Specificity of IgA cold agglutinins: anti-Pr 1

Monoclonal cold agglutinins have usually been found to be IgM(k) and to be directed against the I/i antigens. Three IgA(k) cold agglutinins were tested for antibody specificity. Based on the following criteria, they were shown to be directed against the Pr₁ antigen of the Pr₁/Pr₂/Pr_a antigen system. The antigens reacting with the IgA(k) cold agglutinins were inactivated by protease and treatment of human red cells with neuraminidase and were demonstrable on human erythrocyte glycoproteins. The terminal sugar of the determinant group of these antigens is N-acetylneuraminic acid (NANA), which is not involved in I/i, but also in Pr₂ antigenicity. The antigen activity of these antigens and the Pr₂ and I antigen activities were distributed to different glycopeptide fractions obtained from red cell glycoproteins by ficin cleavage and Sephadex G-50 separation. Periodate oxidation of red cell glycoproteins, which causes a shortening of the polyhydroxy side chain at C-6 of C-9 NANA to a C-7 and/or C-8 NANA derivative, resulted in inactivation of these antigens, while the I antigen remained unchanged and the Pr₂ antigen was increased 16–64-fold. Dog red cells gave no or diminished reactions with the three IgA(k) cold agglutinins, in contrast to increased reaction with anti-Pr₂. Based on the different reactions of the IgA(k) cold agglutinins with dog red cells, a Pr₁ heterogeneity Pr_1h/Pr_1d was demonstrated. In spite of the determination of the Pr_1h/Pr_1d/Pr₂ and the MN antigens by NANA, both antigen systems were shown to be unrelated. Blocking of free ε-lysine amino groups by acetylation or blocking of the NANA carboxyl groups by amidation of red cell glycoproteins resulted in MN inactivation, while the antigens reacting with the IgA(k) cold agglutinins (like Pr₂) remained unaffected. The anti-Pr₁ specificity of IgA cold agglutinins and the predominance of anti-I/-i specificity of IgM cold agglutinins was discussed with respect to inter-relations between immunoglobulin classes and antibody specificities of cold agglutinins.     

Chemical differences between individual human cold agglutinins

The μ- and κ-chains from four human cold agglutinins having anti-I specificity have been analysed:

The κ-chains have either Asp or Glu, but not both, at the N-terminus. This provides additional support for the view that cold agglutinins are of monoclonal origin.

Both heavy and light chains from cold agglutinins vary as much in amino acid composition as do monoclonal chains of a given type chosen at random. The chemical individuality of cold agglutinin μ-chains is shown also by differences in hexose, fucose and glucosamine content.

The apparent lack of correlation between overall antibody activity and chemical composition of constituent peptide chains indicates the need for close definition of combining specificity in structural studies of cold agglutinins.

     

Complement and cold agglutinins. II. Interactions of the components of complement and antibody within the haemolytic complex

Immune haemolysis by cold agglutinins allows the study of the reactions of the components of complement after elution of antibody from the haemolytic complex. Data are presented which indicate: (1) Elution of cold agglutinin from EAC'_1a,4,2a,3 is accompanied by elution of C'₁ esterase and apparent loss of activity of C'_1a from the complex. The remaining EC'_4,3 does not haemolyse in whole complement, but will lyse in either whole complement or R₄ providing antibody is replaced. Lysis of EC'_4,3 in R₄ was performed with anti-A and the Donath–Landsteiner antibody as well as with cold agglutinins. It was concluded that C'₄ is bound firmly to a cell receptor site during complement reactions, that these sites are shared by at least three antigenic systems, and that C'₄ remains functionally active on the cell. (2) The presence of the components of complement, especially C'_1a, exert a weak binding or stabilizing effect upon cold agglutinin within the haemolytic complex. (3) The presence of C'₄ and C'₃ on cells is associated with decreased number of antigenic sites for cold agglutinins and a decrease in sensitivity of the cells in immune haemolysis. (4) Cold agglutinin elutes from EAC'_1a,4,2a,3 unchanged physicochemically or in haemolytic activity. Complexing of complement with cold agglutinin during immune haemolysis did not occur.     

A common idiotype on human macroglobulins with anti-I and anti-i specificity.

The reactions of an idiotypic antiserum prepared against an anti-I cold agglutinin (Da) were studied in radioimmunoassays which were designed to detect partial rather than complete idiotypic cross-reactions among cold agglutinins with anti-I and anti-i activity. Proteins showing strong idiotypic cross-reactions by gel precipitation could not inhibit the binding of 125I-labelled Da to anti-Da (individual specificity). It was necessary to use 125I-labelled cross-reactive proteins in order to demonstrate cross-idiotypic specificity and even the choice of the reference cross-reactive protein was important. The use of the 125I-labelled Fab fragment of a cross-reactive anti-I cold agglutinin (Low) allowed the detection of an idiotypic determinant common to at least 73% of cold agglutinins with specificity for the I-i antigen complex. Among these were two transiently occurring cold agglutinins following M. pneumoniae infection and infectious mononucleosis. This idiotypic determinant was not detected on cold agglutinins with anti-Pr specificity or on isolated monoclonal IgM or IgG lacking cold agglutinin activity, but it was present in low concentrations in pooled IgG. The common idiotypic determinant was found on IgM kappa cold agglutinins belonging to different VK subgroups and on an IgM lambda protein. A knowledge of the structural basis of such idiotypic cross-reactions among proteins from unrelated individuals should provide important information about the genes controlling the synthesis of site-related regions of antibodies.     

Complement and cold agglutinins. I. Complement coating of normal human erythrocytes by cold agglutinins

The antiglobulin test has been used to study the interaction of cold agglutinins and human complement with human erythrocytes. In confirmation of others, it was found that the 11S component, C'₁ and C'₄ must react in sequence with the EA complex for the antiglobulin test to become positive. 11S and C'₁ reacted maximally at 1°C and were unreactive at 37°C. In contrast, C'₄ failed to react at 1°C, a feature believed to be uniquely associated with reactions of cold agglutinins. C'₄ reacted maximally at 20°C, but failed to react, or reacted very weakly, at 37°C. Calcium ions were required for the reaction of C'₁ while neither calcium nor magnesium was required for 11S or C'₄. Although the presence of C'₁ in the EAC'₁ complex gave an increased degree of `stability' to the involved cold agglutinin at 20°C, the cold agglutinin could be removed totally from the EAC'₁ complex by repeated washing or from the EAC'_1,4 complex by raising the temperature to 37°C. The fact that the cold agglutinin is a poor haemolysin, except under unusual circumstances, and yet is easily manipulated, offers an advantage in the study of the mechanisms of action of complement in immune systems.     

Glycosphingolipid receptors for anti-Gd and anti-p cold agglutinins

Anti-Gd and anti-p cold agglutinins exhibit similar serological properties: neuraminidase treatment of erythrocytes greatly reduces their agglutinability by these antibodies and protease treatment enhances their agglutination. We reported previously that an anti-p cold agglutinin was inhibited by sialosyllactoneotetraosylceramide, NeuAc(α2–3)Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)Glc-Cer, the most abundant ganglioside of human eryhrocytes. We now report that two less abundant gangliosides are more potent inhibitors of this antibody, and of the anti-Gd antibodies, than sialosyllactoneotetraosyl-ceramide. These two gangliosides have the same carbohydrate chain, NeuAc(α2–3)Gal(β1–4)GlcNAc(β1–3)Gal(β1–4) GlcNAc(β1–3)Gal(β1–4)Glc (SNH), but they differ in their ceramide moiety. The principal fatty acid of SNH-1 is C_16:0, whereas SNH-2 contains a predominance of C_22:0, C_24:0 and C_24:1. No inhibition was produced by the ganglioside, NeuAc(α2–6)Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)Glc-Cer.Another monoclonal cold agglutinin, Sa, which shares some serological properties with anti-Gd cold agglutinins, was not inhibited by any of these gangliosides.     

Physico-Chemical Characterization of the Cold Auto-Antibodies of Acquired Haemolytic Anaemia

Cold agglutinins were separated from the sera of eleven patients suffering from the cold-antibody type of acquired haemolytic anaemia by the dissociation of the specific antigen-antibody complexes. The electrophoretic mobility of the cold antibody was found to correspond to that of γ₁ globulin in each case.

Ultracentrifugal studies carried out on eluted cold antibodies prepared from the sera of seven patients demonstrated that the antibodies were macromolecules (S_20.w~18.1). In one instance the sedimentation coefficient of the cold antibody was demonstrated to be similar to that of electrophoretically separated `γ₁ globulin' prepared from the same serum, indicating their identical physical state.

The results of treating the sera of nineteen patients with the cold-antibody type of acquired haemolytic anaemia with mercapto-ethanol further confirmed that the cold antibodies were all macromolecules, whether derived from idiopathic cases or cases secondary to some other known disorder.

     

Contributions to the study of Salmonella. Immunological specificity of proteins separated from Salmonella typhi

Rabbits were immunized with live organisms and with proteins separated by various methods from Salmonella typhi (acetone-treated cells); anti-extract and antibacterial sera were investigated in agglutination and gel diffusion tests with various antigens.

The results show that the determinant groups of polysaccharides are not the sole factors involved in O agglutination of the bacteria. Apart from large amounts of H agglutinins and precipitins, anti-extract sera, containing no antibodies to polysaccharides, have been found to contain O agglutinating and precipitating antibodies.

In tests performed with extracts containing the whole bacterial antigen complex, the antibodies precipitated from antibacterial sera by polysaccharides were found to account for about one-third to one-half of the total antibody content of these sera.

Antibacterial sera absorbed with polysaccharide still contain O agglutinins which may be removed by subsequent saturation with protein.

Gel diffusion tests illustrate the results obtained.

The proteins separated from veronal buffer extracts by precipitation with trichloracetic acid are immunologically identical with the protein separated from the bacteria by means of dilute hydrochloric acid. From the serological and physicochemical points of view they behave like the flagellins isolated by Ada, Nossal, Pye and Abbot (1964) from flagella.

As in the case of flagellins, attempts to purify the proteins isolated from the bacterial organisms did not result in separation of the factors involved in O and H agglutination. The peptide determinants involved in both types of agglutination may well coexist on the same molecule.

     

Purification of cold agglutinins from patients with chronic cold haemagglutinin disease. Evidence of their homogeneity from starch gel electrophoresis of isolated light chains

A method is described for the large scale purification of serologically active high-titre cold agglutinins from the sera of patients with chronic cold haemagglutinin disease. Eighteen purified cold agglutinins have been reduced and alkylated and separated into heavy and light chain pools by Sephadex G-100 filtration. The isolated light chains were examined by alkaline urea starch gel electrophoresis and found to be far more homogeneous than normal light chains and comparable in some cases to Bence Jones light chains. However, light chains from different cold agglutinins with the anti-I specificity gave different banding patterns; this might be caused by the fact that they are directed against different parts of the I antigen.     

Antigen-induced suppression of agglutinins following the repeated high dose administration of bacteria (Pseudomonas species) at various ages

Rabbits ranging from newborn to 24 weeks old were injected repeatedly with 10⁹ intact Gram-negative bacterial cells (Pseudomonas N.C.M.B. 406) either intraperitoneally or intravenously. Intraperitoneal antigen evoked a similar response in all age groups; average titres after 7 weeks immunization being of the order of 200,000. Intravenous antigen induced marked suppression of agglutinins in newborn and 3-week-old animals. The two older age groups initially showed elevated titres which declined as further doses of antigen induced immunosuppression.

The nature of the suppression of agglutinins was investigated in 3-week-old rabbits. The phenomenon showed all the characteristics of specific high dose antigen-induced suppression of reactivity. This brings the intact bacterial cell firmly into line with other antigens as far as high dose tolerance or antigen-induced immunosuppression is concerned. It is suggested that the suppression of agglutinins observed in these experiments might best be explained on the basis of immunological exhaustion aided by specific absorption of antibody by antigen at certain stages.

     

Heterogeneity of cold haemagglutinins

All of seven sera with high titre cold agglutinins agglutinated rabbit red cells at 4° to similar titres as were recorded with human red cells. At 20° all the sera showed almost optimal activity against rabbit red cells, while the activity against human red cells was very weak. Two of the sera agglutinated rabbit red cells as strongly at 37° as at lower temperatures.

The results of absorption and elution experiments indicated that the same agglutinin molecules were responsible for the agglutination of human red cells at 4° and of rabbit red cells at 37°. Apparently the reaction with rabbit red cells was more specific than the reaction with human red cells. The variation in thermal amplitude observed when testing cold agglutinins with rabbit red cells may be an expression of variation in specificity even when the different cold agglutinins appear to have identical specificity, anti-I, in tests with human red cells.

     

A human monoclonal IgG reactive with a private idiotope of a monoclonal IgM with autoantibody activity against myelin-associated glycoprotein.

One hundred and thirteen sera from patients with monoclonal IgG were tested for reactivity against a panel of 13 human monoclonal IgM having various autoantibody activities: 6 to myelin-associated glycoprotein (MAG), 2 to vimentin intermediate filament protein and 5 to red blood cell antigens [cold agglutinins with specificity directed to I antigen (3 cases), i antigen (1 case) or Pr antigen (1 case)]. One IgG was found to react with a monoclonal IgM with anti-MAG activity. This reactivity was characterized as idiotypic and directed against a private idiotope of the monoclonal IgM. This work provides further evidence for the existence of anti-idiotypic antibody activity of monoclonal Ig occurring in human B cell neoplasias.     

Light chain homogeneity of post-infective cold agglutinins

Three post-infective cold agglutinins, two of which were associated with human infection with Mycoplasma pneumoniae were purified and compared with cold agglutinins isolated from three patients with chronic autoimmune haemolytic anaemia (CHA) as regards their immunoelectrophoretic patterns, their light chain types and the appearances of their light chain zones (after reduction and alkylation) in starch gel electrophoresis. One of the M. pneumoniae-induced cold agglutinins was indistinguishable from CHA cold agglutinins for it was monotypic (type-K), had a restricted mobility in immunoelectrophoresis and a narrow, well-demarcated light chain zone in urea formic acid starch gel electrophoresis. The other two post-infective cold agglutinins though bitypic with respect to light chains showed a considerable degree of homogeneity in immunoelectrophoresis and in starch gel electrophoresis they had light chain bands which were sharper than the light chain zone of normal human IgG.     

Catfish antibodies to blood group substances: I. Response of the Australian freshwater catfish, Tandanus tandanus (Mitchell), to the injection of human A, B and H secretor materials

Injection of Australian freshwater catfish, Tandanus tandanus (Mitchell), with human ABH secretor fluids mixed with adjuvant, resulted in the production by these fish of potent ABH specific haemagglutinating and precipitating antisera. No ABH haemagglutinins or precipitins were produced by catfish injected with non-secretor fluids.

Fractionation of the agglutinins by absorption, together with immunodiffusion studies, revealed that in most cases, the predominant response was an anti-H response. However, in some fish injected with B or A₁B secretor salivas, high titres of cross-reacting A and B agglutinins were formed.

The nature of the ABH agglutinin response in these fish is discussed with particular reference to three points. (1) The specificities of the ABH agglutinins formed. (2) The possibility that the variety and magnitude of the response are under some form of genetic control. (3) The possibility of the production in Australian freshwater catfish of anti-carbohydrate antibodies of limited heterogeneity.

     

Autoimmune haemolytic anaemia. VI. 51Chromium survival studies in patients with different kinds of warm autoantibodies

The results of ⁵¹Cr red cell survival and organ sequestration studies in seventysix patients with autoimmune haemolytic anaemia are described.

In patients with incomplete IgG and IgA warm autoantibodies increased red cell destruction to a varying degree and mainly spleen sequestration were found. In patients with IgM incomplete warm autoantibodies sequestration of red cells mainly in the liver was seen. Liver sequestration was also present in patients with cold agglutinins/haemolysis, sometimes accompanied by a significant spleen sequestration. In a number of patients with haemolytic autoantibodies, i.e. warm haemolysins and cold agglutinins/haemolysins, a biphasic ⁵¹Cr elimination curve was obtained. The cause of this type of survival curve is discussed.

In patients in whom a positive direct anticomplement test was the only serological abnormality a normal ⁵¹Cr red cell life-span was found.

     

Humoral antibody responses in the gar,Lepisosteus osseus

Lepisosteus osseus possesses high titres of natural agglutinins to human group A (HuA) erythrocytes and to Salmonella typhosa `O'' (STO) antigens. Moderate natural haemagglutinin titres to sheep red blood cells (SRBC) were noted. Cold agglutinin (4°) titres exceeded agglutinin titres recorded at 25°.Immunization with STO antigen resulted in a marked increase in corresponding anti-STO agglutinins, as well as increases in anti-HuA and anti-SRBC agglutinins. Animals immunized with SRBC antigen produced elevated anti-SRBC agglutinin titres, while immunization with HuA erythrocytes produced no changes in agglutinin levels.Immunization with STO following previous injections with HuA resulted in increases of corresponding anti-STO agglutinins, as well as marked increases in anti-HuA agglutinins. Absorption studies indicated that anti-STO agglutinins were not cross reactive against HuA antigens. Natural and immune antibodies were all of the same macromolecular class. Rises in antibody titres were reflected in both cold and warm agglutinins. Animals maintained for long periods at low temperatures exhibited preferential ability for increased synthesis of cold agglutinins to HuA.Natural haemolytic titres, as measured by an SRBC indicator system, were also elevated as compared to mammalian systems. These titres increased with stimulation with SRBC. Results suggest that the high levels of natural haemolytic and agglutinin activity in this species are of immunogenic origin.     

Structural characterization of human monoclonal cold agglutinins: evidence for a distinct primary sequence-defined VH4 idiotype

Cold agglutinins that bind the developmentally regulated I red cell determinant occur naturally among human monoclonal IgM proteins. These autoantibodies are known to use light chains that derive mainly from the minor kappa III (kappa III) variable region subgroup. The kappa III subgroup is also highly expressed in monoclonal rheumatoid factors. However, while most monoclonal rheumatoid factors use structurally homologous heavy chains that derive from the VH1 family, information regarding the structure of the cold agglutinin heavy chains remains fragmentary. We demonstrate here that the kappa III cold agglutinin autoantibodies exclusively use heavy chains that derive from the VH4 family. Furthermore, these autoantibody heavy chains all express the same primary sequence-defined idiotype, corresponding to the second hypervariable region. These data indicate that cold agglutinins use a remarkably homogeneous subset of heavy chain variable regions. Moreover, unique patterns of preferential VH and VL pairing clearly distinguish the anti-I cold agglutinins from all other known monoreactive autoantibodies.     

Variable-Region Subgroup and Specificity of Cold Agglutinins

The variable-region subgroup determined by amino acid sequence analysis of heavy and light chains of two monoclonal cold agglutinins with the new anti-Gd specificity is reported. Both proteins belong to the VHIII subgroup of heavy chains; one light chain falls into the V kappaI subgroup, the other has a blocked N-terminus which so far has not been observed in human kappa chains. The comparison of anti-Gd with anti-I/-i or anti-Pr cold agglutinins indicates that anti-Gd differs from other cold agglutinins with respect to variable-region subgroup. The data extend previous findings on the restriction of certain antibodies to distinct variable-region subgroups.     

Agglutinins against human erythrocytes modified by Newcastle disease virus in Mycoplasma pneumoniae infections

Agglutinins against human group O erythrocytes modified by Newcastle disease virus (NDV-O agglutinins) showed a four-fold or greater rise in titre in 43% of sera from patients with Mycoplasma pneumoniae infection as opposed to 4% of patients with miscellaneous virus infections. The highest titres in Mycoplasma infection were only slightly lower than the highest titres in infectious mononucleosis, a disease known to be associated with NDV-O agglutinins. In Mycoplasma infection, both NDV-O agglutinins and cold agglutinins were more frequent in the younger patients. Analysis of the results by age groups suggested some additional association between NDV-O agglutinins and cold agglutinins independent of age.     

Inverse Correlation Between IgG-Antihinge Region and Antierythrocyte Autoantibody in Chronic Benign and Malignant Cold Agglutination

Previous reports provided evidence of an immunosuppressive role of natural anti-F(ab)₂ antibodies. If suppressive anti-F(ab)₂ antibodies also regulated the autoantibody production in cold agglutination, one would expect high titers of anti-F(ab)₂ to be associated with low titers of cold agglutinins. Indeed, our previous studies revealed an inverse correlation between IgG-anti-F(ab)₂ and cold agglutinins. Many previous experiments focused on anti-F(ab)₂ of an antiidiotypic nature. Recent epitope mapping showed that anti-F(ab)₂ of healthy persons is not an antiidiotype but recognizes a hinge region sequence. We attempted to answer the question whether this IgG-antihinge antibody is responsible for the previously described association between anti-F(ab)₂ and cold agglutinins. IgG-antihinge and IgG-anti-F(ab)₂ antibody was determined and statistically analyzed in the serum of 334 patients with cold agglutination. Our experiments revealed a strong correlation between the concentrations of antihinge and the previously described anti-F(ab)₂ antibody. The anti-F(ab)₂ activity was competitively inhibited by a synthetic hinge peptide. Moreover, patients with high antihinge titers had low cold agglutinin titers, and vice versa. A stratification according to cold agglutinin specificity and disease etiology showed that the inverse correlation is present only in anti-I and anti-i patients suffering from monoclonal B-lymphocyte proliferation. In conclusion, our results confirm the correlation previously described for anti-F(ab)₂ antibody and antierythrocyte autoantibody and define for the first time an association between an idiotype-independent anti-IgG autoantibody and cold agglutinin.