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.

     

Haemolytic plaque formation by mouse peritoneal cells, and the effect on it of Friend virus infection

Peritoneal cells from non-immunized mice, when incubated in vitro with sheep red cells and complement in a film of carboxymethyl-cellulose gum, form plaques of haemolysis after a latent phase of 15–20 hours. Plaques are also produced by the free cells of the pleural cavity but not by lymph node, spleen, thymus and bone marrow cells. Plaques are not produced at room temperature, nor when the complement has been inactivated or the peritoneal cells have been heat-killed. The phenomenon is age-dependent: the peritoneal cells reach the highest activity when the donor mice are about 10 weeks old.

By testing purified populations of lymphocytes and macrophages the cell type responsible for plaque production has been identified as the lymphocyte.

Plaque formation is suppressed in the presence of an anti-mouse immunoglobulin serum without any detectable effect on cell viability or anticomplementary action. This suppressive effect is destroyed by prior precipitation of the antiserum with normal mouse serum.

A technique which facilitates the study of peritoneal cells from individual mice has been developed and applied to peritoneal cells from Friend virus-infected mice. The activity of peritoneal cells 10 days after intravenous or intraperitoneal infection is similar to that of their uninfected counterparts. Peritoneal cells from mice killed 17 days after intravenous infection or 19 days after intraperitoneal infection form significantly reduced numbers of plaques. The reduced activity is accompanied by a decrease in the percentage of lymphocytes present in the peritoneal population of the infected mice.

     

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.     

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.     

Observations on pH and haemolytic complement

The effect of pH on the haemolytic activity of guinea-pig complement was studied over the range 6.0–8.3 using veronal—acetate and bicarbonate buffers of essentially constant ionic strength and osmolality. Complement in both buffer systems showed maximum haemolytic activity at a pH value close to 6.8.

The reaction EA → EAC′₁₄₂ took place optimally at pH 6.8 at 1° but the reaction in which EAC′₁₄₂ was lysed by C′3 at 37° was scarcely affected by pH over the range 6.0–8.2. Fixation of complement by heat-aggregated γ-globulin, by a globulin—anti-globulin precipitate and by polio vaccine plus antibody increased steadily as the pH was raised from 6.15 to 8.5. Beyond pH 8.5 complement itself was unstable. A four-fold increase in the sensitivity of the complement-fixation test was obtained by raising the pH from 7.0 to 8.3.

     

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.     

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.

     

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 generation of antibody diversity. II. Plaque morphology as a simple marker for antibody specificity at the single-cell level

This paper investigates the validity of plaque morphology as a simple marker for distinguishing single cells producing antibody with different specificities. Plaques on an indicator mixture of related erythrocytes may be clear, with both red cell types lysed, or partial with only one lysed, or “sombreros” where both target erythrocytes are lysed but to different extents. The technique can be adapted for use with defined antigens by testing cells against indicator erythrocytes half of which are coupled at high density with the antigen and half at low density: again the population is split into clear and partial plaques. Various arguments and control experiments have been put forward to support the claim that plaque morphology depends on antibody specificity, and hence V region structure, and not on amount of antibody or C region changes: morphology remains the same at different temperatures of incubation, and as a plaque grows at 37 °C, and is not affected by different concentrations of complement in the medium.     

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.     

The induction of an immune response in vitro to a protein antigen (rabbit Fab′2)

Unprimed mouse spleen cells were cultured in vitro with or without rabbit Fab′₂ and after harvesting were tested for the number of plaque forming cells on horse red blood cells coated with rabbit Fab′ and on horse red blood cells alone. It was found that a concentration of 0.2 mg/ml rabbit Fab′₂ in the culture medium gave a maximum number of plaque forming cells on Fab′ coated horse red blood cells, but did not increase the number of plaque forming cells to horse red blood cells.

This response is dependent on cell division in culture, and cannot be induced in spleen cells taken from mice tolerant to rabbit Fab′₂.

     

Identification of the Components of Complement Participating in the Antiglobulin Reaction

Red cells sensitized with a complement-binding antibody and then incubated with fresh serum have been shown to be coated with β_1C and β_1E globulin, two components of the complement system that have been isolated recently. Red cells presumably in the state EAC′_1,4 reacted with anti-β_1E, and cells presumably in the state EAC′_1,4,2,3a reacted with anti β_1E and with anti-β_1C. Agglutination of complement-coated cells by `broad spectrum' antiglobulin sera was effectively inhibited by purified β_1E and β_1C globulin. Red cells from certain patients with acquired haemolytic anaemia were found to be coated in vivo with β_1E and β_1C globulin. The function and significance of the two serum components have been discussed.     

Heterogeneity of human lymphocyte Fc receptors. I. Differential susceptibility to proteolysis

To study the possible heterogeneity of human lymphocyte Fc receptors, isolated human peripheral blood lymphocytes (PBL) were enzymatically altered (`stripped') by exposure to pronase or papain. Pronase treatment markedly increased the percentages of PBL binding IgG-sensitized erythrocytes (EA), while simultaneously removing or inactivating their receptors for heat-aggregated IgG (aggG). Papain treatment markedly diminished the ability of PBL to bind both EA and aggG. Essentially identical results were obtained utilizing EA composed of either human Rh-positive type O erythrocytes sensitized with the human anti-Rh serum Ripley (HRBC-A Ripley) or with chicken erythrocytes sensitized with rabbit anti-CRBC IgG (CRBC-A). CRBC sensitized with Fab'₂ fragments of rabbit anti-CRBC IgG were incapable of forming rosettes with normal or with pronase- or papain-stripped PBL. Pre-treatment of normal lymphocytes with aggG totally ablated their ability to rosette with EA.

Incubation of pronase-stripped PBL for 18–20 hr in 5% CO₂-air at 37°C resulted in diminution (to levels originally present) in the percentages of lymphocytes binding EA, but no regeneration of aggG receptors. Similar incubation of papain-stripped PBL resulted in significant reappearance of receptors binding EA, but no regeneration of aggG receptors. These results strongly suggest that: (1) lymphocyte receptors that bind EA complexes differ from those that bind aggG; (2) some lymphocytes possess cryptic receptors for EA that are expressed after proteolysis with pronase; (3) PBL having receptors for EA also have aggG receptors; and (4) there is no evidence that proteolytic stripping of PBL results in the generation of functionally different receptors for complexed IgG, since the Fc specificity of this receptor remains unchanged.

     

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.     

The use of passive haemolysis in the quantitative estimation of anti-protein antibodies

The conditions for the establishment of a method for the quantitative assay of antiprotein antibodies by the use of passive haemolysis were studied by using an anti-BSA—BSA system as a model.

When haemolytic assaying of antibody was carried out under standard conditions with a fixed concentration of optimally sensitized red cells, in the presence of excess complement (10 C′H₅₀) reproducible titrations could be performed to detect 0.12 μg. N Ab with an error of ±10 per cent.

Factors affecting the specific lysis of optimally sensitized cells were studied and some parameters fixed. The amounts of coated erythrocytes and complement and the length of time of reaction were seen to influence the final degree of lysis given by a fixed amount of antibody.

The conditions of optimal sensitization of erythrocytes with protein antigen by the use of bis-diazotized benzidine (BDB) are indicated. It was found that, when conjugation was carried out at 0° with proper amounts of a standardized cell suspension, BDB and antigen, the resulting coated cells were highly sensitive to immune haemolysis and highly resistant to `spontaneous' lysis.

     

Heterogeneity of human lymphocyte Fc receptors. II. Relationship to antibody-dependent, cell-mediated cytotoxicity

Human peripheral blood lymphocytes (PBL) were incubated (stripped) with pronase or papain and compared with unstripped lymphocytes for their ability to mediate antibody-dependent, cell-mediated cytotoxicity (ADCC). Despite marked removal or inactivation of receptors for heat-aggregated IgG (aggG) by proteolytic digestion, and pronounced changes in the percentages of cells rosetting with IgG-sensitized erythrocytes (EA) (decreased by papain, increased by pronase), stripped PBL functioned normally in ADCC.

Stripped and unstripped lymphocytes were pre-treated with aggG to determine the role of aggG receptors in ADCC. AggG almost totally abolished ADCC by unstripped PBL, but inhibited ADCC by enzyme-stripped lymphocytes relatively poorly. Neither untreated nor stripped PBL were able to induce cytotoxicity of chicken erythrocyte (CRBC) target cells sensitized with the Fab'₂ fragment of anti-CRBC IgG antibody (CRBC-A).

Exposure of PBL to EA monolayers composed of CRBC-A or of sheep erythrocytes (SRBC) sensitized with rabbit anti-SRBC IgG antibody (SRBC-A) depleted PBL of cells that rosetted with CRBC-A and with human Rh-positive, type O erythrocytes sensitized with the human anti-Rh serum Ripley (HRBC-A Ripley). Non-adherent cells were incapable of binding aggG and had markedly diminished cytotoxicity in ADCC. Similarly, exposure of PBL to HRBC-A Ripley monolayers resulted in non-adherent cells that were incapable of rosette formation with HRBC-A or CRBC-A, failed to bind aggG, and exhibited significantly diminished ADCC activity.

These studies indicated that: (1) cytotoxic effector PBL active in ADCC (K cells) have receptors for aggG and for EA; (2) PBL deficient in functional aggG receptors (enzymatically inactivated or removed) are capable of inducing normal levels of ADCC; (3) aggG and EA receptors appear to be closely associated on native K-cell membranes; (4) there is no clear-cut relationship in a given lymphocyte population between the presence of either aggG or EA receptors and ADCC activity; and (5) populations of PBL binding HRBC-A Ripley overlap with, and may be identical to, those binding aggG and other types of EA complexes.

     

异基因骨髓细胞在致敏模型体内归巢示踪与植入分析

目的: 探讨异基因供者骨髓细胞在致敏模型体内的归巢示踪与植入分析。方法: 以异基因脾细胞输注方法建立致敏的BALB/c小鼠模型,同时取正常BABL/c小鼠作为非致敏模型。致敏或非致敏模型经8 Gy 照射后分别经尾静脉移植1×10⁷ C57BL/6小鼠骨髓细胞。用绿色荧光染料CFSE标记供者骨髓细胞,并分别在移植后不同时点(2 h、12 h及48 h),通过组织细胞悬液动态示踪供者细胞在致敏受者各组织的分布。移植后记录各组的生存情况,每周监测造血重建与骨髓恢复情况。予H-2D^b进行标记移植后受者骨髓细胞,检测供者嵌合百分比。结果: CFSE能标记供者骨髓细胞并用于体内示踪实验。动物体内归巢示踪实验表明,与非致敏组相比,异基因供者骨髓细胞在致敏受者体内的外周血、脾脏及股骨的分布均明显减少。植入分析结果发现,非致敏受者于移植后能长期存活,外周血及骨髓细胞均能迅速恢复;而致敏组中,小鼠均于移植后2周左右全部死亡,生存中位数为13 d,外周血及骨髓细胞均随时间推移呈进行性减少。嵌合分析显示移植后第7 d,非致敏受者与致敏受者的供者骨髓细胞百分比分别为(48.07±4.70)％和(0.77±0.11)％,两者差异显著(P<0.01)。结论: 异基因供者骨髓细胞在致敏受者体内脾脏及股骨等部位被清除,不能有效植入。     

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.     

In vitro stimulation of antibody formation by peritoneal cells: III. Effect of active immunization on the subsequent in vitro performance of peritoneal and spleen cells

Male CBA mice were given a single intraperitoneal injection of sheep red blood cells (SRBC) or horse red blood cells (HRBC). They were killed at intervals of 1–10 days thereafter, and micro-cultures of spleen cells or peritoneal cells (PC) were prepared. These consisted of a thin film of tissue culture medium containing carboxymethyl cellulose (CMC), mouse lymphoid cells, guinea-pig complement and either SRBC or HRBC, held at 37° under liquid paraffin. Cultures were read repeatedly for appearance of haemolytic plaques.

PC from SRBC-immunized mice showed an altered reactivity on SRBC monolayer cultures. The peak plaque count achieved in vitro fell progressively for 4 days after immunization, and then returned to normal by day 7. The actinomycin D resistant component of the PC response rose rapidly; at 1 day after immunization it was equal to the total response. Over the next 3 days after immunization it fell again to normal levels. The results suggested that the in vivo injection sets in train events locally in the peritoneal cavity which resembled those following in vitro culture of normal PC in SRBC monolayers. The effects were immunologically specific as only marginal changes followed the injection of HRBC.

Spleen cells from SRBC-immunized mice, when cultured in SRBC monolayers, yielded many cells capable of giving plaques after 5–60 minutes incubation, as expected. These were deemed to be cells forming antibody at the moment of killing of the animal. In addition, such cultures developed new plaques over the subsequent 23 hours in culture. These were produced by cells not initially forming antibody which switched into antibody secretion at some time during culture. At early time points after immunization, this second type of cell was much more numerous than the first type. The switch from non-secretor status could occur in the presence of a high concentration of actinomycin D. Operationally these non-secretors in immunized spleens resembled an important fraction of PC from unimmunized retired breeder mice. The progressive conversion of non-secretor cells into secretors, if it occurs in vivo, would have a major influence on the kinetics of appearance of PFC in a spleen after immunization.

While spleen cells from mice immunized with HRBC performed on HRBC monolayers much as described above, PC from HRBC-immunized mice could not be induced to cause significant lysis in HRBC monolayers. The same was true of PC from mice chronically fed with HRBC. In fact, no method has yet been found to persuade PC to produce lytic plaques active against erythrocytes other than SRBC.

     

Cellular basis for synthesis of the fourth component of guinea-pig complement as determined by a haemolytic plaque technique

Cells from several tissues of the guinea-pig were assayed by a modified Jerne plaque technique for production of the fourth component of complement. Formation of haemolytic plaques was interpreted as in vitro production of C4 by those cells observed in the centres of plaques. C4 production was detected in mononuclear cells from peritoneal exudates, lung washings, and liver. Production of C4 was detected with less frequency in mononuclear cells from spleen, lymph node, bone marrow and thymus. Plaque formation was inhibited by incubating cells with puromycin or cycloheximide. This indicated that plaque formation was the result of in vitro synthesis of C4. The results of this study are compatible with the concept that the reticulo-endothelial system is involved in synthesis of complement components.