The class of surface immunoglobulin on virgin and memory B lymphocytes.

The class of surface immunoglobulin receptors for antigen on B cell precursors of different classes of antibody-forming cells was determined by utilizing a technique of class-specific antigen suicide. Spleen cells are first treated with a class-specific antiserum under conditions that result in the stripping of that class from the cell surface. The cells are then permitted to bind a highly radioactive trinitrophenyl (TNP)-conjugated protein, which leads to lethal irradiation of all TNP-specific B cells except those whose TNP receptors had been removed by the class-specific stripping of surface immunoglobulin. In this way, the class of antibody-forming cells resulting from TNP stimulation of B cells with different classes of surface immunoglobulin can be examined. It was found that the virgin B cell precursors of IgM-producing cells are two types: cells bearing IgM receptors only and those bearing both IgM and IgD receptors. All virgin B cells that gave rise to IgG1 antibody-forming cells had both IgM and IgD on their surfaces, demonstrating that an antigen-dependent switch from IgM and IgD to IgG1 production is a common feature of B cell maturation. In contrast, memory B cell precursors of IgG1 antibody-forming cells had predominantly IgG1 as their surface antigen receptor. The implications of these findings on current models of B cell maturation are analyzed.     

Cell surface immunoglobulin XXI. appearance of IgD on murine lymphocytes during differentiation.

The differentiation of Ig-bearing lymphocytes in adult mice was studied by monitoring the appearance of IgD relative to IgM on the surface of splenocytes obtained from lethally irradiated animals reconstituted for various periods of time with adult bone marrow cells, neonatal splenocytes, or Ig- adult splenocytes. It was found that IgM appears before IgD on differentiating lymphocytes. Furthermore, the rate of appearance of IgD during differentiation of adult cells is similar to that observed with neonatal cells.     

C-reactive protein antigenicity on the surface of human peripheral blood lymphocytes. Characterization of lymphocytes reactive with anti-neo-CRP

Previously, we have shown that antibodies specific for C-reactive protein determinants, not present on the native molecule, termed neo-CRP, also react with a significant percentage of PBL. In the present study, cells were evaluated by flow cytometry using alpha-neo-CRP antisera and mAb specific for lymphocyte subsets. With use of either monocyte-depleted PBL or Percoll-enriched large granular lymphocytes, we observed an overlap between cells reactive with alpha-neo-CRP and cells bearing the surface markers CD16, CD11b, Leu-7, and/or Leu-19, which are expressed on NK cells. In addition, we showed co-expression of the neo-CRP antigen with CD19, CD20, and HLA-DR, cell surface markers which are expressed on B lymphocytes. The major proportion of CD3+ cells failed to exhibit co-expression of neo-CRP. Single parameter flow cytometric analyses demonstrated that cells reactive with alpha-neo-CRP exhibited a bimodal staining pattern based on fluorescence intensity: high intensity neo-CRPbright and low intensity neo-CRPdim. Two-color analysis revealed that neo-CRPbright cells co-expressed CD19, CD20, and HLA-DR, whereas neo-CRPdim cells co-expressed CD16, CD11b, Leu-7, and Leu-19. Anti-neo-CRP also reacted with PBL obtained from patients with CD16+ lymphoproliferative disorders and from patients with chronic lymphocytic leukemia of B cell origin, but not with cells from patients with T cell or myeloid leukemias. The alpha-neo-CRP cells from patients with NK cell expansions showed dim fluorescence, whereas patients with B cell expansions showed bright fluorescence, consistent with the staining patterns observed with normal PBL. In addition, cell lines of T cell, B cell, NK cell, myeloid, and erythroid origin were evaluated for reactivity with alpha-neo-CRP. The cloned NK cell line NK 3.3 reacted as neo-CRPdim, but the B cell lines BL41, BL41/95, T1, T2, and CESS all reacted as neo-CRPbright. The cell lines K562, Molt-4, Hut-78, HL-60, U-937, and THP-1-0, which lack characteristic NK and B cell markers, did not react with alpha-neo-CRP. Additional study of the two-color histograms revealed a distinct diagonal staining pattern that was observed only when cells were co-stained with alpha-neo-CRP and either alpha-CD16 (alpha-Fc gamma RIII) or antibody IV3 (alpha-CDw32; alpha-Fc gamma RII). This finding suggests a 1:1 relationship between Fc gamma R on both NK and B cells and determinants recognized by alpha-neo-CRP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of immunoglobulin determinants on the surface of Xenopus laevis splenic lymphocytes.

The distribution of surface immunoglobulin (Ig) determinants on Xenopus laevis splenic lymphocytes after combination with divalent rabbit anti-Ig coupled to ferritin was studied. The electron micrographs showed the presence of immune complexes in 67% of lymphocytes treated at 0 degrees C-4 degrees C. The complexes were located all around the membrane and uniformly distributed in a random fashion. The variation of ferritin grain counts on cell sections is such, that the existence of two major subclasses of Ig-positive cells may be suggested. Raising the temperature produced a rapid interiorization of the complexes in vesicles without any previous aggregation to form a "cap" having occurred.     

Effects of morphine on the phenotypic expression of cell surface markers on murine lymphocytes.

There is a growing literature indicating that opioid abuse by human addicts and opioid administration to animals have profound effects on the immune system. In the present study, implantation of morphine pellets in mice was associated with reduced phenotypic expression of the cell surface antigens specific to T-lymphocytes and to helper and cytotoxic/suppressor T-lymphocyte subtypes. The effect of morphine, as measured by flow cytometry using monoclonal antibodies specific for antigens expressed by these cells, was dose-dependent. The decrease in expression of antigens was apparent as early as 24 h after morphine pellet implantation and continued for 3 days. In addition, a time-dependent increase in the expression of these antigens was observed in placebo- and morphine-treated mice, suggesting that the pellets had a small antigenic effect. However, at all times studied, morphine-treated mice had fewer cells expressing the antigens than placebo-treated mice. Our results provide additional evidence that the use of opioids by IV drug abusers compromises their immune function.     

T-independent but not T-dependent antigens maintain surface Ig of lymphocytes.

The effect of T-independent (TIA) and T-dependent (IDA) antigens on the surface Ig of 24-hr cultured rabbit spleen cells was investigated by two techniques: the proportion of cells bearing surface Ig was determined by direct rosette formation with anti-light chain allotype-coated erythrocytes; the total amount of surface Ig was estimated by labeling the cells with anti-allotype ¹²⁵I-labeled Fab fragments. The addition of TIA resulted in the maintenance of the proportion of Ig-bearing cells almost to the initial level, an effect which could not be obtained with any of the TDA tested. The same type of effect was observed when the total amount of surface Ig was measured, i.e., there was a slight reduction (about 24%) in the amount of surface Ig in cultures to which TIAs were added and an almost sixfold reduction (about 70%) in cultures to which TDA, Con A, or no antigen was added. Some but not all of the TIA were able to induce [³H]TdR incorporation in 3-day spleen-cell cultures. We concluded that the common feature of TIA is the ability to stimulate the turnover of B-cell surface Ig, a feature that can be used for an easy screening of TIA.     

EBV-transformation of surface IgA-positive human lymphocytes

Surface IgA-positive human lymphocytes were selected, separated, and immortalized with Epstein-Barr virus (EBV). Selection was carried out by rosetting the cells with ox erythrocytes, coated with rabbit anti-human IgA immunoglobulin, followed by separation on Ficoll-Isopaque. Stable monoclonal lines were established by cloning in agarose. This shows that EBV-transformation is not restricted to the IgM-positive B cell category but can be extended to minority classes of B lymphocytes. The method provides the means to establish other minority categories as continuous lines, depending on the efficiency of the preselection procedure.     

Beta-adrenoreceptors on the surface membranes of lymphocytes and macrophages]

In experiments in vivo and in vitro on sensitized and intact guinea pigs and Wistar rats the effect of beta-adrenergic stimulants (adrenaline and izoproterenol) and a beta-adrenergic blocker (propranalol) on lymphocytolysis and the reaction of macrophage adherence inhibition was studied. Adrenaline and izoproterenol were shown to inhibit the antigen interaction with both sensitized and intact cells. Restoring the sensitivity of cells to the antigen, propranalol destroys the defensive action of adrenaline and isoproterenol.     

The glycoprotein surface coat on different classes of murine lymphocytes

Both thymus and spleen lymphocytes of mice have been shown to possess a surface coat visible when the cells are stained with ruthenium red. Measurements on high-magnification photographs showed that the coat on thymus lymphocytes is significantly thicker than on spleen lymphocytes from genetically athymic nu/nu mice, which form a pure B cell population. Most of the coat on thymus cells is removed by treatment of the cells with neuraminidase or with trypsin, indicating that the coat is glycoprotein in nature. Functional implications of the difference in the cell coats of thymus and B cells are discussed.     

Occurrence of two fucosyltransferase activities at the outer surface of rat lymphocytes.

To demonstrate the existence of ectofucosyltransferase activities on the outer surface of rat lymphocytes, we measured fucosyltransferase activities on whole cells using procedures enabling us to exclude the possibility of misleading results due to precursor hydrolysis and intracellular utilization of the free fucose, and to take into account the contamination by intracellular enzymes freed by the small percentage of broken cells. The described ectofucosyltransferases are able to catalyze the transfer of fucosyl residues from GDP-fucose to the endogenous membrane acceptors but the transfer activity towards exogenous acceptors is restricted to low molecular weight compounds. Use of galactose and di-N-acetylchitobiose as exogenous acceptors and concomitant study of the specific inhibition by N-ethylmaleimide enabled us to detect both types of ectofucosyltransferases: a GDP-fucose: galactoside ectofucosyltransferase and a GDP-fucose: N-acetylglucosaminide ectofucosyltransferase.     

CD38 is expressed as noncovalently associated homodimers on the surface of murine B lymphocytes.

CD38 is a transmembrane glycoprotein that functions as an ectoenzyme and as a receptor. Based on the structural similarity between CD38 and ADP-ribosyl cyclase from Aplysia californica, it was hypothesized that CD38 is expressed as a homodimer on the surface of cells. Indeed, CD38 dimers have been reported, however, the structural requirements for their stabilization on the plasma membrane are unknown. We demonstrate that the majority of CD38 is assembled as noncovalently associated homodimers on the surface of B cells. Analysis of CD38 mutants, expressed in Ba/F3 cells, revealed that truncation of the cytoplasmic region or mutation of a single amino acid within the alpha1-helix of CD38 decreased the stability of the CD38 homodimers when solubilized in detergent. Cells expressing the unstable CD38 homodimers had diminished expression of CD38 on the plasma membrane and the half-lives of these CD38 mutant proteins on the plasma membrane were significantly reduced. Together, these results show that CD38 is expressed as noncovalently associated homodimers on the surface of murine B cells and suggest that appropriate assembly of CD38 homodimers may play an important role in stabilizing CD38 on the plasma membrane of B cells.     

Membrane-associated interleukin 2 epitopes on the surface of human T lymphocytes

Analysis of surface fluorescence with flow cytometry has revealed the presence of membrane-associated interleukin 2 (IL-2) epitopes on the surface of long term human T cell clones. These IL-2 epitopes could not be accounted for by soluble IL-2 binding to its specific receptor or adsorbing nonspecifically to the cells. The level of surface IL-2 antigenic determinants on the T cell clones was decreased in the presence of phorbol esters and increased in the absence of an exogenous source of IL-2. It was completely lost upon stimulation of the clones to produce the soluble lymphokine. Surface IL-2 epitopes were also detected on the Jurkat tumor cell line which secretes IL-2 upon stimulation and on another T cell tumor line MOLT 4. MLA-144 produces IL-2 constitutively; however, it did not possess membrane-associated epitopes. Tumor lines of other lineages were negative. A subpopulation of peripheral blood T lymphocytes demonstrated some membrane-bound IL-2, whereas non-T peripheral blood mononuclear cells were negative. Thus, cells with the potential of producing and secreting IL-2 upon stimulation possessed the surface epitopes of the lymphokine and cells either actively secreting IL-2 or without the potential for secretion were negative for surface expression. Membrane-associated IL-2 antigenic determinants appear to represent a T lymphocytic surface marker of potential cellular function. The relationship of this marker to the secreted lymphokine is not known. Although it is possible that the epitopes seen were present on a distinct molecule independent of secreted IL-2, the distribution on a variety of T cells and regulation via cellular activation suggest that the surface expression of IL-2 epitopes is in some way related to the soluble lymphokine.