Plasmacytomas and murine immunoglobulins.
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Animals↗
Biomedical subjects
F D Finkelman
Publications and source records attributed to F D Finkelman.
B cells that simultaneously express surface IgM and IgE in Nippostrongylus brasiliensis-infected SJA/9 mice do not provide evidence for isotype switching without gene deletion.
Recently, it has been reported that in SJA/9 mice infected with Nippostrongylus brasiliensis there are increased numbers of lymphoid cells positive for surface IgM and IgE (sIgM+ and sIgE+) even though they fail to secrete IgE, that both the sIgM and sIgE on these cells are intrinsic, and that there has been no deletion of genes for the Ig heavy chain constant region in these cells. These observations support a nondeletional model for Ig isotype switching. We have now reexamined the nature of sIgE on sIgE+ spleen and mesenteric lymph node cells of N. brasiliensis-infected SJA/9 mice, and the following observations lead us to believe that this sIgE is cytophilic rather than intrinsic: (i) Only approximately 50% of the N. brasiliensis-infected SJA/9 mice have detectable percentages of sIgE+ lymphoid cells. All mice with detectable sIgE+ lymphocytes have lymphocytes positive for intracytoplasmic IgE (cIgE+) and secrete IgE in vitro, while cIgE+ cells and IgE secretion are absent from N. brasiliensis-infected SJA/9 mice that lack sIgE+ cells. (ii) SJA/9 B lymphocytes have receptors for IgE: expression of these receptors is increased in N. brasiliensis-infected mice that have sIgE+ lymphocytes, but not in infected SJA/9 mice that lack sIgE+ lymphocytes. (iii) Treatment of sIgM+ sIgD+ sIgE+ cells for 1 min with dilute acid removes most sIgE but does not affect expression of sIgM or sIgD. (iv) The removal of mouse IgE from sIgE+ B cells facilitates the binding of exogenous rat IgE. (v) The small amount of sIgE that is reexpressed during a period of in vitro culture after acid treatment is blocked by inclusion of exogenous rat IgE in the culture medium. These observations show that most sIgM+ sIgE+ B cells in N. brasiliensis-infected SJA/9 mice do not express intrinsic sIgE; thus studies using these cells to determine mechanisms of Ig isotype switching are inconclusive.
Animals↗
Affinity-purified interleukin 2 induces proliferation of large but not small B cells.
Immunoaffinity-purified interleukin 2 (IL2) stimulated proliferation of large but not small B cells. Stimulation was observed even when B cells were cultured at very low cell densities (3 X 10(4) per microwell containing 0.2 ml of medium). Addition of small numbers of purified splenic T cells did not enhance the IL2-induced B-cell proliferative response. These results suggest that IL2 was not operating through contaminating T cells. B cells cultured with anti-Ig antibody in vitro showed enhanced proliferation when cultured with EL4 thymoma-derived B-cell growth factor but not when cultured with IL2. A direct role for IL2 in B-cell activation is discussed.
Animals↗
Preparation and characterization of monoclonal antibodies to the trichothecene mycotoxin T-2.
Two mouse immunoglobulin G1 monoclonal antibodies that bind to the trichothecene mycotoxin T-2 were prepared. These antibodies, designated 12C12 and 15H6, had affinities for T-2 of 3.5 X 10(6) and 5.8 X 10(7) liters/mol, respectively. A competitive inhibition enzyme immunoassay that employed these antibodies had a sensitivity for T-2 of 50 ng per assay. Both antibodies bound to the metabolite HT-2 but not to the related trichothecenes monoacetoxyscirpenol, diacetoxyscirpenol, deoxynivalenol, and deoxyverrucarol. Evidence is presented that T-2-protein conjugates inhibit protein synthesis in lymphoid cells and that this apparent immunotoxicity may be due to the release of T-2 from the protein carrier.
Animals↗
T cell-derived B cell growth factor(s) can induce stimulation of both resting and activated B cells.
The effects of a preparation containing partially purified, EL4-derived B cell growth factor(s) (BCGF) on B cell growth and proliferation have been examined by using B lymphocyte subpopulations separated on the basis of size. BCGF was found to maintain and enhance proliferation of a significant proportion of large activated B cells. In contrast, small resting B cells required the presence of BCGF and a second stimulus such as anti-IgM antibody (anti-mu) to be induced to proliferate. This disparity was not due to a lack of an effect of BCGF on small resting B cells. A factor contained within the partially purified EL4 supernatant produced time-dependent increases in cell size and RNA content in all subpopulations. These effects were independent of possible effects due to contaminating lymphokines such as interleukin 2 (IL 2), concanavalin A (Con A), and phorbol myristate acetate (PMA). Nonmitogenic doses of lipopolysaccharide (LPS) failed to show similar effects. Our data suggest that B cells at all levels of in vivo activation are responsive to stimulation by a growth factor present in EL4 supernatant, as manifested by cell growth and RNA synthesis. This activity has not previously been described for BCGF preparations. However, because the partially purified, EL4-derived supernatant used as BCGF in these studies has not been purified to homogeneity, we cannot conclude whether the factors that induce resting B cells to increase in size are the same as the growth factors that synergize with anti-mu to induce B cell proliferation or that maintain the proliferation of activated B cells.
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Mouse IgD half molecules with shortened IgD heavy chain result from alterations within C delta locus.
An unusually small (51 KD) IgD myeloma protein was isolated from secretions of TEPC 1017 generation (gen) 24. The delta-chain mRNA and the delta-chain gene in this tumor were compared with those of TEPC 1017 of earlier generations. The gen 24 protein contained one normal-sized kappa-type light chain (21 KD) and one unusually short delta-heavy chain (30 KD). The delta-heavy chain was 15 KD shorter than that of TEPC 1017 of earlier generations, owing to a delta-mRNA (1.15 kb) which was 600 bp shorter than that of TEPC 1017 of earlier generations. TEPC 1017 is a tetraploid tumor, and the gen 24 appears to contain at least two different deletions on different chromosomes. The short mRNA was produced from one of these altered delta-chain genes which had a productive VDJ rearrangement but which had lost the C delta 3 domain and perhaps the C delta H domain as well. Despite these genetic insults, RNA splicing produced delta-mRNA with secreted termini and mRNA with membrane-binding termini. It is suggested that the mouse C delta gene has an unusual predilection for deletions because it normally lacks any vestige of C delta 2 and, during i.p. passage, it suffered further deletions or alterations.
Animals↗
Recombinant interferon-gamma inhibits the B cell proliferative response stimulated by soluble but not by Sepharose-bound anti-immunoglobulin antibody.
Recombinant-derived interferon-gamma (reIFN-gamma) was found to inhibit B cell proliferation that was stimulated by soluble goat anti-mouse IgD or goat anti-mouse IgM antibodies, but not that stimulated by Sepharose-bound anti-Ig antibodies. Recombinant IFN-gamma also inhibited the BSF-1-enhanced soluble anti-Ig B cell proliferation but did not block BSF-1 enhancement of Sepharose anti-Ig-stimulated B cell DNA synthesis. Recombinant IFN-gamma concentrations as low as 0.001 U/ml were effective in suppressing the soluble anti-Ig-stimulated B cell proliferative response, and this inhibitory effect could be partially reversed by co-culture with a hybridoma anti-IFN-gamma antibody. Recombinant IFN-gamma appeared not to inhibit action of resting B cells from G0 to early G1, because it did not inhibit the increases in cell size that were stimulated by anti-delta antibody. However, it was effective in partially suppressing the anti-delta-induced increases in expression of B cell surface Ia. For reIFN-gamma to exert its maximum suppressive effect, it had to be added within the first 7 hr after the onset of culture with anti-Ig. Because reIFN-gamma is a lymphokine that can be detected in vivo, we suggest that it may play a key role in influencing physiologic B cell activation that is induced by antigens or immune complex-mediated cross-linking of surface Ig.
Adjuvants, Immunologic↗
Crohn's disease and systemic lupus erythematosus.
A 28-year-old man with inflammatory bowel disease with complex extraintestinal involvement was found to have diagnostic features of both systemic lupus erythematosus and Crohn's disease. Although some of the systemic complications of these diseases may overlap, both diseases may occur as primary disorders. Coexistence of systemic lupus erythematosus should be considered in patients with inflammatory bowel disease and complex extraintestinal manifestations.
Adult↗
Activation of mouse lymphocytes by anti-immunoglobulin. IV. Stimulation with soluble heterologous anti-delta antibodies.
Mouse spleen cells were stimulated to proliferate in vitro by soluble affinity-purified heterologous antibodies to mouse delta. Antibodies from goat or rabbit antisera to TEPC 1017, a mouse IgD myeloma protein, were purified on an affinity column of TEPC 1033, a second mouse IgD myeloma protein. Maximum uptake of [3H]thymidine in the range of 60,000 cpm was obtained after 48 hr of culture with anti-delta at concentrations of 50 micrograms/ml. In contrast, the hybridoma 10-4.22 anti-delta was nonmitogenic at similar concentrations. The proliferative response was not impaired upon removal of T cells by treatment with an anti-thymocyte serum (ATS), nor by removal of adherent cells by passage of spleen cells over Sephadex G-10 columns and counter-flow centrifugation. Splenic lymphocytes isolated on the fluorescence activated cell sorter (FACS) with intermediate-to-high amounts of surface IgD (sIgD) were responsive to soluble anti-delta, while IgD-negative cells, or cells with low amounts of sIgD, were unresponsive. Spleen cells from mice less than 4 weeks of age, or from mice carrying the X-linked B cell defect (xid), were unresponsive to anti-delta. These results indicate that anti-delta acts similarly to anti-mu in stimulating a proliferative response by later maturing B cells, which are characterized by a high density of sIgD.
Animals↗
Surface immunoglobulin phenotype of complement receptor-positive and complement receptor-negative B cells of normal and xid immunodeficient mice.
Single- and dual-parameter fluorescence-activated cell sorter analyses of complement receptor-positive and -negative (CR+ and CR-) splenic B cells of normal and xid immunodeficient mice were performed to determine the frequencies of surface (s) IgM+ and sIgD+ cells, as well as the quantities of sIgM and sIgD on these cells. Single parameter analysis revealed identical frequencies of sIgM+ and sIgD+ cells in the CR+ B cell population of normal mice. In contrast, the percentage of sIgM+ cells exceeded that of sIgD+ cells in the CR- normal and CR+ xid B cell population to a similar extent. CR- xid B cells showed an even greater discrepancy between sIgM+ and sIgD+ cells. Dual-parameter analysis of CR+ normal B cells showed the typical sIg phenotype of mature B cells, i.e. an intermediate density of sIgM and a high density of sIgD. The CR+ xid B cell population displayed an increase in cells with high sIgM and no-to-low sIgD and a decrease in cells with intermediate sIgM and high sIgD when compared to CR+ normal cells. CR- B cells from normal and xid immunodeficient mice were similar in that both consisted largely of a cell population with low sIgM and no-to-low sIgD. However, the CR- normal B cell population also contained cells with intermediate sIgM and high sIgD, which were lacking in the CR- xid cell population. Similar results were obtained when CR+ and CR- B cells from normal and xid mice were purified from a small resting B cell population, isolated by elutriation. This indicates that the sIg phenotype of CR- cells is not due to the presence of large cells which have lost sIgD during activation.
Animals↗
Polyclonal activation of the murine immune system by an antibody to IgD V. Effect on germinal centers.
Heterologous anti-mouse delta chain has been shown to induce T-independent polyclonal B cell proliferation by 24 h after injection into mice and a T-dependent polyclonal increase in the number of IgG-secreting cells in spleen and lymph nodes 6 days after injection. The effect of anti-delta on germinal center cells has been difficult to determine, however, since anti-delta-induced blast transformation of mantle zone cells makes it difficult to distinguish these cells from germinal center cells. To clarify the effect of anti-delta on germinal centers, we stained lymph node sections from control and anti-delta-injected mice with peanut agglutinin conjugated to horseradish peroxidase which clearly differentiates the germinal center cells from other large and small lymphoid cells. For the first 5 days after i.v. injection of goat anti-mouse delta, the germinal center volumes did not differ significantly from those of mice injected with control antibodies. On days 6 and 7 after injection, the lymph node germinal center volumes of anti-delta-treated mice increased on average over 10-fold as compared to those of mice injected with an equal amount of control antibody. Thus, while anti-delta antibody directly induced blastogenesis of mantle layer B cells, it appears to have little direct effect on germinal center cells. In contrast, rapid germinal center development is seen in anti-delta-injected mice during the T-dependent phase of B cell activation and occurs simultaneously with the appearance of large numbers of IgG-secreting cells.
Animals↗
Immuno affinity purified interleukin 2 (IL2) induces B cell growth.
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Animals↗
Polyclonal activation of the murine immune system by an antibody to IgD. VII. Demonstration of the role of nonantigen-specific T help in in vivo B cell activation.
Previous studies from this laboratory have shown that the injection of mice with an affinity-purified goat antibody to mouse IgD (GaM delta) induces T-independent polyclonal increases in: 1) B cell DNA synthesis, and 2) expression of surface Ia antigen and receptors for T helper factors, 1 to 2 days after injection. In addition, T-independent polyclonal increases in B cell number and IgG1 secretion are observed 6 to 7 days after injection. The administration of normal goat IgG (GIgG) along with GaM delta has been shown to augment GaM delta-induced polyclonal IgG1 secretion. To obtain information about the characteristics of the T help that is required for polyclonal antibody production in this model system, we investigated: 1) the length of the period during which GaM delta must be present to induce day 7 polyclonal antibody production, and 2) the kinetics of the induction of splenic T cell DNA synthesis. We found that GaM delta can be neutralized 3 days after injection by the administration of IgD without decreasing day 7 polyclonal IgG1 secretion, as long as mice are given GIgG at the time that GaM delta is neutralized. In contrast, polyclonal IgG1 secretion is greatly inhibited if GaM delta is neutralized 1 to 2 days after injection or if GaM delta is neutralized 3 days after injection, but GIgG is not administered at this time. Because GIgG can stimulate activated GIgG-specific T cells to secrete helper factors, but, unlike GaM delta cannot focus GIgG-specific T help polyclonally onto B cells, these findings suggest that nonspecific T help, rather than antigen-specific T help, is required in this system after day 3 for the induction of polyclonal IgG1 secretion. Determination of the kinetics of the induction of T cell DNA synthesis in this system by in vivo [3H] thymidine incorporation studies, as well as dual laser fluorescence-activated cell sorter analysis of T and B cell DNA content, indicate that T cells are induced to synthesize DNA 2 days after GaM delta injection and reach plateau rates of DNA synthesis 3 days after injection. Taken together, the GaM delta neutralization experiments and DNA synthesis studies suggest that one reason that GaM delta is required for 3 days in this system is to allow maximal activation of GIgG-specific T cells, which when stimulated later by GIgG secrete nonantigen-specific helper factors that induce GaM delta-activated B cells to secrete IgG1.
Animals↗
Size-dependent B lymphocyte subpopulations: relationship of cell volume to surface phenotype, cell cycle, proliferative response, and requirements for antibody production to TNP-Ficoll and TNP-BA.
Murine splenic B lymphocytes were separated into size-dependent subpopulations by using counterflow centrifugation. Spleen cells were rigorously depleted of T lymphocytes to yield a population of cells that were greater than 90% surface immunoglobulin (Ig)-positive and that had a mean cell volume of 136.6 +/- 3.3 microns. From this population, five fractions of cells were obtained with mean cell volumes that ranged from 115.8 +/- 3.7 microns in fraction 1 to 168.0 +/- 6 microns in fraction 5. The cells in these five subpopulations were characterized by analysis on a fluorescence-activated cell sorter after staining with acridine orange to evaluate RNA and DNA content, and with fluorescein-conjugated anti-mu, anti-delta, and anti-Ia antibodies to evaluate their surface membrane phenotypes. DNA analysis revealed that virtually all of the cells in fractions 1 to 4 had 2 N DNA. Between 7 and 21% of fraction 5 cells were either in S-phase or contained 4 N DNA. In contrast, RNA content increased through the fractions, suggesting a transition from G0 to G1 in the subpopulations with increasing B cell size. As another measure of cell activation seen with increasing cell size, we observed a progressive increase in the expression of surface Ia and a decrease in the expression of surface IgD. In the absence of in vitro stimulation, the larger cells showed significantly higher levels of thymidine incorporation. When polyclonal B cell activators such as LPS or anti-Ig antibody were added, peak proliferative responses were similar in all of the fractions, but the time necessary to achieve a maximal response was shorter for the larger-sized cell subpopulations than it was for the smaller-sized cell subpopulations. Unprimed, size-dependent B lymphocyte subpopulations exhibited spontaneous or "background" antibody formation that occurred primarily in the subpopulations containing the largest cells. T cell factors present in EL4 supernatant enhanced the efficiency of in vitro differentiation of these same subpopulations. When cultured in the absence of T cell help, the thymus-independent type 1 (TI-1) antigen TNP-Brucella abortus (TNP-BA) or the thymus-independent type 2 (TI-2) antigen TNP-Ficoll induced the largest anti-TNP plaque-forming cell (PFC) responses in the fractions containing intermediate-sized cells, suggesting that in vitro, antigen-specific responses came primarily from B cells that have been influenced in vivo to leave their small resting state. The subpopulations containing the smallest size B cells required the presence of both a TI antigen and EL4 supernatant for efficient differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)
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Quantitative analysis of Fc gamma receptors on murine spleen cell populations by using dual parameter flow cytometry.
The expression of Fc gamma R on subsets of mouse spleen cells was examined by dual parameter flow microfluorometry. B cells were detected by labeling them with antibodies against sIgM, sIgD, sIgG, or I-A; essentially all B cells expressed Fc gamma R. The number of Fc gamma R per cell on the sIgD+, sIgM+, and I-A+ cells averaged 2 X 10(4) receptors, and no correlation between the levels of expression of Fc gamma R and the B cell markers was evident. The sIgG+ B cells, however, expressed more Fc gamma R (8 X 10(4) receptors/cell) than sIgM+ and sIgD+ B cells. Fc gamma R on splenic macrophages were examined by double labeling spleen cells for Fc gamma R and Mac-1. The Mac-1+ cells (2 to 16% of the spleen cells) were 100% Fc gamma R+ and expressed threefold to fivefold higher numbers of Fc gamma R per cell than the sIgM+ or sIgD+ B cells. The Fc gamma R on T cells were studied on cells double labeled for Fc gamma R and Thy-1, Lyt-1, or Lyt-2. An average of 20% of the T cells expressed Fc gamma R and at least two subsets of Fc gamma R+ T cells were evident: Lyt-2- cells, most of which expressed intermediate (2 X 10(4) Fc gamma R/cell) levels of Fc gamma R, and Lyt-2+ cells, which expressed mainly high (8 X 10(4) Fc gamma R/cell) amounts of Fc gamma R. The levels of expression of Fc gamma R and sIgM increased dramatically in response to infection and were elevated in mice with genetic defects. We conclude that the level of Fc gamma R expression is a characteristic property of subsets of spleen cells from normal and infected mice.
Animals↗
Characterization of murine lymphocyte IgE receptors by flow microfluorometry.
A flow microfluorometric technique has been developed to analyze IgE receptors on splenic and mesenteric lymph node mononuclear cells from BALB/c mice. Our data show that 1) the binding of DIBADL cross-linked IgE dimers to IgE receptors is specific in that it is inhibited by monomeric rat and mouse IgE but not by mouse or rabbit IgG or by the monoclonal anti-Fc gamma R antibody 2.4G2, and conversely, the binding of DIBADL cross-linked IgG dimers is inhibited by monomeric IgG or 2.4G2 but not by rat or mouse IgE; 2) the binding of IgE dimers is saturable on cells from uninfected and Nippostrongylus brasiliensis (Nb)-infected mice; 3) IgE dimer binding is detectable on most splenic B lymphocytes from uninfected and Nb-infected mice, but not on T lymphocytes from uninfected mice, and on few, if any, T lymphocytes from Nb-infected mice; 4) Nb infection causes a parallel increase in the percentages of B lymphocytes and cells expressing IgE receptors and Fc gamma R; 5) Nb infection leads to a marked increase in B lymphocyte IgE receptor expression, has little if any effect on IgE receptor affinity, and causes only minor changes in Fc gamma R expression; and 6) in vivo activation of B lymphocytes by a goat antibody to mouse IgD decreases IgE receptor expression considerably, but has a minimal effect on Fc gamma R expression. Thus, there are separate receptors for IgE and IgG on murine B lymphocytes, and the effect of Nb infection or anti-IgD treatment on their expression is different.
Affinity Labels↗
Changes in the expression of B cell surface markers on complement receptor-positive and complement receptor-negative B cells induced by phorbol myristate acetate.
The ability of phorbol myristate acetate (PMA) to induce changes in the expression of B cell surface markers on CR- and CR+ B cells from normal mice in an in vitro culture system was examined. The markers studied were CR, sIgM, sIgD, and sIa. CR- B cells acquired the CR after overnight incubation with PMA. A twofold increase in sIa expression on CR- and CR+ B cells was also noted, whereas the staining intensity of sIgM and sIgD decreased on both B cell populations. These changes in the expression of surface markers took place without detectable increases in cell proliferation, cell size, or RNA content. Furthermore, the same effects were observed when CR- and CR+ B cells were prepared from a small B cell population purified by elutriation. It therefore appears that PMA can exert its effect directly on small, resting B cells.
Animals↗
In vivo administration of anti-I-A antibody induces the internalization of B cell surface I-A and I-E without affecting the expression of surface immunoglobulin.
Injection of a hybridoma anti-Ia antibody into adult mice results in a dramatic reduction in the expression of B cell sIa without affecting the expression of sIgD or sIgM. This anti-Ia-mediated modulation of B cell sIa occurs within 3 hr and attains it maximum effect within 18 hr after injection of antibody. There is a rapid reexpression of B cell Ia when such sIa- B cells are cultured in vitro. Culture of B cells in vitro with anti-Ia antibody has no discernible effect on the expression of B cell sIa, nor does it prevent the reexpression of sIa on sIa- B cells obtained from anti-Ia-injected mice. Injection of anti-I-A antibody suppresses the expression of both I-A and of I-E, and similarly, injection of anti-I-E suppresses the expression of B cell I-E and I-A antigens. When fluorescein-labeled monoclonal anti-I-A antibody is injected into mice, a significant fraction of B cell sIa can be demonstrated to be internalized by the B cell. The potential immunologic significance of this phenomena of anti-Ia-mediated modulation of B cell sIa is discussed.
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