured under serum-free conditions. Agarose beads preincubated with monocyte cultures and then transferred to new cultures are more easily phagocytosed than native beads. These results indicate that the phagocytosis of agarose beads depends on opsonization of the beads by one or several substances of monocyte origin. By using antihuman C3 antibodies, trypsin treatment, and sodium dodecyl sulphate washing, we were able to demonstrate C3b and iC3b on the agarose beads. The molecules were covalently bound to the surface of the beads. We conclude that in vitro human monocytes produce and secrete the essential factors for activation and propagation of the alternative complement pathway (factors C3, B, D, H and I), which becomes evident with an external activator like agarose beads in the cultures. The activation of complement by agarose beads results in the attachment of C3b and iC3b to the surface of the beads, which are then phagocytosed by means of C3b and iC3b receptors on the monocytes.     

The relationship between macrophages and C5b-9 complement complexes in human atherosclerosis

The relationship between macrophages and the terminal C5b-9 complement complexes was investigated in human arteries affected with atherosclerosis by using monoclonal antibodies and indirect immunoperoxidase, immunogold silver staining, and double-labeling immunohistochemical techniques. Macrophages were found in all the atherosclerotic arteries as immunoreactive deposits with a nucleus, considered as intact cells, or without a nucleus, considered as cell remnants. The double-labeling technique shows C5b-9 deposits partially colocalized on the intact macrophages or on the cell debris of macrophage origin. These data suggest that C5b-9 complement complex may be formed on activated or dying macrophages with subsequent promotion of inflammatory events and progression of the atherosclerotic lesions.     

Secretion of the terminal complement proteins, C5-C9, by human platelets

The terminal complement components, C8 and C9, and to a lesser extent C5, C6, and C7, but minimal amounts of C3, were shown to be associated with washed human platelets. In unactivated platelets, the complement components were detected in the platelet pellet by hemolytic assays after centrifugation and disruption of the platelets by freeze-thawing. However, after platelets had been activated by collagen, thrombin, or aggregated IgG to induce aggregation, the complement components were released into the supernatant. The rank order of hemolytic activity of C9, C8, C7, C6, and C5 detected in the supernatants of activated platelets was quite different from that found in serum from the same donors, in the same assays. In particular, the serum C7 hemolytic titer was more than twice the serum C9 hemolytic titer, whereas the activity of C9 detected from platelets was more than twice that of C7. This argues against a purely nonspecific uptake of these proteins by platelets from plasma. The functional role of terminal complement components released from platelets during activation is unknown, but it is tempting to speculate that these proteins may have a role in platelet-dependent immunological tissue injury. Because the C5b-9 membrane attack complex activates platelets, it is possible that release of terminal complement proteins serves to amplify platelet activation and may also play a role in diseases in which complement membrane attack complexes have been implicated.     

Fibronectin Binds to Complement-Coated Agarose Beads and Increases Their Association to Mouse Macrophages

We have studied the binding of fibronectin to complement (C3b, C3bi, C3d)-coated agarose beads and its effect on cell association of such beads to mouse macrophages. Fibronectin bound to agarose beads preincubated in human serum, whereas no binding occurred after preincubation of the beads with complement-inactivated (50 degrees C for 20 min or ethylenediaminetetraacetic acid) sera. The binding of iodine-labelled fibronectin to beads preincubated in fibronectin-depleted serum (HS-FIB) was about twice that of beads preincubated in normal serum. Unlabelled fibronectin inhibited the following binding of labelled fibronectin to beads pretreated in HS-FIB. A similar amount of fibronectin bound to agarose beads coated with equimolar amounts of C3b, C3bi, or C3d, suggesting that the common domain C3d carries the main binding site(s) for fibronectin. Preincubation of serum-treated and trypsinized agarose beads with fibronectin led to an increased association (22%) of such beads to mouse macrophages. The results indicate that fibronectin promotes binding of complement-coated agarose beads to mouse macrophages, whereas the ingestion of the beads is mediated via complement C3 receptors.