Structural characterization of a human monoclonal IgA protein

The serum of an IgA myeloma patient was found to contain at least 90% of the monoclonal protein as a 10·05S disulphide-bonded dimer. Small amounts of monomeric and higher oligomeric forms were also observed. Light chains were κ type, disulphide-bonded to heavy chains and preferentially released upon reduction with dithioerithritol.

Electron microscopy of the dimer and oligomeric forms showed structures suggesting linkage between Y-shaped monomer units via the terminus of the Fc fragment. The data is discussed in relation to secretion of the different oligomeric forms.

     

Urinary light chain excretion in leukaemia and lymphoma

Immunoglobulins are produced by plasma cells. However, tissue culture and fluorescent antibody techniques have also demonstrated that other leucocytes, mainly lymphocytes, may synthesize immunoglobulins. In the aberrant protein synthesis of myeloma there is often excessive production of electrophoretically homogeneous light polypeptide chains, appearing in the urine as Bence Jones protein. Among a group of seventy-six patients with leukaemia and lymphoma, nineteen showed quantitatively increased excretion of urinary light chains (> 100 mg/24 hr). In all instances the light chain excretion of one dominating light-chain group (κ) was present. Studies of the κ/λ ratio in serum and isolated γG-globulin were made and appeared normal in the expected 2:1 range. By cellulose acetate electrophoresis these light polypeptide chains from the leukaemia–lymphoma group appeared considerably more heterogeneous than Bence Jones proteins from multiple myeloma.     

Recombination of heavy and light chains from human immunoglobulins

The recombination of reduced, alkylated heavy and light chains from three IgG, two IgM and one IgA pathological protein and from normal IgG has been quantitatively studied.

All γ, α and μ chains showed some recombination with all κ and λ chains tested. One of the IgG and two of the IgM proteins showed more than 50 per cent recombination and in these there was evidence of a selective reconstitution of autologous chain pairs; the structural features which determine re-association appear to be independent of the electrophoretic mobility, or antigenic and allotypic specificity of the interacting chains. The chains of normal IgG and of three myeloma proteins showed a low percentage recombination and these autologous chain pairs did not combine preferentially. It appears that immunoglobulin chains show a variable ability to regain their native configuration after isolation.

Two pathological κ chains which were identical in electrophoretic mobility and Inv specificity but not equivalent in re-association behaviour showed well marked differences in tryptic peptide patterns. This indicates that the number of chemically distinct human light chains exceeds the forty variants previously postulated on the basis of antigenic, allotypic and electrophoretic differences.

     

Isolation and partial characterization of immunoglobulin from a urodele amphibian (Necturus masculosus)

The primitive amphibian species, Necturus maculosus, a urodele, possessed serum immunoglobulin characterized by a mol. wt of approximately 900,000. Upon reduction of disulphide bonds and analysis under dissociating conditions, this molecule was resolved into polypeptide chains resembling light chains and μ-type heavy chains and having mol. wt of 22,000 and 70,000 respectively. The Necturus immunoglobulin was antigenically related to the IgM-like immunoglobulins of the toad (Bufo marinus) and the Xenopus. Unlike these anuran amphibians, however, the Necturus did not possess detectable amounts of low molecular weight immunoglobulins.

The finding raises the evolutionary possibility that the γ-like heavy chains of advanced amphibians may represent the results of a gene duplication independent of that which was responsible for the emergence of the γ heavy chain of mammals.

     

Two myeloma globulins, IgG1-kappa and IgG1-lambda, from a single patient (Im). I. Purification and immunochemical characterization

Electrophoretic analysis of the serum from a patient Im suffering from a multiple myeloma revealed the existence of two abnormal bands. Purification of these two fractions on a DEAE—cellulose column showed that both fractions had the same sedimentation coefficient (7S), belonged to the same class (IgG), and subclass (γl) and bore the same allotype (Gm₁). However, the heavy (H) chains of one immunoglobulin were associated with type κ light (L) chains whereas those of the other were associated with type λ L chains.Three individual antigenic determinants were found in these two proteins. One was common to the H chains of both proteins and the other two were each specific to one of the L chains. These results suggest that the myeloma cells of patient (Im) were able to produce at least three polypeptide chains, one H chain and two different types of L chains.     

Interactions between rheumatoid factor and native γG-globulins studied in the ultracentrifuge

Interactions between a rheumatoid factor preparation and native human (normal and myeloma) and animal γG-globulins have been studied in the ultracentrifuge.

Using pooled normal γG-globulin or a myeloma γG-globulin, the extent of reaction has been shown to be dependent upon the reactant concentration employed, a four-fold excess, by weight, of γG-globulin over rheumatoid factor being required to ensure maximum production of 22S complex.

All native myeloma γG-globulins tested reacted to give a 22S complex, the majority showing similar reactivity to the normal γG-globulin control. A small proportion, however, showed significantly different reactivities.

Of the animal γG-globulins tested, only rhesus monkey γG-globulin showed reactivity similar to human γG-globulin. The other species showed decreased reactivity.

The importance of these findings is discussed.

     

Membrane-associated immunoglobulins of human lymphocytes in immunologic disorders

Membrane-associated immunoglobulins of peripheral blood lymphocytes were studied by indirect immunofluorescence for γ, α, μ, κ and λ chains in healthy subjects and patients with immunologic disease.

In healthy subjects, heavy chains were found on 30·7% of lymphocytes (γ 15·3%, α 7·2% and μ 8·2%) and light chains on 32·8% of cells (κ 20·4% and λ 12·4%). Patients with humoral immune deficiencies had fewer immunoglobulin-bearing cells; sarcoidosis or thymectomy patients had normal or decreased immunoglobulin-bearing lymphocytes; cells with light chains were fewer than those with heavy chains on their lymphocytes. In some cases, normal levels of serum immunoglobulins were found in the absence of the corresponding immunoglobulin-bearing cells, and in others normal immunoglobulin-bearing lymphocytes were present in the absence of the corresponding serum immunoglobulins.

These data suggest that (1) immunoglobulin-bearing lymphocytes in blood do not reflect the condition of immunoglobulin-synthesizing cells in peripheral lymphoid tissues, and (2) in certain immunologic disorders, either some B-lymphocytes do not synthesize immunoglobulins, or immunoglobulins are in such a situation that the whole molecule or part of the molecule is not visualized by current methods.

     

The Separation and Antigenic Characteristics of Some Fractions of Papain Digests of Human Serum Gamma Globulins, and the Antigenic Relationships of Some Human Gamma Globulins with the Gamma Globulins of Some Other Mammalian Sera

1. Papain digests of human γ globulin (7S γ globulin) containing slow (S) and fast (F) immunoelectrophoretic components were fractionated by chromatography on diethylaminoethyl cellulose and by salting out with ammonium sulphate. No direct correlation was found between the antigenic characteristics and the chromatographic and electrophoretic properties of the S component fractionated by these methods.

2. β₂M globulin (γ₁ macroglobulin or 19S γ globulin) contained the S component of γ globulin, but the F component was not detected.

3. Rhesus monkey γ globulin more readily absorbed the anti-F antibodies than the anti-S from anti-human γ-globulin sera. Both rhesus and digested human γ globulins showed a stage of inhibition, when they completely prevented the precipitation of intact human γ globulin by its antiserum. Rhesus γ globulin, however, showed a reaction of partial identity (spur formation) on Ouchterlony analyses with human γ globulin and its antiserum, whereas digested human γ globulin showed a reaction of complete identity. The significance of these observations is discussed.

     

Characterization of conformation independent antigenic determinants in the triple-helical part of calf and rat collagen

About 20 per cent of the antibodies in rabbit antisera to native calf or rat collagen exhibited affinity for denatured rabbit collagen and could be isolated by immunoadsorption. Such antibodies reacted with the unfolded α1-chain as well as with the α2-chain of collagen. Inhibition experiments suggested that the two kinds of polypeptide chains are not completely equivalent in their antigenic determinants. These determinants were not significantly influenced by a treatment of native collagen with pronase, a procedure known to remove short, non-helical sequences at both ends of the molecule. The results suggested that the antigenic determinants are conformation independent. They are, however, mainly located in the middle region of collagen, having a rather complex conformational structure.

Cyanogen bromide cleavage of collagen did not impair the serologic activity of these determinants but with one exception none of the individual cyanogen bromide peptides possessed the full activity of the entire α-chain. However, most of the peptides could be agglutinated by the antibodies when put onto tanned red cells. Inhibition studies of these agglutination reactions clearly demonstrated that virtually all of the peptides carry unique antigenic determinants, which occasionally are shared by a few other peptides. Additional evidence for heterogeneity was obtained by further cleavage of the cyanogen bromide peptides with proteases. The minimum number of antigenic determinants thus estimated in calf collagen was nine. Evidence is provided that their structure in most cases does not correspond to sequences of the type Gly-Pro-X.

     

Serum and urine light chain levels in benign monoclonal gammapathies, multiple myeloma and Waldenström''s macroglobulinaemia

Immunochemical quantitative determinations of both serum and urine free light chains were carried out in seventy-five subjects with a serum M-component.

Diagnoses were benign monoclonal gammapathy (BMG) in thirty-four cases, multiple myeloma (MM) in thirty-one and Waldenström's macroglobulinaemia (WM) in ten. The single radial immunodiffusion test was used throughout, employing a special rabbit anti-Bence Jones antiserum, made specific for hidden antigenic sites of light chains by suitable absorptions. The smallest amount of free light chains which could be detected by this method was 0·02 mg/ml for both κ and λ chains.

In MM 84% of sera and 87% of urine revealed detectable amounts of Bence Jones protein, whereas in WM the corresponding figures were 80% for both sera and urines. The values ranged considerably from case to case, and tended to be higher in the urines. In BMG a definitely lower incidence of free light chains was recorded (in the sera: 35·2%; in the urines: 41·1%), and the levels were restricted to a much narrower range.

The significance of these findings is briefly discussed.

     

Two myeloma globulins IgG1-κ and IgG1-λ, from a single patient (Im): Their common cellular origin as revealed by immunofluorescence studies

The patient's (Im) serum contained two myeloma proteins possessing the same heavy chains (γl) and different light chains (κ or λ).     

Immunology light chain determinants on unstimulated and stimulated human blood lymphocytes assayed by direct immunofluorescence

The occurrence of immunoglobulin determinants on the surface of lymphocytes from human blood was assessed by indirect immunofluorescent staining of living cells after cultivation with phytohaemagglutinin or other stimulants. While antisera to γ, μ or α-determinants only stained a few cells, antisera to light chain determinants stained a larger proportion of the cells. Positive staining was recognized as `ring' staining comprising smaller or larger parts of the cell surface. The specificity of staining was ascertained by several types of controls.

After 48 hours of cultivation, anti-κ serum, applied at dilutions of 1:10–1:16 stained about 35–50 per cent and anti-λ serum about 15–20 per cent of the cells in the PHA cultures but only 3–5 per cent in the cultures incubated without PHA. When the antisera were applied at higher concentrations, positive light chain staining was also seen in the unstimulated cultures. At the highest concentrations, which could be used without increasing the non-specific background, the maximum number of κ-positive cells in the unstimulated cultures was approximately 25 per cent. Antiserum titrations showed that about 5–10 times less antiserum was needed to stain the optimal fraction of PHA treated cells. No increased staining of heavy chain determinants was achieved by increasing antiserum concentrations under the present conditions.

Similar results were obtained with lymphocytes stimulated by 3 days of incubation with concanavalin A, or by 6–7 days of incubation under mixed culture conditions. Lymphocytes of a tuberculin positive donor also gave increased staining for light chain determinants after incubation for 6–7 days with antigen (PPD).

The results indicate that lymphocyte stimulation is accompanied by increased amounts of surface bound immunoglobulins. At the present stage of knowledge, several explanations may account for the fact that light chain determinants are primarily accessible for staining.

The above results were obtained under conditions in which no protein was present in the washings performed during processing for immunofluorescence. In the presence of low concentrations of protein more than 60 per cent of both unstimulated and stimulated cells stained for light chain determinants, while staining for heavy chain determinants remained unchanged and at a low level. It is possible that protein-free washing removed a more loosely adsorbed immunoglobulin fraction passed on from producing to neighbouring non-producing cells.

     

Effect of Transplantable Plasma-Cell Tumours of Antibody Response in Mice

The response to primary antigenic stimulation was measured in C₃H mice bearing two types of plasma-cell tumours, the 5563 tumour producing γ-type myeloma proteins and the 5674 tumour producing β-type myeloma proteins. Haemocyanin and pneumococcus type III polysaccharide, which produce 6.5S antibodies, and sheep erythrocytes, which produce 18S haemolysins, were used.

Levels of serum antibody to all antigens were low in mice with the plasma-cell tumours, compared with those in normal mice, or mice with a mammary carcinoma. The larger the tumour, the lower were the antibody levels. Antibody levels were lower generally with the 5563 tumours than with the 5647 plasma-cell tumours of similar weight.

Although rapid catabolism of normal gamma globulins contributes to the low level of serum antibody in mice with the 5563 γ-type tumour, diminished responsiveness of the antigen-susceptible cells appears to be the principal factor in the impaired antibody response in mice with the 5647 β-type plasma-cell tumour.

     

Rat 7S immunoglobulins: characterization of γ2- and γ1-anti-hapten antibodies

Two antigenically distinct populations of rat antibodies to the 2,4-dinitrophenyl group (DNP) have been isolated by chromatography on DEAE-cellulose and named fraction 1 and fraction 3. Both fraction 1 and fraction 3 had sedimentation coefficients of 6.9S, similar hexosamine contents (1.04 and 0.89 per cent) and electrophoretic mobilities similar to the γ₂- and γ₁-immunoglobulins of guinea-pigs and mice. Fraction 1 consisted of two distinct antibody populations with very similar mobilities.

Only fraction 1 was able to sensitize homologous rat skin for passive cutaneous anaphylaxis (PCA). Both preparations were active in haemolysis of passively sensitized sheep red cells in the presence of complement, and fraction 3 was more lytic than fraction 1. In other tests such as PCA in mice, passive haemagglutination and precipitation in gel the behaviour of the two fractions was similar.

Fraction 3 is probably the immunoglobulin designated γA in earlier reports. The results indicate that fraction 1 and fraction 3 correspond to the 7S γ₂- and 7S γ₁-immunoglobulins of guinea-pigs and mice, except for their behaviour in homologous PCA and in passive haemolysis, and that fraction 1 and fraction 3 are rat 7S γ₂- and 7S γ₁-immunoglobulins, respectively.

     

IgA/IgC cryoglobulinaemia with vasculitis

A mixed IgA/IgG cryoglobulin complex was found in the serum of a 61-year-old man suffering from rheumatoid arthritis, Raynaud's phenomenon and vascular purpura. The purified complex was progressively insoluble at temperatures below 37°C. Reversible loss of cryoprecipitability was seen in conditions of extreme pH, in concentrated urea solutions and in 2-mercaptoethanol. Inactivation of complement at 56°C did not affect cryoprecipitability. The complex contained no complement and showed no anticomplementary activity. Analytical ultracentrifugation of the cryoglobulin at 37°C showed 7S and 11S components present in equal concentration.

The component immunoglobulins of the complex were separated by anion exchange chromatography at 37°C. Crossmixing studies with purified normal immunoglobulins indicated that the patient's IgA component was essential for cryoprecipitability of the complex. RA latex tests for anti-IgG activity at 37°C showed strong agglutination with both cryoglobulin complex and the isolated IgA component; this protein was found to be monoclonal with type K light chains.

Vasculitis, induced by skin testing the patient with his own plasma and isolated cryoglobulin was found to be histologically indistinguishable from that occuring spontaneously; reduction of symptoms paralleled reduction of cryoglobulin on treatment. These observations strongly support the hypothesis that the cryoglobulin complex is responsible for the patient's vasculitis.

     

Immune complex cryoglobulinaemia in lepromatous leprosy: a pathogenetic approach to some clinical features of leprosy

Cryoglobulins isolated from the sera of six patients with lepromatous leprosy were extensively studied by various immunochemical techniques.

Immunoelectrophoresis and analytical ultracentrifugation revealed in all cases the presence of two components only, which were identified as IgG and IgM respectively. The IgG component of all six cryoprecipitates and the IgM component of five of them were polyclonal, whereas the remaining IgM component was monoclonal as it produced a narrow-banded electrophoretic spike and contained kappa light chains only.

Anti-γ-globulin activity, detected in all sera and isolated cryoglobulins, was consistently found to be associated with the IgM fraction of the mixed cryoglobulins and was present at very high titres in the IgM monoclonal component. β_1A serum levels were decreased in all cases.

The cryoglobulins that appear to be immune complexes of the IgG/anti-IgG type may play a role in the pathogenesis of some clinical features of leprosy patients.

     

Human peripheral blood T lymphocytes express α-, μ- and F(ab')2-related determinants in their surface membranes

Whereas a mean of 81% of freshly isolated human T cells bound purified chicken anti-F(ab')₂ antibodies to their surface membranes, 14 and 2% bound chicken anti-μ and chicken anti-α antibody preparations respectively as demonstrated by the mixed antiglobulin rosetting reaction (MARR). Neuraminidase treatment of T cells significantly increased their reactivity in the MARR so that an average of 98, 39 and 40% bound anti-F(ab')₂ anti-μ and anti-α reagents respectively. When using anti-F(ab')₂ antibodies, inclusion of Fabγ in the rosetting medium caused greater than 90% inhibition of the MARR whereas Fcγ produced only 25% inhibition; this indicates that the determinants seen by anti-F(ab')₂ antibodies are not carbohydrate in nature since Fabγ and Fcγ fragments prepared from a human IgG myeloma express identical carbohydrate moieties. To discover whether the various immunoglobulin (Ig) related T cell molecules play a role in antigen recognition, each antiglobulin reagent was assessed for its capacity to inhibit T cell binding of a selected antigen, i.e. fluorescein-labelled keyhole limpet haemocyanin. Whereas the frequency of antigen-binding lymphocytes (ABL) varied from 8·1 to 15·1 per 10³ cells, fewer than 1 in 10³ cells were Ig-positive with a rabbit F(ab')₂ anti-light chain reagent. Thus virtually all ABL were T cells. Each antiglobulin reagent produced 50% or greater inhibition of antigen binding thereby suggesting that F(ab')₂-, α-, and μ-related determinants are in close proximity to, or part of, the T cell antigen receptor. More substantial evidence for a direct association of Ig-related surface determinants and the T cell antigen receptor is provided by the finding that under capping conditions, modulation of surface F(ab')₂-related determinants by anti-F(ab')₂ antibodies reduced antigen binding. Further, that anti-F(ab')₂ antibodies induced F(ab')₂-, α- and μ-related determinants to co-cap suggests that F(ab')₂-related determinants are stably associated with α- and/or μ-related determinants in the T cell membrane and are part of the same Ig-related molecule(s).     

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.

     

Guinea-pig reaginic antibody: I. Isolation and purification of an antibody protein with characteristics of IgE

The methods for isolation and purification of a guinea-pig serum protein with homocytotropic antibody activity and characteristics of IgE are described.

By precipitation in the equivalence zone or immunoadsorption and chromatography on DEAE-cellulose, we isolated an homocytotropic antibody, that was not able to give a precipitin line when it was reacted directly with the antigen. It was capable of sensitizing guinea-pig skin for PCA after a latent period of 24–48 hours but not after 3 hours; it was sensitive to treatment with mercaptoethanol. It had antigenic determinants present in the other guinea-pig immunoglobulins and particular antigenic determinants.

All these properties make us believe that this protein belongs to an immunoglobulin different from γ₁ and similar to the reaginic antibody (IgE) described in other species.

     

The catabolism of human γG immunoglobulins of different heavy chain subclasses. III. The catabolism of heavy chain disease proteins and of Fc fragments of myeloma proteins

The catabolism of ¹³¹I and ¹²⁵I paired labelled Fc fragments of myeloma proteins and of H chain disease proteins of different heavy chain subclasses was studied in men and monkeys. In contrast to the previously demonstrated catabolic heterogeneity of intact γG immunoglobulins, the Fc fragments and H chain disease proteins of all subclasses tested were catabolized at a similar rate. These data suggest that structures not present on the Fc fragments are responsible for the faster turnover rate of γG₃ immunoglobulins and for the differences in half-lives of myeloma proteins within a given subclass.The catabolic features of the H chain disease proteins differed from those of intact γG. Although the whole body half-lives of the two proteins were similar, the fractional turnover rate of the H chain disease proteins was higher than that of γG, on the average 8% of the intravascular pool/day as compared to 4% for γG. One-half to 1% of the intravascular pool of the H chain disease protein and less than 0·1% of the γG was excreted into the urine. An average of 24% of the H chain disease proteins and 44% of the γG equilibrated into the intravascular compartment.