C1 esterase inhibitor gene expression in rat Kupffer cells, peritoneal macrophages and blood monocytes: modulation by interferon gamma.

Kupffer cells (KC) represent the main part of the tissue macrophages. Beside phagocytosis of particulate material, involvement of KC in immunological and inflammatory reactions has been supposed. As C1 esterase inhibitor (C1-INH) is a serine protease inhibitor involved in such processes, the aim of this work was to study C1-INH synthesis in KC and, by comparison, in peritoneal macrophages (PM) and blood monocytes (MC) of the rat. C1-INH synthesis was studied on the protein level by biosynthetic labeling, immunoprecipitation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and on the RNA level by Northern blotting of total RNA or by in situ hybridization. KC were found to express C1-INH gene spontaneously. C1-INH synthesis represents 1.3 +/- 0.2% of total protein synthesis at day 1 of culture and the absolute amount each cell synthesis remains constant during the whole time in culture. Transcripts of C1-INH were detected both in freshly isolated and in cultured KC. In contrast, spontaneous C1-INH gene expression was not detectable in freshly isolated PM, but only in cultured PM. In MC, C1-INH was not detectable at any time, whatever. Treatment of the cells with interferon gamma increased C1-INH synthesis in KC and in PM and caused an induction of C1-INH synthesis in MC. The results suggest that constitutive C1-INH synthesis is a functional marker for mature tissue macrophages.

Cloning and molecular characterization of lycC1INH genes in large yellow croaker (Pseudosciaena crocea).

Complement component 1 inhibitor (C1INH) is a crucial protein in controlling activation of many plasma mediator pathways and can directly interact with Gram negative bacteria. The full-length cDNA of lycC1INH gene was identified from the large yellow croaker. It is of 2046 nucleotides (nt) encoding a protein of 599 amino acids, with a 5'-untranslated region of 99 nt and a 3'-untranslated region of 147 nt including the poly (A) tail. The deduced protein contains a C-terminal serpin (serine protease inhibitor) domain, and two N-terminus immunoglobulin domains without significant homology to other species. Western blot analysis of the protein expression showed that the expression of lycC1INH was obviously up-regulated in liver, spleen and head kidney of the fish challenged by attenuated live Vibrio anguillarum strain. This indicated that lycC1INH might be involved in the immune response of large yellow croaker to bacterial challenge.

Synthesis of C1 inhibitor in fibroblasts from patients with type I and type II hereditary angioneurotic edema.

Patients with hereditary angioneurotic edema (HANE) have serum levels of functionally active inhibitor of the first component of complement (C1 INH) between 5 and 30% of normal, instead of the 50% expected from the single normal allele. Increases in rates of catabolism have been documented in patients with HANE and certainly account for some of decrease in C1 INH level. A possible role for a decrease in synthesis of C1 INH in producing serum levels of C1 INH below the expected 50% of normal has not been well studied. We studied the synthesis of C1 INH in skin fibroblast lines, which produce easily detectable amounts of C1 INH. In type I HANE cells, C1 INH synthesis was 19.6 +/- 4.0% (mean +/- SD) of normal, much less than the 50% predicted. In type II HANE cells, the total amount of C1 INH synthesis (functional and dysfunctional) was 98.9 +/- 17% of normal; the functional protein comprised 43% of the total. Thus, type II HANE cells synthesized functional C1 INH at a much greater rate than for the type I cells. In both type I and II HANE cells, amounts of steady-state C1 INH mRNA levels paralleled rates of C1 INH synthesis, indicating that control of C1 INH synthesis occurred at pretranslational levels. Both type I and type II fibroblasts synthesized normal amounts of C1r and C1s. These data suggest that the lower than expected amounts of functionally active C1 INH in type I HANE may be due, in part, to a decrease in rate of synthesis of the protein, and that the expressions of the normal C1 INH allele in HANE is influenced by the type of abnormal allele present.

Behavior in vivo of normal and dysfunctional C1 inhibitor in normal subjects and patients with hereditary angioneurotic edema.

The metabolism of normal C1 inhibitor and two dysfunctional C1 inhibitors (Ta and WeI) was studied in 10 normal subjects and 8 patients with hereditary angioneurotic edema (HANE), 4 with low antigen concentration (type 1) and 4 with dysfunctional protein (type 2). The fractional catabolic rate of the normal C1 inhibitor in normal subjects was 0.025 of the plasma pool/hour, whereas in HANE subjects it was significantly elevated at 0.035 of the plasma pool/hour. The synthesis of normal C1 inhibitor was decreased in patients with type 1 HANE (0.087 mg/ kg per h compared with 0.218 mg/kg per h). The fractional catabolic rate of dysfunctional protein WeI was similar to normal and showed a slightly accelerated catabolism in patients with HANE, whereas the dysfunctional protein Ta had a strikingly decreased fractional catabolic rate in normals and subjects with HANE. The present study is compatible with reduced C1 inhibitor synthesis in patients with type 1 HANE consistent with a single functional C1 inhibitor gene. The lower than anticipated levels of C1 inhibitor in HANE type 1 appears to result from (a) the single functional gene and (b) increased catabolism of the protein, perhaps related to activation of C1 or other proteases.

Transinhibition of C1 inhibitor synthesis in type I hereditary angioneurotic edema.

To ascertain the mechanism for decreased synthesis of C1 inhibitor (C1 INH) in certain patients with the autosomal dominant disorder hereditary angioneurotic edema, we studied expression of C1 INH in fibroblasts in which the mutant and wild type mRNA and protein could be distinguished because of deletion of exon 7 (delta Ex7). In the HANE delta Ex7 cells, the amount of wild type mRNA (2.1 kb) was expressed at 52 +/- 2% (n = 5) of normal, whereas the mutant mRNA was 17 +/- 1% (n = 5) of normal. Rates of synthesis of both wild type and mutant proteins (11 +/- 3 and 3 +/- 1% of normal, respectively) were lower than predicted from the mRNA levels. There was no evidence of increased C1 INH protein catabolism. These data indicate that there are multiple levels of control of C1 INH synthesis in type I hereditary angioneurotic edema. Pretranslational regulation results in < 50% of the mutant truncated 1.9-kb mRNA. In addition, translational regulation results in decreased synthesis of both wild type and mutatn C1 INH proteins. These data suggest a transinhibition of wild type C1 INH translation by mutant mRNA and/or protein.

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.

Hormonal regulation of complement biosynthesis in human cell lines--II. Upregulation of the biosynthesis of complement components C3, factor B and C1 inhibitor by interleukin-6 and interleukin-1 in human hepatoma cell line.

The effect of interleukin (IL)-6 and IL-1 on the biosynthesis of complement components C3, factor B, C2, C4 and C1 inhibitor (C1 inh), as well as that of albumin, was studied in vitro in human hepatoma-derived cell line, HepG2. Measuring the amounts of secreted complement proteins we detected a significant upregulation of C3 by both hormones. The enhancement of the factor B and especially that of C1 inh production was predominant by IL-6. In our experimental system neither IL-1 nor IL-6 affected the biosynthesis of C2 and C4. Albumin secretion was significantly decreased only in the simultaneous presence of IL-1 and IL-6. Detection of the changes in the amounts of C3- and factor B-specific mRNA of HepG2 cells suggests a pretranslational regulation by these cytokines. The secretion of C3 and factor B was markedly potentiated when IL-1 and IL-6 were added together. However only the gene expression of factor B, but not of C3, was found to reveal synergism. IL-6 enhanced the in vitro production of C3 in mouse hepatocytes as well. This effect was greatly potentiated in the presence of histamine.

Hormonal regulation of complement biosynthesis in human cell lines--I. Androgens and gamma-interferon stimulate the biosynthesis and gene expression of C1 inhibitor in human cell lines U937 and HepG2.

C1 inhibitor (C1inh), a member of the serine protease inhibitor gene superfamily, is a glycosylated plasma protein inhibiting the proteolytic activities of C1r and C1s and involved in the regulation of coagulation, fibrinolysis and kinin-releasing systems. In this study, the in vitro effect of androgen hormones, dehydroepiandrosterone (DHEA), testosterone (TEST) and recombinant human gamma-interferon (gamma-IFN), has been determined on the production of C1inh in human cell lines. In both human monocytoid/histiocytoid cell line U937 and in hepatoma derived cell line HepG2, DHEA and TEST upregulated the gene expression and secretion of C1inh. The most pronounced effect was detected in the concn range 10(-7)-10(-9) M of the hormones. Under the same conditions DHEA and TEST had no detectable effect on the biosynthesis of C3, C2 and factor B by these cells, but DHEA at higher concn (10(-4) M) slightly increased that of C4 in HepG2 cells. Both in U937 and in HepG2 cells recombinant gamma-IFN markedly increased the gene expression and secretion of C1inh. This effect of gamma-IFN was abolished by histamine.

Synthesis of complement proteins in amnion.

The amnion is a metabolically active tissue that has been identified as a site of synthesis of numerous products. We report that amnion tissue explants and amnion-derived epithelial cells synthesize and secrete six proteins of the complement system, C1r, C1s, C1 inhibitor, factor B, C3, and factor H. Synthesis of C2 was minimal and variable, and C5 was not detected. The six synthesized proteins had size and subunit composition characteristic of proteins synthesized in HEp2, a long term cell line derived from malignant epithelial cells. Constitutive and regulated synthesis of five of the six proteins was similar in amnion tissue and cells. However, synthesis of factor B was different in tissue and cells; constitutive synthesis was 12-fold higher in tissue than in cells, and interleukin-1 did not alter synthesis in tissue, but increased synthesis by 11.7-fold in cells. These results indicate that amnion may be a source of complement proteins present in the amnion fluid and may contribute to local host defense along with endometrial glandular epithelial cells, which synthesize C3. Furthermore, our results suggest that amnion tissue is stimulated in vivo to synthesize factor B and cannot respond to interleukin-1 with a further increase in the synthesis rate.

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.

Inhibition of plasma kallikrein by C1-inhibitor: role of endothelial cells and the amino-terminal domain of C1-inhibitor.

Activation of plasma prekallikein and generation of bradykinin are responsible for the angioedema attacks observed with C1-inhibitor deficiency. Heterozygous individuals with <50% levels of active C1-inhibitor are susceptible to angioedema attacks indicating a critical need for C1-inhibitor to be present at maximum levels to prevent unwanted prekallikrein activation. Studies with purified proteins do not adequately explain this observation. Therefore to investigate why reduction of C1-inhibitor to levels seen in angioedema patients results in excessive kallikrein generation we examined the effect of endothelial cells on the inhibition of kallikrein by C1-inhibitor. Surprisingly, it was found that a C1-inhibitor concentration of greater than 1 microM was needed to inhibit 3 nM kallikrein. We propose that this apparent protection from inhibition was mediated by kallikrein binding to the cells via the heavy chain in a high molecular weight kininogen and zinc independent manner. Protection of kallikrein from inhibition was not observed when C1-inhibitor truncated in the amino-terminal domain by the StcE metalloproteinase was used, which suggests a novel function for this unique domain. The requirement for high concentrations of C1-inhibitor to fully inhibit kallikrein is consistent with the fact that reduced levels of C1-inhibitor result in the kallikrein activation seen in angioedema.

Increased expression of C1-inhibitor mRNA in patients with hereditary angioedema treated with Danazol.

The attenuated androgen Danazol can partially reverse the biochemical defect and prevent angioedema in patients with inherited C1-inhibitor (C1-INH) deficiency (hereditary angioedema, HAE). Though its clinical effectiveness is independent from significant increase of C1-INH plasma levels, its mechanism of action remains unknown. Since angioedema is a local phenomenon, it could be controlled by restoring tissue levels of C1-INH. We measured the expression of C1-INH mRNA in peripheral blood mononuclear cells (PBMCs) of 13 patients with HAE type 1 (seven untreated and asymptomatic, and six on Danazol at the minimal effective dose) and of eight normal controls. mRNA levels were quantitated by computerized optical densitometry of reverse transcriptase-PCR products, normalized for the amount of glyceraldehyde-3-phosphate-dehydrogenase and expressed as percent of normal pooled RNAs. Each determination represented the mean of three separate experiments. Measurement of C1-INH mRNA in two patients before and after 1 month of Danazol 400 mg per day demonstrated a post-treatment increase of 15 and 21%, respectively. When HAE patients and controls were analyzed as groups, C1-INH mRNA levels of patients untreated and asymptomatic (median 73%, range 65-78) were significantly lower (P=0.001) compared to controls (median 101%, range 87-121) and to patients on Danazol (median 91%, range 82-96); the difference among the last two groups was not statistically significant. Our data demonstrate that minimal effective doses of Danazol increase the expression of C1-INH mRNA in PBMC of HAE patients even in the absence of a significant increase of C1-INH plasma levels.

The effect of WSEWS pentapeptide and WSEWS-specific monoclonal antibodies on constitutive and IL-6 induced acute-phase protein production by a human hepatoma cell line, HEPG-2.

Interleukin-6 receptor (IL-6R) is a member of the cytokine receptor superfamily characterised by the obligatory presence of WSXWS (Trp-Ser-X-Trp-Ser) sequence motif near the transmembrane domain. To more clearly understand the role of this motif, we treated the HepG2 hepatoma cell line with synthetic WSEWS peptide (E is glutamic acid) and checked the spontaneous and IL-6-induced production of acute-phase protein fibrinogen and C1-inhibitor (C1-INH). The peptide revealed a definitely stimulatory effect both on the constitutive synthesis of C1-INH and on the IL-6-induced fibrinogen synthesis of HepG2 cells. Monoclonal antibody specific for WSEWS pentapeptide was stimulatory for the spontaneous secretion of both fibrinogen and C1-INH. However, the IL-6-induced elevations of these acute-phase proteins were oppositely regulated, since the anti-WSEWS monoclonal antibody was inhibitory on the production of fibrinogen induced by IL-6 but strongly augmented the IL-6 induced production of C1-INH. Our study indicates that the WSEWS motif is critical in the effect of IL-6 on the acute-phase protein production influencing either the ligand binding by the WSEWS-containing receptor molecule or the signal transduction.

Endothelial cells cultured from human brain microvessels produce complement proteins factor H, factor B, C1 inhibitor, and C4.

The inflammatory mediators, cytokines and complement proteins are believed to regulate the sequential events during the development of lesions secondary to ischaemia and reperfusion. The endothelial cell monolayer of the brain microvasculature is the critical interface between the blood-borne mediators and brain tissue. The involvement of these cells in complement production and regulation has not been well documented. In the present study, expression of complement proteins (C1 inhibitor, factor H, factor B, C4) by cultured endothelial cells obtained from human brain microvessels has been characterized. Interferon gamma upregulates the production of all the complement factors studied. Serine proteases, plasmin and miniplasmin induce the expression of C4, decrease the level of ELISA detectable C1 inhibitor, and do not affect the production of factors H and B. These data indicate that complement proteins are expressed locally by the brain microvessels, and may modulate the inflammatory responses of brain tissue.

Regulation of C1 inhibitor synthesis.

The primary biologic roles of C1 inhibitor (C1-INH) are the regulation of activation of the classical complement pathway and of the contact system of kinin formation. Heterozygosity for deficiency or dysfunction of C1-INH results in hereditary angioedema (HAE). This deficiency results in loss of homeostasis with unregulated complement and contact system activation. Due to the consequent C1-INH consumption, plasma levels of C1-INH in patients with HAE are decreased below 50% of normal. In addition, diminished synthesis contributes to the lowered levels in some patients. The hepatocyte is the primary source of C1-INH, although a number of other cell types, including peripheral blood monocytes, microglial cells, fibroblasts, endothelial cells, the placenta, and megakaryocytes also synthesize and secrete the protein both in vivo and in vitro. Interferon-gamma and alpha (IFN), colony stimulating factor-1, interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) all induce C1-INH synthesis in a variety of cell types. The IFN-response elements in the 5'-flanking region and in the first intron have been partially characterized, as have several of the promoter elements that direct basal transcription of the gene. However, although androgen therapy, in vivo, results in an increase in C1-INH plasma levels, a direct effect of androgens on C1-INH synthesis has not been convincingly demonstrated. Although the C1-INH gene contains a potential glucocorticoid/androgen response element, this element does not appear to respond to androgen. Continued analysis of the transcriptional regulation of the C1-INH gene may lead to new approaches to therapy of HAE.

Autoimmune angioedema: a new role for autoantibody in disease pathogenesis.

Angioedema may be due to hereditary forms of Cl-Inh deficiency, but recently an autoimmune form of angioedema has been described in which the mechanism is novel. While the peripheral blood monocytes of patients with autoimmune angioedema produce a normal, functionally active, 105 KD Cl-Inh in normal quantities, the Cl-Inh isolated from the patient's plasma exists in a dysfunctional lower molecular weight (96 KD) performance. Rather than bind and biologically inactivate the enzyme, a relatively common phenomenon in autoimmune disease, the autoimmune angioedema cleave the Cl-Inh molecule. The following sequence of events is proposed: structural and functionally normal Cl-Inh is synthesised and secreted, this secreted inhibitor is complexed by autoantibody and following enzyme interaction, denatured 96 KD Cl-Inh is proposed. This process depletes the pool of normal, functional Cl-Inh to critical levels and predisposes patients to episodes of oedema.

Complement C1 inhibitor is produced by brain tissue and is cleaved in Alzheimer disease.

C1 inhibitor was identified in human brain tissue by Western blotting and by immunohistochemistry using multiple antibodies to the native protein. The presence of C1 inhibitor mRNA was identified by reverse transcriptase-polymerase chain reaction analysis of brain mRNA extracts. The mRNA was also detected in cultured postmortem human microglia and in the IMR-32 human neuroblastoma cell line. Immunohistochemically, the native protein was detected in residual serum of capillaries and pyramidal neurons of both control and Alzheimer disease cases, as well as in occasional senile plaques of Alzheimer tissue. The reacted protein was detected on dystrophic neurites and neuropil threads in Alzheimer tissue by 4C3 monoclonal antibody, which recognizes a neoepitope following suicide inhibition. These data indicate that C1 inhibitor, a regulatory molecule controlling multiple inflammatory proteolytic cascades, is produced in normal brain. In Alzheimer disease, C1 inhibitor undergoes a prominent reaction in abnormal neuronal processes, such as dystrophic neurites and neuropil threads.

[Efficient expression of human C1-inhibitor in CHO cells by using a dicistronic expression vector].

The full-length cDNA fragment encoding human C1-INH was obtained by gene recombination techniques and a stable expression plasmid was constructed by inserting the human C1-INH cDNA into an efficient dicistronic expression vector pED. This plasmid was transfected into DHFR-deficient Chinese Hamster Ovary cells (CHO-dhfr-) by Lipofectin method, and the positive CHO-dhfr+ cell clones which stably express C1-INH was generated. Expression of C1-INH mRNA was detected by Northern blot. Expression of C1-INH protein was confirmed by immunoprecipitation and Western blot analysis. The level of expressed C1-INH was estimated at 0.4 microgram/ml by specific ELISA techniques. The biological activity of recombinant C1-INH was assayed by using the inhibition of C1 estrolytic activity.

Characterization of recombinant human C1 inhibitor secreted in milk of transgenic rabbits.

C1 inhibitor (C1INH) is a single-chain glycoprotein that inhibits activation of the contact system of coagulation and the complement system. C1INH isolated from human blood plasma (pd-hC1INH) is used for the management of hereditary angioedema (HAE), a disease caused by heterozygous deficiency of C1INH, and is a promise for treatment of ischemia-reperfusion injuries like acute myocardial or cerebral infarction. To obtain large quantities of C1INH, recombinant human C1INH (rhC1INH) was expressed in the milk of transgenic rabbits (12 g/l) harboring genomic human C1INH sequences fused to 5' bovine αS(1) casein promoter sequences. Recombinant hC1INH was isolated from milk to a specific activity of 6.1 U/mg and a purity of 99%; by size-exclusion chromatography the 1% impurities consisted of multimers and N-terminal cleaved C1INH species. Mass spectrometric analysis of purified rhC1INH revealed a relative molecular mass (M(r)) of 67,200. Differences in M(r) on SDS PAGE and mass spectrometric analysis between rhC1INH and pd-hC1INH are explained by differential glycosylation (calculated carbohydrate contents of 21% and 28%, respectively), since protein sequencing analysis of rhC1INH revealed intact N- and C-termini. Host-related impurity analysis by ELISA revealed trace amounts of rabbit protein (approximately 10 ppm) in purified batches, but not endogenous rabbit C1INH. The kinetics of inhibition of the target proteases C1s, Factor XIIa, kallikrein and Factor XIa by rhC1INH and pd-hC1INH, indicated comparable inhibitory potency and specificity. Recently, rhC1INH (Ruconest(®)) has been approved by the European Medicines Agency for the treatment of acute attacks of HAE.