Monoclonal IgM Kappa Research Articles

Lymphocytic interstitial pneumonia associated with a marked increase in monoclonal IgM-.KAPPA.-type rheumatoid factor and serum CA19-9.

A 62-year-old Japanese female was admitted due to dyspnea. She showed a marked increase in CA19-9 (maximum, 192,000 U/ml) and monoclonal IgM-kappa type rheumatoid factor (RF) activity. The patient died of respiratory failure 3 months later. Autopsy findings revealed an infiltration of IgM-kappa-positive plasma cells in the pulmonary interstitium, and therefore a diagnosis of lymphoid interstitial pneumonia (LIP) was made. The bronchoepithelial cells were stained with monoclonal antibody-reactive with CA 19-9 antigen. This is the first documented LIP, associated with a marked increase in monoclonal IgM-kappa type RF and CA19-9 in the serum.

The characterization of DNA antibody idiotypes—A description

The objective of this workshop was for investigators to characterize the idiotype systems they were employing to evaluate the expression of common idiotype families on the immunoglobulins and anti-DNA antibodies of patients with systemic lupus erythematosus (SLE) as well as other diseases, and in normal individuals. Twenty-four different systems were described, in which the idiotypes identified were expressed in SLE or related disorders (see Table). Each investigator was asked to provide information on the nature and source of the anti-idiotype probes employed to search for the expression of each idiotype amongst other antibodies, and the location (where known) for each idiotype on the heavy or light chain variable region, including any data on the molecular localization of idiotypic determinants. The 16/6 idiotype family and its expression in various systems was discussed by several groups. The prototype SLE peripheral-blood derived hybridoma monoclonal IgM kappa anti-DNA antibody employed to generate anti-16/6 idiotype probes showed higher binding to poly (I), poly (dT) and denatured DNA than to native DNA; it also showed binding to cell membrane antigens (platelets and lymphocytes), glycolipids extracted from the cell membrane of Mycobacteria tuberculosis, Klebsiella polysaccharide K30, and cytoskeletal structures. The expression of this idiotype was probed by a rabbit anti-16/6 polyclonal antibody, and in previous studies in SLE sera, 50% of patients with active disease expressed this idiotype on serum immunoglobulins as well as on 40% of immunoglobulins deposited in SLE skin and kidney lesions. The 16/6 Id has also been identified on paraproteins of G, M and A isotypes and in the serum of patients with mycobacterial infections during the active stages of their disease. Dr Lori Tucker (Boston) described some of the studies she is currently undertaking in Dr Schwartz's laboratory, looking at the molecular aspects of 16/6 expression. The parent 16/6 IgM~c antibody utilizes an Ig heavy chain variable region gene from the VHuI family, and the heavy chain of 16/6 is responsible for the expression of

Benign paraproteinaemia and immune neutropenia

A 65-year-old woman presented in 1981 with a 6-year history of a sore mouth and tongue. Physical examination was unremarkable, but investigation revealed neutropenia. Serum IgM was markedly raised at 23.0 g/l (normal 0.6-2.8) due to the presence of a monoclonal IgM kappa paraprotein; serum IgG and IgA were normal

Pseudothrombocytopenia induced by a monoclonal IgM kappa platelet agglutinin

Pseudothrombocytopenia is an in vitro phenomenon usually associated with anticoagulant (EDTA)-dependent IgG platelet agglutinins. A low Coulter platelet count was investigated in a 63-year-old woman with multiple sclerosis and a monoclonal IgM kappa gammopathy. An unusual type of EDTA- and temperature-independent IgM platelet agglutinin was identified by in vitro agglutination of donor platelets and was removed by immunoabsorption.

Demonstration of a cross-reactive idiotype (IdRQ) in rheumatoid factors from patients with rheumatoid arthritis but not in rheumatoid factors from healthy, aged subjects.

A human monoclonal IgM kappa paraprotein with rheumatoid factor (RF) activity was used to elicit antiidiotypic antibodies in rabbits. The antiidiotypic antiserum thus obtained reacted with samples from 40% of 72 rheumatoid arthritis patients, but not with any of the samples from 22 aged control subjects having serum RF. Our findings suggest that, despite the similarities between RF from rheumatoid arthritis patients and that from healthy individuals, the expression of V region genes may be different in healthy subjects and those with the disease.

The Major Rheumatoid Factor Cross-Reactive Idiotype in Rheumatic Disease

The major rheumatoid factor cross-reactive idiotype (RCRI), a tertiary structure formed by both light and heavy chains, is found on 60% of all monoclonal IgM kappa RFs. To determine if the RCRI is expressed in patients with rheumatic disease, we used polyclonal rabbit anti-idiotypic antibodies to detect RCRI in sera and in pokeweed mitogen cultures of blood mononuclear cells (PBM) from patients with rheumatoid arthritis (RA) and juvenile rheumatoid arthritis (JRA). We detected increased expression of RCRI+, plasma cells in PWM cultures, and in sera from these patients. We have determined that some 7S IgM molecules from RF+RA patients are RCRI+, and can bind IgG in a sensitive RF ELISA. We have also observed that the CD5+ B cell subset, which is responsible for autoantibody production, generates RCRI+ antibodies. We review these data and discuss the relationship of the idiotypic network of interacting antibodies with rheumatic disease.

CD5 positive immunoregulatory B cell subsets

B-chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease often expressed as a clonal expansion of CD5+ B cells. We report the characterization of CD5+ B cells from two unique B-CLL patients. Cells from patient 1 coexpressed CD5 (leu-1), CD19 (Leu-12), CD20 (B1), and HLA-DR; they were CD10 (J5), CD21 (B2), CD22 (Leu-14), CD25 (IL2-R1), PCA-1, surface, and cytoplasmic Ig negative. They suppressed normal peripheral blood lymphocyte (PBL) pokeweed mitogen (PWM) -stimulated immunoglobulin (Ig) synthesis greater than 80%. Cells from patient 2 were CD5 (Leu-1), CD19 (Leu-12), CD20 (B1), CD21 (B2), CD22 (Leu-14), HLA-DR, IgM, and kappa positive. They were negative for CD10 (J5), CD25 (IL2-R1), and PCA-1. These cells did not suppress normal PBL PWM-stimulated Ig synthesis but produced a monoclonal IgM kappa protein with rheumatoid factor-like activity. These observations suggest that there are different CD5+ B cell subsets, one immunosuppressive and the other autoreactive.

High-titer, high-thermal-amplitude cold autoagglutinin not associated with hemolytic anemia.

An example of an unusual cold autoagglutinin is reported. The antibody was monoclonal IgMKappa able to fix complement, and, in the presence of albumin, had both a high titer (greater than 4,096 at 4 degrees C) and a wide thermal range (4-37 degrees C). The patient was closely followed over a 3-year period with no evidence of hemolysis ever documented, despite a persistently positive direct antiglobulin test and the presence of the cold autoagglutinin. In contrast to previous reports regarding cold-agglutinin disease, this case demonstrates that in vivo hemolysis is not always associated with cold autoagglutinins that in vitro show a high thermal range in the presence of albumin.

Two patients Illustrating lymphoma transition and response to therapy in Sjögren's syndrome

Two patients with Sjögren's syndrome (SS) who subsequently developed malignant B-cell lymphomas are reported in detail. The first patient had both benign- and malignant-appearing lymphoid infiltrates on the same submandibular gland specimen and was successfully treated with combined chemotherapy and irradiation. The second patient developed cutaneous lymphoid infiltrates difficult to diagnose by light microscopy but containing a monoclonal IgM-Kappa population revealed by immunoperoxidase staining and immunoglobulin gene rearrangement studies. Her lesions resolved rapidly and completely on cyclophosphamide, recurred rapidly when this drug was discontinued, and resolved again on a second course of cyclophosphamie which is currently maintained at 50 mg daily. Both patients are doing well without recurrence two and three years after initial treatment. This clinical experience is presented to emphasize: (1) the clinical use of molecular biologic techniques to define the earliest appearance of malignant transformation in Sjögren's syndrome, and (2) the successful outcome that can be achieved with prompt institution of appropriate treatment. The phenomenon of lymphoma development in SS is discussed with regard to immunoregulatory abnormalities predisposing to malignancy in the setting of autoimmune disease.

Molecular interactions between human IgG, IgM rheumatoid factor and streptococcal IgG Fc receptors.

Group A streptococci type M15 were previously shown to bind both human IgG via the Fc component and a purified monoclonal IgM kappa rheumatoid factor (IgM RF). Using 125I-labelled IgG and 125I-labelled IgM RF, the present study gave association constants of 2.2 x 10(7) and 2.9 x 10(8) M-1, respectively. The binding of 125I-IgG to the streptococci was inhibited by unlabelled IgG, IgG Fc and fragment D of staphylococcal protein A but not by the IgM RF or F(ab')2 of anti-idiotype antibodies to RF (anti-Id RF). Inversely, unlabelled IgM RF and anti-Id RF inhibited the binding of 125I-IgM RF markedly and unlabelled human IgG and IgG Fc only slightly or moderately, respectively. Thus, group A streptococci type M15 showed different binding sites for IgG Fc and the antibody combining sites of a human monoclonal RF. The findings were still more complex on a background of previous reports showing that streptococcal IgG Fc receptors and RFs bind to the same amino acids on the Fc molecule. This complex pattern may play a role in the pathogenesis of rheumatoid arthritis.

A rat monoclonal antibody 4D7 produced by a hybridoma established from a yolk-sac tumor-bearing rat binds selectively to the stage-specific embryonic antigen SSEA-1.

A monoclonal rat IgM (kappa) antibody (MAb) is produced by a hybridoma obtained by fusion of the rat myeloma Y3 Ag 1.2.3 with spleen cells from a female W/Fu rat bearing a yolk-sac carcinoma isograft. In the rat this antibody (4D7) shows strong selective binding to all tested yolk-sac carcinomas, but no binding to cultured cells of a number of other tumor types or to cells of normal tissues. Immunohistochemical analysis of the specificity of the antibody confirmed the strong binding to yolk-sac carcinomas and also revealed binding to some other rat tissues. These include one colon carcinoma, the embryonic ectoderm and the primitive visceral endoderm of 8.5-day-old embryos, the central nervous system and the oviduct epithelium and some cells in the seminal tubules, the gastrointestinal epithelium and associated mucus and also the distal tubuli and collecting ducts of the kidney. The MAb binds strongly to purified SSEA-1 but not to purified Lewis A glycolipid. It is concluded that the 4D7 MAb recognizes a determinant which is identical to or includes Gal beta 1-4(Fuc alpha 1-3) GlcNAc. The immunogenicity of the SSEA-1 determinant is further confirmed by the demonstration that antibodies binding to purified SSEA-1 but not to Lewis A appear in the sera of some of the rats developing primary yolk-sac carcinoma.

Monoclonal anti-DNA IgM kappa in neuropathy binds to myelin and to a conformational epitope formed by phosphatidic acid and gangliosides.

Anti-DNA antibodies that cross-react with phosphorylated epitopes of other cellular constituents may be involved in the pathogenesis of autoimmune disease. An IgM monoclonal antibody from a patient with chronic lymphocytic leukemia (CLL) and neuropathy bound to denatured DNA and immunostained myelin in peripheral nerve and spinal cord. The monoclonal IgM bound to ELISA microwells coated with a mixture of phosphatidic acid and gangliosides at serum dilutions of up to 1/100,000, but binding to phosphatidic acid alone was observed at dilutions of less than 1/100 only, and there was no binding to gangliosides alone. Incubation with micelles containing phosphatidic acid and gangliosides selectively absorbed the monoclonal IgM and inhibited its binding to denatured DNA and to myelin. These observations suggest that autoantibodies may bind to conformational epitopes formed by two separate molecules, and that autoantibodies that cross-react with phosphorylated epitopes in DNA and neural tissue could be involved in autoimmune neurologic diseases.

Sarcoidosis and immunocytoma

The current literature contains reports of sarcoidosis with polyclonal increases in immunoglobulins. There are also reports of lymphoma in patients with pre-existing sarcoidosis. This is believed to be the first report of a patient in whom a monoclonal IgM kappa paraproteinemia developed after the diagnosis of sarcoidosis. This paraproteinemia was linked with a histologically proved non-Hodgkin's lymphoma. The possible relationships between the two disease entities are discussed.

Reassessment of Malignant “Angioendotheliomatosis”

Malignant angioendotheliomatosis (MAE) is a lethal intravascular proliferation which has been thought to be of endothelial origin. In order to characterize its cellular nature, we studied 15 cases of MAE immunocytochemically, using antisera for factor VIII-related antigen, cytokeratin, epithelial membrane antigen, vimentin, blood group isoantigens, thoracic duct lining cell antigens (TDLCA), common leukocyte antigen, and Ulex europaeus I lectin. In 14 of 15 cases, common leukocyte antigen was observed in malignant intravascular cells. Similar reactivity for factor VIII-related antigen was present in 14 cases, but was largely restricted to cells enmeshed in fibrin-platelet thrombi, and probably represents adsorption of platelet-derived factor VIII by tumor cells. All cases failed to bind Ulex europaeus lectin and lacked immunoreactivity for TDLCA, cytokeratin, epithelial membrane antigen, and blood group isoantigens; two manifested positivity for vimentin. Immunofluorescent microscopy of frozen tissue in one case showed monoclonal IgM-kappa immunoglobulin on the surfaces of tumor cells. Electron-microscopic study of three cases disclosed a predominant cell type lacking features of epithelial or endothelial differentiation; a minor cell population displayed endothelial characteristics and was thought to be reactive. Four patients with typical MAE also had extravascular large-cell lymphoma in lymph nodes, spleen, adrenal glands, stomach, or soft tissues. Six patients showed clinical evidence of autoimmune disease. These results suggest that MAE displays lymphoid rather than endothelial differentiation.

Monoclonal IgM in a patient with paraproteinemic polyneuropathy binds to gangliosides containing disialosyl groups

Monoclonal IgM kappa from a patient with polyneuropathy associated with paraproteinemia was found to bind to several polysialogangliosides. Binding of IgM to gangliosides was shown by enzyme-linked immunosorbent assay (ELISA) and by overlaying thin-layer chromatograms of human brain and peripheral nerve gangliosides with the patient's serum followed by radioiodinated goat antihuman IgM. The latter technique showed that the IgM paraprotein reacted with a number of gangliosides. In an ELISA the IgM paraprotein reacted strongly with GD2, GD3, GD1b, and GT1b, but not with GM1, GM3, and GD1a. Thus, the epitope for the patient's IgM paraprotein appears to involve the disialosyl configurations.

Sequence similarities and cross-idiotypic specificity of L chains among human monoclonal IgM kappa with anti-gamma-globulin activity.

The complete amino acid sequence of five light chain variable (V) regions of human monoclonal IgM kappa rheumatoid factors (RF) was determined, and their cross-reactive idiotypes (CRI) were characterized with antibodies induced by immunization with synthetic peptides PSL2 and PSL3, corresponding to the second and third complementarity-determining regions (CDR) of the SIE light chain. Together with two additional RF studied previously, all seven RF belong to the V kappa IIIb sub-subgroup. The region encoded by the V kappa gene segment (positions 1 to 95) in all seven proteins was virtually identical in primary structure, whereas the sequence from positions 96 to 108 defined the usage of the J kappa 1 gene in three proteins and the J kappa 2 gene in four of them. Position 96 contributed by the recombination of the V kappa and J kappa gene segments showed the presence of four different amino acid residues. Both anti-PSL2 and anti-PSL3 bind efficiently to all separated L chains when analyzed by the Western blot technique, and the binding was inhibited specifically by the corresponding peptides. The results reveal that the majority of human IgM-RF light chains are derived from a single germ line V kappa gene or a family of closely related V kappa III germ line genes, and express two "primary structure-dependent" CRI, which are largely dependent on the amino acid sequence of the second and third light chain CDR.

Noncovalent association of heavy and light chains of human immunoglobulins. IV. The roles of the CH1 and CL domains in idiotypic expression.

A rabbit anti-idiotypic antiserum was raised against a monoclonal human IgM kappa(Me) in order to analyze the possible modulation of idiotypic expression by Fab constant domains. IgM(Me) fragments, subunits, and domains were prepared by chemical and enzymatic cleavages. All molecular species were shown to have a well-defined secondary and tertiary structure by circular dichroism. Full recombination between domains and subunits was ascertained by difference spectroscopy. The expression of the idiotype on native and recombined fragments, domains, and subunits was quantitated in a competitive enzyme-linked immunosorbent assay (ELISA). Reduced and alkylated Fab, isolated H and L chains, purified Fv(Me), intact VH and VL domains and H-L, VH-L, VL-H, and VH-VL recombinants were compared on a molar basis to native Fab(Me) for idiotypic expression. VH-specific determinants were found, whereas the L chains were virtually devoid of idiotypic activity. Both the peptic FV(Me) fragment, which is composed of intact VH and VL domains, and the recombined VH-VL heterodimer were found to be fourfold less active for idiotype expression than native Fab(Me). However, full inhibition was achieved at high molar concentrations, suggesting that all the idiotopes present on Fab(Me) were expressed on FV(Me) but with a reduced antigenicity. Comparison of VH-L and VL-H hybrid molecules revealed that the presence of the C mu 1 domain was sufficient to restore full idiotypic expression as compared with native Fab(Me). These data support the hypothesis that the first constant domain of the mu heavy chain alters the quaternary interaction between the variable domains, and therefore modulates the expression of the idiotype through longitudinal interactions that are not affected by reduction of the inter-H-L chain disulfide bond.

Genetic elements used for a murine lupus anti-DNA autoantibody are closely related to those for antibodies to exogenous antigens.

The mRNAs encoding heavy and light chains of a hybridoma-derived monoclonal IgM kappa anti-DNA autoantibody from lupus-prone MRL/Mp-lpr/lpr mice (Ighj) have been transcribed into cDNA copies and molecularly cloned, and their complete nucleotide sequences have been determined. The mRNA for the heavy chain variable region, including leader peptide and 5' untranslated region, is transcribed from a heavy chain variable region (VH) gene closely related (and possibly allelic) to VH genes of the C57BL/6 (Ighb) nitrophenyl antibody family. The deduced amino acid sequence corresponding to the light chain variable region of this autoantibody shows extensive similarities with non-autoantibody molecules of the V kappa 1 group, suggesting a common variable gene origin. The joining segments, constant regions, and 3' untranslated regions of both the heavy and light chain mRNAs are nearly identical to corresponding sequences of non-autoantibodies from normal mice. Our findings suggest that this anti-DNA autoantibody originated from the same germline repertoire as antibodies to exogenous antigens.

Kinetic study of rheumatoid factor influence on complement-mediated modulation of immune precipitation.

Human monoclonal IgM kappa rheumatoid factor PA (mRF) expressed various effects on complement-dependent modulation of ovalbumin/antiovalbumin lattice formation measured kinetically by laser light scattering (LLS): (1) in the absence of complement, mRF (44.2-177 U/ml) caused enhancement of LLS in a dose-dependent manner; (2) mRF partially prevented expression of complement-mediated inhibition of lattice formation and, at the same time, complement inhibits mRF-dependent LLS enhancement; (3) complement caused disruption of lattice-mRF bonds during solubilization of preformed lattice-mRF complex as demonstrated by specific mRF activity in supernatants. This effect is dependent on complement activity and on both complement and mRF concentrations; (4) ovalbumin/antiovalbumin complexes gradually lost the ability to react with mRF during the first 2 min of complement-mediated inhibition of lattice formation.

Cryoprecipitation of an anti-Pr2 monoclonal IgM cold agglutinin in the presence of GM3 ganglioside.

The mechanism of cryoprecipitation of a monoclonal IgM kappa cryoglobulin (Mou) with a cold agglutinin activity of Pr2 specificity has been studied. By immunodiffusion this cryoglobulin reacted (by its Fab' fragment) with micellar GM3, a ganglioside bearing the Pr2 antigenic determinant. In contrast to previous reports that indicated a possible temperature dependent self-association of IgM molecules via an immunological interaction leading to cold precipitation, we could not detect any affinity of this cryoglobulin for IgM when we used passive hemagglutination or an indirect enzyme-linked immunosorbent assay (ELISA). However, a GM3-like ganglioside could be extracted, by drastic methods, from the cryoglobulin studied at 22 degrees C, whereas no GM3 was extracted from two control cryoglobulins. Some minor gangliosides (representing less than 25% of total amount of bound gangliosides) were also extracted from Mou cryoglobulin and these gangliosides were shown to crossreact with GM3, as they specifically bind to Mou cryoglobulin by ELISA. After cryoprecipitation the serum still contained a monoclonal anti-Pr2 IgM kappa. A GM3-like ganglioside could be extracted from this purified IgM, and cryoprecipitability could be induced by the addition of a minute amount of micellar GM3. These results suggest that Mou cryoglobulin circulates as an immune complex and that cryoprecipitation may depend on unique IgM-GM3 (or IgM-GM3 cross-reacting gangliosides) complexes.