Cell Surface IgD Research Articles

Autoantibodies, T, B and Dendritic cell abnormalities in Postural Orthostatic Tachycardia Syndrome (POTS)

Background: Postural Orthostatic Tachycardia Syndrome (POTS) is the most common form of autonomic dysfunction with a heterogeneous pathogenesis and presentation. A small percentage of patients with POTS show evidence for autoantibodies, which suggests a B cell related immune pathology. Aim: We previously showed expansion of the α/β double negative T cells and a decreased HLA-DR-to-CD69 ratio in approximately 15% of a small cohort of POTS patients. We, therefore sought to study peripheral blood T cell subsets and their correlations to autoantibodies in POTS. Methods: We assayed peripheral blood white blood cells of 100 POTS (75 female and 25 male) and 100 age and sex matched controls for cell surface markers that designate subsets of T, B, NK and dendritic cells using flow cytometry. Results: We found a statistically significant increase in total T cells, and un-switched memory B cells as well as an increase in IL-2 receptor expression in T cells (p < 0.01). Serum autoantibodies were present in 5-28% of our POTS cohort. Patients with high levels of AChR binding, and blocking antibodies had a positive relationship with increase in T cells and their double negative subsets (p = 0.0039), whereas patients with ANA showed correlations with B cell surface IgD and CD8 cells (p = 0.0007). Interestingly in patients with anti-thyroglobulin antibodies, a positive correlation was observed only with plasmacytoid dendritic cells (p = 0.0378). Conclusions: There is a distinct correlation between specific autoantibodies and various T, B and dendritic cell subsets suggesting a heterogeneous nature of autoimmune abnormalities in POTS.

Induction and properties of syngeneic murine anti-immunoglobulin D.

High titers of autoantibodies directed to isotypic determinants of IgD were produced by inoculation of syngeneic monoclonal IgD, conjugated covalently to keyhole limpet hemocyanin, into adult or neonatal inbred mice. Anti-idiotypic antibodies were induced at the same time. The average affinity of the mouse antibodies (Ka approximately equal to 10(7) M-1) is similar to that of rabbit anti-IgD and of syngeneic anti-IgE induced by the same procedure. Results indicate that B cells of the mice are not tolerant to serum IgD and that tolerance is maintained at the level of T cells. Direct interaction of the syngeneic anti-IgD with cell-surface IgD was minimal, and there was no convincing evidence that cell-surface IgD was down-regulated in the anti-IgD-producing mice. Further studies, preferably employing monoclonal anti-IgD, are required to determine whether epitopes on cell-surface IgD can be recognized by syngeneic anti-IgD. The ability to generate in vivo high titers of anti-IgD should facilitate the production of such monoclonal antibodies.

Exposure to crosslinked IgD induces receptors for IgD on T cells in vivo and in vitro.

IgD is a surface immunoglobulin, which is coexpressed with IgM on greater than 90% of mature B cells, but its levels in serum are extremely low compared to those of IgM. It role as a surface receptor has been reemphasized by our recent findings that IgD receptors are induced on helper T cells by exposure to IgD and that such cells have immunoaugmenting properties. The present study shows that crosslinking of soluble IgD or of monomeric cell surface IgD is required and sufficient for the induction of T cells bearing receptors for IgD, both in vivo and in vitro. Effective IgD crosslinking in this respect can be obtained with antigen or with heterologous and immunogenic as well as nonimmunogenic allotype-specific anti-IgD. These results reinforce the concept that the induction of T cells bearing receptors for IgD is an integral component of the normal immune response.

Increased expression of the B lymphocyte receptor for transferrin is stimulated by in vivo crosslinking of cell surface IgD

Expression of a receptor for the serum protein transferrin has been shown to be a characteristic of several cell lineages and increased transferrin receptor (TFR) expression to reflect cellular activation. In vitro studies of human B lymphocytes stimulated with antibodies to IgM have demonstrated that these cells have the ability to express TFR and that increased B-cell TFR expression is seen first sometime after these cells enter the G1 phase of the cell cycle. It also has been shown that TFR expression is necessary for proliferation to occur and may be regulated by a factor produced by mitogen-activated T lymphocytes. To examine expression of TFR by activated B lymphocytes in vivo, and to determine the kinetics of induction of TFR expression, we have studied the effects of injecting mice with an affinity-purified goat antibody to mouse IgD (GaMδ) on TFR expression. This antibody previously has been shown to activate polyclonally mouse splenic B cells in vivo in a T-independent fashion. Results show that (i) there is a small but definite quantity of TFR on resting splenocytes, (ii) at 24 hr after injection nearly all B cells but not T cells express increased amounts of TFR, (iii) TFR is increased to nearly the same extent in congenitally athymic BALB/c nu/nu mice as in their normal nu/+ littermates and therefore GaMδ-induced increased B lymphocyte TFR expression is relatively T independent, (iv) TFR expression increases as early as 3 hr after injection of 800 μg of GaMδ and increases steadily until it peaks 24–48 hr later, and (v) TFR expression in GaMδ-injected mice increases concomitantly with surface Ia antigen and cell size.

Comparison of the clonal diversity of the B cell repertoires in adult mice that differ in the expression of cell surface IgD.

To determine whether the expression of surface IgD (sIgD) influences the extent of the expressed B cell repertoire, the clonal diversity of the B cell population in mice treated chronically with anti-IgD (delta) antibodies has been compared with the B cell repertoire observed in control animals, using the splenic focus limiting dilution B cell assay. The results show that the phosphorylcholine (PC)-specific B cell precursor frequency in anti-delta antibody-treated mice is increased when compared with that of control mice. Isotype and idiotype (T15) analyses of PC clonal products from anti-delta antibody-treated and control mice revealed no distributional differences. Analyses of the 2,4-dinitrophenyl (DNP)- and fluoresein isothiocyanate-specific B cell repertoires confirmed that the equal or increased precursor frequencies observed in anti-delta antibody-treated mice are not specific for the PC antigen. The increased precursor frequency of B cells from anti-delta antibody-treated mice was not the result of increased homing of B cells from anti-delta antibody-treated mice to recipient spleens, since B cells from control mice homed twice as well to recipient spleens as did B cells from anti-delta antibody-treated mice. Other studies demonstrated that (a) on average, antibody-secreting clones were generated more slowly when B cells from anti-delta antibody-treated mice were used as a source of precursors than B cells of control mice and (b) both sIg- spleen cells and sIg+ spleen cells from anti-delta antibody-treated mice generated a higher frequency of specific antibody-secreting clones than did the corresponding populations from control mice. These observations suggest that a population of sIgM+sIgD- B cells exists that resembles sIgD+ B cells rather than neonatal or xid B cells in its ability to generate responses to PC and suggests that the sIgM+sIgD- B cells from anti-delta antibody-treated mice are more responsive than are sIgM+IgD+ B cells, regardless of antigenic specificity, to the stimuli provided in the splenic focus system. Finally, this study suggests that the expression of sIgD does not influence the extent of the expressed B cell repertoire.

Physiology of IgD. VI. Transfer of the immunoaugmenting effect of IgD with T delta-containing helper cell populations.

We show that the IgD-induced augmentation of the immune response to trinitrophenylated keyhole limpet hemocyanin can be transferred to syngeneic mice with spleen cells from IgD-injected donors. The augmenting activity is present in the Lyt-1+2-, L3T4+ T cell population and is absent from B cells. The ability of transferred T cells to augment the immune response correlates with the presence of a high frequency of Lyt-1+2- T cells that form rosettes with IgD-coated sheep erythrocytes (T delta cells). Such rosette-forming cells can also be induced by incubation of spleen cells from normal donors in IgD-coated petri dishes. Injection of normal spleen cells exposed to IgD-coated petri dishes together with antigen also augments the immune response of recipients. The existence of a regulatory circuit based upon interactions between T delta cells, antigen, B cell surface IgD, and serum IgD, is proposed.

Expansion and cell surface Ig isotype switching in the antigen-binding cell population of [formula omitted] mice and [formula omitted] littermates

Swiss-Webster nu nu splenocytes placed in modified Mishell-Dutton culture containing sheep red blood cells (SRC) generated increased numbers of antigen-binding cells (ABC) compared with antigen-free cultures. In constrast Balb/c nu nu cultures did not expand their ABC population in response to SRC, suggesting that strain background may influence the effect of the nu nu gene on T-dependent immune responses. Cell surface Ig isotype analysis indicated that the SRC-induced expanded ABC population exhibited a significant decrease in cell surface IgD and a significant increase in ABC bearing both IgM and IgG. The Swiss-Webster nu/+ littermate cell surface Ig isotype patterns were generally similar to the nu nu ABC patterns, but with different kinetics.

Regulation of IgM and IgD synthesis in B lymphocytes. II. Translational and post-translational events.

Studies investigating the relative rates of biosynthesis of mu- and delta-polypeptide chains in normal resting B lymphocytes have shown that the translation rate of mu m is about sevenfold higher than that of delta m, thus reflecting the relative abundance of mRNA encoding these two chains. The turnover rate of cell surface IgM is faster, however, than that of cell surface IgD, resulting in higher expression of cell surface IgD relative to IgM under steady state conditions. LPS stimulation of B lymphocytes induces the complete cessation of synthesis of the delta-chain, thus accounting for the gradual disappearance of IgD from the cell surface of activated cells.

The reactivities of Bauhinia purpurea and Lens culinaris lectins to mouse B Lymphocytes and their subsets.

The use of lectins for the separation of murine spleen lymphocyte subsets was examined. Bauhinia purpurea agglutinin was found to be effective for the enrichment of B cells by differential agglutination. When the differential agglutination technique using lectins was applied to the separation of B cell subsets, Lens culinaris agglutinin was found to be able to fractionate B cells into high responding and low responding subsets for dextran sulfate. A different expression of cell surface IgD was also observed between the cell populations showing different agglutinability with Lens culinaris agglutinin.

Polyclonal activation of the murine immune system by an antibody to IgD. II. Generation of polyclonal antibody production and cells with surface IgG.

Abstract : In the accompanying paper we shoed that the injection of BALB/c mice with 800 mu g of GaM delta induced a direct polyclonal increase in B cell proliferation as well as an indirect increase in T cell proliferation. This led us to investigate whether the same treatment might lead to polyclonal Ig secretion. We found that 6 to 7 days after GaM delta injection one-quarter to one-half of splenic B lymphocytes lost detectable surface IgM and acquired surface IgG. Surface labeling experiments established that much of this IgG had the electrophoretic characteristics of membrane rather than serum IgG, and was, therefore intrinsic rather than cytophilic. GaM delta also induced a striking increase in Ig secretion 6 to 7 days after injection, as indicated by 1) an approximately eightfold increase in serum IgG1 levels and smaller increases in serum IgM and IgG2a levels; 2) greater than fivefold and 50-fold increases in the in vitro incorporation of 3H-leucine into IgM and IgG, respectively, by spleen cells from GaM delta-injected mice; and 3) three to 10-fold and 20 to 50-fold increases in the percentages of spleen cells with large amounts of intracytoplasmic IgM and IgG, respectively. The great majority of the increase in cell surface and secreted IgG was accounted for by an increase in the IgG1 subclass. Both absorption and plaquing studies indicated that the increase in Ig secretion and plaquing studies indicated that the increase in Ig secretion was polyclonal rather than a specific immune response to goat Ig. The injection of anti-delta antibodies failed to induce B cells from congenitally athymic mice or mice that were tolerant to the injected anti-delta antibody to undergo a switch in cell surface isotype or to differentiate into antibody-secreting cells. We interpret these data and data presented in the companion paper as suggesting the binding to and crosslinking of B cell surface IgD by ligand leads to 1) direct activation of B lymphocytes that can include...

Circulating autoantibodies to IgD in rheumatic diseases.

Most mature B lymphocytes possess IgD on their cell surface. Antibodies to IgD produce an adjuvant-like effect in many experimental systems. Employing sensitive class-specific radioimmunoassays, elevated concentrations of autoantibodies to IgD of the IgA, IgM, and IgG classes were detected in the sera of 55% of patients with adult onset rheumatoid arthritis (RA), of 45% with systemic lupus erythematosus (SLE), of 67% with mixed connective tissue disease syndrome (MCTD), and of only 8% of juvenile RA. Marked differences in the class of anti-IgD were noted. An elevated IgA anti-IgD was detected in 45% (p less than 0.01) of RA sera, 27% of SLE sera, and 58% (p less than 0.01) of MCTD sera. IgG anti-IgD was increased in only 26% of RA, 32% of SLE, and 8% of MCTD sera. IgM anti-IgD was elevated in only one patient. IgA anti-IgD was inhibited greater than 90% by IgD but not at all by IgG. Correlations between the concentration of immunoglobulin G and IgA anti-IgD were noted in RA (rs = 0.37, p = 0.02), SLE (rs = 0.46, p = 0.015), and MCTD (rs = 0.4). These data suggest a potential role for autoantibodies to cell surface IgD as modulators of the immune system in some patients with autoimmune disorders.

Tryptic and plasmic cleavage of a rat myeloma IgD

Rat cell surface immunoglobulins were analysed in sodium dodecyl sulfate polyacrylamide gels. By specific immunoprecipitation a 200,000 dalton IgM and a 165,000 dalton IgD were demonstrated. A molecule similar to the mouse and rabbit Fc receptors was non-specifically precipitated. The digestion of a rat myeloma IgD with trypsin or plasmin was studied. Although the IgD molecule was rapidly converted in Fab and Fc fragments by trypsin (1 min., 0°C) it was relatively resistant to plasmin. (Several hours at 37°C were necessary to obtain a complete conversion.) During the plasmin digestion a Facb fragment was observed. These results are discussed in regard to a possible physiological cleavage of cell surface IgD.

Murine cell surface immunoglobulin: two native IgD structures.

Murine spleen cell surface IgD is found in two forms upon SDS-polyacrylamide gel electrophoresis under nonreducing conditions. Both IgDI and IgDII are present on the surface of intact cells and appear to be native structures. Neither form could be accounted for by proteolytic degradation or disulfide bond rearrangement. IgDII has an apparent m.w. of 96,000, suggesting an HL structure. IgDI has an apparent m.w. of 150,000, approximately 33,000 daltons lower than that expected for an H2L2 structure. Significant variation in the relative amounts of IgDI and IgDII was found when mice of different strains were examined.

Acquisition of cell surface IgD after in vitro culture of neoplastic B cells from the murine tumor BCL1.

Murine BCL1 tumor cells bear large amounts of surface IgM and trace amounts of surface IgD. In the present studies we have shown that cultivation of these cells, in the absence of lipopolysaccharide, results in the acquisition of IgD by virtually all the cells. These results suggest that BCL1 cells can differentiate in vitro into more mature B cells and offer an attractive model for analyzing the factors controlling appearance of IgD on a monoclonal cell line.

Murine Cell Surface Immunoglobulin: Two Forms of δ-Heavy Chain

Abstract The heavy chain of isolated murine cell surface IgD is present in two forms, separable by electrophoresis on SDS-polyacrylamide gradient gels. Both forms of δ-heavy chain are present on the surface of intact spleen cells and have apparent m.w. of approximately 70,000 (δ1) and 68,000 (δ2). Treatment of surface IgD with neuraminidase before isolation results in a single IgD heavy chain band on SDS gels having an apparent m.w. of 65,000, indicating that δ1 and δ2 differ in sialic acid content. Delta2 is removed from the cell surface by papain more readily than δ1, suggesting a possible functional significance for the two forms.

Demonstration of Mouse Serum IgD

Abstract Although the mouse would provide an excellent experimental model for the study of serum IgD function, previous investigations have failed to detect serum IgD in this species. We have developed an immunofluorescence inhibition assay that has allowed us to identify and quantitate mouse serum IgD. Mouse sera or serum fractions were assayed for their abilities to inhibit staining of mouse spleen cells with an allotype-specific fluorescein isothiocyanate-labeled rabbit antibody against mouse cell-surface IgD (FITC-RaMδa), as analyzed with a fluorescence-activated cell sorter. FITC-RaMδa stains surface IgD+ spleen cells of mouse strains that have surface(s) sIgD of the a allotype, such as BALB/c, but not strains that have surface IgD of the b allotype, such as C57BL/6 or C.B20, which have the C57BL/6 CH genome on a BALB/c background. The staining of BALB/c lymphocytes by FITC-RaMδa was inhibited by BALB/c serum, but not by C57BL/6 or C.B20 serum. The staining was also not inhibited by BALB/c sera that had been adsorbed with rabbit anti-mouse Ig or a mouse hybridoma protein anti-mouse δ, or by BALB/c IgG, IgM, or IgA plasmacytoma proteins. These data strongly suggest that mice have IgD in their serum. Mouse serum IgD is probably secreted rather than shed from B cells, since amounts of serum IgD and splenic IgD in the same mice did not correlate with each other and serum IgD and shed sIgD had different density profiles.

B-cell tolerance. III. Effect of papain-mediated cleavage of cell surface IgD on tolerance susceptibility of murine B cells.

Under defined conditions, papain removes IgD from cells while leaving IgM, H-2, Ia, Lyb-2, and complement receptor intact. The effect of such treatment with papain on the induction of tolerance in murine splenic B cells was determined in an in vitro system. Treatment of the cells with papain has no effect on subsequent antibody responsiveness presumably because surface receptors regenerate before and during incubation with immunogen. Removal of increasing amounts of IgD results in increasing susceptibility of thymus-dependent responsive cells to tolerance induction. The tolerance susceptibility of thymus-independent responsive cells, which we have previously suggested are immature cells that bear only IgM, is unaffected by cleavage of IgD. If cells are incubated for 24 h after treatment with papain, cell surface IgD and tolerance resistance return. These results indicate that a surface molecule affects susceptibility of B cells to induction of tolerance and suggest that this molecule may be IgD.

Mouse immunoglobulin receptors on lymphocytes: identification of IgM and IgD molecules by tryptic cleavage and a postulated role for cell surface IgD

The two mouse immunoglobulin receptors on lymphocytes (IgM and IgD-like) were individually digested by trypsin. The tryptic susceptibility, and the products released, were similar to those of their human counterparts. Evidence for a structural homology between human IgD and its presumed mouse conterpart has been provided by the ramarkably similar profile of fragments resulting from digestion. More definitive homology awaits sequence determination. The extreme susceptibility of surface IgD to proteolysis contrasted with the resistance of surface IgM. We therefore propose that the major role of IgD is to release a fragment (Fabdelata) following exposure to antigen and then elicit a regulatory anti-idiotype response which acts through recognition of the protease-resistant IgM idiotype remaining on the cell surface.

Structure and Biological Function of Human IgD

Human peripheral lymphocytes were stimulated to incorporate tritiated thymidine when cultured with anti-sigma. The stimulation of lymphocytes by anti-sigma inversely correlates to PHA-induced lymphocyte transformation. In addition, lymphocytes from individuals with low serum IgD levels exhibited a significant response to anti-sigma, whereas, those with normal or slightly elevated levels of serum IgD showed minimal stimulation. This study is the first to provide evidence that cell surface IgD may regulate metabolic functions of lymphocytes and is consistent with the idea that IgD is a 'triggering' receptor.