Experiments with Labelled Complement: I. Complement Labelled in vivo

Guinea pigs were injected with ³⁵S-amino acids and their sera were subsequently used as a source of labelled complement. The adsorption of radioactivity from such sera by specific immune precipitates and by insoluble, suspended, denatured proteins was studied under various experimental conditions. It was found that heat-inactivation of the serum sharply reduced the radioactivity carried down as did the addition of ethylene diamine tetraacetic acid to fresh serum. The latter effect could be overcome by the addition of calcium and magnesium ions. The addition of fresh unlabelled guinea-pig serum to heat-inactivated labelled guinea-pig serum increased the radioactivity adsorbed by the immune precipitates. This indicated that, when the heat-labile components of complement are thus supplied, the labelled heat-stable components can again be adsorbed. This effect of unlabelled complement could not be obtained with labelled serum that had been treated with hydrazine. It was also shown that the heat-stable, hydrazine-sensitive component was present in midpiece and absent from endpiece, indicating that it was not C′₄, but probably a component of C′₃.

The adsorption of active C′₁ by some heat-denatured suspended serum protein preparations was demonstrated.

     

Inhibition of complement by carrageenin: mode of action, effect on allergic reactions and on complement of various species

Carrageenin prevents the reaction of guinea-pig C′ with sensitized red blood cells, probably by a direct inactivation of the C′₁. The anti-complementary effect of carrageenin is antagonized by protamine sulphate. Marked reduction of circulating C′ activity in guinea-pigs resulting from intravenous injection of carrageenin, does not alter their susceptibility to acute or protracted anaphylactic shock. The C′ activities of human, guinea-pig, rabbit, rat and monkey sera are all inhibited in vitro by carrageenin.     

Studies on the antigenic properties of complement: III. Specific inactivation of cell-fixed components of guinea-pig complement by anti-complement*†

Evidence is presented to show that on EAC′₁ cells the activity of cell-fixed guinea-pig C′₁ can be abolished by exposure to an agglutinating anti-C′₁ serum. The function of cell-fixed guinea-pig C′₄ on EAC′_1,4 cells can be blocked by an agglutinating anti-C′₄. It is concluded that the activities of both fixed C′₁ and fixed C′₄ are bound to the structural integrity of two distinct antigenic substances. No functional blocking and no agglutination could be observed with respect to fixed guinea-pig C′₂. A specific treatment with anti-C′₄ not only inactivates EAC′_1,4 cells with respect to C′₂ but also abolishes the reactivity of EAC′_1,4,2 cells with respect to C′₃. These findings suggest that C′₂ may act upon EAC′_1,4 like an enzyme, i.e. without being bound materially; as a result of the reaction, fixed C′₄ may be converted chemically into a form which itself acts as a receptor site for the fixation of C′₃.     

Preparation and partial characterization of sheep erythrocyte—antibody—complement intermediate EAC′1a,4,2a,3,5,6,7

A reagent has been prepared which contains C′₄, C′₂, C′₃, C′₅, C′₆ and C′₇ by chromatography of guinea-pig serum on CM-cellulose. With this reagent, when supplemented with additional C′₅, EAC′_{1a,4,2a,3,5,6,7} was prepared from EAC′_1a. The reactivity and stability of cells possessing active C′₇ sites prepared with this reagent appeared to be the same as EAC′_{1a,4,2a,3,5,6,7} prepared from EAC′_1a,4 and mixtures of individual functionally pure components. The reactivity of cells possessing active C′₇ sites has been measured by initial velocity of generation of C′₈ sites, and an inhibitor of this reaction, Antrypol, has been described. A method for the estimation of the average number of C′₇ sites was investigated and a correlation appears to exist between the average number of sites per cell and the initial velocity of reaction with C′₈. Finally, a provisional method for the assay of C′₈ and C′₉ has been described.     

: VI. : Fowl Antibody • The Fixation of Complement by Antibody to Soluble Antigens. The Effect of Fresh Normal Fowl Serum on the Precipitation Reaction

The uptake of haemolytic fowl C′ by fowl anti-BSA and BSA has been detected and measured with sheep red cells sensitized with fowl haemolysin. It was, however, very small compared with the amount of guinea-pig C′ taken up by similar amounts of rabbit anti-BSA, and not directly related to the amount of precipitating antibody in any particular antiserum; nor did it occur when preformed resuspended specific precipitates were added to the C′ solution. Fixation of guinea-pig C′ by fowl anti-BSA could not be demonstrated, either with fresh antiserum, or with heated antiserum in the presence of normal fresh fowl serum.

Uptake of haemolytic fowl C′ was shown to coincide with a great increase in specific precipitation when this was done in 0.9 per cent NaCl. This increase was produced partly by the addition of a non-specific serum factor, and partly by more complete precipitation of the antibody. Specific precipitates formed in 0.9 per cent NaCl in the presence of fresh normal fowl serum (NFS) contained much larger amounts of a macroglobin component than precipitates formed in the absence of NFS or in 8 per cent NaCl.

     

Studies of the Reaction Between the Sensitized Erythrocyte and the First Component of Complement

When sensitized erythrocytes (EA) possessing the 1st (C′₁), 4th (C′₄) and 2nd (C′₂) components of haemolytic complement were treated with 1,4-butanediamine, C′₁ but not C′₄ or C′₂ was removed from the cell. All the straight chain diamines of 3 to 8 carbons, with the amino groups on either end, removed C′₁ with about equal facility; the diamines were considerably more active than the corresponding monoamines. C′₁ could be recovered in solution following its removal from the red cell by the diamine. The rate of detachment of C′₁ was quite high for the first 10 minutes, after which it was slower and followed first order kinetics. It appears that C′₁ and the diamine compete for the same site on the sensitized erythrocyte. The following observations suggest that Ca⁺⁺ has a role in the EAC′₁ bond: (a) An increased Ca⁺⁺ concentration diminishes the effect of the diamine. (b) The EAC′₁ bond dissociates at low bivalent cation concentration. (c) A number of bivalent cations will preserve the EAC′₁ bond, Ca⁺⁺ being the most effective.     

Inhibition of the C′1 component of complement by amino acids

Studies were performed to determine whether epsilon-amino caproic acid (EACA) and related compounds known to inhibit plasminogen activation might also similarly affect immune haemolysis by inhibition of C′₁ proesterase activation. Long-chain amino acids (six to eight carbon atoms), diamino amino acids and amino alkanes inhibited immune haemolysis, whereas dicarboxylic and fatty acids did not. Inhibition of immune haemolysis by the former group was reversed by the addition of complement reagents R₂, R₃ and R₄ (lacking C′₂, C′₃ and C′₄ components), but not by reagents R₀ and R₁ (lacking components C′₀ and C′₁). These results suggested that the amino group was necessary for inhibition of immune haemolysis by EACA and that EACA inhibited complement activity by reacting with the C′₀ and C′₁ components.

Since the C′₁ component of complement was inhibited, the activation of C′₁ proesterase was studied with the pH stat. The following observations were made: (1) The reaction between C′₁ proesterase and the sensitized red cell produced hydrogen ion and therefore was considered to be enzymatic. (2) This activation reaction was inhibited by EACA and ethylenediamine tetraacetic acid (EDTA). (3) C′₁ esterase hydrolysed p-tosyl-1-arginine-methyl-ester (TAMe) provided that the system was free of serum or that TAMe was added to the sensitized red blood cells before the complement source (C′₁). (4) C′₁ esterase activity, in contrast to C′₁ proesterase activation, was not inhibited by EACA or EDTA. (5) Addition of increasing amounts of TAMe to the intact haemolytic system resulted in decreased rates of immune haemolysis, together with increased rates of hydrolysis, suggesting that TAMe was competing for the active site of C′₁ esterase with a natural substrate (C′₂) essential for haemolysis.

     

The preparation and properties of alexinated intermediates that react with conglutinin: I. Guinea-pig complement

Evidence has been advanced confirming earlier work that conglutinin reacts solely with a determinant exposed in fixed C′_3a. The reactivity is not influenced by the decay of earlier components or the activity of later ones. Heterologous sources of C′_3a (rabbit and human) reacting with guinea-pig EAC′₁₄₂ give conglutinable intermediates.

The stability of EAC′_1423a for conglutination is much greater than for lysis. The study of decayed intermediates which still react with conglutinin suggests that the bulk of the C′_3a fixed has after some time no haemolytic function. Possible explanations of this phenomenon are discussed.

The `long-term' functions of the bulk of the fixed C′_3a may therefore be associated with manifestations of complement activities other than haemolysis.

     

in vitro and in vivo : Inhibition of Guinea-Pig Complement • by Carrageenin

The sulphated polysaccharide carrageenin is a powerful inhibitor of the haemolytic action of guinea-pig serum complement. In vitro carrageenin appears to act at a stage prior to the fixation of C′₁ to sensitized red blood cells. Intravenous administration of carrageenin to guinea-pigs markedly reduces the level of serum complement activity.     

The Relationship of the Haemolytic Activity of Complement to the Concentration of Sensitized Erythrocytes

The relationship of the haemolytic activity of complement (C′) to the concentration of sensitized erythrocytes (EA) has been found subject to precise mathematical definition, on the basis of data obtained from simultaneous C′ titrations carried out with twelve different EA concentrations ranging from 10⁷ EA/ml. to 2×10⁹ EA/ml. Increase in EA concentration is accompanied by increase both in the amount of C′ required for 50 per cent haemolysis (RD₅₀ C′), and in the value of σ (reciprocal of the slope of the curve relating probit percentage haemolysis to log C′). The relationship of RD₅₀ C′ to EA concentration is an exponential one; the equation expressing this relationship contains a parameter which is interpreted as consistent with the existence of a threshold value of C′ which should be taken into account in assessing RD₅₀ C′. An explanation of the increase in RD₅₀ C′ and σ with increase in EA concentration is offered.     

Fowl Antibody: V. The Interactions of Fresh and Heated Fowl Serum and of Guinea-Pig Complement Measured by the Lysis of Sensitized Red Cells

Dilutions of fresh fowl serum up to about 1:100 inhibit the lysis of sensitized red cells by guinea-pig complement; this inhibition is decreased by treatments of the serum that also reduce its own haemolytic activity. Studies of this effect are complicated by the fact that fresh fowl serum, when diluted up to 1:32–1:64, lyses sheep red cells with no added haemolysin; with cells sensitized with added haemolysin this lysis is seen in even higher dilutions. Lysis by fowl complement is in turn inhibited by guinea-pig complement: the lytic action of 1–2 C′H₅₀ of fowl C′ is blocked by guinea-pig serum 1:1000. Thus mixtures of sheep red cells sensitized with a mammalian haemolysin, fresh fowl serum and guinea-pig complement possess two distinct haemolytic activities which are mutually inhibitory, giving rise to effects that simulate specific fixation.

Heated fowl serum, if present in excess, inhibits lysis by fowl C′; when added in small amounts it supplies a limiting heat stable factor and enhances lysis.

     

Complement components of a haemolytically deficient strain of rabbits

A strain of complement (C′) deficient rabbits was studied. No C′₆ haemolytic activity was found in the sera of these rabbits. Both haemolytic and leucocidal activities were stored upon adding purified C′₆ isolated from either normal rabbits or normal guinea-pigs. The mixture of deficient serum with sensitized sheep cells resulted in the generation of active C′₅ sites, SAbC′_1,4,2,3,5, since lysis occurred after the addition of highly purified C′₆, C′₇, C′₈ and C′₉ to the washed cells. These rabbits possess a heat labile substance which is considerably heavier than C′₆. This C′₆ inactivator reacts specifically with the intermediate complex, EAbC′_1,4,2,3,5,6. The product of the reaction appears to be incapable of combining with the next component in the haemolytic sequence, C′₇. The inactivator is not depleted from the fluid phase during interaction with cells possessing active C′₆ sites.

The elution characteristics from DEAE- and CM-cellulose and the sedimentation constants C′₆ and of the inactivator, as well as of certain other rabbit C′ components, were determined. C′₆ inactivator was also found in normal rabbit serum. A mechanism to explain the variability in results of homotransplant rejection in C′₆ deficient rabbits is offered.

     

A sedimentation pattern technique for measuring conglutination: its application to demonstrating immunoconglutinins to C′4

The usual method of measuring the conglutination phenomenon is to use a centrifugation and resuspension technique to assess the clumping of alexinated cells. This method is designed only to detect powerful clumping and immunoconglutinins measured in this way have always been found to react with fixed C′_3a.

A disadvantage of this method is that only antibodies to complement-antigens present in large amount on the alexinated cell will be detected. This may explain why immunoconglutinins (I-Ks) to other complement components have not been found.

A method of measuring I-Ks by a sedimentation pattern has been devised. A macroglobulin fraction of an antiserum to purified Forssman hapten is used to sensitize the erythrocyte. This is complement-fixing much beyond its agglutination titre and is almost free of reactivity with rheumatoid factors. Guinea-pig complement is used to alexinate and various defined intermediates have been tested—namely EA (sensitized sheep cells) EAC′₄, EAC′₁₄₂, EAC′_1423a, EAC′_43a.

It has been found that immunoconglutinins reacting with EAC′₄ are commonly found in human sera. Reaction with EAC′₄ is usually as good as and sometimes much better than with EAC′₁₄₂.

However the reactivity of most I-Ks with fixed C′_3a has been confirmed as has the exclusive reactivity of bovine conglutinin with fixed C′_3a.

Some level of I-K has been found in all the human sera tested. Particularly high levels have been found in trypanosomiasis.

     

Studies on chemotaxis. XII. Generation of chemotactic activity for polymorphonuclear leucocytes in sera with complement dificiencies

The significance of the C′ system for chemotaxis of rabbit PMNs was studied in vitro. C′6-deficient rabbit sera and C′5-deficient mouse sera were compared with normal rabbit and mouse sera for their ability to generate chemotactic factors in the presence of antigen—antibody complexes and endotoxins. No difference could be detected between these C′-deficient and normal sera. Full chemotactic activity was observed even when a rabbit anti-C′6 serum was incubated with an antigen—antibody complex. These results are incompatible with C′ (5, 6, 7)_a being the chemotactic factor in serum for polymorphonuclear leucocytes.     

Interactions of the complement system with endotoxic lipopolysaccharide: the generation of an anaphylatoxin

The incubation of endotoxin derived from Veillonella alcalescens or Serratia marcescens with fresh guinea-pig serum leads to the production of a factor which contracts guinea-pig ileum. This factor has been identified as an anaphylatoxin since it causes tachyphylaxis and its activity is abolished by antihistamines. The activity is not generated if the serum has been heat inactivated or if the reaction is carried out at 0° or in the presence of EDTA. The complement system (C′) is implicated in the generation of this anaphylatoxin by the above data and by static and kinetic C′ consumption studies which show a correlation between the disappearance of the late acting C′ and the appearance of gut contracting activity. Endotoxoid preparations which do not utilize C′ are not able to generate anaphylatoxin. A further parallel between classical anaphylatoxin and the endotoxin-generated factor is found in the pattern of mammalian sera which support the reaction. In view of the similarities between the biological properties of anaphylatoxin and the syndrome of endotoxic shock, it is suggested that some of the manifestations of the latter might result from an interaction between endotoxin and the C′ system leading to the generation of an anaphylatoxin.     

The Inhibition of Immune Haemolysis by Salicylaldoxime

It has been shown that haemolysis of sheep erythrocytes by antibody and either guinea pig or human complement is inhibited by salicylaldoxime (0-hydroxybenzaldoxime.) Salicylaldoxime differs in its action on immune haemolysis from such previously described inhibitors as ethylenediaminetetraacetic acid and diisopropylfluorophosphate in that it specifically inhibits the reaction between the intermediate complex EAC′_1,4,2 and C′₃. The inhibition does not appear to be reversible by divalent cations. To be effective the inhibitor must be present during the course of the reaction, since haemolysis proceeds unimpaired if the inhibitor is added to complement and removed by dialysis, or if added to either red cells or EAC′_1,4,2 and removed by centrifugation. The experimental evidence suggests that salicylaldoxime acts by preventing the effective combination of C′₃ with EAC′_1,4,2, and not by the destruction of C′₃.     

Formation of C′6 by rabbit liver tissue in vitro

Various rabbit tissues such as liver, spleen, peritoneal macrophages and kidney, were cultured in vitro at 37° for periods of up to 72 hours. The combined content of C′₆ in tissue and culture medium was determined at the end of the incubation period. Production of C′₆ activity was found in liver cultures and possibly in spleen and macrophage cultures. The rate of inactivation of serum C′₆ at 37° in culture medium was also determined.     

Physico-chemical characteristics of the third and fourth component of complement after dissociation from complement—cell complexes

The radioactivity labelled third (C′₃) and fourth (C′₄) components of human complement were dissociated from erythrocyte membrane—complement complexes. The differential effectiveness of various reagents in causing dissociation suggests that C′₃ and C′₄ are bound to membrane sites by hydrophobic bonds. Density gradient ultracentrifugation of the dissociated components showed that they are bound in macromolecular form. Their electrophoretic mobility was greater than that of their native counterparts. In physical and immunochemical properties, dissociated C′₃ and C′₄ closely resembled the respective haemolytically inactive conversion products that, during immune haemolysis, accumulate in the fluid phase. It is concluded that in immune haemolysis C′₃ and C′₄ become linked to membrane sites only following modification by their respective converting enzymes.     

Nilsson and H. J. : Studies on the mode of action of the fifth, sixth and seventh component of human complement in immune haemolysis: Müller-Eberhard*

The sixth (C′₆), seventh (C′₇) and eighth (C′₈) components of human complement were separated from each other by chromatography on hydroxyl apatite and obtained in functionally pure form. Density gradient ultracentrifugation revealed that C′₆ and C′₇ interact with the fifth component of complement (C′₅) to form a reversible protein—protein complex. Evidence was obtained that this complex occurs also in unfractionated, fresh human serum. Kinetic analysis of the reaction sequence in immune haemolysis disclosed that C′₅, C′₆ and C′₇ act, in this order, subsequent to the third component (C′₃) and prior to C′₈. Exploration of the mode of action of C′₅, C′₆ and C′₇ led to the formulation of two alternative hypotheses. The three components act either sequentially or as one functional unit. Their fluid phase interaction, the shape of dose—response curves and the encountered difficulty to isolate intermediate reaction products favour the concept that they act as a functional unit.     

The Inhibition of the Haemolytic Action of Guinea-Pig Complement by Heated Guinea-Pig Serum

Haemolysis of sensitized erythrocytes (EA) can be carried out by incubating them successively with R2, followed by R1. When heated guinea-pig serum is present during the incubation of R2 with EA it inhibits haemolysis, though it has no effect when present during the incubation with R1. This inhibition is stronger with aged preparations of R2 which have much lower titres of C′₄ than fresh preparations, though this increased inhibition does not occur because of the inactivation of C′₄. Pre-incubation of EA with heated serum does not cause inhibition of their subsequent haemolysis by R2 and R1. Guinea-pig serum that has been decomplemented with an antigen-antibody precipitate and then heated is still inhibitory. Heated serum has no inhibitory action when haemolysis is carried out with whole guinea-pig serum in which all the components of complement are present simultaneously.