Fluid phase destruction of C2hu by C1hu. II. Unmasking by C4ihu of C1hu specificity for C2hu.

It has been demonstrated that C1 isolated in the unactivated form fails to inactivate C4 or C2 in the fluid phase, while the activated molecule, C1 rapidly converts C4 to hemolytically inactive C4i, but does not efficiently inactivate C2. The production and presence of C4i now confers on C1 the ability to rapidly inactivate C2. After heating at 56 degrees C, so as to destroy the hemolytic activity, heat inactivated C1 is still capable of inactivating C4 but the presence of C4i no longer confers an ability to inactivate C2. Studies with the subunits of C1-C1q, C1r, C1s, indicate that the action of C1s on C2 can be inhibited by C1r and that this inhibition is reversed by the presence of homologous C4. These studies indicate that the interaction of C4i with a heat labile receptor conformation in C1 uncovers a masked specificity for C2.

Fluid phase destruction of C2hu by C1hu. I. Its enhancement and inhibition by homologous and heterologous C4.

The fluid phase inactivation of C2(hu) by C1(hu) is markedly enhanced by the presence of C4(hu). The enhancement is afforded by C1 inactivated C4(hu), namely C4i(hu), and requires the simultaneous presence of enzymatically active C1. Heterologous C4 of guinea pig origin protects C2(hu) from the inactivation by C1(hu). Thus, in both the fluid phase and on the cellular intermediate, C4(hu) is essential to the specific action of C1(hu) on C2(hu). It is possible that C4i alters C2 so as to present a more suitable substrate to the C1 enzyme or that C4i acts on the C1 to uncover a specificity for native C2.

Human C4-binding protein. I. Isolation and characterization.

C4-binding protein (C4-bp), a new component of the complement system, was isolated from human plasma by precipitation with polyethyleneglycol, followed by chromatography on ion exchangers. C4-bp was identified on sodium dodecyl- sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by two independent criteria: its ability to bind to C4b, and immunoprecipitation with a monospecificantiserum. Purified C4-bp is a 10.7 s glycoprotein. It consists of several disulfide bonded subunits of mol wt 70,000 daltons. Under nonreducing conditions, its mol wt has been estimated on SDS-PAGE as 540- 590,000 daltons. C4-bp moves as a slow B-globulin at pH 8.6 in the absence of free divalent cations, but when the buffers contain Ca(++)-lactate, C4-bp is a gamma globulin. Purified C4-bp binds to purified C4b. The reaction proceeds in the presence or absence of divalent cations and is not inhibited by diisopropylfluorophosphate. The C4b/C4-bp complexes have sedimentation coefficients between 15 and 17 s on sucrose gradient ultracentrifugation, and can be readily identified by crossed immunoelectrophoresis (CIE). The complexes move faster toward the anode than either protein. C4-bp is multivalent. Saturation is reached at molecular ratios of C4b/C4- bp of between 4 and 5. The interaction between C4b and C4-bp may complicate the electrophoretic patterns of these proteins in normal human serum, if the complement system is activated before or during the run. However, in EDTA-plasma, native C4 and C4-bp do not form stable complexes and can be identified in separate peaks after CIE.

Human C4-binding protein. II. Role in proteolysis of C4b by C3b-inactivator.

We recently described the isolation from human serum of a high molecular weight protein with specific binding affinity for fluid-phase activated C4. We show here that the C4-binding protein (C4-Bp) functions as an essential cofactor in the proteolysis of C4b in the presence of C3b-inactivator (C3bINA). C4-bp, together with C3bINA, cleave the alpha'-chain of C4b into three fragments called alpha2, alpha3, and alpha4, with mol wt of 47,000, 25,000, and 17,000 daltons, respectively. The alpha2 fragment was dissociated from C4b without reduction, whereas the alpha3 and alpha4 fragments were disulfide bonded the other chains of C4b. The reaction did not occur when either C4-bp or C3bINA were omitted, nor in the presence of either protein in combination with beta1H. Native C4 was not affected by C3bINA aand C4-bp. C4b was not cleaved when incubated in serum of a patient with genetic deficiency of C3bINA. However, when purified C3bINA was added, the alpha'-chain of C4b was cleaved and fragments with the same molecular weight as alpha2, alpha3, and alpha4 were generated.

Structural and functional differences between the H-2 controlled Ss and Slp proteins.

Based on functional and structural data, it is concluded that the Ss protein in the mouse expresses the activity of the fourth component of complement. Removal of the Ss, but not of Slp, antigen correlates with a high degree of significance (P less than 0.001) with decrease of C4 hemolytic activity. In phenotypically Slp negative mice the plasma/serum levels of Ss correlate with the C4 activity (P less than 0.001). Structurally, Ss is a 209,000-mol wt protein, consisting of three covalently linked polypeptide chains (alpha,beta,gamma). Treatment of Ss with C1 cleaves a 7,000-8,000-mol wt fragment from the alpha-chain. Slp is also a three chain covalently linked protein of 209,000 daltons, however its three chains differ in size from those of the Ss protein. Slp does not express hemolytic activity and its alpha-chain is not cleaved by C1.

Activation of the guinea pig alternative complement pathway by mouse IgA immune complexes.

Activation of the complement system by IgA was investigated with immune complexes containing a mouse IgA myeloma protein with specificity for phosphorylcholine linked to bovine serum albumin (PC-BSA). These IgA anti-PC-BSA immune complexes activated the alternative complement pathway in mouse and guinea pig serum, while human complement was not affected. The activation proceeded with consumption of C3 but little or no consumption of C5. C3 did not bind to the IgA immune complexes during complement activation although it did bind covalently to IgG immune complexes. It is suggested that IgA immune complexes do not supply a suitable surface for C3 binding and effective alternative pathway convertase assembly; therefore, cleavage is limited and occurs primarily in the fluid phase. Without C3 binding, C5 cleavage does not occur nor can the alternative pathway activation proceed to the amplification step.

Modulation of function of the activated first component of complement by a fragment derived from serum. I. Effect on early components of complement.

An activity designated Kf can be separated from human serum and shown to give a 100-300% enhancement in the hemolytic activity of fully activated, fractionated C1. The enhancement of C1 activity is not because of activation of precursor C1 and it is not attributable to an effect on C1 binding. EAC42 or EAC4 intermediates interacted with C1Kf exhibit a greater T(max) and shorter Z(max) than when such intermediates are reacted with the same number of hemolytic units of C1. C3 consumption by the EAC1Kf42 intermediate greatly exceeds that of the EAC142 intermediate produced from the same EAC4 cells by comparable inputs of the other two complement components. Taken together, these findings suggest that Kf-treated C1 achieves more efficient utilization of C4 and C2 to create a larger number of 42 sites as appreciated on the intermediates by shorter T(max) and a greater Z(max), and an increased capacity to utilize C3. The capacity of Kf to enhance C1 upon introduction into whole serum of a patient with hereditary angioedema (HAE) in a manner comparable to its effect on fractionated C1 suggests that the effect of Kf may be pertinent to certain pathophysiologic conditions of man.

Mechanism of action of the C4 nephritic factor. Deregulation of the classical pathway of C3 convertase.

Three mechanisms that regulate the formation and function of the classical pathway C3 convertase (C4b2a) have been elucidated: (a) an intrinsic decay of the enzyme that is temperature dependent; (b) an extrinsic decay mediated by the effect of the serum protein C4b binding protein (C4-bp); and (c) inactivation of C4b by the proteolytic action of C4b/C3b inactivator (C4b/C3bINA), which cleaves that alpha' chain of C4b to yield C4d (alpha 2) and C4c (alpha 3, alpha 4, beta, and gamma chains). A fourth mechanism described here is based on the observation that the IgG fraction of the serum of certain patients with glomerulonephritis contains a protein termed C4 nephritic factor (NFc), which prevents the intrinsic decay of C4b2a. This protein, which prolongs the half-life of surface-bound C4b2a from 7.5 min to greater than 5 h, increases the use of C3 and C5. It also inhibits the decay produced by C4-bp by preventing the dissociation of C2a from the C4b2a complex. Additionally, the C2b/C3bINA alone, or in the presence of C4-bp, fails to cleave the alpha' chain of C4b in the surface-bound stabilized C4b2a complex. This protective property of NFc requires the presence of C2a, because C4b was not protected unless it was bound to C2a. Thus in the presence of NFc, the three natural controls of the function of the classical pathway convertase, intrinsic decay, extrinsic decay, and proteolytic cleavage, are bypassed.

Effect of glucocorticosteroids on epidermal Langerhans cells.

The effects of topical and systemic administration of various glucocorticoids on the density of epidermal Langerhans cells (LC) were studied in guinea pigs. Glucocorticoids, such as betamethasone dipropionate and valerate, caused a marked decrease in LC demonstrable by staining for cell membrane ATPase activity and Ia antigens. By electronmicroscopy, LC also showed morphologic alterations. The observed decrements in LC density correlated with the concentration and known vasoconstrictive potency of the glucocorticoids administered. The anti-inflammatory action of glucocorticoids in skin disorders may, at least in part, be through their ability to alter epidermal LC, thus interfering with the antigen-presenting functions of these cells.

Short communication: casein haplotype variability in sicilian dairy goat breeds.

In the Mediterranean region, goat milk production is an important economic activity. In the present study, 4 casein genes were genotyped in 5 Sicilian goat breeds to 1) identify casein haplotypes present in the Argentata dell'Etna, Girgentana, Messinese, Derivata di Siria, and Maltese goat breeds; and 2) describe the structure of the Sicilian goat breeds based on casein haplotypes and allele frequencies. In a sample of 540 dairy goats, 67 different haplotypes with frequency >or=0.01 and 27 with frequency >or=0.03 were observed. The most common CSN1S1-CSN2-CSN1S2-CSN3 haplotype for Derivata di Siria and Maltese was FCFB (0.17 and 0.22, respectively), whereas for Argentata dell'Etna, Girgentana and Messinese was ACAB (0.06, 0.23, and 0.10, respectively). According to the haplotype reconstruction, Argentata dell'Etna, Girgentana, and Messinese breeds presented the most favorable haplotype for cheese production, because the casein concentration in milk of these breeds might be greater than that in Derivata di Siria and Maltese breeds. Based on a cluster analysis, the breeds formed 2 main groups: Derivata di Siria, and Maltese in one group, and Argentata dell'Etna and Messinese in the other; the Girgentana breed was between these groups but closer to the latter.

Lysis of complement-sensitive Entamoeba histolytica by activated terminal complement components. Initiation of complement activation by an extracellular neutral cysteine proteinase.

Activation of complement by Entamoeba histolytica may be initiated by the extracellular 56-kD neutral cysteine proteinase which cleaves the alpha chain of C3. To determine the relationship between the fluid-phase activation of complement and our observation that only strains isolated from patients with invasive disease are resistant to complement-mediated lysis, we investigated the fate of C3 with recent amebic isolates. When 125I-C3 was incubated with trophozoites in serum, C3 in the fluid phase was cleaved to C3b or C3bi, but the alpha chain of the C3 molecules on the cell surface appeared intact. Since the lysis of nonpathogenic strains takes place in the absence of bound C3b, we demonstrated that this reaction occurs by reactive lysis initiated in the fluid phase: (a) the killing of nonpathogenic strains was enhanced when alternative pathway activation was accelerated by the addition of cobra venom factor; (b) non-pathogenic strains were lysed by purified terminal components; and (c) sera incubated with pathogenic E. histolytica produced passive lysis of chicken erythrocytes. These results demonstrate for the first time that complement-sensitive E. histolytica are lysed by activation of the terminal complement components in the fluid phase where the 56-kD neutral cysteine proteinase cleaves C3, and not by the surface deposition of activated C3.

Mechanism of killing of Giardia lamblia trophozoites by complement.

Only antibodies of the IgM class support the lytic effect of complement on Giardia lamblia (GL). We sensitized GL trophozoites (SGL) at 4 degrees C with serum containing anti-GL antibodies or IgM purified from this serum, and either normal human serum (NHS), complement 2-deficient human serum (C2d-HS), or C4-deficient guinea pig serum was used as source of complement. SGL were killed by NHS (86%) and by the deficient sera (50 and 40%, respectively), suggesting activation of the alternative pathway. However, the reaction was inhibited by Mg-EGTA. These observations led to studies of the role of C1. The lytic effect of NHS and C2d-HS on SGL was abolished by immunochemically depleting C1 from these sera, and reconstituted by adding purified C1q plus C1r and C1s. Factor B-depleted C2d-HS also lost its capacity to mediate killing, but reconstitution with factor B led to a dose-dependent increase in the killing of SGL. We next investigated the participation of the membrane attack complex in this system. SGL carrying C5b to C7 were lysed when incubated with C8 alone (56%); the addition of C9 further increased killing (98%), while C9 in the absence of C8 had no effect. We concluded that although activation of the classical pathway produces lysis of SGL, lysis may also proceed through a unique pathway of complement activation that requires C1 and factor B, but is independent of C4 and C2. Lysis of SGL can be accomplished by C5b to C8 in the absence of C9.

Role of complement and polymorphonuclear cells in demethylchlortetracycline-induced phototoxicity in guinea pigs. Inhibition by decomplementation in vivo.

In this study, demethylchlortetracycline was used as a prototype of exogenous phototoxic substances. In vitro, exposure of serum containing demethylchlortetracycline to ultraviolet-A irradiation resulted in the diminution of total complement hemolytic activity and C4, C2, C3, and C5 activities. In addition, chemotactic activity for human polymorphonuclear cells was generated, which was thermostable and antigenically related to human C5 but not human C3. In vivo, phototoxic lesions were induced in guinea pigs upon intradermal injections of demethylchlortetracycline solution, followed by ultraviolet-A irradiation. On a scale of 0-3+, the animals developed a maximal response of 2.5 at 20 h. This clinical response was associated with cellular infiltrate in the dermis, consisting of 29 +/- 2% of neutrophils at 24 h. The participation of the polymorphonuclear cells was evaluated in guinea pigs rendered neutropenic by treatment with cyclophosphamide. In these guinea pigs, demethylchlortetracycline and ultraviolet-A induced a maximal response of 0.75 +/- 0.5, which was associated histologically with 1.2 +/- 0.5% neutrophils in the dermis. The role of complement in this process was studied in guinea pigs congenitally deficient in C4, and in guinea pigs decomplemented by treatment with cobra venom factor. In contrast to normal guinea pigs, C4-deficient animals exhibited a maximal reaction of 0.83 +/- 0.16 at 6 h, which subsided within 24 h. Cobra venom factor-treated guinea pigs developed a maximal response of 0.5 at 0.5 and at 6 h. These clinical changes were associated with the development of an increased vascular permeability, as demonstrated by studies using guinea pigs injected intravenously with Evans blue solution. In animals with a normal complement system, there was intense localized bluing at the sites of phototoxic lesion. In contrast, only minimal bluing was observed in decomplemented guinea pigs. These data indicate that a normal number of polymorphonuclear cells and an intact complement system are required for the full development of demethylchlortetracycline-induced phototoxic lesions.

Expression and localization of proteins of the complement system in human skin.

The complement system participates in the immune recognition of foreign antigens, many of which may penetrate the skin by physical injury or transcutaneous adsorption. In this study, we examined the presence of complement components and complement regulatory proteins in the human skin and cultured human keratinocytes. Immunofluorescence studies showed C3, Factor B, decay accelerating factor, the C3b receptor (CR1), and C3d receptor (CR2), distributed among cells of the epidermis as well as on cultured keratinocytes. Immunoblot analysis of keratinocytes supernatants showed the presence of C3 with a molecular weight of approximately 180 kD. The decay accelerating factor was localized as previously reported on elastic fibers; additionally it was observed in the basement membrane zone. In situ hybridization studies suggest the expression of CR1 and CR2 mRNA in human epidermis. These results show the presence in the human epidermis of complement components that are capable of generating the initial C3 convertase of the alternative pathway. The presence of complement regulatory proteins could endow keratinocytes with immune functions such as the regulation of complement activation and endocytosis of C3 opsonized particles.

Activation of the complement system in patients with porphyrias after irradiation in vivo.

Irradiation of the forearms of two patients with erythropoietic protoporphyria and one patient with porphyria cutanea tarda resulted in an in vivo activation of the complement system, as assessed by diminution of the hemolytic titers of the third component of complement by 23-57%, and of the fifth component of complement (C5) by 19-47%. Such treatment also generated chemotactic activity for human polymorphonuclear cells; the chemotactic activity was stable at 56 degrees C and antigenically related to human C5. On Sephadex G-75 chromatography the chemotactic activity eluted with an apparent molecular weight of 15,000. These in vivo results extend our previous in vitro observation of photoactivation of complement in sera from patients with erythropoietic protoporphyria and porphyria cutanea tarda, and suggest that the complement system may participate in the pathogenesis of cutaneous phototoxicity in these patients.

Killing of human melanoma cells by the membrane attack complex of human complement as a function of its molecular composition.

The efficiency of the membrane attack complex (MAC) in killing M21 melanoma cells was determined varying the molar ratio of cell-bound C9:C8. It was found that C5b-8 produced functional channels as evidenced by 86Rb release and propidium iodide uptake; cell killing occurred in the absence of C9 with greater than 5 X 10(5) C5b-8/cell; the maximal molar ratio of C9:C8 was 6.6:1; using nonlytic numbers of C5b-8 (4.7 X 10(5)/cell), greater than 90% killing ensued at a C9:C8 molar ratio of 2.8:1 at which approximately 9,000 poly C9/cell were formed, and 50% killing at a ratio of 1:1; (e) when the MAC was assembled on cells at 0 degree C, consisting of C5b-8(1)9(1), and unbound C9 was removed before incubation at 37 degrees C, killing was similar to that observed when poly C9 formation was allowed to occur. Thus, MAC lytic efficiency toward M21 cells may be enhanced by but does not depend on poly C9 formation.

Erythropoietic protoporphyria. Photoactivation of the complement system.

The complement system was analysed in 14 asymptomatic patients with erythropoietic protoporphyria. In the majority of the sera studied the levels of complement components C1, C4, C2, and C3 were within the normal range. Upon ultraviolet light (330--460 nm) irradiation of the serum samples in vitro, a marked decrease in total hemolytic activity accompanied by reduction of C1, C4, C2, and C3 levels was observed. The loss of total hemolytic activity can be directly correlated with the levels of protoporphyrin (PP) and similar changes can be obtained in normal serum upon addition of PP followedf by ultraviolet light irradiation. It is postulated that after irradiation the excited PP develops the capacity to activate the complement sequence with the production of cleavage products, which may contribute to the skin changes observed in these patients upon sun exposure.

Complement-derived chemotactic activity is generated in human serum containing uroporphyrin after irradiation with 405 nm light.

Patients with porphyrias have varying degrees of photosensitivity, associated with elevated levels of porphyrins in plasma, erythrocyte, urine and/or feces. To investigate the role of complement in the pathogenesis of cutaneous lesions, varying amounts of uroporphyrin were added to normal human serum (0.1-10 microgram/ml), and the mixtures were then exposed to 405 nm irradiation. Such treatments result in the diminution of total hemolytic complement activity and hemolytic titers of C1, C4, C2, C3, and C5; furthermore, cleavage products of C3 and C5 were detected. Chemotactic activity for human polymorphonuclear leukocytes was generated that was inhibitable by incubation with anti-C5, but not with anti-C3 antisera. No chemotactic activity was generated in Mg++-EGTA treated serum nor in C4-deficient guinea pig serum. These data indicate that irradiation with 405 nm light of normal human serum containing uroporphyrin results in activation of the complement system via the classical pathway, and the generation of complement (C5)-derived chemotactic activity for human polymorphonuclear leukocytes.

Altered erythrocyte C3b receptor expression, immune complexes, and complement activation in homosexual men in varying risk groups for acquired immune deficiency syndrome.

We studied levels of erythrocyte C3b receptors (E-CR1) and correlated them to the level of circulating immune complexes (CIC) and complement activation in patients with or at risk for acquired immunodeficiency syndrome (AIDS). A significant reduction was found in patients with AIDS (185 +/- 93 CR1/cell), AIDS-related complex, and generalized lymphadenopathy, whereas healthy male homosexuals or normal controls had 434 +/- 193 and 509 +/- 140 CR1/cell, respectively (P less than 0.001). Family studies indicate that this defect is acquired. Reduction in E-CR1 was associated with increased levels of CIC when assayed by binding to Raji cells, but not when tested by C1q binding. Complement activation was assessed by levels of C3bi/C3d-g in plasma, measured with a monoclonal antibody specific for a neoantigen in C3d. AIDS patients had increased C3 activation (2.68 +/- 1.67%) when compared with normal controls (0.9 +/- 0.22%) (P less than 0.01). The decreased E-CR1, the presence of CIC, and C3 activation suggest that complement activation by immune complexes may play a role in the clinical expression of the disease.

Enhancement of random migration and chemotactic response of human leukocytes by ascorbic acid.

Incubation of human leukocytes with ascorbic acid at neutral pH and at concentrations 10-50 times that of normal blood levels augmented both the in vitro random migration and chemotaxis of the cells by 100-300% without influencing their phagocytic capacity. Enhancement of mobility by ascorbate was evident for isolated neutrophils, eosinophils, and mono-nuclear leukocytes and was independent of the specific chemotactic stimulus. Stimulation by ascorbate of the hexose monophosphate shunt of adherent neutrophils and augmentation by ascorbate of neutrophil mobility had comparable dose-response relationships, could be reversed by washing the cells, and were both suppressed by preincubation of the neutrophils with 6-aminonicotinamide, but not with the neutrophil-immobilizing factor. Glutathione, the proposed intermediate for ascorbate action, similarly stimulated hexose monophosphate shunt activity and enhanced migration. The enhancement in vitro of leukocyte mobility by ascorbate at concentrations found in some normal tissues, therefore, appears to be dependent upon stimulation of the leukocyte hexose monophosphate shunt.