The effects of defibrination with ancrod in experimental allergic glomerular injury.

Quantitative studies of the effects of defibrination (with ancrod) have been undertaken in two forms of allergic glomerular damage, nephrotoxic serum nephritis and acute serum sickness in rabbits. No differences in intrarenal fixation of nephrotoxic antibody, complement activation or host antibody response were detected between defibrinated and untreated rabbits with nephrotoxic serum nephritis. Defibrination prevented intraglomerular fibrin deposition in this disease; but some glomerular damage as shown by a rise in blood urea and endothelial proliferation still occurred in defibrinated animals. No differences in immune elimination of BSA, circulating immune complex formation or intrarenal localization of immune complexes were noted in defibrinated animals with acute serum sickness. No intraglomerular fibrin deposition was detected in treated or untreated animals in this disease model. It is concluded that the protective effects of ancrod are directly related to defibrination, and not to any other modification of allergic events.     

: III. : Fowl Antibody • Its Haemolytic Activity with Complements of Various Species and Some Properties of Fowl Complement

Red cells sensitized with naturally occurring or `immune' fowl antibody were lysed by fresh fowl serum. This fowl complement could not be replaced by that of human, rabbit or guinea pig.

Both naturally occurring and `immune' antibodies to red cells in the mammalian species tested did not lyse with fowl complement. Lysis by fresh fowl serum of sheep red cells sensitized with mammalian antibody is due to the presence of naturally occurring antibody to these cells in the fowl serum; the enhancement of this lysis by the mammalian antibody will be described more fully in the next paper.

Naturally occurring antibody to sheep red cells present in turkey serum and `immune' antibody to fowl red cells were not lytic with fresh turkey serum, with fowl complement, with guinea-pig complement or with any combination of these.

Some properties of fowl complement are given.

     

The potentiating effect of rheumatoid arthritis serum in the immediate phase of nephrotoxic nephritis

Nephrotoxic nephritis induced in rats was employed as an experimental model to investigate the possible effects of rheumatoid factor on in vivo antigen–antibody reactions. Rats injected simultaneously with rheumatoid arthritis serum and rabbit nephrotoxic globulin showed a three-fold increase in immediate proteinuria compared with rats injected with nephrotoxic globulin alone. This potentiating effect of rheumatoid arthritis serum was evident even when the serum was injected 48 hr after the nephrotoxic globulin and was also apparent to a lesser extent in rats decomplemented by a prior injection of aggregated human IgG. Normal human serum had no effect on the proteinuria produced by a standard dose of nephrotoxic globulin while rheumatoid arthritis serum injected with normal rabbit globulin did not increase urinary protein excretion above baseline levels. In rats injected with rheumatoid arthritis serum and nephrotoxic globulin, human IgM (presumably rheumatoid factor) was detected by immunofluorescence on the glomerular basement membrane along with the nephrotoxic globulin and rat complement and persisted at this site for as long as 42 days after the initial injections. Rheumatoid factor activity was also recovered by elution from glomeruli isolated from rat kidneys 24 hr after the injection of rheumatoid arthritis serum and nephrotoxic globulin.     

A kinetic study of the bacteriolytic action of human serum

The killing and lysis of Escherichia coli by human serum have been measured simultaneously at frequent intervals for periods of 30–60 minutes. The kinetic effects of varying the amounts of lysozyme, antibacterial antibody and complement have been studied.

The rate of lysis is largely controlled by the lysozyme concentration but complement is also necessary. Killing is closely related to complement concentration. Antibody is needed in such small amounts that it is rarely a limiting factor.

Inhibition of serum lysozyme by anti-human lysozyme prevents lysis and reduces killing but both are restored to normal by addition of egg-white lysozyme. Both lysis and killing are stopped by bentonite absorption of serum and complete return to normal is not attained by subsequent addition of egg-white lysozyme. In a lysozyme-free system lysis does occur after some delay probably due to the action of complement and antibody alone.

In the presence of adequate complement and antibody the loss of complement (measured haemolytically) in bentonite treated serum is inadequate to account for the fall in bactericidal activity. A new bentonite absorbable factor (BAF) essential for complete serum bactericidal power is postulated.

     

Naturally occurring anti-tissue antibodies in rat sera

Seventy per cent of normal rat sera have been shown to contain heat labile serum component(s) active against various rat organ homogenates as demonstrated by haemolytic complement fixation and passive haemagglutination tests. The main antigenic activity in rat liver has been found in the mitochondrial fractions.

It was also demonstrated by the indirect fluorescent antibody technique that both guinea-pig complement and high molecular weight rat globulins were fixed to rat organ sections.

Chemotactic activity has also been observed with rat serum and rat liver mitochondria and it is suggested that these naturally occurring antibodies may be implicated in the removal of tissue breakdown products.

     

Interactions of Anti-glomerular Basement Membrane Antisera

An in vitro assay procedure for evaluating the antibody response to glomerular basement membranes, lung basement membranes and tendon fibrils indicated a sharing of identical or similar antigenic components.

At least 70 per cent of the glomerular basement membrane-binding antibodies are absorbable by tendon fibrils, and eluates from such tendon preparations are not nephrotoxic.

Within the species employed (man, rabbit and dog) for glomerular basement membrane sources, there appears to be no species specificity as regards the presence of the nephrotoxic component.

Immunization of sheep with either canine or rabbit glomerular basement membrane leads to the elaboration of a haemagglutinin for the respective species.

     

Circulating conversion products of C3 in liver disease. Evidence for in vivo activation of the complement system

Circulating conversion products of the third component of human complement (C3) were sought for in different forms of liver disease by the method of antigen–antibody crossed electrophoresis. Immunochemical determinations of C3 and C4 by the single radial immunodiffusion method were performed simultaneously.

The conversion product C3b was found in four out of twelve patients with chronic active hepatitis (CAH) and in seven out of nine with primary biliary cirrhosis (PBC). C3b was also seen in a patient with the special form of acute hepatitis characterized by arthritic prodromas and a high titre of hepatitis-associated antigen (HAA) in serum in the acute phase.

The group of CAH patients had low serum C3 and C4 regardless of whether C3 breakdown products could be demonstrated or not. In PBC normal serum levels of C3 and C4 were generally found.

It is concluded that in some patients with CAH and in a majority of the patients with PBC activation of complement takes place in vivo, possibly on immune complexes deposited in the liver. The serum level of C3 is not a good parameter of immunologic activity in liver disease.

     

Localization of intrinsic factor and complement fixing intrinsic factor–intrinsic factor antibody complex in parietal cell of man

In an attempt to localize intrinsic factor in the human parietal cell, and to study its intracellular union with the intrinsic factor antibody and complement, intrinsic factor antibody was separated from coexisting parietal cell antibody in pernicious anaemia sera by gel filtration. Intrinsic factor antibody of both `binding' and `blocking' type was also produced in rabbits by immunization with semi-purified human intrinsic factor–[⁵⁷Co]B₁₂ complex.

Intrinsic factor antibody obtained from both sources produced fluorescence in the human parietal cells in the indirect Coons' test in the presence of fluoresceinated anti-human IgG. The fluorescence was localized peripherally, at the cell membrane. When instead of the fluoresceinated anti-human IgG a fluoresceinated anti-human complement (C) serum and normal complement containing serum were used, intrinsic factor antibody from both sources produced fluorescence of the entire parietal cell cytoplasm of the human mucosa.

Thus, intrinsic factor was localized at highest concentration at the membrane of the parietal cell in man, the intrinsic factor antibody–intrinsic factor complex was demonstrated within the human parietal cell, and evidence was obtained that this antigen–antibody complex fixes complement (C). The possible role of the intrinsic factor–intrinsic factor antibody–complement complex in the development of gastric atrophy in pernicious anaemia has been considered.

     

Serial complement component alterations in acute glomerulonephritis and systemic lupus erythematosus

Serial determinations of whole serum complement (C') and absolute weight concentrations of the C'1q (11S) portion of the first and the third (C'3), fourth (C'4) and fifth (C'5) components of human complement were carried out in patients with post-streptococcal acute glomerulonephritis, systemic lupus erythematosus and other types of nephritis including Henoch–Schönlein (anaphylactoid purpura), Goodpasture's syndrome and chronic glomerulonephritis.

Total haemolytic complement was decreased in patients with acute glomerulonephritis and active systemic lupus erythematosus, but distinctly different component patterns occurred which were characteristic for each disease. In acute glomerulonephritis the concentration of C'1q was normal and C'4 was decreased only during the initial phase of illness. More prolonged depressions of C'3 and C'5 occurred which paralleled the whole complement titres. In contrast subjects with active systemic lupus erythematosus had normal C'5 concentrations and decreases in C'1q, C'4 and C'3 with prolonged depression of C'4 being the most consistent finding. Recent evidence has suggested that both post-streptococcal acute glomerulonephritis and systemic lupus erythematosus share a similar immunopathogenic mechanism involving the deposition in renal glomeruli of immune complexes formed by non-glomerular antigen and specific antibody. If so, the physico-chemical characteristics of the immune complexes differ enough to cause distinctive component alterations. Entirely normal whole complement and component levels were observed in patients with other types of nephritis.

In post-streptococcal acute glomerulonephritis the early return to normal of fourth component concentrations may be a favourable prognostic sign while the persistent depression of this component in systemic lupus erythematosus may be an indication of occult disease activity.

     

The relationship between complement and antibodies of different animals in the immune-adherence phenomenon

A comparison is made of the titres of complement from different animals when reacting with antisera from different species, in the immune-adherence phenomenon. A failure of reaction between rabbit antibody and horse and pig complement is noted. This may be overcome by the addition of heated guinea pig serum. Whereas a similar failure of reaction between rabbit antibody and horse complement in immune haemolysis is also overcome by the addition of ammonia-treated guinea pig serum, this does not occur in immune-adherence. This is interpreted as meaning that there is a further heat-stable ammonia-sensitive co-factor necessary for immune-adherence in addition to the four components of complement. This co-factor is also inactivated by zymosan.

The behaviour of mouse complement in immune-adherence indicates a relative deficiency in C′2 as in immune haemolysis.

Conglutinin is shown to inhibit immune-adherence irrespective of the species of complement used.

     

The participation of complement in the parietal cell antigen–antibody reaction in pernicious anaemia and atrophic gastritis

Indirect evidence suggests that the parietal cell antibody circulating in the serum of pernicious anaemia patients is a complement fixing antibody. In this work, we have presented direct evidence using an immunofluorescent technique, that the antigen–antibody union occurring in the gastric mucosa between this antibody and the parietal cell antigen binds complement (C'). We have further adduced data to indicate that serum C' activity was decreased in more than one-third of our patients with pernicious anaemia and in one-fourth of those with advanced atrophic gastritis. Eighty-five per cent of the patients with lowered serum C' had parietal cell antibody in the serum and some of them also had intrinsic factor antibody.

These findings support the concept of the autoimmune mechanism in the development of the gastric atrophic lesion in a proportion of patients with pernicious anaemia and atrophic gastritis. This mechanism includes the participation of complement in the antigen–antibody reaction at the parietal cell level.

     

Agglutinating and non-agglutinating antibodies in rabbits inoculated with a particulate antigen (Salmonella typhimurium)

Agglutinating and non-agglutinating anti-Salmonella typhimurium antibodies were specifically purified from the sera of immunized rabbits. Both types of antibody had the same electrophoretic mobility and were localized in the IgG fraction. It was not possible to find antigenic differences between agglutinating and non-agglutinating antibodies by immunodiffusion.

Agglutinating antibody activated the complement system, while non-agglutinating antibody lacked this capacity. Only the former increased clearance of antigen from the blood. When serum samples with different antibody titres determined by agglutination (agglutinating antibody) and Coombs test (non-agglutinating antibody) were injected in mice, clearance of antigen from the blood showed changes. These results were similar to those previously observed by us when different precipitating: co-precipitating antibody ratios were used, and indicated that competition of both antibodies for the antigen depends on their respective amounts.

When mice protection tests were set up by injection of agglutinating and non-agglutinating antibody before the inoculation of 10 LD₅₀ S. typhimurium, non-agglutinating antibody was found to be less effective than agglutinating antibody.

Non-agglutinating antibody was detectable during the whole course of immunization. Its serum concentration was higher than that of the agglutinating antibody.

Non-agglutinating antibody behaves in a similar way to co-precipitating antibody. The initially proposed hypothesis that such antibodies could interfere with immunity to certain chronic infections was extended to include the non-agglutinating antibodies demonstrated here.

     

Proteins adhering to Escherichia coli after exposure to guinea-pig serum

The constituents of guinea-pig serum that attach to the surface of a serum-sensitive strain of Escherichia coli have been determined. Antisera from rabbits immunized with bacteria exposed to serum (`sensitized' bacteria) were analysed by means of immunoelectrophoresis. It is concluded that at least seven constituents from the serum of the normal adult guinea-pig adhere to the E. coli during sensitization. These bind tightly enough to resist removal by at least six washings of the sensitized bacteria. Two of these constituents (IgG and IgM) are known to possess antibody activity. A third constituent, β_1C, is known to be the third component of the complement system of the guinea-pig. Another is probably the fourth component of this complement system. The other three serum components remain unidentified, although one of them is shown to possess esterase activity, and thus may be related to the first component of complement.

Evidence is presented suggesting that normal guinea-pigs may have little or no antibody against E. coli present in the IgG fraction of the serum.

If, before sensitization of the E. coli, the serum is heated to inactivate the activity of bactericidal complement, the serum does not kill this organism, but no change is detectable in the serum proteins adhering to these bacteria. Similarly, if, before sensitization of E. coli, the bactericidal antibody to the E. coli is absorbed from the serum, while bactericidal complement is left intact, there is no change in the serum proteins adhering to the bacteria. When the serum used for sensitizing E. coli is from guinea-pigs that have been actively immunized with the same strain of E. coli, rabbits immunized with these sensitized bacteria appear to form increased amounts of antibody directed against the 7S γ₂-globulin of the guinea-pig serum.

     

Studies of bactericidal activity to Escherichia coli of porcine serum and colostral immunoglobulins and the role of lysozyme with secretory IgA

Gel filtration and immune inhibition techniques were used to study bactericidal activities of IgG, IgM and IgA against smooth strains of Escherichia coli 0141 and 08 in sow serum and colostrum and post-colostral piglet serum. Bactericidal activity in sow sera was primarily associated with IgM and a low molecular weight IgG component, 7S IgG activity was less frequently observed. In colostral whey fractions and post-colostral piglet sera, in the absence of lysozyme, bactericidal antibody activity was associated with IgM and 7S IgG. In post-colostral serum bactericidal antibody was also attributable to a low molecular weight form of IgG.

IgA in serum from the sow and neonate showed no bactericidal activity, even in the presence of lysozyme, whereas in colostrum secretory 11S IgA had bactericidal activity, but only in the presence of complement and lysozyme.

     

Inhalation challenge and skin testing in farmer's lung

An immunologic basis for episodes of farmer's lung is shown by the response of susceptible individuals to inhalation challenge with extracts of Micropolyspora faeni, the main sensitizing agent in moldy hay dust. However, if the complement fixing capacity of the farmer's serum for M. faeni antigen is used as a quantitative assessment of sensitivity, it does not predict whether an individual will respond to inhalation challenge. A positive immediate skin test to M. faeni antigens was observed in all cases with over 40 CH50 consuming units of antibody per milliliter of serum. A similar correlation was seen between complement consumption and precipitins.     

The demonstration of the involvement of antibody in the in vitro lysis of chicken erythrocytes by allergized spleen cells

The experiments described demonstrate that the in vitro lysis of chicken erythrocytes (CRC) by allergized mouse spleen cells (ASC) is an antibody-dependent phenomenon. Supernatants obtained from cultures of ASC were shown to contain antibody which could effect antibody-dependent cell-mediated cytotoxicity.

The cytolysis observed with ASC was inhibited by both an IgG fraction and a F(ab')₂ portion of rabbit anti-mouse IgG. No inhibition was obtained by pretreating the ASC with AKR anti-C3Hθ serum and guinea-pig complement.

     

Metabolism of radio-labelled C3: effects of in vivo activation in rabbits

Turnover studies were performed in rabbits using biologically screened, highly purified, radio-labelled human C3 and C3c. Experiments were also carried out using agents known to activate the complement system in vivo—cobra venom factor, human nephritic serum and nephrotoxic antibody to rabbit glomerular basement membrane. Activation of labelled C3 by cobra factor provided information regarding the metabolic behaviour of C3d.     

Properties of protective malarial antibody

Properties of protective malarial antibody have been studied in cultures of P. knowlesi giving average parasite multiplication rates of sixfold in 24 hours. Parasite growth was assessed by incorporation of [³H]-leucine into protein.

Immune serum has little effect upon the growth of intracellular parasites, but prevents reinvasion of red cells and inhibits the succeeding cycle of parasite development. Protective antibody is present in relatively low titre in immune sera even after long immunization and this may explain certain characteristic features of malarial immunity.

Protective antibody in the sera studied is associated with IgG and IgM; its action is not complement dependent, but requires at least two combining sites per molecule. Anti-malarial antibody has several features in common with viral neutralizing antibody.

     

The adherence of leucocytes and platelets induced by fixed IgG antibody or complement

Human, rabbit and guinea-pig neutrophils and rabbit and guinea-pig macrophages were shown to adhere to sheep erythrocytes sensitized with isologous IgG antibody (EAIgG). IgM antibody, however, was less active and use of rabbit IgM antibody to fix complement from different species allowed the examination of neutrophil, macrophage and platelet adherence to alexinated erythrocytes (EAIgMC′).

Complement from twelve mammalian species induced the adherence of isologous neutrophils. On the other hand, platelets showed some variability. Ox, goat, sheep and pig platelets, like those from man and the baboon, did not adhere to EAIgMC′, although adherence of platelets from the rabbit, guinea-pig, rat, mouse, horse and cat was observed. C′3 was the complement component implicated in these reactions.

Treatment of the leucocytes with trypsin prevented the adherence to EAIgMC′ but not to EAIgG. Chelation of free magnesium with EDTA inhibited the adherence of rabbit neutrophils, but not of human neutrophils, to EAIgMC′. EDTA had no effect on the adherence to EAIgG.

Rabbit and guinea-pig neutrophils exhibited some species specificity towards the antibody and complement which would induce their adherence. This was not shown by human neutrophils or by rabbit and guinea-pig macrophages.

The adherence of particles to phagocytes comprises the first stage of phagocytosis and the role of antibody and complement in this process is discussed.

     

Xanthine oxidase contributes to lung leak in rats subjected to skin burn

We found that rats subjected to thermal skin injury (skin burn) had increased serum xanthine oxidase (XO) activities, increased serum complement activation (decreased serum CH₅₀ levels), increased erythrocyte (RBC) fragility, increased lung neutrophil accumulation, and increased lung leak compared to sham-treated rats. Treatment of rats with allopurinol (an XO inhibitor) not only decreased serum XO activity, but also decreased complement activation, RBC fragility, lung neutrophil accumulation, and lung leak abnormalities in rats subjected to skin burn. We conclude that XO may contribute to acute lung injury and a number of events associated with the development of acute lung leak following skin burn.