Complement components, but not complement inhibitors, are upregulated in atherosclerotic plaques.

Complement activation occurs in atherosclerotic plaques. The capacity of arterial tissue to inhibit this activation through generation of the complement regulators C1 inhibitor, decay accelerating factor, membrane cofactor protein (CD46), C4 binding protein (C4BP), and protectin (CD59) was evaluated in pairs of aortic atherosclerotic plaques and nearby normal artery from 11 human postmortem specimens. All 22 samples produced mRNAs for each of these proteins. The ratios of plaque versus normal artery pairs was not significantly different from unity for any of these inhibitors. However, in plaques, the mRNAs for C1r and C1s, the substrates for the C1 inhibitor, were increased 2.35- and 4.96-fold, respectively, compared with normal artery; mRNA for C4, the target for C4BP, was elevated l.34-fold; and mRNAs for C7 and C8, the targets for CD59, were elevated 2.61- and 3.25-fold, respectively. By Western blotting and immunohistochemistry, fraction Bb of factor B, a marker of alternative pathway activation, was barely detectable in plaque and normal arterial tissue. These data indicate that it is primarily the classical, not the alternative pathway, that is activated in plaques and that key inhibitors are not upregulated to defend against this activation.

Prostatic luminal cell differentiation and prostatic steroid-binding protein (PBP) gene expression are differentially affected by neonatal castration.

BACKGROUND: Although normal prostatic development is androgen-dependent, the prostate continues to grow in the neonate despite castration. However, the manner in which neonatal growth of the prostate occurs, in the absence of the testis, remains largely unknown. The purpose of this study was to examine the differentiation of prostatic epithelial cells after neonatal castration. METHODS: Immunohistochemistry was utilized to detect the expression of differentiation products: basal-cell cytokeratin (CK 5), luminal-cell cytokeratin (CK 18), and prostatic steroid-binding protein (PBP), a ventral prostate-specific marker indicative of secretory function in luminal cells. The reverse transcription-polymerase chain reaction was used to detect transcription products of the three polypeptide subunits of PBP, designated C1, C2, and C3. Rats were castrated on day 5 after birth, and ventral prostates were collected on day 14. Dihydrotestosterone was injected (100 microg/animal every 2 days) in castrated animals to determine if PBP expression could be initiated by androgen. RESULTS: Although no major effects of castration were detected on the differentiation of stromal or basal cells (which differentiate prior to day 5), castration had a pronounced effect on luminal-cell differentiation. Castration inhibited PBP protein expression, but did not affect the expression of luminal-cell cytokeratin (CK 18) protein. Furthermore, castration reduced C1, C2, and C3 transcription. Androgen replacement to castrated animals allowed for the initiation of PBP expression, although its onset was delayed. CONCLUSIONS: These observations indicate that the testis is not necessary for prostatic luminal-cell differentiation, but is necessary for full expression of luminal-cell secretory phenotype. Furthermore, our study suggests that factors of testicular origin, in addition to androgen, are needed for proper timing of PBP expression. This investigation establishes that the cytological and the physiological differentiation of the rat prostate are differentially regulated.

Complement C1s activation in degenerating articular cartilage of rheumatoid arthritis patients: immunohistochemical studies with an active form specific antibody.

OBJECTIVE: The first complement component C1s was reported to have novel functions to degrade matrix components, besides its activities in the classic complement pathway. This study explores participation of C1s in articular cartilage degradation in rheumatoid arthritis (RA). METHODS: Normal articular cartilage (n = 6) and cartilage obtained from joints with RA (n = 15) and osteoarthritis (OA, n = 10) were immunostained using anti-C1s monoclonal antibodies PG11, which recognises both active and inactive C1s, and M241, which is specifically reactive to activated C1s. The effects of inflammatory cytokines on C1s production by human articular chondrocytes were also examined by sandwich ELISA. RESULTS: In normal articular cartilage, C1s was negative in staining with both PG11 and M241. In contrast, degenerating cartilage of RA was stained with PG11 (14 of 15 cases), and in most of the cases (13 of 15 cases) C1s was activated as revealed by M241 staining. In OA, C1s staining was restricted in severely degrading part of cartilage (5 of 10 cases), and even in that part C1s was not activated. In addition, C1s production by chondrocytes in vitro was increased by an inflammatory cytokine, tumour necrosis factor alpha. CONCLUSION: These results suggest that C1s activated in degenerative cartilage matrix of RA but not in that of OA. C1s is thought to participate in the pathogenesis of RA through its collagenolytic activity in addition to the role in the classic cascade.

Immunological and genetic studies on hereditary angioedema.

Ca. eighty patients with hereditary angioedema due to C1-inhibitor deficiency were diagnosed and treated in Poland during last the 15 yrs, mainly in centers from Warsaw, Cracow and Bydgoszcz. The detailed description concerns 17 patients, 14 females (82%) and 3 males (18%). Important diagnostic points, response to treatment, and critical serum values of C1-inhibitor were evaluated. In the experimental part, we have established fibroblasts cell lines from 5 patients with type I HAE, with characteristic profound deficits of C1-INH (antigenic and functional) and C4. Fibroblasts from all the patients synthesized only ca. 20% of the normal values of C1-INH. We have confirmed the data from other authors that C1-INH synthesis can be substantially increased (11 to 20-fold) by IFN-gamma at a pretranslational level.

Assembly of the C1 complex.

The C1 complex of complement is a Ca(2+)-dependent complex protease comprising two loosely interacting subunits. C1q, the recognition subunit, is an hexameric protein with six peripheral globular domains, each connected through collagen-like "arms" to a central fibril-like "stalk". The catalytic subunit, C1s-C1r-C1r-C1s, is a Ca(2+)-dependent tetrameric association of two serine protease zymogens, C1r and C1s, that are sequentially activated by cleavage of a single peptide bond, upon binding of C1 to activators. Each monomeric protease is comprised of six structural motifs which form at least four domains, distributed in two functionally distinct regions, alpha (N-terminal) and gamma-B (C-terminal). The catalytic (gamma-B) regions of C1r and C1s are respectively located in the centre and at each end of the isolated tetramer, and the Ca(2+)-dependent C1r-C1s associations are mediated by the interaction (alpha) regions, which contain one Ca2+ binding site each. Physicochemical and electron microscopy studies indicate that the tetramer, which is highly elongated, folds into a more compact conformation upon interaction with C1q. Various models for C1 have been proposed, in which the tetramer either interacts with the outside part of the C1q arms (O- and W-shaped models), or is folded within the C1q arms (S- or 8-shaped models). These models are discussed in light of available information and in consideration of the structural requirements of C1 activation and function.

Regulation of the synthesis of C1 subcomponents and C1-inhibitor.

We have investigated the synthesis of C1q, C1r, C1s and C1-inhibitor in HepG2 cells, human umbilical vein endothelial cells (HUVEC), fibroblasts (skin and synovial membrane), chondrocytes and monocytes. C1q was only synthesised by monocytes, although the mRNAs for the C1qA and C1qC chains were expressed in HUVEC. C1r, C1s and C1-inhibitor were synthesised by all cell types. The secretion rates of C1r and C1s were approximately equimolar in fibroblasts and chondrocytes whereas the secretion rate for C1s exceeded that for C1r in the other cell types. Molar ratios of C1s to C1r were approximately 2:1 for HepG2 cells, 5:1 for monocytes and 10:1 for HUVEC. Stimulation with interferon-gamma resulted in increased expression of all four proteins. The C1s:C1r ratio did not alter in chondrocytes or fibroblasts, but approached unity in HepG2, monocytes and HUVEC, due to relatively greater stimulation of C1r gene expression.

Chemical characterization and location of ionic interactions involved in the assembly of the C1 complex of human complement.

The C1 complex of human complement comprises two loosely interacting subunits, C1q and the Ca(2+)-dependent C1s-C1r-C1r-C1s tetramer. With a view to gain information on the nature of the ionic interactions involved in C1 assembly, we have studied the effects of the chemical modifications of charged residues of C1q or the tetramer on their ability to reconstitute the C1 complex. Treatment of C1q with pyridoxal-5'-phosphate, acetic anhydride, and citraconic anhydride, as well as with cyclohexanedione and diethylpyrocarbonate, inhibited its ability to associate with C1s-C1r-C1r-C1s. Treatment of the collagen-like fragments of C1q with the same reagents yielded the same effects. Treatment of C1s-C1r-C1r-C1s with 1-ethyl-3-[-3-(dimethylamino) propyl] carbodiimide also prevented C1 assembly, through modification of acidic amino acids which were shown to be located in C1r. Further studies on the location of the interaction sites within C1q, using ligand-blotting and competition experiments with synthetic peptides, were unsuccessful, suggesting that these sites are contributed to by two or three of the C1q chains. It is concluded that C1 assembly involves interactions between acidic amino acids of C1r and lysine (hydroxylysine) and arginine residues located within the collagen-like region of C1q. Sequence comparison with mannan binding protein, another collagen-like molecule which binds the C1s-C1r-C1r-C1s tetramer, suggests Arg A38, and HyL B32, B65, and C29 of C1q as possible interaction sites.

Perioperative alterations in polymorphonuclear leukocyte function of gastrointestinal surgery.

To characterize the alteration of perioperative polymorphonuclear leukocytes (PMNs) function in surgical stress, we studied twenty six patients undergoing gastrointestinal surgery. Seventeen patients with thoracic esophageal cancer underwent total thoracic esophagectomy through a right thoracotomy (severe surgical stress group). Nine patients underwent cholecystectomy (slight surgical stress group). Phagocytic oxygen-dependent microbicidal activity in the esophagectomy patients significantly increased postoperatively, by measuring O2- production (35.3 +/- 7.0 nmol/10(6) cells/ml/20 min on postoperative day 1 vs. 28.6 +/- 6.2 preoperatively, p < 0.01) and luminol-dependent chemiluminescence (99.5 +/- 29.9 x 10(5) cpm/10(5) cells on postoperative day 3 vs. 67.5 +/- 12.8 preoperatively, p < 0.01). On the other hand, only a slight change was seen in the cholecystectomy patients. We conclude that the postoperative PMN function in terms of oxygen-dependent microbicidal activity significantly increases when the degree of surgical stress is sufficient. In order to gain insight into the mechanism of PMN activation, we specifically analyzed the expression of complement receptors. Up-regulation of complement receptors were seen in the esophagectomy patients, which parallels the activation of PMN microbicidal activities. It was suggested that phagocytic PMN function in severe surgical stress significantly increases postoperatively, in part, based on the upregulation of cell surface complement receptors.

Identification of a human non-interferon lymphokine activating monocyte complement biosynthesis.

A monocyte-stimulating activity produced by mitogen-induced mononuclear cells has been defined by its ability to enhance the synthesis in vitro of complement C1 subcomponents, C2 and C3. A lymphokine responsible for this activity was purified from culture supernatants of peripheral blood mononuclear cells activated by staphylococcal enterotoxin A. From 0.5 litre of supernatant the purification procedure [(NH4)2SO4 precipitation, phenyl-Sepharose chromatography and preparative electrofocusing] yielded about 100 pmol of purified lymphokine. Its pI is 7.9 and its Mr, estimated by SDS/polyacrylamide-gel electrophoresis, is 14,600, 27,000 and 56,000, the high-Mr species representing oligomeric forms of the Mr-14,600 molecule. Its amino acid analysis reveals a high percentage of hydrophobic amino acids (34%); the absence of histidine residues suggests that it is a novel monocyte-activating lymphokine. It enhances C1r and C1s biosynthesis at a pretranslational level. From its structure and activity this lymphokine appears different from gamma-interferon.

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.

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.

Synthesis of complement by macrophages and modulation of their functions through complement activation.

During the last decade considerable progress has been made to characterize intimate functional links between macrophages, a major cellular component of immunoinflammatory responses, and the complement system representing the major humoral mediator of inflammation. Macrophages of various species and tissue sites have been shown to synthesize and release most of the complement components providing these cells with their own "pericellular" complement system. Circumstantial evidence for the assembly of both classical and alternative pathway convertases has been adduced. An intricate network of feedback loops involving endogenous and extrinsic factors operates to adjust complement production to acute requirements, for example augmenting production in the face of accelerated turnover at sites of inflammation, and returning it to baseline levels once the inflammatory stimulus has subsided, in order to maintain a fine-tuned balance. The molecular mechanisms underlying regulation of complement synthesis by macrophages are beginning to be elucidated by use of gene technology. On the other hand, complement activation products exert a number of effects on macrophages via specific surface receptors causing internalization of offending agents, microbes, and immune complexes, promotion of intracellular killing, controlling migration behavior, inducing release of potent biologic substances such as lysosomal enzymes, arachidonic acid metabolites, and interleukin 1. In these interactions, two important humoral mediator systems of inflammation, the complement system and the arachidonic acid cascade, are functionally linked at the level of the macrophage. Stimulation of the release of immunomodulating compounds from macrophages invoke a role for complement in immune regulation. This multifaceted interplay is of particular importance considering the mobility of macrophages that allows them to gain almost unrestricted access to sites of ongoing immunoinflammatory responses. The time seems to have come to abandon the petrified thinking in socalled systems as, for instance, humoral versus cellular, specific versus unspecific, and to proceed to interlocking functions guided by physiology proper.

Production of first component of complement by corneal fibroblasts in tissue culture.

Corneal fibroblasts were studied to determine if they have the ability to synthesize and secrete complement components in tissue culture. Culture media were assayed for functional complement activity of C1, C4, C2, C3, C5, C6, and C7 with the use of 50% hemolysis of sensitized sheep RBCs. Only C1 showed a progressive increase in hemolytic activity at days 3, 5, 7, 9, and 11 of tissue culture. This increase in hemolytic C1 could be reversibly inhibited by cycloheximide, an inhibitor of protein synthesis. Corneal fibroblasts may be a potential source of C1 in the cornea.