C3b inactivator accelerator (A-C3bINA) was isolated from human plasma. An antiserum produced against the purified protein gave a reaction of identity with beta 1 H, a well-documented contaminant of C3 preparations. Beta 1 H appears to be composed of a single polypeptide chain containing a significant quantity of carbohydrate, and having a sedimentation coefficient of 5.6 on analytical, and 6.4 on sucrose density gradient ultracentrifugation. Its mol wt based on SDS polyacrylamide gel electrophoresis and equilibrium sedimentation is approximately 150,000, whereas it elutes from Sephadex G200 with an apparent mol wt of 300,000, suggesting that beta 1 H is an asymmetric molecule. Beta 1 H potentiates the inactivation of C3b by C3b inactivator, binds to EAC43 to limit the formation of EAC43bB and EAC43bBP, and in contrast to C3b inactivator, it increases the rate of loss of hemolytic sites from EAC43bB and EAC43bBP. For the C3b inactivator-potentiating effect, beta 1 H and C3b inactivator must necessarily be simultaneously present. The kinetics of inactivation of C3b by C3b inactivator and beta 1 H are first order, suggesting that potentiation is not a multistep process. The mechanisms of binding to C3b and inhibition of the alternative pathway convertases C3bB and C3bBP are currently unknown.

Activation of the classical complement pathway occurs when immune complexes interact with the first component, C 1, to convert it from its inactive precursor form to an active esterolytic enzyme, CI. 1 CI by producinglimited proteolysis of C4 and C2 produces the classical pathway (23 convertase, C42 (1). The alternative pathway is activated when the complex surface polysaccharides of bacteria or yeasts come into contact with serum (2). As a consequence of this contact, C3b, the major cleavage product of C3; factors B (B) 2 and D (17)); and properdin (P) interact to assemble the alternative pathway C3 and C5 convertases (3, 4). Activation of the complement system results in the generation of phlogistic molecules from C2, B, C3, and (35 which act on the membranes of lymphocytes, macrophages, polymorphonuclear leukocytes, mast cells, platelets, and smooth muscle cells (5), all of which may be involved in the inflammatory response.Modulation of complement activation is achieved by at least three plasma proteins: Cl-inhibitor which stoichiometrically inhibits (31 (6), C3b inactivator (C3BINA), which enzymatically inactivates C3b (7,8), and fllH globulin which by binding to C3b, displaces Bb from the alternative pathway (33 and (35 convertases, prevents (33b from interacting with B and C5, and potentiates the inactivation of C3b by C3bINA (9, 10).In human serum there is usually good correlation between concentrations of the *" SuppOrted by HERT grants 532 and 564 and funds from the University of Glasgow and the Arthritis and Rheumatism Council of Great Britain.l A bar over a symbol indicates that the component is in its active form. 2 Abbreviations used in this paper." B, factor B; C3bINA, C3b inactivator; C-rat, rat serum diluted 1 to 15 in 0.086 M EDTA diluted in isotonic veronal-buffered saline containing 0.01% gelatin; D, factor E); D5W ++, 5% dextrose containing 0.0005 M Mg ++ and 0.00015 M Ca++; DFP, diisopropyl fluorophosphate; DGVB ++, equal volumes of D5W ++ and veronal-buffered saline containing 0.01% gelatin, 0.0005 M Mg ++ and 0.00015 M Ca++; DGVB', three volumes 5% percent dextrose and one volume isotonic veronal-buffered saline containing 0.01% gelatin; E '*b, rabbit erythrocytes; 0.01 M EDTA.DGVB =, 0.086 M EDTA diluted in DGVB=; 0.04 M EDTA.GVB', 0.086 M EDTA diluted in isotonic veronal-buffered saline containing 0.01% gelatin; 0.02 M EDTA.GVB=, 0.04 M EDTA.GVB= diluted in isotonic veronal-buffered saline containing 0.1% gelatin; 0.01 M EDTA.GVB', 0.02 M EDTA.GVB= diluted in isotonic veronal-buffered saline containing 0.01% gelatin; GVB +÷, isotonic veronal-buffered saline containing 0.01% gelatin, 0.0005 M Mg ++, and 0.00015 M Ca++; GVB =, GVB ++ without Mg ÷÷ and Ca++; MEM, Eagle's minimum essential medium; Mg.EGTA, one volume 0.1 M EGTA diluted in nine volumes of DGVB', containing 0.005 M Mg ++, pH 7.4; P, properdin; PBS, phosphate-buffered saline; RD, serum depleted of I7); RPMI.FCS, RPMI-1640 containing 20% heat-inactivated fetal calf serum. J. ExP. MED.

No abstract

A human plasma protein binds to cell-bound C3b, the major cleavage product of the third component of complement. Consequent upon this binding, C3b no longer functions in either the classical or alternative pathways. This C3b inhibitory activity is a property of a protein previously designated beta 1H on the basis of its electrophoretic mobility.

We have studied the expression of the complement components C2, C3, factor B, C1 inhibitor (C1-inh), C4-binding protein (C4-bp) and factor H in human peripheral blood monocytes, skin fibroblasts, umbilical vein endothelial cells (HUVEC) and the human hepatoma cell line G2 (Hep G2) in the absence and the presence of interferon-gamma (IFN-gamma). E.l.i.s.a. performed on culture fluids, run-on transcription assays, Northern blot and double-dilution dot-blot techniques confirmed that monocytes expressed all six components, whereas fibroblasts, HUVEC and HepG2 each expressed five of the six components. Fibroblasts and HUVEC did not synthesize C4-bp, and Hep G2 did not produce factor H. In addition to these differences, the synthesis rates of C3, C1-inh and factor H were not the same in all cell types. However, the synthesis rates of C2 and factor B were similar in all four cell types. The half-lives of the mRNAs were shorter in monocytes than in other cell types. Monocyte factor H mRNA had a half-life of 12 min in monocytes, compared with over 3 h in fibroblasts and HUVEC. The instability of factor H mRNA in monocytes may contribute to their low factor H secretion rate. IFN-gamma produced dose-dependent stimulation of C2, factor B, C1-inh, C4-bp and factor H synthesis by all cell types expressing these proteins, but decreased C3 synthesis in all four cell types. Cell-specific differences in the response to IFN-gamma were observed. The increased rates of transcription of the C1-inh and factor H genes in HUVEC were greater than in other cell types, while the increased rate of transcription of the C2, factor B and C1-inh genes in Hep G2 cells was less than in other cell types. IFN-gamma did not affect the stability of C3, factor H or C4 bp mRNAs, but increased the stability of factor B and C1-inh mRNAs and decreased the stability of C2 mRNA. Although these changes occurred in all four cell types studied, the half-life of C1-inh mRNA in monocytes was increased almost 4-fold, whereas the increases in the other cell types were less than 30%. These data show that the constitutive synthesis rates of complement components may vary in the different cell types. They also show that the degree of change in synthesis rates in response to IFN-gamma in each of the cell types often varies due to differences in transcriptional response, sometimes in association with changes in mRNA stability.