An initiator element and a proximal cis-acting sequence are essential for transcriptional activation of the complement factor I (CFI) gene.

Human complement factor I (CFI) is a serine protease which regulates the complement system by inactivation of C3b and C4b in the presence of appropriate cofactors. In this study, we have analyzed the mechanism controlling the constitutive transcriptional activation of the CFI gene. Using deletion analysis and transient CAT expression assays, we have mapped the minimal promoter to the region located between -46 and +160 bp relative to the major transcription start point (tsp), and also shown that cis-acting elements both upstream and downstream of the tsp are important for promoter activity. A silencer element was also found between -71 and -46 bp. The sequence surrounding the tsp was related to the mouse terminal deoxynucleotidyltransferase initiator element (Inr) and point mutations in this sequence, from -3 to +4, drastically reduced CFI promoter activity. Mutations in a -9 to -5 bp CTGAA sequence immediately upstream of the tsp also reduced promoter activity. Gel mobility shift analysis demonstrated the binding of nuclear factors to a CTGAA repeat located at -9 to -5 and +101 to +105. Our results suggest that CFI promoter contains a functional Inr element that is essential for promoter activity, and the interactions of the CTGAA element located between -9 and +5 with nuclear factor(s) may be part of the machinery required for CFI Inr-dependent transcription.

Transcriptional and post-transcriptional regulation of complement factor I (CFI) gene expression in Hep G2 cells by interleukin-6.

We have investigated the effects of IL-1 and IL-6 on human complement factor I (CFI) production by Hep G2 cells. IL-6 treatment caused a dose- and time-dependent increase in CFI secretion while IL-1 did not demonstrate such effects. The increase in CFI synthesis correlated with increase in CFI mRNA levels. The half-life of CFI mRNA in untreated cells was approx. 23 h and this was increased to 31 h (26% increase) following induction with IL-6. The IL-6 induced increase in CFI gene expression was inhibited by actinomycin D indicating regulatory effects at the level of transcription. Nuclear run-on experiments showed that IL-6 increased the rate of CFI gene transcription 4.2-fold. Transient transfection analysis of chloramphenicol acetyltransferase reporter gene constructs containing truncated segments of the 5'-flanking region of CFI gene showed that the cis-acting sequence(s) controlling the IL-6 inducible transcription resides in an 83 bp region located between -738 bp and -655 bp relative to the transcription start site. Our results indicate that the upregulation of CFI gene expression by IL-6 involves a coordinate effort at the level of transcription and mRNA stability, with the enhanced rate of transcription being the principal mechanism.

Cloning and characterization of the promoter for the human complement factor I (C3b/C4b inactivator) gene.

Complement factor I is a serine proteinase that regulates the classical and alternative pathways of complement by cleaving C3b and C4b and preventing the assembly of C3 and C5 convertase enzymes. In order to understand the regulation of factor I gene expression in liver cells, 4kb of the 5' flanking region of the gene was cloned, and the 1474-bp 3'-end was sequenced and shown to contain a number of transcription factor consensus sequences. A major and two minor transcription start sites were identified, respectively, at 152, 178, and 198bp upstream of the translation start site by primer extension analysis. The transcriptional activity of the 1474-bp fragment was analyzed by fusion of 5' deletion constructs to a cat-encoding gene expression vector and transient transfections into Hep G2 cells. A 273-bp fragment located at -112 to +161 relative to the major transcription start site was sufficient for promoter activity. The 3' fragment spanning +3 to +161 and containing a TATA-like element did not demonstrate promoter activity, suggesting that the core promoter resides in a 115-bp sequence located between -112 and +3. This region contains an Inr-like element overlapping the major cap site and a CTF-NF1 element, two potential CCAAT boxes and an AP-2 element partially overlapping an Sp-1 site. Thus, factor I promoter may belong to the TATA-less Inr-driven class II promoters whose transcription is regulated by Sp-1. The transcriptional activity of the 1474-bp 5' flanking fragment was upregulated by PMA, IL-6 and TNF-alpha, suggesting that factor I may be an acute phase reactant.

Biosynthesis of complement factor P (properdin) by the human pre-monocyte cell line (U-937).

The human monocyte-like cell line, U-937, is known to differentiate into macrophage-like cells following stimulation with phorbol myristate acetate (PMA) or interferon-gamma (IFN-gamma). The activated cells have been reported to have enhanced capacity to synthesize C2, C3, Factors B and H. Here, U-937 cells were used as a model system to investigate the effects of immunomodulatory agents on the biosynthesis of Factor P by monocytoid cells. Non-stimulated U-937 cells progressively secreted increasing amounts of Factor P over a 72-hr culture period. The secreted Factor P was hemolytically active. The daily production of Factor P was nearly linear (approx. 2.1 +/- 0.2 ng/10(6) cells; mean +/- SEM). Factor P synthesis was reversibly inhibited by cycloheximide indicating de novo synthesis. Both secreted Factor P and Factor P in normal plasma contained Factor P of heterogeneous molecular sizes and eluted from Sephacryl S-300 gel filtration column as a broad peak (mol. wt 250-800 kDa). The synthesis of Factor P by U-937 cells was augmented 1.8-, 2.1- and 2.5-fold respectively following induction with PMA (30 ng/ml), IFN-gamma (100 U/ml) and LPS (0.1 microgram/ml). Metabolic labeling of U-937 cells and autoradiograms of SDS-PAGE analysis of Factor P immunoprecipitates demonstrated a 54 kDa band in the culture supernate, co-migrating with purified 125I Factor P. Intracellular Factor P however had an apparent mol. wt that was 4000 kDa smaller than secreted Factor P. Thus U-937 cells synthesize a precursor Factor P subunit polypeptide chain which undergoes post-synthetic glycosylation and polymerization to give rise to the oligomers characteristic of native Factor P in fresh plasma. Our data also demonstrate that Factor P synthesis by monocytic cells can be enhanced by immunomodulatory factors or mediators that are generally found at sites of inflammation and immune response.

Regulation of complement factor H synthesis in U-937 cells by phorbol myristate acetate, lipopolysaccharide, and IL-1.

The capacity of the human monocyte cell line U-937 to synthesize complement factor H was examined. The kinetics of secretion of factor H into cell culture supernatant were followed by a sensitive solid phase RIA capable of measuring 0.1 ng of protein. Daily secretion of factor H was low and almost linear and was approximately 3 ng of factor H per 10(6) cells. Factor H synthesis was inhibited by cycloheximide but returned to the levels seen in untreated cultures after removal of the inhibitor. LPS and IL-1 both effected a time- and dose-dependent enhancement of factor H synthesis. Induction of U-937 cells with PMA to differentiate into macrophage-like cells also resulted in increased factor H synthesis. RIA of cell lysates, immunofluorescence microscopy, as well as FACS analysis all revealed that factor H Ag was also associated with the U-937 cell membrane. The population of U-937 cells bearing membrane-associated factor H was decreased from 77 to 43% after stimulation for 48 h with LPS (1 microgram/ml). [35S]Methionine metabolic labeling and SDS-PAGE analysis of factor H immunoprecipitates from unstimulated and stimulated culture supernatants and cell lysates demonstrated a major polypeptide, m.w. 150,000, and a minor component, m.w. 42,000. Western blot analysis of factor H in fresh normal plasma also detected both 150,000 and 42,000 m.w. factor H proteins. This is in agreement with the recent demonstration of a 4.4- and 1.8-kb mRNA for factor H in human liver. These data demonstrate that U-937 cells synthesize factor H that is structurally and antigenically similar to factor H in normal plasma. The exact nature of the membrane-bound factor H and its functions and mechanism of attachment to the cell membrane remain to be elucidated.

Biosynthesis of the third component of complement by the human monocyte-like cell line, U-937.

The human monocyte-like cell line, U-937, was shown to synthesize and secrete C3 by hemolytic assays, radioimmunoassays and metabolic labeling experiments. The daily synthesis of antigenic C3 by unstimulated U-937 cells was low (about 3 ng/10(6) cells/24 hr) over a 3 day period. Induction of the cells to differentiate into macrophage-like cells with phorbol myristate acetate (PMA), resulted in 5-fold augmentation of C3 synthesis and secretion into the culture medium. Using a plaque assay for enumerating C3 production by single cells, approx. 5% of unstimulated U-937 cells were found to secrete hemolytically active C3. The proportion of C3-plaque forming cells was increased about 6-fold in PMA stimulated cells. The synthesis of C3 by U-937 cells was reversibly inhibited by cycloheximide. Data from SDS-PAGE analyses showed that U-937 cells synthesized C3 as a precursor polypeptide chain and was capable of processing this pro-molecule into the secreted two chain form. C3 antigen immunoprecipitated from stimulated U-937 cell lysates showed an increased amount of low mol. wt material as compared to C3 antigen immunoprecipitated from the lysates of unstimulated cells. This may be attributable to increased intracellular proteolytic activity in the PMA stimulated cells. The studies show that the U-937 cell line provides a useful model for studies on the synthesis and processing of complement proteins and the physiological regulation of complement production.

Effects of anti-inflammatory and anti-rheumatic drugs on the activities of purified and membrane-bound Na+/K+ adenosine triphosphatase.

We have examined the effects of anti-inflammatory and anti-rheumatic drugs on membrane-bound and purified Na+/K+-ATPase activity in vitro. Only the gold-containing compounds (gold sodium thiomalate and auranofin) were found to inhibit the enzyme activity in a dose-dependent manner. Sodium thiomalate and triethylphosphine, the ligand compounds for gold sodium thiomalate and auranofin, respectively, had no effect on ATPase activity. The antagonistic properties was abolished by preincubation of the gold compounds with dithiothreitol. Lineweaver-Burke analysis of the inhibitions of purified ATPase by the gold compounds was found to follow uncompetitive kinetics. Inhibition of ATPase by gold may cause disruption of transmembrane cation transport and thus result in impairment of several metabolic processes and cellular functions.

In vitro induction of cytologic and functional differentiation of the immature human monocytelike cell line U-937 with phorbol myristate acetate.

The tumor-promoting ester 4 beta-phorbol 12-myristate 13-acetate (PMA) has been shown to induce the differentiation of the immature monocytelike cell line U-937 c in vitro into a heterogeneous population of cells, including small "dense" cells, large vacuolized or "foamy" cells, spindle-shaped cells, and cells with multiple filopodia ("stellate" cells). The effect of PMA was dose- and time-dependent, the optimal conditions being 40-162 nM PMA for 48 hours. The minimum time of exposure to PMA to ensure further differentiation of U-937 cells was about 5 hours. The PMA-stimulated cells acquired morphologic, ultrastructural, and functional characteristics typical of cells of the monocyte/macrophage lineage. The PMA-treated U-937 cells became adherent, ceased to proliferate, and exhibited increased expression of monocyte-specific antigens (Leu-M2, - M3, HLADr), surface receptors (FcR, C3bR), enzymes (nonspecific esterase, transglutaminase), and ability to mediate chemotaxis, phagocytosis, superoxide anion production, and antibody-dependent cytotoxicity reactions. The induced cells lost their morphologic differentiation and ability to attach to surfaces and regained proliferative capacity upon repeated subculture in PMA-free media.

Neutrophil function in the immotile cilia syndrome.

Patients with ICS have a microtubular abnormality of cilia, which is thought to contribute to frequency respiratory tract infections. Neutrophils have a cytoplasmic microtubular system that, if abnormal and malfunctioning, could also contribute to recurrent infections. Neutrophil function has been assessed in 14 patients with ICS and compared with that of controls. The ICS patients included those with the dynein defect, the radial spoke defect, and microtubular transposition. Cell migration in each patient has been examined under agarose and by Boyden chamber techniques. No significant abnormality was detected in random or directed migration of neutrophils. Six patients representing the three different types of abnormal cilia had more extensive studies of neutrophil function performed. These included assessments of bacterial phagocytosis and killing, lysosomal degranulation, and oxidative pathway activity. Phagocytosis and killing of Staphylococcus aureus 502A in vitro was normal. Hexose monophosphate pathway activity, tetrazolium dye reduction, and lysosomal degranulation by neutrophils at rest and during phagocytosis were similar to those of control neutrophils. Our findings suggest that an abnormality of neutrophil function does not play an important role in the respiratory infections of patients with ICS.

Inhibition of vitamin D2-induced arteriosclerosis in rats by depletion of complement with cobra venom factor.

Widespread calcerous deposits developed in the aorta, heart and kidneys of rats fed for 4 days with purina chow and high doses of vitamin D2 (200,000 IU/kg body wt/day). Decomplementation of rats with highly purified cobra venom factor (CoF) prior to vitamin D2 feeding, almost completely prevented calcium deposition in the aorta and arteritis. The mortality rate in the CoF-treated vitamin D2-fed rats was much lower than in untreated rats. These findings suggest that the complement system may be recruited in the pathogenesis of vitamin D2-induced arteriosclerosis.

Purification and characterization of a single chain precursor C3-protein (pro-C3) from normal human plasma.

Fresh human plasma was treated with proteinase inhibitors and passed through an immunoadsorbent column of Sepharose anti-C3 globulin. The insolubilized C3 was eluted with 5 M guanidine and, after extensive dialysis, was reduced with 0.2 M 2-mercaptoethanol and electrophoresed on SDS-polyacrylamide gels. The bulk of the eluted C3 dissociated into two major protein bands, m.w. 125,000 and 75,000 corresponding to the alpha- and beta-chains of C3. In addition, a nonreducible single polypeptide chain (SPC) with a m.w. value of 197,000 +/- 2,000 similar to the apparent m.w. of unreduced C3 was consistently present. SPC has been purified by elution from SDS (SPC) and found to remain a single polypeptide chain upon re-electrophoresis on SDS gels in the presence of 0.2 M 2-mercaptoethanol. The purified SPC reacted with antisera to denatured C3, C3alpha, and C3beta chains. Additionally, antisera to SPC, also reacted with denatured C3, C3alpha-, and C3beta-chains, revealed a reaction identity between SPC and C3, and detected partial identity between SPC and C3alpha- as well as C3beta-chains. This suggested that SPC and C3 are antigenically related. The amino acid compositions and tryptic peptide maps of SPC and C3 were similar. Based on these findings, it is suggested that SPC must represent a single-chain precursor C3 (pro-C3) in plasma that escaped post-synthetic proteolytic cleavage into a two-subunit chain C3 molecule.

Cleavage of human C5 by trypsin: characterization of the digestion products by gel electrophoresis.

Human C5 is composed of two nonidentical polypeptide chains, alpha and beta (m.w. 130,000 and 80,000, respectively) linked together by disulfide bonds and noncovalent forces. Cleavage of C5 by trypsin fragments with increased anodic mobilities. Limited digestion of C5 by trypsin (substrate to enzyme ratio 10:1 w/w at 37 degrees C for 1 min) resulted in the release of a small terminal alpha-chain peptide (alpha1, m.w. 15,000) probably analogous to C5a, from a large fragment, C5b (m.w. 195,000) composed of an intact beta-chain disulfide linked to an alpha-chain that has a lower m.w. (alpha' 115,000). Further digestion (37 degrees C, 5 min) resulted in cleavage of the alpha-chain at multiple sites with the production of three peptides from the alpha'-chain (alpha2I, 23,500; alpha2II 15,700 and alpha2III 10,200) and a residual fragment, C5c (m.w. 144,000). The alpha1 and alpha2 peptides are not covalently linked to the beta-chain nor to one another. The C5c fragment on the other hand is composed of small peptides of the alpha'c chain (alpha3 14,000; alpha4I 9,000; ALPHA 4II 11,000; alpha 5 23,000 to 30,000) which are linked to the beta-chain and also probably to one another by covalent bonds. Secondary cleavage occurred upon prolonged digestion with trypsin (37 degrees C, 20 min), and this resulted in the progressive erosion of the alpha'c peptides and the conversion of C5c to smaller C5c-like species.

Kinetic studies on the fragmentation of the third component of complement (C3) by trypsin.

The kinetics of cleavage of C3 by trypsin was analyzed by electrophoresis in agarose and in polyacrylamide gels containing sodium dodecyl sulfate and the data obtained were used to construct an anatomical model for C3 showing the sites of tryptic attack, the fragments generated, and their composition. Trypsin was shown to cleave C3 in a stepwise fashion. The attack was initially directed at the alpha-polypeptide chain and resulted in the generation of C3a and C3b. Further cleavage of the alpha-chain of C3b, converted it into C3b1 and then into C3d and C3c. Cleavage of the beta-chain by trypsin occurred only at the C3c stage with the release of a small peptide (m.w. 12,000) from C3c and the formation of C3c'. On immunoelectrophoresis, C3c' had a less anodal mobility compared to the beta1A mobility of C3c. C3a, once formed could be further cleaved to give residual fragments with decreasing net positive charge. Exposure of C3 to acid conditions, pH 5.0 or below, rendered the molecule exceedingly susceptible to tryptic degradation.

Chemotactic migration of leucocytes through corneal layers: an in vitro study.

The ability of polymorphonuclear leucocytes (PMNs) to migrate into the different corneal layers in the presence or absence of a chemotactic stimulus was investigated in rabbits. The epithelial and/or endothelial surfaces were damaged in some corneas and in others they were intact. One side of the cornea was kept in contact with a viable population of rabbit PMNs and the other side with a chemotactic agent (zymosan activated human serum). The migration of PMNs into the cornea was traced histologically. The PMNs could not penetrate intact epithelium and Descemet's membrane even under the influence of a chemotactic stimulus. The stroma allowed PMN migration only when the chemotactic agent was present. The endothelium offered no resistance to PMN invasion whether the chemotactic stimulus was present or not.

Effect of proteolytic digestion on the structure and function of human properdin.

Human properdin (P) was found to be sensitive to the action of trypsin, chymotrypsin, pepsin, and Streptomycetes caesipitosus protease. Incubation of P with these enzymes resulted in loss of its functional activity and the production of antigenically deficient components compared to untreated P. Upon incubation with trypin, P was initially cleaved into a minor fragment and a major fragment. Further degradation ot the fragments occurred with prolongation of inculation time. The minor fragment was highly susceptible to further proteolysis compared to the major fragment which contained the carbohydrate moiety of the molecule. SDS-polyacrylamide gel electrophoretic analysis of trypsin-digested P suggested that the subunit polypeptide chains were initially cleaved at similar points to produce the major and minor fragments. The sedimentation velocity of the major fragment was higher than that of the intact molecule. The implications of these observations of the configuration of P are discussed.

Glomerulonephritis with mesangial deposits of IgA unassociated with systemic disease.

Typical features of IgA-associated nephritis were found in renal biopsies from 16 of 355 consecutive patients. Generalized segmental mesangial proliferation was noted in biopsies from most patients, and dense deposits were detected by electron microscopy in mesangial regions of approximately 50% of biopsies. Immunofluorescent studies showed IgA to be the predominant immunoglobulin in glomueruli; IgG was present in less than 50% of biopsies and IgM in only 12%. The serum IgA value was significantly increased (P les than 0.001) in 50% of patients and the mean IgA/IgG ratio was significantly increase (P less than 0.001) for the patient group as a whole, which suggests a selective increase in IgA. Mesangial deposits of C3 were present in 15 of 16 biopsies and properdin was noted in all biopsies tested; C4 was not demonstrated in any biopsy. This suggests activation of the alternative complement pathway. The results of this study support the concept that IgA-associated nephritis is a unique condition that in some patients gives rise to idiopathic recurrent hematuria. Although the prognosis is good in the majority of patients, the renal disease may progress.