Antibody restores human alternative complement pathway activation by mouse erythrocytes rendered functionally deficient by pretreatment with pronase.

Activation of the human alternative pathway of complement (C) by surfaces requires the initial deposition of C3b by fluid-phase C3 convertase and sustained C3 cleavage by C3 convertases fixed to the surface. Nonactivating particles have previously been characterized by an inability to sustain the function of C3 convertases on their surfaces because these sites were susceptible to the regulatory action of the control proteins. Pronase converts the mouse erythrocyte (E) from an activator to a nonactivator by markedly decreasing the ability of the cell to affix C3b generated by a fluid-phase C3 convertase; this conversion is unrelated to the action of the control proteins on bound C3b.

Clinical and biochemical effects of stanozolol therapy for hereditary angioedema.

Stanozolol, an inexpensive anabolic steroid with a 30:1 anabolic:androgenic ratio, was administered to 12 male and 15 female patients with biochemically proven hereditary angioedema over a 2-yr period to obtain a systematic assessment of the relationship between drug dosage and clinical response, incidence of side effects, and amelioration of complement abnormalities. All 27 patients attained the minimal effective dose, ranging from 0.5 to 2 mg daily, which controlled the frequency and intensity of symptoms with minimal side effects.

Membrane distribution and adsorptive endocytosis by C3b receptors on human polymorphonuclear leukocytes.

C3b receptors on human polymorphonuclear leukocytes (PMN) were nonrandomly distributed in small clusters on the plasma membranes of these cells when assessed by indirect immunofluorescence at 0 degree C using monospecific rabbit Fab' or F(ab')2 anti-C3b receptor and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat IgG anti-F(ab')2. When PMN were incubated with the bivalent anti-C3b receptor at 37 rather than at 0 degree C, almost no immunofluorescence was observed, which indicates that the C3b receptor-F(ab')2 complexes had been rendered inaccessible to TRITC-IgG anti-F(ab')2. Endocytosis of the anti-C3b receptor ligand was quantitated by measuring the binding 131I-IgG anti-F(ab')2 by PMN that had previously taken up 125I-F(ab')2 anti-C3b receptor at 0 and at 37 degree C, respectively.

Structural determinants of the capacity of heparin to inhibit the formation of the human amplification C3 convertase.

The ability of heparin glycosaminoglycan to prevent formation of the properdin-stabilized amplification C3 convertase is independent of antithrombin binding activity and requires substitution of the amino sugar and a degree of oxygen (O)-sulfation which could be on the uronic acid or the amino sugar. Preparations of heparin glycosaminoglycan isolated by different techniques from different species (rat, human, and porcine) exhibited an equivalent capacity to inhibit generation of the amplification C3 convertase. Hyaluronic acid, which is devoid of O-sulfation, had no inhibitory activity; chondroitin 4-sulfate of rat and whale origins, chondroitin 6-sulfate of rat and shark origins, and dermatan sulfate from porcine skin are O-sulfated on the galactosamine and had minimal activity.

Lateral distribution and diffusion of the C3b receptor of complement, HLA antigens, and lipid probes in peripheral blood leukocytes.

Fluorescence microscopy and fluorescence redistribution after pattern photobleaching have been used to measure the distribution and motion of a number of fluorescent molecules bound to the plasma membranes of human leukocytes. The fluorescent molecules include fluorescein-labeled F(ab')2 and Fab' fragments of an anti-C3b receptor antibody, fluorescein-labeled IgG and Fab fragments of a monoclonal anti-HLA antibody, and the two lipid probes 3,3'-dioctadecylindocyanine and N-4-nitrobenzo-2-oxa-1,3-diazole L-alpha-dimyristoyl phosphatidylethanolamine. From these studies we have concluded that the C3b receptors on human polymorphonuclear leukocytes and monocytes are predominantly present in discrete clusters.

Identification of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte, and monocyte.

A human erythrocyte membrane glycoprotein of 205,000 mol wt (gp205) has been identified as the C3b receptor of the erythrocyte, polymorphonuclear leukocyte (PMN), B lymphocyte, and monocyte. Initially, gp205 was sought and characterized as a constituent of the human erythrocyte membrane that can impair activation of the alternative complement pathway by inducing loss of function of the properdin-stabilized amplification C3 convertase (C3b,Bb,P) through displacement of Bb from C3b and by promoting cleavage-inactivation of C3b by C3b inactivator. These inhibitory activities of gp205 suggested that this membrane glyeoprotein had an affinity for C3b and prompted an analysis of its possible identity as the C3b receptor of human peripheral blood cells.

Surface-associated heparin inhibits zymosan-induced activation of the human alternative complement pathway by augmenting the regulatory action of the control proteins on particle-bound C3b.

Discrimination by the human alternative pathway between activating and nonactivating particles occurs after deposition of C3b by the continuous low-grade interaction of the alternative pathway components in biologic fluids and is dependent on the modulation by surface constituents of the interaction of bound C3b with the control proteins, beta 1H, and C3b inactivator (C3bINA). When heparin glycosaminoglycan was coupled to activating particles, such as zymosan or Sepharose, by cyanogen bromide activation, their capacity to activate the human alternative pathway was inhibited. The loss of alternative pathway-activating capacity was directly correlated to the number of heparin molecules bound/zymosan particle, whether the ratio was varied by increasing the amounts of heparin in the initial coupling reactions or by treating a fully inhibited particle with incremental concentrations of heparinase.

Regulation of the amplification C3 convertase of human complement by an inhibitory protein isolated from human erythrocyte membrane.

An activity that is inhibitory to the properdin-stabilized amplification C3 convertase (C3b,Bb,P) was solubilized from human erythrocyte (E(hu)) membranes by Nonidet P-40 and purified to homogeneity. The inhibitory membrane glycoprotein had an apparent M(r) of 1-1.2x10(6) on gel filtration in the presence of Nonidet P-40.

Clinical and biochemical effects of impeded androgen (oxymetholone) therapy of hereditary angioedema.

Daily therapy and alternate-day therapy with the attenuated androgen oxymetholone were compared in patients with hereditary angioedema (HAE). Fifteen of 16 patients who experienced at least monthly attacks of HAE without treatment were asymptomatic on administration of 5 mg oxymetholene daily. When 13 of the patients who had been maintained asymptomatically on 5 mg oxymetholone daily were advanced to a treatment schedule of 5 mg every other day, seven attacks occurred during a cummulative 50 mo of therapy.

[Alternative complement pathway (author's transl)].

The dual role of the alternative complement pathway in recognition of foreign substances by a non-immune host and in the intrinsic regulation of the complement sequence is now well recognized. Activation of this pathway occurs through escape from its regulatory mechanisms induced by the activating principle; its functional expression depends on the respective levels of the component proteins C3, factor B, factor D and properdin, and on the control proteins beta 1H and C3bINA. This article presents recently acquired knowlege on the molecular mechanisms of activation, regulation and behaviour under pathological conditions of the alternative complement pathway.

Action of the C3b-inactivator on the cell-bound C3b.

The action of C3bINA and beta 1H on cell-bound C3b is described in this paper. The alpha-polypeptide of C3b that binds covalently to cell surfaces is cleaved by the C3bINA and beta 1H into two fragments: one of 60,000 (C3b alpha-60) and another of 40,000 (C3b alpha-40) daltons. The beta-chain of C3b is unaffected by the C3bINA and beta 1H.

A molecular basis of activation of the alternative pathway of human complement.

The fluid phase interaction of native C3, B, D and P continuously generates C3b; C3b complexes with B to permit cleavage-activation by D, thereby generating C3b, Bb, the amplification C3 convertase. C3b, Bb formed in the fluid phase or on a non-activating surface for the alternative pathway undergoes decay-dissociation through release of Bi, and the residual C3b undergoes cleavage inactivation by the C3b inactivator (C3bINA). The capacity of P to stabilize C3b, Bb and therby augment C3 cleavage is counterbalanced by beta 1H, which inactivates the convertase by displacing Bi and facilitates the inactivation of residual C3b by C3bINA.

Autosomal locus regulates inverse relationship between sialic acid content and capacity of mouse erythrocytes to activate human alternative complement pathway.

The observation that mouse erythrocytes (E(m)) from 21 inbred strains had variable capabilities to activate the human alternative complement pathway permitted the demonstration that membrane sialic acid content was inversely related to activating capacity and was regulated by codominant alleles of a single autosomal locus. Linear regression analysis also demonstrated a significant inverse correlation between the sialic acid content of E(m) from four inbred strains and the concentration of beta1H required for decay-dissociation of the properdin-stabilized amplification convertase on the E(m). E(m) from F(1) hybrids derived from strains with high and low alternative pathway activating capacities and from their backcrosses exhibited the alternative pathway activating capacities expected if the activity were regulated by alleles of a single autosomal locus.

Membrane sialic acid on target particles modulates their phagocytosis by a trypsin-sensitive mechanism on human monocytes.

Monolayers of human peripheral blood monocytes in the absence of exogenous proteins ingest a variety of natural particulate activators of the human alternative complement pathway. Sheep erythrocytes, which do not ordinarily activate the human alternative complement pathway or initiate a direct monocyte phagocytic response, can be modified to exhibit both functions by the deletion or alteration of membrane sialic acid residues. Enzymatic removal of the sialic acid residues with sialidase or their conversion to heptulosonic acid derivatives by limited oxidation with NaIO4 and reduction with BH4- have equivalent dose-response effects on the capacity of the altered sheep erythrocytes to initiate the phagocytic response by human monocytes or to activate the alternative pathway in human serum.

Regulation by membrane sialic acid of beta1H-dependent decay-dissociation of amplification C3 convertase of the alternative complement pathway.

Sheep erythrocytes in their native state did not activate the alternative complement pathway, as measured by lysis in dilutions of normal human serum containing [ethylenebis(oxyethylenenitrilo)] tetraacetic acid but acquired this capacity after membrane sialic acid residues had been removed (by sialidase) or modified (by NaIO(4)). Activation of the alternative pathway by sheep erythrocytes required removal or modification of at least 40% of the membrane sialic acid to reach threshold, and it increased proportionately when larger amounts of sialic acid had been affected. Studies with isolated proteins of the alternative pathway demonstrated that the altered erythrocyte membranes resembled natural activators in protecting bound C3b from inactivation by C3b inactivator and beta1H and protecting bound amplification C3 convertase (C3b,Bb) from decay-dissociation by beta1H.

Modulation of the formation of the amplification convertase of complement, C3b, Bb, by native and commercial heparin.

Native rat mast cell macromolecular heparin proteoglycan and commercial hog heparin glycosaminoglycan chains inhibit generation of the amplification convertase, C3b, Bb. The inhibitory action of heparin is not due to chelation of magnesium. Heparin is most active in inhibiting convertase formation on cellular intermediates formed with the lowest C3b input and developed with the highest B concentration, thereby suggesting the receptor site for B on C3b as the point of heparin action.