Membrane receptors of mouse leukocytes. I. Two types of complement receptors for different regions of C3

Mouse leukocytes were studied for membrane receptors for the third C component by rosette formation with C coated erythrocytes (EAC). Methods were devised for the preparation of EAC complexes containing either mouse C3b or mouse C3d. EAC 1-3dmo were prepared from EA treated with whole mouse serum while EAC 1-3bmo were produced from EAC 142hu treated with whole mouse serum containing sodium suramin. The specificity of the EAC complexes for mouse leukocytes was confirmed by inhibition experiments using fluid phase human C3d. Low concentrations of fluid phase human C3d inhibited EAC1-3dmo rosettes but failed to inhibit EAC 1-3bmo rosettes. Eight-fold higher concentrations of fluid phase C3d caused partial inhibition of EAC1-3bmo rosette formation with lymphocytes, but not with other types of murine leukocytes. Thus mouse leukocytes apparently contain the same two types of C receptors as do human and guinea pig leukocytes. Mouse CR1 is specific for a non-C3d region of C3b, (possibly analogous to human C3c) whereas mouse CR2 is specific for both C3d and the C3d region of C3b.     

Trypsin-activated complex of human factor B with cobra venom factor (CVF), cleaving C3 and C5 and generating a lytic factor for unsensitized guinea pig erythrocytes. I. Generation of the activated complex.

A complex formed between cobra venom factor (CVF) and isolated human factor B (B) was found to be converted by trypsin to a stable enzyme, CVF-B which cleaved the third component (C3) and the fifth component (C5) of human complement. The formation of CVF-B by trypsin required divalent cations, whereas the formation of the lytic factor from human serum occurred even in the presence of EDTA. CVF-B purified by gel filtration could initiate the hemolysis of unsensitized guinea pig erythrocytes when incubated with human complement components C5 to C9 in 0.01 M EDTA buffer. C3 was not required for the lysis of guinea pig erythrocytes initiated by CVF-B because of the beta1C precipitation line formed between human serum and anti-beta1C antibody did not inhibit the hemolysis by CVF-B in agarose gel. Treatment of beta1C and beta1F globulins in whole human serum with CVF-B in the presence of 0.01 M EDTA converted them to components with higher mobilities on immunoelectrophoresis.     

Alternative complement pathway activation by C4b deposited during classical pathway activation

Sheep erythrocytes (E) sensitized with anti-E antibody (A) were reacted with guinea pig C1 (C1gp) and human C4 (C4hu) or guinea pig C4 (C4gp) to prepare EAC1, 4b. Treatment of the EAC1, 4b with a buffer containing EDTA removes C1rgp and C1sgp, resulting in the formation of EAC4b. EAC4b prepared in this way were found to be lysed by human or guinea pig serum in a gelatin Veronal-buffered saline containing 2 mM MgCl2 and 8 mM EGTA (Mg-EGTA-GVB). In the hemolytic sensitivity of EAC4bhu, essentially no difference was noted whether IgG or IgM antibodies were used for preparation of EAC4bhu. The extent of the hemolysis of EAC4bhu was dependent on the dose of C4bhu. Because EAC4bhu were lysed even by C2-deficient human serum, C3 convertase of the classical complement pathway would not be involved in the hemolysis of EAC4bhu. Furthermore, the reactivity of EAC4bhu with serum in Mg-EGTA-GVB remained even after treatment of the intermediate cells with 1 mM PMSF, indicating that any remaining C1gp was not responsible for the hemolysis. Therefore, the hemolysis of EAC4b by sera in Mg-EGTA-GVB was considered to be mediated via activation of the alternative complement pathway (ACP). Pretreatment of EAC4bhu with anti-C4hu antibody or C4-binding protein suppressed the hemolysis of EAC4bhu via the ACP activation. Furthermore, EAC4bhu were more sensitive to hemolysis by the reaction with a mixture of C3, B, D, and H followed by rat serum in EDTA-GVB than EAC1qgp were. These results indicate that C4b molecules on the cell membrane participate in the activation of ACP.     

A simple one-step hemolytic assay for C2 with C2-deficient human serum.

A simple one-step procedure has been developed for the molecular titration of C2 by utilizing the ability of the test material to restore the hemolytic activity of human serum selectively deficient in C2 (C2D serum). In this assay, equal volumes of EA (10(8) cells/ml), C2D serum (1/20), and a suitable dilution of a source of C2 were incubated at 37 degrees C for 60 min and the fraction of cells lysed was used to calculate the effective molecules of C2/ml test material. The assay can be used to titrate C2 in human, guinea pig, rat, mouse and rabbit sera, but not C2 in dog serum. The assay is simple and reproducible, and comparable in sensitivity to the conventional two-step assay with EAC14 cells and Cgp-EDTA.     

EAC4 and EAC14 production without purified Ci.

EAC4 and EAC14 of high activity were prepared by treating sensitized red cells with human or guinea pig serum in the presence of TTHA, a chelating agent whid at low pH to maximize C1 and C4 activity and to minimize C3 contamination. Cells prepared with guinea pig complement were contaminated with C2, which could be decayed away at 37 degrees C. Cells sensitized with IgG antibody were more reactive than those sensitized with whole serum or with IgM, and preparations made with TTHA-complement were more reactive than those prepared with purified C1 plus EDTA-complement. EAC14 stored at 0 degrees C for 3 weeks lost very little activity.     

Role of TraT protein, an anticomplementary protein produced in Escherichia coli by R100 factor, in serum resistance.

Escherichia coli K12 strain W3110/SM bearing a plasmid containing the traT gene (traT+ strain) was more resistant to the bactericidal activity of guinea pig serum than the same strain bearing this plasmid without the traT gene (traT- strain). A murine mAb was generated against synthetic TraT peptide (86-99). This antibody reacted only with denatured TraT protein, but it was used for monitoring TraT protein by immunoblotting during purification of the protein. Six mAb were then generated against partially purified traT protein from the solubilized membrane fraction of the traT+ strain. These mAb reacted with the native protein even on living cells, and their F(ab) fragments were found to suppress the inhibitory effect of the TraT protein on the bactericidal activity of serum. TraT protein was purified from solubilized membranes of the traT+ strain by ion exchange and gel filtration chromatographies. The purified TraT protein inhibited the lysis of sensitized erythrocytes by serum complement. Its inhibitory action was mainly on the C6 step. It strongly inhibited the reaction of C6 with EAC14b2a3b and excess C5, C7, C8, and C9. TraT protein also inhibited the reaction of C7-deficient human serum with guinea pig erythrocytes when it was activated by cobra venom factor. It did not inhibit the reaction of preformed C5b6 complexes. However, TraT did not have any effect on the cleavage of 125I[C5] to 125I[C5b] in similar conditions. It also partially inhibited the reaction steps of C4, C5, and factor B and limited guinea pig complement serum in 0.1% gelatin veronal buffered saline, pH 7.4, containing 10 mM EDTA with their respective preceding intermediate cells. It had no effect on either the binding of C3 to EAC14b2a or the cleavage of C3b by factors H and I. TraT protein probably inhibits the formation of C5b6 complex or causes structural alteration of the complex to a nonfunctional form.     

Cytotoxic effects of normal sera on lymphoid cells. I. Antibody-independent killing of heterologous thymocytes by guinea pig, rabbit, and human sera: role of the alternative pathway of complement activation.

The mechanisms whereby normal sera may cause the death of xenogeneic lymphoid cells in vitro have been reviewed in this study using guinea pig, rabbit and human sera as the source of activity and rat and mouse thymocytes as target cells. In all of the combinations analyzed the cytotoxic reactions were found to be mediated by complement (C) as evidenced by sensitivity of the sera towards either heat inactivation (56 °C, 30 min) or treatment with cobra venom factor or sodium ethylenediaminotetraacetate (EDTA). C activity was provided via the alternative pathway in every instance: (i) both C4-deficient guinea pig serum and C2-deficient human serum displayed cytotoxicity on the target cells; (ii) sera from all three sources were active in the absence of free Ca²⁺, which is required to activate C via the classical pathway; and (iii) GPS incubated at 50 °C for 20 min to destroy the activity of factor B of the alternative pathway lacked significant cytotoxic activity while still able to lyse sensitized sheep red blood cells, a reaction proceeding via the C142 pathway. Two independent lines of evidence appeared to exclude the possible role of antibodies in nonspecific serum cytotoxicity. First, the cytotoxic capacities and the titers of guinea pig and rabbit sera were not significantly affected after absorption with target cells in the presence of EDTA, i.e., in the absence of free divalent cations, a condition which does not interfere with antigen antibody binding. By contrast, the activity was eliminated when absorption was performed in the absence of chelating agents or in the presence of a selective Ca²⁺ chelator, sodium ethyleneglycoltetraacetate, plus excess Mg²⁺ These observations also highlight the Mg²⁺-dependence of the removal of activity by absorption. Second, γ-globulins isolated from a highly cytotoxic guinea pig serum were not toxic for rat thymocytes when tested in the presence of rat C. These results suggest that conventional antibodies, whether of “natural” origin or otherwise, are unlikely to play a role in serum-produced nonspecific cytotoxicity. Furthermore, and since incubation of human serum with rat or mouse thymocytes produced conversion of factor B, “absorption” of cytotoxic activity would seem to be more likely a consequence of the consumption of C activity via the C3 shunt than of the removal of any antibodies.     

Activation of the alternative complement pathway by Leishmania promastigotes: parasite lysis and attachment to macrophages

When exposed to normal human or guinea pig sera, promastigotes of Leishmania enriettii and L. tropica activate the complement cascade by the alternative pathway and fix C3 on their surfaces. In high (25%) serum concentrations, the result of complement activation is parasite lysis. At lower concentrations (4%), complement fixation results in enhanced parasite binding and uptake into murine peritoneal macrophages. Parasites are lysed in normal guinea pig, C4-deficient guinea pig, normal human, and C2-deficient human sera when they are incubated at 37 degrees C for 30 min. Fetal calf and normal mouse sera are poorly lytic. Lysis requires Mg++ but not Ca++, is mediated by heat labile (56 degrees C, 30 min) component(s), and does not occur when the incubations are maintained at 4 degrees C. Guinea pig serum preadsorbed with promastigotes of L. tropica in EDTA at 4 degrees C for 30 min is fully lytic. Immunofluorescence studies with anti-C3 antibodies show that under these conditions C3 is deposited on the surface of the parasite. The serum-dependent binding of parasites to macrophages is also mediated by heat-labile, nonadsorbable factor(s) present in normal guinea pig and mouse sera, as well as C2-deficient and C4-deficient sera. The serum-dependent macrophage recognition mechanism is trypsin sensitive but relatively resistant to chymotrypsin. Parasites but not macrophages can be presensitized at room temperature with low levels (8%) of serum to enhance their binding to macrophages. Presensitization does not occur at 4 degrees C. These results show that Leishmania promastigotes of several species can fix complement by activating the alternative complement pathway. This may then result either in parasite lysis or in an accelerated uptake of the parasite into phagocytic cells. In vivo, the biologic outcome of infection may reflect a balance between extracellular lysis and enhanced uptake into phagocytic cells.     

Number of hits necessary for complement-mediated hemolysis

The number of hits necessary for the C8 and C9 steps of immune hemolysis was reexamined with a previously unemployed experimental design, in which various numbers of EAC1-7, excess of the supplementary component and a constant amount of the component tested were incubated in a constant volume (Inoue et al. 1976. Infect. Immun. 13: 337). Our results were consistent with previous findings; the steps of guinea pig C8 and C9, the human C8 each followed a one-hit mechanism, while that of human C9 showed ka multi-hit response. When lysis of sensitized erythrocytes (EA) by normal human serum was analysed in a similar way, one-hit curves were obtained. This result, taken together with the above results, suggests that immune hemolysis occurs by a single lesion including a single C8 and multiple C9 in the case of human complement and that normal human serum contains sufficient excess of C9. On the other hand, when C9-deficient human serum was used for lysis of EA, multiple-hit curves were obtained. The mechanism of lysis by C5b-8 may differ from that by C5b-9.     

Collagenase of human skin basal cell epithelioma.

Collagenase of human basal cell epithelioma was purified by sequential ammonium sulfate precipitation, Sephadex gel filtration and affinity chromatography on collagen-polyacrylamide gel. The collagenase, when partially purified, was found to have an approximate molecular weight of 50,000. The purified enzyme contained no caseinolytic activity. On polyacrylamide gel electrophoresis, the purified enzyme gave a single protein band. The purified collagenase cleaved native acid-soluble guinea pig skin collagen at 37 degrees C with a pH optimum of 8. The enzyme was inhibited by EDTA, cysteine, and human serum but not by soybean trypsin inhibitor. Heparin did not stimulate the enzyme activity. Purified collagenase reduced the specific viscosity of native acid-soluble guinea pig skin collagen to 50 per cent of its original value at 27 degrees C. Polyacrylamide gel disc electrophoresis of the reaction products showed bands corresponding to alphaA, betaA, and alphaB fragments. Electron microscopic examination of SLS aggregates of the reaction products showed that the cleavage site by the enzyme was at a point 75 per cent from the "A" end (TCA75) and 25 per cent from the "B" end (TCB25) of the collagen molecule.     

Identification of RNA as a complement inhibitory component in an extract of Ehrlich ascites tumor cells.

A factor capable of inhibiting complement was obtained from intact Ehrlich ascites tumor cells by mild extraction with phosphate-buffered saline (PBS). The inhibitor caused a decrease in extent of lysis of EAC14 with a concomitant extension of Tmax. EA, EAC1, EAC4 and EAC142 were all less susceptible to complement-mediated lysis after treatment with the tumor cell extract. Partial purification of a complement inhibitor was accomplished. The inhibitor was rich in RNA and its activity was totally destroyed by RNAase but not DNAase. RNA from mouse tissues, yeast, and Escherichia coli also inhibited complement hemolytic activity. The partially purified material only inhibited lysis of EAC1 and EAC14. Slow inhibition of fluid phase C1 was also demonstrated. In addition, RNA-rich partially purified tumor cell extract was capable of precipitating with purified human C1q.     

The role of the first component of the bovine complement system in the haemolytic activity of a bovine derived lytic intermediate

A stable lytic intermediate (LI) which is lysed in the presence of guinea pig C3-C9, can be prepared from sensitized sheep erythrocytes (EA) and bovine serum. The lytic activity is destroyed by 0.01M EDTA at physiological ionic strength, but may be restored by partially purified bovine C1 or human C1 devoid of any C4 or C2 activity. The presence of C1 appears to be essential for the activity of the LI. Compounds such as DFP and PMSF which effectively inhibited the esterase activity of bovine C1 also inhibited the capacity of C1 to reform an active LI from the decayed lytic intermediate (DLI). The esterase inhibitors had no apparent effect on the active LI. Previous exposure of the DLI to DFP-inactivated C1 did not inhibit the subsequent and effective binding of active C1. It is suggested that the C1 forms an intimate and essential relationship with either or both of the C4 and C2 components for the activity of the bovine derived LI. This is supported by comparing the uptake of tritium labelled DFP-inactivated C1 by EA and the DLI.     

Rapid removal of mycoplasma from cell lines mediated by a direct effect of complement

Mycoplasma can be removed from the surface of contaminated human and murine cell lines by incubation for 4 h with human, rabbit, guinea pig, or mouse sera. Several lines of evidence suggest the involvement of complement in this process: (1) The activity can be abrogated by heat treatment (56 degrees C for 45 min). (2) Using monoclonal antibodies directed against C3a and C3b, the deposition of C3b fragments on the surface of mycoplasma-positive cells can be demonstrated after 1 h incubation with human serum. (3) Ca2+ depletion ablates the ability of serum to remove the activity. (4) C2def' sera are inactive while addition of purified C2 reconstitutes the activity. The latter two findings implicate that activation of the classical pathway of complement is responsible for the effect. Antibody, however, is not required as demonstrated by the uncompromised activity of Ig-deficient sera from bursectomized chicken. Treatment with human serum or rabbit serum was used successfully to permanently cleanse 10/10 tumor cell lines of human and of murine origin. The complete removal of mycoplasma was monitored over at least 8 weeks by direct DNA staining and confirmed by agar culture and transfer of supernatants to mycoplasma-free Vero cells followed by DNA staining. Thus the direct interaction of mycoplasma and complement appears to be an effective and rapid means of curing cell lines from mycoplasma.     

Complement-mediated hemolytic activity of succinylated concanavalin A: preparation and activity of cell intermediates

Succinylated concanavalin A (SCon A) lyses sheep erythrocytes (E) in the presence of complement, whereas the native tetravalent lectin, Con A, is inactive. We have studied the ability of E-SCon A (ES) to interact with early acting guinea pig (gp) or human (hu) complement components (C1, C2, C4) and found that cell intermediates ESC1, ESC4, ESC14, and ESC142 can be generated that are analogous to intermediates conventionally prepared with E and rabbit IgM (pentameric) anti-Forssman antibody. Titration of gp or hu C1, C4, and C2, and quantification of the number of activated C1 molecules bound to ESC4 by the C1 fixation and transfer test showed in each case that an average of one effective site per cell was sufficient to cause cell lysis. Determination of tmax for optimal formation of ESC142 sites depended on the species combination of components used to make the intermediates, and the decay of ESC142 and EAC142 sites or sites generated with ESC4, EAC4, and trypsin-activated C2 were similar. The sugar alpha-D-methylglucopyranoside (alpha-MGP) inhibited binding of SCon A to E and eluted the lectin from ES, whereas galactose was nearly inactive, consistent with lectin sugar-binding selectivity. In contrast, both sugars were ineffective in eluting SCon A or C4hu from ESC4hu, indicating that C4hu blocked the interaction between lectin and alpha-MGP, perhaps by steric hindrance. SCon A is a divalent functional analogue of IgM anti-Forssman antibody that may be a uniquely suited reagent specific for cell membrane glycoconjugates for studying the mechanism of binding and activation of complement components.     

Evidence that bovine conglutinin reacts with an early product of C3b degradation, and an improved conglutination assay.

When EAC43b were treated with heated serum in EDTA, reactivity with bovine conglutinin appeared rapidly, even at 0 degrees C, and almost simultaneously with the loss of C3b rosetting capacity. At the time conglutinability first appeared, there was no detectable decrease in I-A or hemolytic C3 activity, and no detectable C3 antigen release from the cells. With prolonged exposure to heated serum in EDTA, I-A (immune adherence) and hemolytic C3 activity were lost. If this exposure was at 37 degrees C, C3 antigen became strongly detectable in the supernatant fluid, and eventually conglutinability was markedly reduced or lost, whereas C3d rosettes were unaffected. We suggest that bovine conglutinin reacts with some early product of C3b degradation, rather than with C3d, and propose that this intermediate be designated C3k. We have developed a semi-quantitative assay for bovine conglutinin, utilizing a Coulter Counter to register the decrease in total particles due to red cell aggregation. By using this method, we have detected conglutination with mouse complement (C) as well as with that from man and the guinea pig.     

Identification of thymus-dependent areas in frozen sections of guinea pig lymphatic tissue by adherence of rabbit erythrocytes

Untreated rabbit erythrocytes adhere to thymus-dependent areas of guinea pig lymphatic tissues as shown with frozen sections. The adherence reaction is temperature dependent. Optimal results were obtained by incubation of the tissue section with the erythrocytes at temperatures between 0 ° and 4 °C. At 37 °C no adherence of erythrocytes was observed. Out of other erythrocytes tested (human, sheep, chicken, rat, mouse) only rat and mouse cells showed weak adherence to guinea pig thymus sections.     

Serum-induced inhibition of isotonic fluid absorption by the kidney proximal tubule. III. Further evidence that complement-mediated cell lysis is involved.

The results of our previous studies have suggested that serum-induced inhibition of proximal tubular fluid absorption is due to complement-induced lysis of the tubular cells. The present study provides further evidence in support of this idea as well as other information pertinent to the mechanism of complement activation in vivo. 1. The electrical resistance of the luminal brush border membrane is reduced drastically concomitantly with a drop in cell potential difference when serum is perfused intraluminally. 2. Human C1 inhibitor (30-50 units/ml) does not significantly affect the inhibitory activity of human serum on fluid absorption, suggesting the non-involvement of the classical pathway. 3. Reactive lysis reagents (C56, C7, C8 + C9) partially inhibit fluid absorption when infused into the lumen. 4. In contrast to our previous report (Sato, K. and Ullrich, K.J. (1974) Biochim. Biophys. Acta 354, 182-187), very fresh serum, 10-times diluted can inhibit fluid absorption if perfused for 10 min. 5. Both mouse and guinea pig serum, which are normally inactive, are activated to attack the tubular cells if 1/100 volume rat or rabbit serum is added to them No such activation occurs by mixing guinea pig serum and mouse serum. The available data suggest that the presence of the later complement components but not the heat-labile factor (Factor B) or C3PA or C1 in the added serum is a prerequisite for mouse and guinea pig sera to be activated to inhibit fluid absorption.     

A self determinant recognizing T cell hybridoma

A T cell hybridoma (53(113)) obtained by fusion of BALB/c spleen cells and the BW 5147 lymphoma T cell line is described. This hybridoma recognizes mouse RBC (MRBC) and rat RBC, but not human, rabbit, guinea pig, or SRBC. The culture supernatant possesses hemagglutinating activity for the same indicator RBC. In addition to this, 53(113) cells are able to form protein A plaques in the presence of guinea pig complement and normal mouse serum (NMS) or purified mouse immunoglobulins (Ig). Because mouse Ig as well as sonicates from MRBC are able to inhibit the rosettes between the hybridoma cells and the MRBC, and because the sonicates inhibit protein A plaque formation, it seems likely that the same product can recognize a similar determinant expressed on MRBC and mouse Ig. The hypothesis that a 53(113) structure recognizes identical or cross-reactive carbohydrate determinants shared by murine Ig and C is considered.     

Ixodes dammini: salivary anti-complement activity

Saliva of the tick Ixodes dammini prevents hemolysis of rabbit erythrocytes by the human alternative pathway of complement. Deposition of C3b to activating surfaces and concomitant C3a release are inhibited. C3b deposition to activating surfaces is inhibited regardless the origin (humans, rat, mouse, guinea pig, and hamster) of the serum. The inhibitor elutes as a single peak upon gel filtration, with an apparent molecular weight of 49,000. Salivary anti-complement may contribute to successful feeding of I. dammini in their natural hosts.     

Complement inhibitor(s) released by leukocytes. III. Evidence for a "new" C1 inhibitor in the supernatants of short-term cultures of mouse spleen and thymus cells.

Mouse spleen or thymus cells in short-term culture release a factor, designated S, that binds to sheep erythrocytes (E). Supernatant-treated sheep erythrocytes (SE) are capable of fixing and transferring the activated first component of guinea pig complement. SEC1, however is not capable of initiating hemolysis by the rest of the complement components. SE is capable of binding but not activating native C1; native C1 bound to SE seems irreversibly inhibited. Evidence is presented that S may be the same factor as the previously described inhibitor released by mouse spleen or thymus cells that inhibits the utilization of C2 by EAC14.