Daniel Levitt

Human B-lymphocyte subpopulations: I. Differentiation of density-separated B lymphocytes☆

Abstract Human peripheral blood mononuclear cells, B-cell-enriched populations, and T cells were separated according to their buoyant densities using discontinuous gradients of Percoll. Lymphocyte subpopulations were stimulated with various mitogens and polyclonal activators. Low-density T-cell fractions responded better than total T cells or high-density fractions when stimulated with phytohemagglutinin, concanavalin A, and pokeweed mitogen. A low-density B-cell fraction differentiated better than other fractions after stimulation with PWM and total T cells, while high-density B cells (with or without T cells) gave the greatest plasma cell response when treated with lipopolysaccharide. Differential contamination of individual fractions by monocytes do not explain these findings. Thus, both T- and B-lymphocyte subpopulations can be separated by their buoyant densities into subpopulations displaying distinct reactivities toward mitogens and polyclonal activators.

A radioimmune study of the effect of bromodeoxyuridine on the synthesis of proteoglycan by differentiating limb bud cultures

Abstract A radioimmune assay has been developed for the quantitative determination and the qualitative identification of core protein of cartilage chondroitin sulfate proteoglycan. Utilizing this method it has been shown that during differentiation of chick limb bud mesenchyme to cartilage, there is a marked augmentation of synthesis of core protein. Treatment with 5-bromo-2′-deoxyuridine results in an irreversible inhibition of synthesis of cartilage-specific chondroitin sulfate proteoglycan.

Immunoglobulin Isotype Expression of Normal Pre-B Cells as Determined by Immunofluorescence

We have undertaken an immunofluorescent analysis of the immunoglobulin heavy and light chains expressed by pre-B cells from normal human fetal and adult bone marrow using purified mouse monoclonal and goat antibodies to human immunoglobulin isotypes. Our results indicate that (i) the great majority of normal pre-B cells in both fetuses and adults expresses intracytoplasmic μ chains only, (ii) immunoglobulin light-chain synthesis may be initiated during a late stage in pre-B-cell development, and (iii) heavy-chain isotype switching at the pre-B-cell stage may not occur during normal B-cell development.

Epstein-Barr virus-induced lymphoblastoid cell lines derived from the peripheral blood of patients with X-linked agammaglobulinemia can secrete IgM.

We have established lymphoblastoid cell lines (LCL) from the peripheral blood of three individuals with X-linked agammaglobulinemia as well as three of their immunodeficient first-degree relatives. Lines could be induced with Epstein-Barr virus only when T lymphocytes were depleted from total mononuclear leukocytes. The LCLs derived from XLA patients expressed characteristics of IgM-secreting plasmacytes, including intense cytoplasmic fluorescence after staining with anti-μ, easily detectable amounts of IgM in culture supernatants, and radiolabeled IgM with both heavy and light chains in culture media and cell lysates. The cell lines induced from blood of the first-degree relatives of these patients were more varied. They often exhibited multiple isotypes for both heavy and light chains in different cells or expressed a B-lymphocyte phenotype (easily detectable surface Ig but no Ig secretion). These studies suggest that B-cell precursors are present in peripheral blood of individuals with classical XLA. Differentiation of such cells to mature cells of the B lineage can be accomplished using Epstein-Barr virus after removal of T cells.

Methanol fixation permits flow cytometric analysis of immunofluorescent stained intracellular antigens

Fixation and immunofluorescent staining methods were developed for analyzing intracellular antigens with the cell flow cytometer. Fixing cell suspensions with 100% methanol provided best preservation of morphology, lowest fluorescent background staining and most intense specific immunofluorescence. Immunoglobulins present in B cell lines that were representative of different developmental stages could be distinguished quantitatively. Fluorescence histograms were compared with fluorescence microscope presentation of stained cells. Intracellular antigens that reacted with monoclonal antibodies could also be evaluated by flow cytometry. This method was utilized to assess plasmacyte development in mouse spleen cell cultures after stimulation with lipopolysaccharide.

Effect of 5-bromodeoxyuridine on ultrastructure of developing limb-bud cells in vitro ☆

Abstract Limb-bud cells differentiate into chondrocytes, myocytes, fibrocytes and epithelial cells in vivo. While detailed morphological descriptions of developmental events have been presented, mechanisms governing differentiation of cells in vivo are not yet apparent. Limb-bud cells can be grown in tissue culture and will differentiate into the same cell types as occur in vivo. Chondrogenesis in vitro appears to mimic differentiation in vivo as evaluated both biochemically and ultrastructurally. Incorporation of H235SO4 into glycosaminoglycans increases markedly on the fourth day in culture. At the same time, cells assume the morphology of mature chondrocytes and synthesize extracellular matrix composed of 250–400A proteoglycan granules and poorly cross-banded collagen fibers. Myocytes can be detected as early as 21 hr after initiation of cell cultures and contain giant polysomes and thick and thin filaments. When early embryonic limb-bud cells are treated with 5-bromo-2′-deoxyuridine (BrdUrd), myogenesis is diminished and differentiation into chondrocytes is prevented irreversibly. Incorporation of H235SO4 remains depressed throughout the culture period despite removal of the drug after the second day of growth. Development of organelles associated with synthesis of extracellular material (rough endoplasmic reticulum and Golgi apparatus) is not prevented by treating limb-bud cells with BrdUrd. Extracellular matrix consists primarily of bundles of collagen fibers.

Human B-lymphocyte subpopulations: II. Plasma cell differentiation of isotype-specific B lymphocytes from peripheral blood☆

Abstract Human B lymphocytes were separated into subpopulations according to their surface isotype by rosetting with anti-μ- or anti-α-coated bovine red blood cells. Anti-isotype-separated B cells were cultured with T cells and pokeweed mitogen (PWM); plasma cells and immunoglobulin secretion were quantitated after 7 days in vitro. B cells expressing IgM on their surface differentiated almost exclusively into IgM-producing plasma cells. In B lymphocyte populations depleted of sIgM+ cells, the vast majority of plasma cells produced and secreted IgG or IgA. When sIgA+ B cells were cultured for 7 days with T cells and PWM, IgA plasma cells were predominant. Anti-immunoglobulin antibodies (anti-μ, γ, or α) added to B cells or B-cell subpopulations suppressed plasma cell development in an isotype-specific manner; anti-μ inhibited IgM plasma cell development far greater than it affected IgG or IgA plasma cell differentiation. Similar results were seen with anti-γ and anti-α for IgG or IgA plasma cell development. These results are consistent with the idea that PWM, a T-dependent activator, normally triggers a mature, committed population of B lymphocytes expressing only μ + δ or γ or α on their surface. Such cells would either be in the process of or subsequent to surface isotype switching and therefore posses a mature B-cell phenotype.

Separation of lymphocyte subpopulations using biotin-avidin erythrocyte rosettes

The major method for isolating murine lymphocyte subpopulations involves negative selection using antibody plus complement-mediated cytolysis. We have developed an efficient rosette method for enriching murine B and T cells using biotin-conjugated antibodies and avidin-coated sheep erythrocytes. Rosetted and non-rosetted subpopulations are separated rapidly on Percoll cushions. In systems employing rabbit anti-mouse immunoglobulin or monoclonal rat anti-mouse Thy-1.2 conjugated to biotin, positively-selected cells are greater than 90% pure while negatively-depleted populations possess less than 2% contamination with unwanted cells. Recoveries from starting spleen cell populations range between 50 and 75%. This method provides an easily performed alternative for obtaining positively and negatively selected cell populations and can be used with any biotin-conjugated antibody protein.

Chlamydia trachomatis (L2 serovar) binds to distinct subpopulations of human peripheral blood leukocytes

We have previously shown that infants with pneumonitis caused by Chlamydia trachomatis, an obligate intracellular bacterium, possess increased percentages of B lymphocytes but not T lymphocytes in their peripheral blood. It was then demonstrated that chlamydiae induce proliferation in vitro of human peripheral blood B lymphocytes and, in the presence of T cells, differentiation of B cells to immunoglobulin-secreting cells. In this study, we show that C. trachomatis (L2 serovar) binds preferentially to 50% of human B lymphocytes from peripheral blood but only to a small percentage, if any, of T cells. Both monocytes and granulocytes bind and ingest chlamydiae. Despite chlamydial binding to B cells and ingestion by monocytes, no uptake by B cells and limited growth (fewer than 0.5% inclusion-containing cells) in monocytes occur. There is a dramatic decrease in the percentage of cells associated with the bacteria after culture. These results are the first demonstration of binding of C. trachomatis (L2 serovar) to lymphocytes and represent a direct step toward correlating physical interactions between bacteria and lymphocytes with specific immunostimulatory activities in vitro.

Excessive numbers and activity of peripheral blood B cells in infants with Chlamydia trachomatis pneumonia

The peripheral blood lymphocytes of seven infants who had lower respiratory infections caused by Chlamydia trachomatis (chlamydial pneumonia) were studied for abnormalities that may be related to the hyperimmunoglobulinemia characteristic of this infection. Both proportions and numbers of B cells and plasma cells were strikingly elevated in these infants, as indicted by the percentage of peripheral blood mononuclear cells (PBMC) that reacted with fluorochrome-labeled antibodies to human immunoglobulins. Cells expressing IgM and IgD on their surface, and cells possessing IgM and IgG in their cytoplasm were especially increased above levels found in normal adults, infants, and a group of infants with other infections. Cells from infected infants secreted exceptionally large amounts of IgM, IgG, and IgA when cultured in the absence of added mitogens. These data suggest that chlamydial pneumonia induces substantial B-cell activation during a period of development when antibody responses are normally difficult to stimulate.