Unreleased intracellular monoclonal macroglobulin in chronic lymphocytic leukaemia

In two patients presenting as chronic lymphocytic leukaemia, immunofluorescence studies have demonstrated the presence of an accumulated intracytoplasmic material reacting with antisera to μ chains and only to one type of light chains, whereas their serum contained neither monoclonal IgM nor free μ chains.

In the first case, the IgM lambda inclusion bodies were crystals detected in the cytoplasm of small lymphocytes and were not clearly lined by rough endoplasmic reticulum. IgM λ molecules were present at the surface of the lymphocytes.

In the other patient, who elaborated free κ light chains, the inclusions found in marrow plasma cells and in peripheral blood lymphocytes were Russell bodies.

The practical and theoretical importance of non-secretory immunoproliferative disorders, characterized by an unreleased monoclonal immunoglobulin marker, is outlined.

     

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.

     

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.

     

Synthesis of immunoglobulins by biopsied tissues and cell lines from Burkitt's lymphoma

Cell suspensions of Burkitt''s lymphomas and cell lines derived from the same tumours were compared for immunoglobulin synthesis by analysis of the culture fluid. Thirty-one out of fifty tumour cell suspensions (i.e. twenty-one out of thirty-five patients) synthesized IgG (γ-chains) with type κ and/or type λ light chains; IgM synthesis was found in only five cases. Of the Burkitt''s lymphoma cell lines, twelve out of nineteen synthesized IgG (γ-chains); type κ light chains were produced by ten of these cell lines and type λ light chains by three. Only three cell lines synthesized μ chains. IgA synthesis was not detected in any of the biopsied tissues or cell lines.Comparison of the immunoglobulin synthesis by the cells of the biopsied tissue and the derived cell line showed very good agreement. This leads to the conclusion that the pattern of immunoglobulins synthesized by Burkitt''s lymphoma cell lines is representative of the original tumour.Investigation of cells from repeat biopsies, and serial testing of the derived cell lines showed that the capacity to synthesize particular immunoglobulins and chains remained constant.The fact that many of the biopsied tumour tissues and cell lines synthesized more than one immunoglobulin, or different classes of heavy chains and types of light chains, raises the question whether these immunoglobulin-producing cells originate from one or more cells.     

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.

     

Serological heterogeneity of the IgM components of mixed (monoclonal IgM–polyclonal IgG) cryoglobulins

Mixed IgG–IgM cryoglobulins were isolated from the sera of seven patients with macroglobulinaemia or cryoglobulinaemia. The IgM components of all seven cryoproteins were monoclonal, containing κ light chains only, whereas the IgG components were polyclonal, containing both κ and λ light chains. Despite their apparent immunological homogeneity, the IgM components showed a wide range of antiglobulin activity. The data indicate that serological specificity may vary from one mixed cryoglobulin to another and that the monoclonal IgM components of different mixed cryoglobulins represent a heterologous group of antiglobulins.     

Studies on the specificities of two IgM lambda cold agglutinins

The reactions of two monoclonal IgM lambda cold agglutinins, Sch and Sher, have been studied in detail with human and animal erythrocyte antigens. Although they were unusual in having lambda and not kappa polypeptide chains, they could be assigned to the anti I and anti-Pr₁ groups of cold agglutinins.

The findings with serum Sher indicated that the Pr₁ antigen may be more complex than previously thought. The occurrence of unexpectedly large numbers of specificities among monoclonal anti-I, anti-i and anti-Pr antibodies is discussed and it is suggested that each monoclonal antibody may recognize only a limited portion of a complex red cell antigen.

     

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.

     

Immunological studies of an atypical (myeloma) immunoglobulin

An 8S myeloma component, isolated from serum of a patient with myelomatosis is described, which appears to have no antigenic determinants in common with human, α-, δ-, γ- or μ-polypeptide chains as revealed by immuno-electrophoresis and Ouchterlony gel diffusion analysis.

The myeloma protein migrates in the fast γ-region on electrophoresis at pH 8.6 and has an elution volume on Sephadex G-200 similar to that of 6.5S IgA.

The isolated myeloma component has an approximate molecular weight of 200,000 and a total carbohydrate content of 10.7 per cent.

Reduction with β-mercaptoethanol and acid dissociation yields light polypeptide chains of Type L and a carbohydrate-rich component, in the ratio of 1:4.

Antisera specific to determinants on the heavy chains of the myeloma protein showed no reaction with the immunoglobulins A, D, G or M. Instead unique determinants were found on the heavy polypeptide chains.

     

The lymphoid follicles of the human palatine tonsil

The distribution of immunoglobulin (Ig) in the lymphoid follicles of the human palatine tonsil has been re-examined by applying the unlabelled antibody peroxidase–anti-peroxidase (PAP) sequence and a two-stage fluorescein isothiocyanate (FITC) technique to trypsinized paraffin sections and cryostat sections. Large numbers of cells containing Ig were demonstrated in the follicle centres, γ, μ, δ and α heavy chains being present. Cells containing κ and λ chains were found in all follicle centres, a finding incompatible with the monoclonality of the centres. Extracellular Ig (γ, α, μ and δ heavy chains, κ and λ light chains) was also present in the follicle centres. The lymphocytes of the mantle (zone C) appeared to have μ and δ chains (and κ and λ chains) on their surfaces, and were considered to be immature B cells, thereby providing support for our earlier hypothesis that these cells constituted a pool of B memory cells. Trypsinized paraffin sections proved superior to cryostat sections for the demonstration of intracellular Ig, but the reverse was true for surface Ig. Extracellular Ig was equally evident in both types of section. The metalophil method of Marshall (1948) revealed dendritic histiocytes in the follicle centre and lymphocyte mantle (zones B and C). The follicles were supplied by one or more prominent arterioles, around which the dendritic cells were located, an arrangement which may function as a `traffic area' for trapping circulating B cells on the antigen-bearing dendritic cells.     

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.

     

Structural peculiarities of a truncated VκIII immunoglobulin light chain in myeloma with light chain deposition disease

We report on the primary sequence of the monoclonal immunoglobulin light chain (LC) REV involved in myeloma-associated light chain deposition disease (LCDD). This sequence was deduced from that of the corresponding complementary (c)DNA in bone marrow plasma cells. Products of three independent amplifications by polymerase chain reaction (PCR) were sequenced and found to be identical. The κ mRNA encoding this N-glycosylated LC showed an overall normal structure consisting of a Vκ_III segment rearranged to Jκ_II. Direct N-terminal amino acid sequencing of the circulating monoclonal IgA2,κ showed identity with the bone marrow-derived sequence. The κ-chain presented several unusual features affecting both the leader sequence and the variable (V) region. Four unique amino acid substitutions were found at positions -8, -3, -2 and -1 in the leader sequence and probably resulted in an unusual cleavage by signal peptidase, thus making the LC truncated by one residue and accounting for its unique hydrophobic N-terminus: Ile-Ile-Leu. Additional peculiarities were observed in the V region, including a Thr74 → Asn substitution creating a N-glycosylation site, and Thr53 → Ile, which was only reported once among human κIII chains, in another LCDD case, and may be of special significance at a position usually harbouring a polar amino acid.     

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.     

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.

     

Surface immunoglobulins on blood lymphocytes of normal and immunodeficient persons studied by the mixed antiglobulin method

Surface immunoglobulins on human peripheral blood lymphocytes were investigated by the mixed antiglobulin technique—using the single layer mixed antiglobulin method as originally described (SLMA), and a modification employing a double layer of antibody (DLMA). Lymphocytes isolated from the blood of normal individuals had a mean of 7.8 and 18.4 per cent Ig + cells by the SLMA and DLMA techniques respectively. The DLMA data are similar to results obtained by other methods of detecting membrane Igs indicating that the mixed antiglobulin method is comparable in sensitivity. When the total numbers of Ig + cells, obtained by separate κ and λ testing, were compared with results obtained using single anti-light chain antisera, there was no significant difference, suggesting that most positive lymphocytes carry a single variety of light chain.

Lymphocytes from the blood of seventeen patients with primary immunodeficiency were analysed. Four patients with variable immunodeficiency and four others with absent serum IgA all had normal surface Igs including α chains. All members of a family having an X-linked immunodeficiency had normal surface Igs including the affected members and a presumed carrier.

Four cases of immunodeficiency associated with thymoma proved to have disparate findings. One patient exhibited a selective absence of μ antigens on the membranes of blood lymphocytes of over 2800 tested cells. Two other cases had normal surface Igs while a fourth patient, previously reported, lacked all surface Igs.

     

Temperature changes produced by the injection of catecholamines and 5-hydroxytryptamine into the cerebral ventricles of the conscious mouse

1. Changes in temperature were determined following injection of noradrenaline, adrenaline, isoprenaline, dopamine and 5-hydroxytryptamine (5-HT) into the cerebral ventricles of the conscious mouse.

2. Noradrenaline (1-20 μg) and dopamine (10-160 μg) caused falls in body temperature. Adrenaline (1-20 μg) caused a slight and transient rise in body temperature followed by a fall. Isoprenaline (5-20 μg) caused a rise in body temperature, hypothermia only occurring after very high doses (200 μg) of this catecholamine.

3. α- and β-adrenergic blocking agents, phentolamine (> 2 μg) and propranolol (> 5 μg) respectively, caused falls in body temperature when injected into the cerebral ventricles of the mouse.

4. Specific drug antagonism studies were limited owing to the intrinsic effects of the α- and β-adrenergic blocking agents. However, some evidence was obtained to indicate that noradrenaline mediated its effects through a central α-type adrenergic receptor.

5. 5-HT (10-160 μg) caused a fall in body temperature. The action of this indoleamine and the catecholamines in regard to thermoregulatory function is discussed.

     

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.

     

Antigen-binding thymus-derived lymphocytes: II. Nature of the immunoglobulin determinants

Thymus-derived `rosette'-forming lymphocytes which have been separated from other SRBC-sensitive cells by means of cotton wool columns were examined for the presence of immunoglobulin. This was carried out by inhibition of rosette formation by anti-immunoglobulin sera. Inhibition was effected by a number of anti-IgM sera shown to contain antibodies with specificities directed towards the `hinge' region of the μ chain. No other heavy chain specific antisera were inhibitory.

The ratio of rosette inhibition by anti-κ and anti-λ light chain sera varied during the course of the response to SRBC, the latter inhibiting by 89 per cent 3 days post-immunization.

     

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.

     

Studies on the structural and biological functions of the Cμ3 and Cμ4 domains of IgM

The development of methods for the production of intact Cμ3 and Cμ4 domains of IgM have made possible the assessment of some of their structural and biological functions. Antiserum against Fcμ fragment detected both domains and illustrated their complete antigenic non-identity. Circular dichroism spectroscopy and the retention of antigenicity indicated that both domains had retained most of their native structure. No interaction of the type Cμ3—Cμ3, Cμ4—Cμ4 or Cμ3—Cμ4 could be detected under non-dissociating conditions by analytical ultracentrifugation or molecular exclusion chromatography experiments. These results lead us to believe that the transmission of effector messages between the Fab and Fc parts of IgM takes place through structural changes at the quaternary level.

C[unk]1-fixation experiments with IgM and several of its fragments and domains show that (a) the Cμ4 domain contains the C[unk]1-fixing site; (b) the high C[unk]1-fixing capacity of IgM or Fc₅μ cannot be explained on the basis of a simple accumulative model of complement fixing domains; (c) the C[unk]1-fixing site is independent of the native structure of the Cμ4 domain; (d) the C[unk]1-fixing site does not contain carbohydrate.

Examination of the IgM receptor on the surface of human T lymphocytes show that (a) Cμ4 domain is primarily responsible for the reaction and Cμ3 domain has very little affinity; (b) native structure is essential for the reaction because reduction and alkylation of the Cμ4 domain destroyed both its original conformation and affinity for this receptor; (c) IgM and Fc₅μ had a much greater affinity for the receptor than monomeric subunits: (d) carbohydrate on Cμ4 domain is not involved in the affinity reaction.