Anticomplementary Effect Research Articles

Alterations of complement system following thermal injury: use in estimation of vital prognosis.

Complement system has been investigated in the first 15 days postburn. Complement titer (measured by a kinetic method of immune hemolysis) decreases immediately after thermal injury. This depression is mainly explained by mechanical leakage and is related to burn surface. No anticomplementary effect has ever been found in this period. After 48 hours complement titer rises. Magnitude and rate of rise are inversely related to the severity of burn trauma (depth and surface). Infection occurrence is statistically related to but somewhat independent of complement levels and interferes with the rate of increase of complement levels. The basis of the rise to normal or elevated complement levels remains unexplained.

Humoral immunity in malignant skin melanoma. Isolation of melanoma specific IgG from melanoma metastases

Three M potassium chloride extracts of malignant melanoma, hypernephroma and colonic carcinoma were found to consume complement in the absence of patient sera. Treatment of the tumour extracts with papain abolished the anti-complementary effect. IgG and IgA present in tumour extracts were identified in cross-over electrophoresis by means of monospecific antibodies. Purified melanoma-derived immunoglobulins were reacted in complement fixation test against the 3 M KCl extract of an established melanoma cell line with positive result. The presence of specific antimelanoma IgG as well as native common melanoma antigen was thus established. It is concluded that specific antimelanoma antibodies are present within the clinical tumour whereas the actual localization of these antibodies, i.e., whether present on tumour cell membranes or on infiltrating lymphocytes, remains an open question. Sera from 93 patients suffering from stage I and II skin melanoma were assayed for circulating antibodies against antigen present in the extract of an established melanoma cell line. A positive CFT was established in 27% of the patients. The discrepancy between this finding and that of other investigators evades good explanation.

Study of the interaction between monoclonal IgM proteins and the complement system

The capacity to activate the complement system and to fix isolated C1 of different monoclonal IgM proteins and various fragments was studied. All the 8 different IgM proteins tested fixed isolated C1, but the dose of the IgM preparations necessary for the fixation of the 50% of the available C1 activity strongly differed from each other. The complement activating (anti-complementary) effect of the IgM preparations was weak. (Fcμ)5 fragments prepared by tryptic digestion from 4 different IgM proteins and separated by gel filtration at pH 8.0 could fix C1 and their anti-complementary effect significantly increased with respect to the original IgM preparations. It was demonstrated that the C1-fixing ability of the (Fcμ)5 fragment was lost after gel filtration at pH 3.0 and a C1-fixing low mol. wt peptide fraction was released from the (Fcμ)5. This peptide recombined with the non-C1-fixing (Fcμ)5 and restored its ability for the C1-fixation. It was shown, furthermore, that the (Fcμ)5 preparation which could not fix isolated C1, activated the complement system via the alternative pathway.

The failure to show a necessary role for C3 in the in vitro antibody response.

The in vitro antibody response of mouse spleen cells to TNP coupled to both T-dependent and T-independent carriers as well as to sheep erythrocytes has been studied to investigate the possible role of complement activation in the induction of antibody formation. The following has been found. (1) In vitro responses of both IgM and IgG can be obtained to both T-dependent and T-independent antigens in serum-free media, although they are smaller than those found in serum-containing media. This shows that no exogenous source of complement is necessary for in vitro antibody formation by spleen cells. (2) Similarly, normal antibody responses are obtained if the cultures are grown in human serum depleted of C3b-inactivator, which contains high concentrations of C3b. (3) In the presence of antibody to mouse C3 the response to the T-independent antigen is reduced, the IgM responses being more affected than the IgG. However, purified F(ab')2 anti-C3 has no inhibitory effect and it therefore seems likely that it is the formation of intact immune complexes containing Fc rather than the interference with C3 function that is responsible for the inhibition seen. (4) The conventionally purified anti-complementary factor from cobra venom has no effect on the antibody response in serum-free culture or when human or fetal calf sera are used. In no experiment was any potentiation of T-dependent responses observed. However, the presence of quite small concentrations (2%) of cobra veno

Specific role of magnesium ion in protecting the anticomplementary effect of DNA in immune hemolysis.

When a constant quantity of denatured DNA required for C-fixation reaction was employed as the antigen, an anticomplementary action of DNA was frequently observed. In order to cope with this difficulty in the analysis of C-fixation, the relationship between DNA, Mg2+ and C was investigated. It was found that this phenomenon was closely related to the flexible conformation of DNA which is highly influencd by Mg2+. Further, this effect could be eliminated in the presence of 5MM MgCl2. The mechanism of the elimination by Mg2+ of the anticomplementary effect of DNA was discussed.

Occurrence of a complement activator in human urine.

It is known that in rats, heterologous nephrotoxic serum (rabbit antirat kidney) produces an acute hemorrhagic pneumonitis. Administration of anticomplementary substances such as heat aggregated human γ-globulin and zymosan, protects the animal from the pneumotoxic activity of nephrotoxic serum. Previous work has disclosed the presence of an anticomplementary substance isolated from normal urine by gel filtration that proved also effective in this regard. The present communication deals with some of the work developed in order to study the mechanism of the anticomplementary activity exhibited by the urinary substance. The preliminary findings are suggestive that the anticomplementary effect is the result of an activation of the complement system provoked by the urinary substance.

Hypocomplementemia of membranoproliferative nephritis. Dependence of the nephritic factor reaction on properdin factor B.

Membranoproliferative nephritis in children is frequently associated with a hypocomplementemia produced at least in part by C3 breakdown mediated by a circulating anticomplementary factor known as C3 nephritic factor (C3NeF). C3 breakdown by this factor in vitro requires the presence of a pseudoglobulin cofactor and magnesium. The present study provides evidence that properdin factor B (C3 proactivator) is activated in the nephritic factor reaction and is the direct mediator of C3 breakdown by C3NeF. Depletion of factor B from mixtures of normal human serum (NHS) and plasma from a patient with membranoproliferative nephritis (MPP), either by heating or by immune equivalence absorption, blocks C3 breakdown by C3NeF. Addition of purified factor B to these mixtures restores the anticomplementary effect. When purified factor B is added to mixtures of MPP and purified C3, breakdown also occurs. Associated with the C3 breakdown is a change in the electrophoretic mobility of factor B from the beta to the gamma position, a shift which has been associated with cleavage activation of the molecule. Further, serum factor B levels are often low in patients with membranoproliferative nephritis and bear a rough inverse correlation with C3NeF levels. It thus appears that factor B is the previously described heat-labile C3NeF cofactor. Whether the C3NeF reaction proceeds via a pathway comparable to that activated by the cobra venom factor or via that activated by zymosan or inulin cannot be determined from the present data.

Effect of Hypertonic Sucrose Upon the Immune Bactericidal Reaction

This study was performed to determine the mechanism whereby hypertonic sucrose inhibits the immune bactericidal reaction. Other investigators had postulated that the initial attack of complement (C) on the cell wall was followed with lysozyme-containing whole serum by an enzymatic reaction upon the peptidoglycan substrate resulting in cell death. In the absence of serum lysozyme, secondary lethal changes might occur from damage to the cell's inner membrane as a result of osmotic forces in the presence of a defective cell wall. Hypertonic sucrose giving rise to plasmolysis and protection of the inner membrane was presumed to differentially inhibit the immune response mediated by lysozyme-free serum. The experimental results observed in this investigation have indicated, however, that the inhibitory effect of sucrose upon the bactericidal reaction may be explained simply by its anticomplementary effect and not by any effect on the bacterial cell. This view was supported by the following observations: (i) the comparability of the inhibitory effect of sucrose upon the immune hemolytic and bactericidal reactions, (ii) the comparable percentage loss in bactericidal activity of whole serum and lysozyme-free serum resulting from hypertonic sucrose, (iii) bactericidal antibody titrations were relatively unaffected and C titrations markedly inhibited by sucrose, (iv) the inhibitory effect of sucrose on the bactericidal reaction was unaffected by prior growth of the organism in the presence of sucrose, (v) the kinetics of the bactericidal reactivity of lysozyme-free serum in hypertonic sucrose, compared with whole serum, did not reveal a prolonged lag phase with lysozyme-free serum, but simply diminished reactivity at all times. These observations are compatible with the view that the C attack upon the outer surface of gram-negative bacteria, which plays a part in the cell's permeability control, may account for cell death. In this regard, the immune bactericidal reaction is quite comparable to the lysis of red cells or nucleated cells by C despite the lack of overt lysis in bacteria, probably because of their underlying supporting structures.

Critical Variables of the Jerne Plaque Technique As Applied to Rodent Antibody-Forming Systems Responding to Heterologous Red Cell Antigens

Summary Improved conditions were developed for the Jerne plaque technique; a simple test (“hemolytic spot-testing”) allows a study of interspecies antigen-antibody systems and interspecies complement effects as well as ionic requirements for hemolysis in agar. These optimal conditions were applied to the Jerne plaque test with parallel results. The Jerne technique can easily be applied to study the hemolytic antibody response of C3BF1 mice, Golden Syrian hamsters, and Wistar rats immunized with heterologous RBC. Optimal results depend on the type and dilution of complement used for a certain heterologous combination: guinea pig complement gives best results in the mouse anti-sheep system; rabbit complement is superior in the mouse anti-rat system; and human complement is hemolytically most active in the mouse anti-hamster, hamster anti-mouse, and rat anti-mouse combination. Each C′ requires different ionic conditions of the reaction medium. It is demonstrated that optimal results of the Jerne plaque test depend on adequate concentrations of Ca++ and Mg++ in the agar. The data suggest also that the anticomplementary effect of agar is ionic. This hemolysis-reducing or inhibiting factor is overcome by DEAE dextran and higher cation concentrations of the agar. The mechanism of action of the polycation and Ca++ and Mg++ cations is discussed. The hemolytic activity of complement of different species is not only reflected in variation of the number of plaques but also in average plaque size. A quantitative complement-antibody correlation is considered as a possible explanation for this observation, and suggests that this method may provide a model for the study of the dynamics of cell lysis.

Mixed IgA-IgG cryoglobulinemia: Immunological studies and case reports of three patients

Immunologic studies in three patients with mixed IgA-IgG cryogobulinemia are presented. In one of the isolated cryoglobulins IgA and IgG only were detected, whereas two contained small amounts of β 1A in addition to IgA and IgG. The latter two cryoglobulins caused strong latex agglutination. A number of other tests for rheumatoid factor activity gave negative results in all three instances. All three cryoglobulins possessed a strong anticomplementary effect, like that shown under the same experimental conditions by known specific precipitates of human and rabbit origin. The anticomplementary activity of the cryoglobulins was abolished by treatment at +56 °C. The possibility of the three IgA-IgG cryoglobulins being circulating antigenantibody complexes is discussed.

IgG Cryoglobulinaemia

Large amounts of cryoglobulin, about 20 mg/ml, were discovered in the serum of a woman suffering from recurrent aphthous stomatitis. The cold precipitable protein was found to be a polyclonal immunoglobulin of type G. It contained some beta1A‐beta1C‐globulin, and possessed a heat labile anticomplementary effect. Most of the tests for rheumatoid factor activity gave negative results. On ultracentrifugation at +36°C, the cryoglobulin sedimented as a homogeneous 7.8 S°20w peak. Although the ulcerative stomatitis was painful, the patient otherwise remained in good health. Sometimes she was a little anaemic. The granulocyte count was low in the peripheral blood. The bone marrow was hypocellular, with increased numbers of plasma cells and lymphocytes.

Relationship of the anti-complementary effect of fowl serum to its own haemolytic activity.

THE marked, anti-complementary effect of fresh fowl serum on guinea-pig complement and its reduction by heating to 56° C are well known1,2. Treatments of fowl serum by methods other than heating which reduced this effect also resulted in a decrease in its own haemolytic activity3, suggesting that the anti-complementary effect of fowl serum may be related to its own complement content. More recently, it has been shown that the euglobulin fraction of fowl serum, corresponding to the midpiece or first component of fowl complement, is the fraction which reduces the haemolytic titre of guinea-pig complement4,5. This heat-labile euglobulin fraction appears to be the same as the ‘normal chicken factor’ reported to be necessary for the specific fixation of guinea-pig end-piece by fowl antibody/antigen complexes4,6. Its anti-complementary effect on guinea-pig complement was also mentioned, but it was stated that this could be removed by dissolving the fraction in heated normal fowl serum to give a ‘concentrated normal chicken factor’5. Orlans, Rose and Clapp3 investigated this factor and found that, with certain concentrations of fowl serum, it could produce reductions in the haemolytic activity of guinea-pig complement, simulating specific fixation by antigen/antibody mixtures. We have now compared the effects of fowl serum, before and after various treatments, on the haemolytic activities of both fowl and guinea-pig complements.

ANTICOMPLEMENTARY PHENOMENA IN MONOCLONAL HYPERGLOBULINEMIA.

Abstract Investigations of the mechanism of anticomplementary activity of 5 strongly anticomplementary sera from patients with monoclonal hypergammaglobulinemia have been carried out. Evidence has been presented showing an increased tendency of some gamma myeloma globulins, both in the whole serum and in the isolated state, to become aggregated by heat. The complement inactivation caused by these proteins occurs in a manner identical to that produced by normal gamma globulin or antigen-antibody complexes. Low levels of a normal serum inhibitor of C′ 1 esterase have been found in these strongly anticomplementary sera. The increased capacity to bind complement resulting from the increased aggregation and a decrease in C′ 1 esterase inhibitor level seem to be the inciting factors responsible for the anticomplementary effect seen in gamma myeloma and benign monoclonal hyperglobulinemia.

Studies of Anaphylaxis: Effect of Decomplementation with Aggregated γ-Globulin

Summary The intravenous administration of aggregated human γ-globulin (AHGG) to guinea pigs depressed serum complement (C′) levels and resulted in diminished responses to passive cutaneous anaphylaxis (PCA). The data suggest that C′ may, under some circumstances, mediate PCA. Modification of passive systemic anaphylaxis by AHGG appeared not to be mediated by its anticomplementary effect.

Alleged anticomplementary effect of aspirin.

VAN OSS et al.1 recently reported that aspirin at concentrations of 2.2 × 10−3 M (40 mgm./100 ml.) inhibited the lysis of red cells in a simple complement-haemolysis reaction, but that sodium salicylate was inactive. This was interesting in view of the therapeutic action of aspirin against diseases such as the rheumatic disorders which are thought to involve immunological phenomena2. Two other related compounds, salicylaldoxime and salicylaldehyde, were earlier reported3 to inhibit red cell lysis in a similar reaction. I therefore investigated the complement-haemolysis technique since it might usefully predict anti-rheumatic activity in other salicylate derivatives.

The anticomplementary effect of kidney tissue; its association with ammonia production.

In studying the problem of the peculiar susceptibility of the kidney to coliform bacterial infection it was found that kidney tissue, unlike that of other organs, interferes with the ability of normal serum to destroy these organisms. The effect was attributable to strong anticomplementary activity, 5 to 15 times greater than that of other organs. Inactivation of complement by kidney tissue was found to have characteristics of a chemical reaction, the active principle being heat-labile, non-dialysable, and difficult to separate from tissue particles. Attempts to purify it or to obtain it in a soluble form usually resulted in great loss in activity. The component of complement affected was the fourth; i.e., that which is characterized by susceptibility to injury by ammonia. Similarities were found to exist between the conditions of ammonia formation and complement inactivation by kidney homogenates, the most notable being enhancement by phosphate and glutamine. The possibility is suggested that these findings may help to explain the vulnerability of the kidney to certain infections, especially those due to bacteria which are destroyed by the combined action of complement and antibody.

Normal components of guinea pig serum which may cause antigens to show an apparent anti-complementary effect.

NORMAL guinea pig serum is the most common source of complement for the complement-fixation test, and may be used fresh or, more commonly, preserved either by freeze-drying or by Richardson's method1. Such ‘complement’ is used in the presence of a great variety of antigens, and the possibility of a reaction between components of this serum other than complement and the antigen is usually either ignored or allowed for as an anti-complementary effect of the antigen acting alone.

Studies on Serum Bactericidal Activity

Summary The intravenous administration of heparin into rabbits caused a temporary depression in the normal serum bactericidal activity. Serum and plasma exhibited less bactericidal activity than normal if either heparin or citrate was added to the blood in vitro immediately after withdrawal and prior to coagulation. In contrast, neither heparin nor citrate had a demonstrable effect on the serum bactericidal activity when added to the separated serum in vitro. The concentrations of citrate that inhibited bactericidal activity were anticomplementary when the complement titrations were carried out in saline. The heparin concentration used did not show a measurable anticomplementary effect with the test system employed. The depression of bactericidal activity caused by the in vitro or in vivo addition of heparin to blood was partially reversed by the addition of protamine sulfate. The x-irradiation induced depression of bactericidal activity was not reversed by single or multiple injections of protamine sulfate.

Complement Fixation Test with a Triple Antigen for Syphilis, Tuberculosis, Leprosy or Chagas' Disease in Blood Banks

SummaryA quantitative complement fixation test using a triple antigen, made up of cardiolipin No. 72 for syphilis, T. cruzi extract for Chagas' disease and a tubercle bacillus extract for tuberculosis and leprosy, is presented. Experimental work has been done to establish the basic principles governing the reaction by demonstrating that the specific systems react independently of the presence of other antigens.Sera from 786 blood donors were tested against the triple antigen and against each one of the specific antigens, in complement fixation tests. The 599 sera that did not react with the triple antigen did not show any reactivity with the antigens for syphilis, tuberculosis-leprosy or Chagas' disease; no false negatives occurred with the triple antigen. The 103 sera which reacted with any one of the specific antigens, reacted also with the triple antigen. Four sera were anti-complementary, simulating “reaction” with the triple antigen; 76 sera gave “reaction” with the triple antigen but did not show any specific reaction in the tests for syphilis, Chagas' disease, tuberculosis or leprosy. These results could be due to the cumulative anticomplementary effect of these sera plus that of the antigens composing the triple antigen.The preceding results indicate that a screen test employing the triple antigen should be used instead of the regular test for syphilis, in areas where syphilis, tuberculosis, leprosy, and Chagas' disease are endemic.

THE ACTIVATING EFFECT OF MAGNESIUM AND OTHER CATIONS ON THE HEMOLYTIC FUNCTION OF COMPLEMENT

1. The evidence presented indicates that Mg(++), or other cation such as Ca(++), Ni(++), or Co(++), is essential for the hemolytic action of C'. Ca(++), Ni(++), and Co(++) are less effective than Mg(++). The hemolytic system usually does not contain sufficient Mg(++) for optimal hemolytic activity so that a marked enhancement can be obtained by addition of extra Mg(++). 2. The enhancing action of tissue fluids can be ascribed to their contribution of Mg(++). 3. Substances which bind Mg(++) and Ca(++) are anticomplementary when added to the usual hemolytic system which contains only a small quantity of Mg(++). This type of anticomplementary effect can be overcome by addition of extra Mg(++). 4. Ca(++) may also be essential to the lytic process but its action is much less pronounced than that of Mg(++).