Solubilization of immune precipitates by six isolated alternative pathway proteins.

Abstract

Antigen-antibody precipitates can be dissolved by the complement cascade (1). The complement-mediated solubilization of immune precipitates requires activation of C3 via the alternative pathway (2) but not participation of the late-acting components, C5 through C9 (1, 3). The classical pathway alone, in the absence of the alternative pathway, is not sufficient to solubilize immune complexes (3).It has recently been demonstrated that the alternative pathway can be assembled from six isolated plasma proteins: C3, factors B and D, properdin, C3b inactivator (C3bINA), x and fllH (4, 5). Cells such as rabbit erythrocytes (6), human lymphoblastoid (Raji) cells (7), and Escherichia coli (8) have been shown to be lysed by the isolated alternative pathway proteins and membrane attack pathway proteins. The question therefore arose as to whether the composite mixture of the isolated alternative pathway proteins is capable of solubilizing immune precipitates. In this communication we report that, indeed, the six isolated alternative pathway proteins can solubilize immune precipitates. In addition, we report on the role of the three regulatory factors (C3bINA, fllH, and properdin) in the solubilization process. Materials and MethodsBuffers. PB: isotonic phosphate-buffered saline, pH 7.4; Mg-GPB: PB containing 0.1% gelatin and 1.2 mM MgC12; EDTA-GPB: PB containing 0.1% gelatin and l0 mM EDTA; DGVB: isotonic veronal buffered saline, pH 7.4, containing 0.1% gelatin, 0.15 mM CaCI2, 0.5 mM MgCI2, and 2.5% dextrose.Immune Precipitates. Egg albumin (grade V) was purchased from Sigma Chemical Company, St. Louis, Mo. Rabbit antiserum to egg albumin was prepared by injecting a rabbit with egg albumin incorporated in complete Freund's adjuvant. IgG antibody to egg albumin was isolated by DEAE-cellulose column chromatograph y25and by affinity chromatography on Sepharose 4B-egg albumin. The IgG was labeled with I by the chloramin T method (9). The specific activity was 5.5 × 106 cpm/p.g protein.Immune complexes were prepared at equivalence with egg albumin and labeled IgG antibody to egg albumin. The mixture was incubated at 37°C for 30 min, then at 4°C overnight. The resulting precipitates were washed three times by centrifugation in the cold and resuspended in Mg-GPB. * Supported, in part, by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture, the Governmenl of Japan.~Abbreviations used in this paper: Ab, antibody; C3bINA, C3b inactivator; DGVB, isotonic veronal buffered saline, pH 7.4, containing 0.1% gelatin, 0.15 mM CaCI2, 0.5 mM MgCI2, and 2.5% dextrose; EDTA-GPB, PB containing 0.1% gelatin and 10 mM EDTA; Mg-GPB, PB containing 0.1% gelatin and 1.2 mM MgCI2; PB, isotonic phosphate-buffered saline, pH 7.4; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis.J. Ex~,. M r~n.

The Role of Mannose-Binding Lectin-Associated Serine Protease-3 in Activation of the Alternative Complement Pathway

“…Recombinant proenzyme of mouse Df (proDf) containing a five-residue activation peptide at its N terminus was prepared as described previously (9). Human MASP-1 and human Bf were purified from the serum (12,13). Mouse rMASP-3 with a C-terminal V5-His tag was prepared using the Drosophila expression system (Invitrogen) (14).…”

Deterioration of Immune Complex Solubilization Activity of Serum by Increased Concentration of Factor D

“…3). Somewhat similar conditions may arise in the absence of regulators such as factor H and factor I [15]. In this study, we did not investigate the kinetics and effects of other complement components involved in the solubilization of IC.…”

“…Complement-mediated solubilization of precipitated immune complexes (IC) was first reported by Miller and Nussenzweig [8], The alternative pathway was thought to be essential for solubilization [8][9][10][11][12][13][14], and isolated alterna tive pathway proteins [15], including factor D [16], were found to be involved in solubilization. Furthermore, Tak ahashi et al [13] distinguished three stages which occur during the solubilization of immune precipitates.…”

Formation of soluble immune complexes by complement in sera of patients with various hypocomplementemic states. Difference between inhibition of immune precipitation and solubilization.

“…In vitro, complement can bring about the formation of soluble complexes either by inhibiting their aggregation at the time of the antigen-antibody reaction or by solubilizing preformed immune precipitates (2,3). Inhibition of immune precipitation (IIP)' relies predominantly on an intact classical pathway of complement activation, whereas solubilization (SOL) is dependent on the presence of normal alternative pathway function (4)(5)(6). Both processes result in the formation ofsoluble complexes by the covalent binding of C3 fragments (7,8).…”