Activation of the lectin complement pathway by H-ficolin (Hakata antigen).

Ficolins are a group of proteins which consist of a collagen-like domain and a fibrinogen-like domain. In human serum, there are two types of ficolins named L-ficolin/P35 and H-ficolin (Hakata Ag), both of which have lectin activity. We recently demonstrated that L-ficolin/P35 is associated with mannose-binding lectin (MBL)-associated serine proteases (MASP) 1 and 2 and small MBL-associated protein (sMAP), and that the complex activates the lectin pathway. In this study, we report the characterization of H-ficolin in terms of its ability to activate complement. Western blotting analysis showed the presence of MASP-1, MASP-2, MASP-3, and sMAP in H-ficolin preparations isolated from Cohn Fraction III. The MASPs in the preparations had proteolytic activities against C4, C2, and C3 in the fluid phase. When H-ficolin preparations were bound to anti-H-ficolin Ab which had been coated on ELISA plates, they activated C4, although no C4 activation was noted when anti-MBL and anti-L-ficolin/P35 were used. H-ficolin binds to PSA, a polysaccharide produced by Aerococcus viridans. C4 was activated by H-ficolin preparations bound to PSA which had been coated on ELISA plates. These results indicate that H-ficolin is a second ficolin which is associated with MASPs and sMAP, and which activates the lectin pathway.

Structural and functional evidence for microglial expression of C1qR(P), the C1q receptor that enhances phagocytosis.

Microglial activation has been associated with several degenerative diseases of the central nervous system (CNS). One consequence of activation is the induction of a more efficient phagocytic response, and it is therefore important to determine what factors regulate microglial phagocytosis and whether this capacity influences the progression of neurodegenerative changes. Previous studies have demonstrated that complement component C1q enhances Fc receptor- and CR1-mediated phagocytosis in cells of the myeloid lineage via a cell surface receptor, C1qRp. Because C1q has been found in the area of lesions in several degenerative CNS diseases, the current investigations were carried out to characterize the effects of C1q on microglial phagocytosis. Neonatal rat microglia were shown to express C1qRp, as assessed by flow cytometry and immunocytochemistry. Interaction of these cells with substrate-bound C1q was shown to enhance both FcR-and CR1-mediated phagocytosis two- to fourfold. In addition, introduction of an antibody raised against the carboxy-terminal, cytoplasmic domain of C1qRp into microglia by electroporation markedly diminished the ability of C1q to enhance uptake of IgG-coated targets, whereas nonspecific IgG had no such effect. These results suggest that C1q in areas of active degeneration may promote the phagocytic capacity of microglia via interaction with microglial C1qRp.

cDNA cloning and primary structure analysis of C1qR(P), the human C1q/MBL/SPA receptor that mediates enhanced phagocytosis in vitro.

The complement protein C1q, mannose-binding lectin (MBL), and pulmonary surfactant protein A (SPA) are structurally similar molecules that enhance phagocytic function in vitro. Monoclonal antibodies R3 and R139, which inhibit the enhancement triggered by these three ligands, were used to purify a 126,000 M(r) cell surface protein designated C1qR(P). Amino acid sequence was obtained and the corresponding cDNA was cloned. C1qR(P) is a novel type I membrane protein with the following putative structural elements: a C-type carbohydrate recognition domain, five EGF-like domains, a transmembrane domain, and a short cytoplasmic tail. All peptides identified by amino acid sequencing are encoded by the cDNA. Additionally, an anti-peptide antiserum was generated, which is reactive with C1qR(P). The data indicate that the cloned cDNA encodes the receptor that plays a role in C1q/MBL/SPA-mediated removal or destruction of pathogens and immune complexes by phagocytosis.

Reconstitution of murine resident peritoneal macrophages for antibody-dependent cellular cytotoxicity by homologous serum Clq.

Mouse resident peritoneal macrophages (PM) were reconstituted in their response to activation for antibody-dependent cellular cytotoxicity (ADCC) for sheep erythrocyte targets (SRBC) by subhemolytic dilutions of homologous or autologous sera. ADCC-responsive inflammatory PM were largely unaffected in their activation by exogenous serum. Augmentation of resident PM for ADCC by homologous serum was correlated with the complement-activating potential of the mouse monoclonal anti-SRBC IgG isotype in that serum augmented IgG gamma 2a greater than IgG gamma 2b much greater than IgG gamma 1. The active component of mouse serum was heat-labile at 56 degrees C for 30 min and was present in both C5-deficient AKR and C5-sufficient homologous C3H mouse sera. Western blot analysis of the cell lysates for Clq confirmed that oil-elicited and thioglycollate-elicited inflammatory PM had greater levels of endogenous Clq than did resident PM which correlated with their innate responsiveness for ADCC activation. Depletion of Clq from serum by immunoprecipitation with IgG antibody to Clq or by ion exchange chromatography removed the active reconstituting activity for ADCC. Purified mouse Clq (0.4 microgram) partially replenished the ADCC augmenting activity of Clq-depleted AKR mouse serum. SRBC targets preopsonized with IgG gamma 2a and purified mouse Clq (0.075-5.0 microgram/ml) fully reconstituted the ADCC response of resident PM similar to homologous serum indicating that the major active component of serum was Clq. Thus resident PM with low endogenous levels of Clq were reconstituted for ADCC by the addition of exogenous Clq, whereas inflammatory PM with sufficiently high endogenous levels of Clq were not further enhanced by exogenous Clq. Our findings indicate that Clq may provide an essential second signal in concert with Fc receptor binding of IgG to initiate ADCC activation of macrophages.

Human pulmonary surfactant protein (SP-A), a protein structurally homologous to C1q, can enhance FcR- and CR1-mediated phagocytosis.

C1q, a subunit of the first component (C1) of the classical complement pathway, and the pulmonary surfactant protein SP-A are structurally homologous molecules, each having an extended collagen-like domain contiguous with a non-collagenous domain. It is the collagen-like region of C1q that binds to mononuclear phagocytes and mediates the enhancement of phagocytosis of opsonized particles by these cells. Because SP-A enhances the endocytosis of phospholipids by alveolar type II cells and alveolar macrophages, we examined whether these two molecules were functionally interchangeable. The phagocytosis of sheep erythrocytes opsonized with IgG or with IgM and complement was enhanced by the adherence of monocytes or macrophages, respectively, to SP-A. The enhanced response was dependent on the concentration of SP-A used for coating the surfaces, similar to that seen when monocytes were adhered to C1q-coated surfaces. Both the percentage of cells ingesting the opsonized targets and the number of targets ingested per cell increased with increasing concentrations of SP-A. No such enhancement was seen with cells adhered to albumin, iron-saturated transferrin, or uncoated surfaces. However, SP-A did not substitute for C1q in the formation of hemolytically active C1. C1q did not stimulate lipid uptake by alveolar type II cells or alveolar macrophages and had only a slight inhibitory effect on the binding of SP-A to alveolar type II cells. Thus, these results suggested that a function which requires interactions of both the collagenous and the non-collagenous regions (i.e. initiation of the classic complement cascade) could not be mimicked by a protein sharing structural macromolecular similarity but lacking sequence homology in the non-collagen-like region. However, SP-A could substitute for C1q in stimulating a function previously shown to be mediated by the collagen-like domains of the C1q molecule.

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.

Absence of induction of IL-1 production in human monocytes by complement fragments.

The ability of C fragments to induce IL-1 production in human monocytes was examined by using various approaches to carefully exclude the role of contaminating endotoxin. The presence of IL-1 activity in monocyte supernatants and lysates was assayed by the augmentation of PHA-induced proliferation of murine thymocytes. SRBC were opsonized with IgM rabbit antibodies and various human C components to prepare EAC reagents that contained less than 25 pg LPS/ml of EAC at 5 x 10(8) cells/ml. EAC1q, EAC4b, EAC4b2aoxy, EAC4b2aoxy C3b, EAC4b2aoxyC3bi, and EAC4b2aoxyC3d all failed to induce IL-1 production when incubated at 10- to 100-fold excess with adherent human monocytes. Similarly, LPS-free purified C3a, C5a, and C5a des Arg all showed no IL-1-inducing activities at concentrations up to 25 micrograms/ml. However, the same C5a preparations were active on human monocytes in the induction of chemotaxis, and C3a and C5a both induced skin-blueing in guinea pigs. Fragment Ba and Bb preparations purified by gel filtration chromatography contained approximately 100 pg LPS/micrograms Ba or Bb. These Ba and Bb preparations at 10 and 50 micrograms/ml, respectively, induced IL-1 production in the presence of 5 micrograms/ml polymyxin B (PMB). However, Ba and Bb preparations purified by affinity chromatography and HPLC contained lower levels of endotoxin contamination and displayed IL-1-inducing activities at Ba and Bb concentrations of 50 and 100 micrograms/ml, respectively, that were almost completely inhibited by PMB. To explore further the role of contaminating endotoxin, a Bb preparation was adsorbed with PMB-4B in the presence of a dialyzable detergent to remove LPS bound to the Bb. This LPS-free Bb preparation failed to induce IL-1 production while maintaining intact enzymatic activities. These results indicate that various solid phase or soluble C fragments, including C3b, iC3b, C3d, C3a, C5a, Ba or Bb do not induce IL-1 production in human monocytes in the absence of contaminating endotoxin.

C1q induces chemotaxis and K+ conductance activation coupled to increased cytosolic Ca2+ in mouse fibroblasts.

Cultured mouse fibroblasts (L cells) respond to whole C with a slow hyperpolarization. Among the C components tested, C1q was found to be most effective. In contrast, the cell did not respond to C1, in which the collagen-like region of the C1q molecule is masked. The C1q-induced hyperpolarizing response was inhibited by collagen or C1q-specific antisera. Human diploid skin fibroblasts (Flow 1,000 cells) also exhibited similar membrane potential changes in response to whole C or C1q. After repeated applications of C1q, the cell membrane became unresponsive (desensitized). The treatment of L cells with pronase E inhibited the C1q-induced response, whereas the response to ATP, which is known to interact to its own receptor, was still preserved. The reversal potential of C responses was close to the K+ equilibrium potential. The hyperpolarizing response was inhibited by a blocker of Ca2+-activated K+ channels in fibroblasts (quinine), by deprivation of extracellular Ca2+ or by a Ca2+ channel blocker (nifedipine). By means of Ca2+-selective microelectrodes, the cytosolic free Ca2+ concentration was found to increase from 126 to 206 nM upon stimulation of L cells with C1q. Using an agarose-well method, L cells were observed to migrate predominantly toward C1q or whole C. It is concluded that the fibroblasts have the C1q receptor sensitive to pronase E and that activation of C1q receptors gives rise to Ca2+ influx, triggering an increase in the cytosolic free Ca2+ ions, which in turn induces a hyperpolarizing response as a result of the stimulation of Ca2+-activated K+ channels and initiates chemotaxis to C1q.

On the interaction of the first complement component C1 and its subunit C1q with solid-phase IgM immune complexes.

The interaction of C1 and C1q with solid-phase anti-dextran MOPC-104E IgM was studied. An enzyme-linked immunosorbent assay (ELISA) was used to detect bound C1q. The results revealed that immobilized IgM is converted to the functionally active 'staple' conformation by the specific polyvalent ligand dextran (B 1355/S). C1q is fixed to IgM dependent on the antigen concentration, and its binding might be explained by assuming a functional binding constant (K) of approximately 10(9) M-1. Molecules bound with a K in the range of 10(7) M-1 cannot be detected by this ELISA procedure. The fixation of C1q saturated with an excess of the C1r2S2-tetramer differs from that of free C1q. C1q incorporated in the C1 complex rapidly dissociates independently of the antigen concentration. Since the complement binding sites are located at definite positions on the IgM molecule because of its pentameric structure, it is suggested that the distinguishable association properties of C1 and C1q are brought about from the altered flexibility of the C1q molecule complexed with C1r2S2.

C1q enhancement of antibody-dependent granulocyte-mediated killing of nonphagocytosable targets in vitro.

A possible role for C1q in antibody-dependent granulocyte-mediated killing of nonphagocytosable targets was investigated utilizing IgG-dependent granulocyte cytotoxicity directed against microfilariae of Dirofilaria immitis. Granulocyte-mediated killing of microfilariae is enhanced by addition of fresh serum. Lack of C4 did not significantly reduce the observed increase in cytotoxicity. The addition of highly purified monomeric human Clq (0.2 microgram/ml) in the presence of immune IgG resulted in a two- to fivefold enhancement of killing (P less than 0.025). C1q enhancement of killing occurred in the absence of fluid-phase IgG, but killing was significantly less than when both fluid-phase IgG and C1q were present. The effect of C1q was inhibited by the addition of solubilized type I collagen (44-92% inhibition of killing, P less than 0.05). Significant 125I-Clq binding to microfilariae occurred only in the presence of immune IgG. In addition, C1q in concentrations ranging from 0.5 to 2.0 micrograms/ml resulted in a dose-dependent increase in binding of 125I-immune IgG to microfilariae. Finally, when purified C1q was added to preopsonized, washed microfilariae, granulocyte production of superoxide was increased from 0.25 +/- 0.07 to 0.68 +/- 0.07 nm/10(6) cells.10 min (P less than 0.01). These results describe a novel functional role for C1q in enhancement of antibody-dependent cellular cytotoxicity towards nonphagocytosable targets.

C1q enhances the phagocytosis of Cryptococcus neoformans blastospores by human monocytes.

We investigated whether C1q, a subunit of the first component of C, could modulate human peripheral blood monocyte-mediated phagocytosis of Cryptococcus neoformans (CN). Adherence of monocytes to C1q-coated surfaces induced a significant enhancement of ingestion of CN blastospores that had been opsonized with specific anticapsular IgG (IgG-CN). Additionally, C1q enhanced the monocyte-mediated phagocytosis of CN opsonized with C (CN-absorbed, nonimmune, normal human serum; C-CN). Ingestion of IgG- and C-CN by control and C1q-stimulated monocytes was maximal by 1 h of incubation. The monocyte-mediated enhancement of phagocytosis caused by C1q was paralleled by a proportionate increase in fungicidal activity, an effect which was maximal by 3 h of incubation. Human serum albumin-adherent, control monocytes exhibited only a low level of killing after 3 h of incubation. C1q enhancement was blocked by preincubation of the surfaces with a goat, polyclonal F(ab')2 anti-C1q. This study describes a new cellular function for the cell surface C1q receptor: the enhancement of phagocytosis of a pathogenic organism by monocytes.

Solubilization of immune precipitates by complement in the absence of properdin or factor D.

Various experiments have demonstrated that immune precipitates (IPs) are not solubilized by complement in the absence of alternative pathway function. To determine whether the characteristics of the IPs were responsible for these observations, we studied the solubilization (Sol) of IPs formed by bovine serum albumin (BSA)-rabbit antiBSA and tetanus toxoid (TT)-human antiTT. Sera deficient in properdin solubilized a fraction of BSA-antiBSA precipitates, although only when the IPs were formed in antibody excess. The same sera solubilized TT-antiTT precipitates with some delay but almost as efficiently as normal serum. Factor D-depleted serum solubilized a fraction of TT-antiTT precipitates too, indicating that Sol may proceed through activation of the classical pathway only. Thus, the requirements for complement-mediated Sol depend on the characteristics of the IPs and do not necessarily include alternative pathway function.

Inhibition of C1q functions by RHP, a protein elevated in sera from patients with rheumatoid arthritis.

We have previously shown that serum levels of C1q, unbound to C1r X C1s, are elevated in rheumatoid arthritis. We have also shown that RHP, a newly described serum protein which affects the C1q-anti C1q precipitin reaction, is also present at elevated levels in rheumatoid arthritis. We now show that RHP inhibits the hemolytic activity of C1q, disaggregates C1, and inhibits the ability of C1q bound to latex beads or to aggregated IgG to enhance the oxidative metabolism of neutrophils.

C1q production and C1q-mediated immune complex retention in lymphoid follicles of rat spleen.

Involvement of C1q in retaining immune complexes in germinal centers in rat spleen was studied in vivo and in vitro. C1q production was found in fibroblastic reticulum cells in the peripheral mantle zone, in follicular dendritic cells in germinal centers, and in transitional forms between these two cells in the inner mantle zone. In passively immunized animals, immune complexes were found transiently on fibroblastic reticulum cells, then on the transitional forms and follicular dendritic cells. Extracellular C1q was detected by the presence of immune complexes on both the transitional forms and follicular dendritic cells, but not on fibroblastic reticulum cells. Thus, the fibroblastic reticulum cell appeared to trap immune complexes but not to retain either immune complexes or C1q. The morphology and function of the fibroblastic reticulum cell and the follicular dendritic cell suggest that they belong to the same lineage. Immune complexes were bound in vitro to germinal centers in cryostat spleen sections in the same manner as those retained in vivo. The binding required no complement in the incubation medium and was inhibited by C1q-suppressing factors. The extracellular C1q originating from the follicular cells may therefore play a role in retaining immune complexes in the germinal center.