Complement activation in the follicular light zone of human lymphoid tissues.

A comparative immunohistochemical study of the distribution pattern of complement components and regulatory proteins within secondary lymphoid follicles was performed by the immunoperoxidase technique. Fifteen lymphoid tissues including appendices. Peyer's patches and tonsils were analysed. Sixty secondary lymphoid follicles with evident polarity, that is, the distinct coexistence of a light zone, dark zone and mantle zone in the same lymphoid follicle, were tested with single antibodies. The light zones were consistently immunostained in a dendritic meshwork pattern with all antibodies. The immunostaining patterns were classified into two major groups based on the immunoreactivity of the dark zone. One immunostaining pattern was characterized by no immunostaining of the dark zone to the majority of the antigens. The second group was characterized by a diffusely weak to moderate dendritic meshwork pattern of the dark zone to some of the immunostainings of C9 (monoclonal), S-protein, and DF-DRC1, and all immunostainings of CR1 (CD35), Ber-Mac-DRC (CD35), CR2 (CD21), and R4/23. All four complement regulatory proteins were localized by immunoelectron microscopy attached to the cell surface of the cells, including follicular dendritic cells, in the light zone. Our data indicate that there is an evident functional difference between the light zone and the dark zone, and that complete activation of the complement system occurs only in the light zone.

Synthesis and regulation of C1 inhibitor in human skin fibroblasts.

Proteins of the C1 complex, C1q, C1r, and C1s, of the classical pathway of complement activation are known to be synthesized in human skin fibroblasts. Using metabolic labeling with [35S]methionine, immunoprecipitation, and SDS-PAGE, we demonstrate that human skin fibroblasts synthesize and secrete C1 inhibitor with an apparent molecular mass of 78 kDa in the cell lysate and 102 kDa in the extracellular medium. This C1 inhibitor had the capacity to bind activated C1s. Fibroblasts synthesized 30- to 50-fold more C1 inhibitor than was synthesized in monocytes. As previously reported, fibroblasts also synthesized C1r and C1s. IFN-gamma, IFN-beta 1, and TNF had significant, but distinct, effects on synthesis of C1 inhibitor, C1r, and C1s. Incubation of the cells with IFN-gamma, 1000 U/ml, for 24 h induced increases in the synthesis of C1 inhibitor, C1r, and C1s by 4.2-, 1.9- and 1.6-fold, respectively. IFN-beta 1 had effects similar to IFN-gamma, although smaller in magnitude. TNF, 12.5 ng/ml, induced increases in the synthesis of C1 inhibitor, C1r, and C1s by 1.5-, 1.4- and 2.6-fold. IL-1, IFN-beta 2 (IL-6), and LPS did not affect synthesis of C1 inhibitor, C1r, or C1s. Fibroblasts are present in large amounts in most tissues. Synthesis of C1 inhibitor, C1r, and C1s by these cells could provide a source of these important proteins in body tissues. In addition, fibroblasts should be a good model for the in vitro study of genetic diseases involving the synthesis of these proteins.

Biosynthesis of normal and low-molecular-mass complement component C1q by cultured human monocytes and macrophages.

High levels of low-molecular-mass complement component C1q (LMM-C1q), a haemolytically inactive form of C1q, are found in serum of individuals with inherited complete (functional) C1q deficiency and in serum of patients with systemic lupus erythematosus, whereas lower levels are present in normal serum [Hoekzema, Hannema, Swaak, Paardekooper & Hack (1985) J. Immunol. 135, 265-271]. To investigate whether LMM-C1q is a (by-)product of C1q synthesis or the result of degradation of C1q, cultures of blood monocytes and of alveolar macrophages, which secrete functional C1q, were studied. A considerable portion of C1q-like protein secreted by these cells was found to be LMM-C1q. In contrast with the C1q fragments that resulted from degradation of normal C1q during phagocytosis, culture-derived LMM-C1q appeared to be identical with LMM-C1q found in serum, as judged by sedimentation behaviour, subunit structure and recognition by poly- and mono-clonal antibodies raised against C1q. The presence of LMM-C1q in cytoplasmic organelles compatible with the Golgi apparatus and the inability to generate LMM-C1q by impeding hydroxylation and triple-helix formation of C1q further argues against degradation as its source. Monocyte cultures of homozygous probands from two families with complete functional C1q deficiency reflected the abnormalities in serum, i.e. absence of functional C1q, but increased levels of LMM-C1q. By contrast, secretion of C1q and LMM-C1q by cells from healthy individuals was clearly co-ordinate, indicating that LMM-C1q in serum may provide a unique marker of C1q synthesis in vivo.

Guinea pig macrophages synthesize a low molecular weight form of C1q with affinity for the C1r2C1s2-complex but which does not bind to Fc in immunoglobulin aggregates.

Biosynthetically labelled C1q secreted by guinea pig peritoneal macrophages was analysed by sedimentation through sucrose gradients followed by SDS-PAGE. In addition to the haemolytically active C1q of mol. wt 460,000 Da a low mol. wt (LMW) form of C1q was identified which had no detectable affinity for Fc of aggregated immunoglobulin, but which retained the ability to associate with the C1r2s2-complex. This LMW-C1q was covalently associated with two additional polypeptides of mol. wt 46 and 50 kDa.

Selective synthesis of mRNA and proteins by human peripheral blood neutrophils.

Human peripheral blood polymorphonuclear neutrophils (PMN) have been considered to be capable of little if any protein biosynthesis. We evaluated the ability of PMN to synthesize both mRNA and proteins. Using in vitro [35S]methionine pulse-chase labeling of purified PMN, followed by immunoprecipitation of cell lysates with immobilized mAb and analysis by gel electrophoresis, PMN were shown to synthesize CR1, FcR, CR3 alpha-chain, MHC class I, and actin. In contrast, incorporation of [35S]methionine into either CR3 beta-chain or the secondary granule protein lactoferrin was not detected. Purification of mRNA from PMN and analysis by Northern blots demonstrated the presence in PMN of CR1, actin, and MHC class I transcripts. However, despite the apparent lack of CR3 beta-chain biosynthesis, specific beta-chain message was detectable in PMN RNA. Inhibition of mRNA synthesis in PMN with actinomycin D resulted in decreased synthesis of nascent CR1, FcR, MHC class I, and actin compared with control cells. Thus, PMN continue to transcribe and translate the genes for certain membrane and cytoskeletal proteins. In contrast, the lack of detectable synthesis of either lactoferrin or CR3 beta-chain suggested that biosynthesis in circulating PMN is selective.

Induction of prolyl hydroxylase activity in a nonadherent population of human leukocytes.

A nonadherent population of human monocytes has been shown to express the collagen hydroxylating enzyme prolyl hydroxylase in vitro. Enzyme levels present in freshly isolated nonadherent cells were induced 300% during the first 72 hours of culturing, which could be suppressed by cycloheximide. Maximum induction required both a feeder layer of adherent leukocytes, and 10-15% autologous plasma. Biosynthesis of Clq, a protein which also is hydroxylated by prolyl hydroxylase, by the nonadherent cells was significantly less than the adherent monocytes. Therefore, this collagen biosynthetic marker enzyme was not associated with Clq synthesis, which suggests that the enzyme is present for collagen biosynthesis.

The effect of vitamin C nutriture on complement component C1q concentrations in guinea pig plasma.

This study shows that guinea pigs fed 100 times the amount of vitamin C needed for growth and for prevention of scurvy have elevated levels of complement component C1q. C1q is a plasma protein rich in hydroxyproline, an amino acid whose biosynthesis requires ascorbate. C1q is essential for host defense against pathogens, both as a component of the classical complement pathway and as an opsonin in the phagocytosis process. We measured C1q in vitamin C-depleted guinea pigs that had been repleted for 4 wks with the following daily doses of ascorbate (mg/100 g body wt): 0.50 (suboptimal), 2.0 (adequate), 10 (ample) and 50 (tissue saturating). We measured C1q in three ways: indirectly by quantifying protein-bound hydroxyproline and directly by hemolytic assay and by immunodiffusion against anti-C1q. Regardless of the method, plasma C1q was 30-50% higher in animals fed tissue-saturating ascorbate than in those fed adequate or suboptimal amounts of the vitamin (p less than 0.05, one-way analysis of variance, least significant difference test). These data confirm and significantly extend earlier work that provided indirect evidence for a relationship between C1q and ascorbate nutriture in the guinea pig. They are consistent with a possible relationship between ascorbate nutriture and host defense.

[Formation of classical C3 convertase during the alternative pathway of human complement activation]. / Obrazovanie klassicheskoi C3-konvertazy v khode aktivatsii al'ternativnogo puti komplementa cheloveka.

The fluid phase C3 convertase of the alternative pathway of human complement activation has been constructed from the isolated C3 component and from purified factors B and D. The enzyme was able to activate the isolated components C4 and C2 in the presence of C4 but had no effect on C2 in the absence of C4. The C4 and C2 activation was monitored by the loss of their hemolytic activity during the incubation with the alternative fluid phase C3 convertase. The activation of C4 and C2 components by the membrane-bound alternative C3 convertase formed on red cells (EC3bBb) was followed by the formation of C3 convertase of the classic pathway--EC4b2a. This resulted in the enhancement of hemolysis.

Effects of inhibitors of C1q biosynthesis on macrophage Fc receptor subclass-mediated antibody-dependent cellular cytotoxicity and phagocytosis.

We examined the effects of the inhibitors of C1q or collagen biosynthesis, 2,2'-dipyridyl (DP), and 3,4-dehydro-DL-proline (DHP) on murine macrophage (M phi) FcR subclass-mediated antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis of sheep erythrocyte targets. Oil-elicited peritoneal M phi from C3HeB/FeJ mice which were cultured for 24 hr with DP (0.08 or 0.10 mM) or DHP (0.8 or 1.0 mM) showed a significant decrease in FcR subclass-mediated ADCC for murine monoclonal IgG2a (FcRI) and IgG2b/IgG1 (FcRII) as well as for heterologous polyclonal IgG. These collagen inhibitors also blocked phagocytosis mediated by both IgG2a- and IgG2b-opsonized erythrocytes. DP was more potent than DHP in blocking FcR effector functions in a reversible fashion and neither inhibitor affected M phi C3b receptor function. Pretreatment of M phi with collagenase resulted in significant reduction in FcR-mediated ADCC and phagocytosis. The inhibition of M phi FcR subclass-mediated ADCC and phagocytosis by collagen C1q synthetic inhibitors or by collagenase treatment further confirms a functional relationship between cell-associated C1q and FcR-dependent functions.

A cobra venom factor (CVF)-induced C3 convertase activity in the hemolymph of Galleria mellonella.

A cobra venom factor (CVF)-induced C3 convertase has been generated from the hemolymph of Galleria mellonella. CVF was immobilized on Sepharose 4B and treated with cell-free hemolymph obtained from either unvaccinated G. mellonella larvae or larvae immunized with formalized Pseudomonas aeruginosa. The C3-cleaving activity was detected by the ability to cleave the alpha-chain of bovine C3 in a manner analogous to the CVF-induced mammalian C3 convertase, CVF,Bb. The insect-derived C3 convertase formed at 28 degrees C but not at 37 degrees C, then once formed was active at both 28 degrees C and 37 degrees C. EDTA did not inhibit the formation and action of the insect derived C3 cleaving activity.

In vitro inhibition of the classical pathway of human complement by a natural microbial product, colistin sulphate.

Colistin sulphate was found to be an inhibitor of the classical pathway of the complement system. The main sites of inhibition were the interaction of EAC14 with C2 and EAC142 with C3. It also inhibited EAC14 formation from EA and C2-deficient serum, EAC1-7 formation from EAC1-3, C5, C6 and C7 and the interaction of EAC1-7 with C8 and C9, though less efficiently. It did not inhibit formation of C3/C5 convertase of the alternative pathway. The inhibition of the classical pathway was reversible since hemolytic activity was completely restored after dialysis.

Biosynthesis of the subcomponents C1q, C1r and C1s of the first component of complement (C1) by guinea pig hepatocyte primary cultures.

Thus far, the synthesis of C1q by liver cells has not been demonstrated. To investigate this possibility, viable hepatocytes were isolated from the liver of guinea pigs and primary cultures were established. The cells (10(6) cells/ml) were cultured under serum-free conditions for 8 days and the culture medium was changed every 24 h. The few contaminating Kupffer cells were lysed by preincubating the cell cultures with a monoclonal (22C4-8) antibody directed against a nonpolymorphic Ia determinant and preabsorbed rabbit serum. The hemolytic activity of C1 and its subcomponents C1q and C1r/C1s was tested in the supernatants. Guinea pig hepatocyte primary cultures synthesize and secrete up to 3 X 10(3) effective C1q molecules/cell/24 h and 34 X 10(3) effective C1r/C1s molecules/cell/24 h. The synthesis of C1q and C1r/C1s could be reversibly inhibited by cycloheximide (50 micrograms/ml). Furthermore, to demonstrate de novo synthesis of the C1q subcomponent, endogeneous labeling with 3H-proline (or 14C-proline) was performed. The immunoprecipitated C1q from cellular lysates and culture medium was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Compared to biosynthetically labeled guinea pig C1q from peritoneal macrophages, three corresponding bands (30, 28 and 24 kDa, respectively) were detectable in the fluorograph. The data show that guinea pig hepatocytes are able to synthesize C1 subcomponents, whereby the synthesis of C1q and C1r/C1s occurs independently.

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.

Membrane factors responsible for homologous species restriction of complement-mediated lysis: evidence for a factor other than DAF operating at the stage of C8 and C9.

Species-restricted lysis of complement refers to the relative inefficiency of complement to lyse cells from the homologous species. Restriction occurs at least at the steps involving C3/C5 convertase formation and the C9 insertion phase of the complement cascade, and is presumed to be mediated by inhibitory factors in the target cell membrane. In this study, we have examined whether decay accelerating factor (DAF), a membrane protein known to modulate C3/C5 convertase activities on cell surfaces, acts as a regulatory protein in species-restricted lysis of human erythrocyte (E). The role of DAF was assessed in homologous lysis by the classic pathway, in reactive lysis, and in lytic steps requiring C8 and C9. The results indicated that DAF participated in regulating C3/C5 deposition on the surface of homologous E, but had no effect on homologous restriction in reactive lysis and in the reaction of C8 and C9 with antibody-sensitized E C1-7. Treatment of E with pronase or with dithiothreitol (DTT) abolished the restricting effect of homologous C8/C9, indicating that species-restricted lysis by C5b-9 involves membrane factor(s) sensitive to pronase and DTT.

[Biosynthesis of the complement component C1q in mouse peritoneal macrophage cultures]. / Biosintez komponenta komplementa C1q v kul'turakh peritoneal'nykh makrofagov myshei.

Biosynthesis of C1q complement component by resident peritoneal macrophages from (CBA X C57BL/6)F1 mice has been studied in in vitro experiments. Using anti-mouse C1q antibodies immobilized on CNBr Sepharose it has been demonstrated that 14C glycine incorporates both into intracellular C1q and C1q secreted into the medium. The maximum radioactivity of intracellular C1q was observed 48 h after cultivation, with it dropping drastically between hours 72-96. Kinetics of radiolabelled C1q was similar, but 24 hours delayed. Cell viability during 96 h of cultivation remained unchanged. These data can be considered as the indication of feedback regulation of C1q biosynthesis at the cellular level.

Complement subcomponent C1q secreted by cultured human monocytes has subunit structure identical with that of serum C1q.

An enzyme-linked immunosorbent assay (e.l.i.s.a.) that is capable of quantifying C1q concentrations as low as 2 ng/ml and a sensitive haemolytic assay were used to study the appearance of material that cross-reacts with human serum C1q as well as C1q haemolytic activity in human monocyte culture media. This material was detected in the medium after 10-14 days and continued to be secreted through to day 28 of culture, at which time the cultures were terminated. Material specifically immunoabsorbed with Sepharose-anti-C1q antibody from a culture medium of cells that was metabolically labelled with [3H] proline or [35S] methionine demonstrated a polypeptide pattern identical with that of serum C1q on SDS/polyacrylamide-gel electrophoresis. Under non-reducing conditions two protein bands were detected migrating with the same Rf values as the serum C1q A-B and C-C dimers. On reduction three bands were evident, which migrated identically with the A, B and C chains of serum C1q. The amount of radioactivity in these bands increased with time in culture, consistent with the e.l.i.s.a. and haemolytic C1q assays. These bands were reactive with monospecific anti-C1q antibody after transfer to nitrocellulose.

Characteristics of complement subcomponents C1r and C1s synthesized by Hep G2 cells.

The association and activation states of complement subcomponents C1r and C1s biosynthesized by Hep G2 cells were studied. C1r and C1s are secreted in stoichiometric amounts; in the presence of Ca2+ they are associated in a complex that sediments similarly to plasma C1r2-C1s2. Both compounds are synthesized as monomer proteins of apparent Mr 86 000. C1r is secreted as a dimer. Secreted C1r is not autoactivatable but undergoes proteolysis by exogenous C1r; secreted C1s is also proteolysed by exogenous C1r. In the presence of immune-complex-bound C1q, secreted C1r and C1s are able to reconstitute C1, but normal activation requires extrinsic C1r2-C1s2.

Biosynthesis of complement protein D by HepG2 cells: a comparison of D produced by HepG2 cells, U937 cells and blood monocytes.

The biosynthesis of complement protein D of the alternative pathway by HepG2 cells, a human hepatocyte cell line, was studied and compared to the biosynthesis of D by U937 cells and blood monocytes. Increasing amounts of antigenic D were detected in HepG2 cell culture supernatants by radioimmunoassay. The kinetics of D synthesis and secretion by HepG2 cells was followed in a pulse-chase study using [35S]cysteine. As analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography, only a single D band was seen intra- and extracellularly and both forms had the same apparent molecular weight as D synthesized by U937 cells or purified from serum. Treatment of HepG2 and U937 cells with canavanine, an arginine amino acid analog, to inhibit intracellular processing resulted in slight depression of the apparent molecular weight of D synthesized by these cells. D synthesized by blood monocytes had an apparent molecular weight similar to that synthesized by HepG2 and U937 cells, suggesting that these cell lines do not synthesize and process D differently than normal monocytes. The data demonstrate that the hepatocyte is a site of D synthesis and suggest that D is not synthesized as a precursor molecule.

Characterization of C1q, C1s and C-1 Inh synthesized by stimulated human monocytes in vitro.

C1q, C1s and C1 Inh synthesized and secreted by human monocytes were characterized by SDS-PAGE. C1q is formed of three chains A (Mr approximately 35 000), B (Mr approximately 33 000) and C (Mr approximately 25 000) which are associated in two subunits A-B and C-C. It appears identical to C1q purified from plasma. C1s is secreted as a non-activated, monocatenar protein of Mr approximately 87 000 identical to proenzymic C1s from plasma. Secreted C1 Inh (Mr approximately 100 000) has a slightly higher Mr than purified plasmatic C1 Inh. Monensin treatment of the cells favours the intracytoplasmic accumulation of products at various glycosylation stages.