Beta 2-microglobulin is selectively associated with H-2 and TL alloantigens on murine lymphoid cells.

We have used a rabbit antiserum prepared against purified rat beta2-microglobulin to immunoprecipitate molecules from lysates of radioiodinated murine thymocytes and splenocytes. All the molecules that are reactive with this serum have subunits of 44,000 and 12,000 and can be identified as H-2 and TL antigens. Thus, the anti-beta2mu serum can deplete lysates of the majority of the TL and H-2 atigens which can be subsequently recognized by alloantisera. If TL and H-2 are precipitated from the lysates before the addition of anti-beta2mu, no beta2mu-reactive molecules remain. Our results indicate that Ia antigens cannot be depleted from the lysates with anti-beta2mu. The studies also suggest that TL and H-2 heavy chains can exist as both monomers and dimers. These observations are discussed with regard to previous studies concerning the native structure of H-2 and TL antigens.

The relationship between 2 -microglobulin and immunoglobulin in cultured human lymphoid cell lines.

beta(2)-microglobulin was detected on the cell surface and in the medium of human lymphoid cells established in long-term culture. The secretion of beta(2)-microglobulin was relatively uniform when different cell lines were compared, whereas IgG production varied widely. kappa- and micro-membrane antigens were modulated by specific antibody; beta(2)-microglobulin was not modulated. Anti-kappa and anti-micro antisera had no effect on the expression of membrane beta(2)-microglobulin, nor had anti-beta(2)-microglobulin antiserum any effect on the expression of kappa- and micro-membrane antigens.

Molecular similarities between the Qa-2 alloantigen and other gene products of the 17th chromosome of the mouse.

The alloantigen Qa-2, whose gene is located on the 17th chromosome between H-2D and Tla, is identified as a molecule of 43,000 daltons which is associated with beta 2-microglobulin. Qa-2 comprises approximately 0.15% of the iodinateable cell surface protein of lymph node cells. Sequential precipitations demonstrated that Qa-2 is distinct from H-2D and H-2K molecules.

Initiation of protein synthesis at an unusual position in an immunoglobulin gene?

The amino acid sequence of urinary beta(2)-microglobulin has been partially determined and found to be related to the constant region of IgG immunoglobulin heavy chain. beta(2)-Microglobulin is present in normal individuals. Its gene may have evolved from an immunoglobulin gene by the use of an unusually located start signal for initiating synthesis of the polypeptide.

Aggregation of HL-A Antigens at the Lymphocyte Surface Induced by Antiserum to beta2-Microglobulin.

Rabbit antiserum to human beta(2)-microglobulin followed by goat antiserum to rabbit immunoglobulin induces aggregation of beta(2)-microglobulin at the lymphocyte surface, as shown by immunofluorescence and by acquisition of resistance to lysis by complement. This treatment also affects all HL-A antigens of the cell, which cap together with beta(2)-microglobulin, so that the cell becomes resistant to lysis upon addition of antibodies to HL-A in the presence of complement. These findings suggest that a physical linkage exists between these two classes of polypeptides at the surface of the living cell and is in agreement with recent biochemical data obtained by others with cell membrane preparations.

Beta 2-microglobulin: association with lymphocyte receptors.

beta(2)-Microglobulin (beta(2)m) is a low-molecular-weight protein constituent of lymphocyte membranes. Amino acid sequence analysis has revealed a high degree of homology between the beta(2)m and certain regions of immunoglobulin molecules, suggesting a possible recognition function for the beta(2)m, in analogy with the immunoglobulins. The data presented demonstrate that highly specific antiserum against beta(2)m blocks lymphocyte reactivity against allogeneic cells in mixed leukcocyte cultures and against phytohemagglutinin, both of which processes presumably function via a cell surface receptor on thymus-derived (T) lymphocytes. There is very little inhibition of T lymphocyte rosette formation with sheep red blood cells. The findings suggest a possible relation between the beta(2)m and recognition units on the T lymphocyte surface.

Low molecular weight urinary proteins. I. Partial amino acid sequences of the retinol-binding proteins of man and dog.

Human and dog retinol-binding proteins were isolated and their physico-chemical characteristics compared. Partial amino acid sequences of the first 50 residues were determined for both proteins and found to be remarkably similar. Only five residues were shown to be different; all could be due to single base pair mutations. However, immunological cross-reactivity was not detected between the two proteins with specific antisera prepared in rabbits against the human and dog retinol-binding proteins.

Turkey beta 2-microglobulin--II. Physiological parameters investigated by radioimmunoassay.

A competitive binding radioimmunoassay (RIA) was developed utilizing 125I-labeled turkey beta 2-microglobulin (beta 2m), rabbit anti-turkey beta 2m serum and polyethylene glycol for separation of bound and free antigen. The antibody-antigen reaction was demonstrated to be monospecific by immunofixation electrophoresis. Characteristic parameters of the RIA were: nonspecific binding, 5.5%; linear dose-response range, 0.24-64.0 ng turkey beta 2m; dose-response correlation coefficient, -0.995; assay sensitivity, 0.52 ng; upper limit of precision, 40.4 ng. RIA analysis of adult turkey tissue and organ homogenates revealed highest amounts (greater than 10 micrograms turkey beta 2m/g) in liver and kidney and intermediate levels (2-10 micrograms/g) in lymphoid organ (thymus, spleen, caecal tonsil, bursa), skin and lung extracts. Erythrocytes and peripheral blood lymphocyte extracts contained 8.0 and 5.1 micrograms/10(9) cells respectively whereas all other tissues examined expressed greater than 2.0 micrograms/g. The beta 2m content in turkey serum was 192.5 micrograms/ml. The molecular weight distribution of turkey beta 2m in serum determined by gel permeation chromatography consisted of a minor component of approximately 60,000 daltons and a major fraction eluting at a position characteristic of free beta 2m. A cross-reaction between turkey and mammalian beta 2ms was not observed.

Turkey beta 2-microglobulin--I. Isolation, properties and amino acid analysis.

Turkey beta 2-microglobulin (beta 2m) was purified from pooled serum by successive steps of ultrafiltration, gel filtration chromatography, lectin affinity chromatography, anion exchange chromatography, isoelectric focusing and a second step of gel filtration. Identification of turkey beta 2m was based upon NH2-terminal primary structure analysis. The NH2-terminal primary structure of turkey beta 2m is: NH2-Lys-Ile-Glu-Val-Tyr-Ile-Lys-. The purity of the isolated protein was confirmed by two-dimensional polyacrylamide gel electrophoresis, immunodiffusion and immunoelectrophoresis. Physicochemical parameters of turkey beta 2m are: mol. wt, 10,500 (observed), 9959 (calculated); beta electrophoretic mobility; pI, 4.7, 5.2; E1%280, 10.9; absence of terminal D-mannopyranosyl and D-glucopyranosyl residues. Amino acid composition analysis demonstrated similarities between turkey and chicken beta 2ms that distinguished them from mammalian beta 2ms.

Association of human and bovine beta 2-microglobulins with detergent solubilized HLA-A,B antigens.

The association of human and bovine beta 2-microglobulins with detergent solubilized HLA-A,B antigens was analyzed by a direct binding assay using radiolabeled beta 2-microglobulin and an immunoadsorbent containing a monoclonal antibody to the HLA-A,B heavy chains. Binding of beta 2-microglobulin to HLA-A,B heavy chains could be saturated with respect to the amount of membrane glucoprotein in the system and reached steady state after 6 h at 37 degrees C. Inhibition of [125I]beta 2-microglobulin binding to HLA-A,B heavy chains by beta 2-microglobulin purified from human urine or bovine colostrum resulted in identical inhibition curves and apparent dissociation constants of 1 X 10(-8) M. This evidence suggests that beta 2-microglobulins from different species have similar binding sites for HLA-A,B heavy chains.

Quantification of effector/target conjugation involving natural killer (NK) or lymphokine activated killer (LAK) cells by two-color flow cytometry.

Precise estimates of the frequency of NK- and LAK-target conjugates were obtained by two-color flow cytometry using hydroethidine and calcein as intracellular labels for target cells and effector cells, respectively. These two dyes can easily be used with a standard single-laser flow cytometer with excellent signal separation and dye retention. Hydroethidine labeling did not alter target susceptibility, and calcein labeling did not significantly alter NK function. Excellent agreement was obtained between this flow cytometric method and visual estimation of the frequency of fresh or IL-2-activated human lymphocytes that form conjugates with K-562 target cells. The percentage of cloned NK or LAK cells that form conjugates with K-562 target cells was dependent on the E:T ratio, with extrapolated maximum conjugate frequencies (alpha max) of 40-50%. However, the frequency of lymphocytes forming conjugates with K-562 cells did not closely correlate with the cytolytic activity of a given lymphocyte population. This two color flow cytometric method employing a pair of fluorochromes that do not modify cell membranes or alter cell function in cytotoxicity assays should facilitate further studies of mechanisms involved in the initial stages of target cell recognition by NK and LAK cells.

A flow cytometric method to detect anti-pyruvate dehydrogenase antibody in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is an autoimmune disease characterized by the presence of anti-mitochondrial antibodies specifically directed against the M2 group of mitochondrial antigens. Recently, the E-1, the E-2, and protein X components of pyruvate dehydrogenase enzyme complex have been identified as the major antigens within the M2 group of autoantigens. An immunoassay using pyruvate dehydrogenase enzyme complex as a specific antigen for the diagnosis of PBC was developed. Pyruvate dehydrogenase enzyme complex was attached to polystyrene microbeads, incubated with sera from PBC patients (n = 18), normal controls (n = 50), or patients with other autoimmune diseases (n = 26), followed by incubation with a second fluorescein isothiocyanate conjugated goat anti-human immunoglobulin and then analyzed by flow cytometry. High numbers of fluorescence channels (mean, 1,693 +/- 846) were obtained for all PBC sera except for two patients. Compared to the conventional anti-mitochondrial antibody assay, the assay had a sensitivity rate of 94% and a specificity rate of 100%. The reactive antibodies are predominantly of the immunoglobulin G3 subclass. Their levels could be correlated with the histopathologic stages of PBC. These results were corroborated by immunoblotting. Sera from patients with later stages of PBC strongly reacted with pyruvate dehydrogenase enzyme complex components, E1 alpha, and protein X.

Post-gamma globulin : tissue distribution and physicochemical characteristics of dog post-gamma globulin.

An animal model (dog) was established to investigate physicochemical and biological parameters of a basic low molecular mass protein---post-gamma globulin (p-gamma). In this study the distribution of this 11 000 daltons (11 kdaltons) protein was determined in extracts of the number of tissues. Extracts of parotid gland, brain and kidney contained high amounts of "free" p-gamma globulin (11 kdaltons). Two higher molecular mass forms (34k and 27 kdaltons) of this protein were demonstrated in the kidney and brain extracts. Extracts of ovary and uterus were devoid of the 27 k moiety and kidney extracts lacked the "free" p-gamma globulin (11k). These studies indicate that p-gamma globulin may be a part of a much larger molecule or that p-gamma globulin may be associated with another yet unknown protein. These findings may be important for clinical and pathological studies in human kidney and central nervous system (CNS) diseases.

beta 2-microglogulin levels in cancerous and other disease states.

Serum beta2-microglobulin levels were measured, by radioimmunoassay, in patients suffering from a variety of benign and malignant clinical disorders. Elevated beta 2-microglobulin values were found in neoplastic and non-neoplastic disorders affecting a variety of organs. The most striking increases in beta 2-microglobulin are found in the plasma cell dyscrazias and several solid tumors, particularly those affecting the lung. Lymphoid neoplasms demonstrate a spectrum of changes of serum beta 2-microglobulin. At the one end of this spectrum were the plasma cell tumors, which show a high incidence of raised beta 2-microglobulin levels, while patients with Hodgkin's disease rarely show such increases in circulating beta 2-microglobulin.

Post-gamma globulin. II. Radioimmunoassay determination of levels of post-gamma globulin and beta 2-microglobulin.

Post-gamma globulin previously isolated and partially sequenced in this laboratory was used for production of polyclonal and monoclonal (hybridoma) antibodies. A radioimmunoassay method was developed for quantitation of post-gamma globulin with either antibody. The titration curves obtained were treated statistically and found practically indistinguishable. The sensitivity of the method adopted for the quantitation of post-gamma globulin in a variety of biological fluids was 0.13 ng/ml and the upper limit of precision was 2.5 ng/ml. The following results were obtained (mean +/- 1 SD): normal sera, 0.96 +/- 0.20 microgram/ml; pregnancy sera, 1.08 +/- 0.28 micrograms/ml; cord blood 2.08 +/- 0.33 micrograms/ml; hospitalized patient's sera, 1.3 +/- 0.54 micrograms/ml; geriatric subjects' sera 2.26 +/- 1.10 micrograms/ml; cerebrospinal fluid, 5.37 +/- 3.36 micrograms/ml; saliva, 1.22 +/- 0.67 micrograms/ml; synovial fluid, 1.27 +/- 0.41 micrograms/ml and urine, 0.11 +/- 0.125 microgram/ml. To shed light on the catabolism of post-gamma globulin the levels of beta 2-microglobulin were also measured radiometrically. Correlative statistical analysis of all the data have shown that renal handling of post-gamma globulin and beta 2-microglobulin may be very similar but not necessarily identical.

Red cell hypoplasia and monoclonal gammopathy in a patient with lymphoproliferative disorder.

A 69-year-old male was observed to have red cell hypoplasia. Two years later monoclonal gammopathy IgG-I, K, Gma+ and InV (1-) was documented in this patient. Persistent lymphocytosis, abnormal response to phytohemagglutinin, and at autopsy multiple lymphoid nodules occurring in the bone marrow, suggestive of lymphoproliferative disorder, were observed. A review of the literature indicates that this clinical presentation is rare. The possibility that red cell aplasia may be associated with lymphoproliferative disorder in some instances must be considered inasmuch as this may have an important bearing in the management of such patients.