Biomedical subjects

C M Snapper

Publications and source records attributed to C M Snapper.

At least 37 records · Page 2Linked to original sources

PubMed1996

B cells lacking RelB are defective in proliferative responses, but undergo normal B cell maturation to Ig secretion and Ig class switching.

A number of distinct functional abnormalities have been observed in B cells derived from p50/ NF-kappa B or c-rel knockout mice. RelB, another member of the NF-kappa B/Rel family of transcription factors, is expressed during the latter stages of B cell maturation and can bind to regulatory sites within the Ig heavy chain locus. Therefore, we tested the ability of B cells from relB knockout mice (relB-/-) to proliferate, undergo maturation to IgM secretion, and switch to the expression of downstream Ig isotypes in response to distinct activators including LPS, anti-CD40 mAb or CD40 ligand, and/or dextran anti-IgD antibodies in combination with various cytokines, including IL-4, IL-5, IFN-gamma, and TGF-beta. B cells lacking RelB showed up to 4-fold reductions in DNA synthesis in response to LPS, CD40, and membrane Ig-dependent activation relative to controls. However, relB-/- B cells were comparable to control B cells in their ability to undergo maturation to IgM secretion and switch to the expression of IgG3, IgG1, IgG2b, IgG2a, IgE, and/or IgA under all activation conditions tested. Thus, RelB, like c-Rel and p50/NF-kappa B, plays a role in B cell proliferation. However, in contrast to c-Rel and p50/ NF-kappa B, it is not critically involved in maturation to Ig secretion or expression of Ig isotypes.

Animals↗

PubMed1996

A model for induction of T cell-independent humoral immunity in response to polysaccharide antigens.

We provide a model for induction of T cell-independent, polysaccharide-specific Ig secretion in response to bacterial challenge. Two predominant pathways are defined that require the concerted action of multivalent membrane Ig cross-linking by the polysaccharide Ag with 1) various B cell-activating moieties contained within the bacterial pathogen and/or 2) cytokines, such as IFN-gamma and granulocyte-macrophage colony-stimulating factor produced by NK cells and macrophages, that become activated in a T cell-independent manner during bacterial infection.

Animals↗

PubMed1996

Regulation of 3' IgH enhancers by a common set of factors, including kappa B-binding proteins.

Targeted disruption of the p50 subunit of NF-kappa B resulted in isotype class switch defects resembling those observed in mice in which the downstream IgH enhancer 3'alpha E(hs1,2) was deleted. We postulated that kappa B binding proteins may regulate class switching by interacting with 3'alpha E(hs1,2) or with other IgH 3' enhancers with which 3'alpha E(hs1,2) synergizes. kappa B binding sites were identified in 3'alpha E(hs1,2) and 3' alpha-hs4, the distal 3' IgH enhancer. A kappa B binding site within 3'alpha E(hs1,2) contributes to at least half the activity of the enhancer in plasma cells, while the same kappa B binding site participates in the complex repression of the enhancer in B cells. In the case of 3'alpha-hs4, a kappa B binding complex activates the enhancer in pre-B, B cells and plasma cells. Additional binding sites within 3'alpha-hs4 for factors known to regulate 3'alpha E(hs1,2), including Oct-1 and BSAP, were identified, and their contribution to 3'alpha-hs4 regulation during B cell development was assessed. Oct-1 positively regulates the enhancer in pre-B and B cells, while BSAP is a repressor in pre-B cells and an activator at the B cell stage. These studies identify kappa B binding proteins as key modulators of 3'alpha E(hs1,2) and 3'alpha-hs4, and suggest coregulation of the two enhancers by a common set of factors.

Animals↗

PubMed1996

B cells from p50/NF-kappa B knockout mice have selective defects in proliferation, differentiation, germ-line CH transcription, and Ig class switching.

To better understand the role of NF-kappaB in normal B cell physiology, we used a purified population of resting B cells from p50/NF-kappa B knockout (p50-/-) mice to determine their ability to proliferate, secrete Ig, express germ-line CHRNA, and undergo Ig isotype switching in vitro in response to a number of distinct stimuli. p50-/- B cells proliferated normally in response to dextran-anti-IgD Abs (alpha delta-dex) and membrane-bound, but not soluble, CD40 ligand (CD40), and they were virtually unresponsive to LPS when compared with control B cells. p50-/- B cells secreted markedly reduced Ig in response to alpha delta-dex or mCD40L in the presence of IL-4 + IL-5, despite their relatively normal proliferative rates, whereas normal Ig secretion was restored by the combination of alpha delta-dex and CD40L. p50-/- B cells expressed normal steady-state levels of germ-line CH gamma 1 and CH alpha RNA but markedly reduced germ-line CH gamma 3 and CH epsilon RNA upon appropriate stimulation. Although p50-/- B cells underwent substantial switching to IgG1, a marked reduction in the switch to IgG3 and IgE, as IgA, was observed. These data are the first to demonstrate key, independent roles for p50/NF-kappaB in normal B cell maturation to Ig secretion, germ-line CH gene activation, and Ig class switching, as well as mitogenesis, and provide a powerful and well-defined in vitro model system for studying the role of p50/NF-kappaB in a wide range of normal cellular functions.

Animals↗

PubMed1996

IL-10 selectively regulates murine Ig isotype switching.

A role for IL-10 in regulating Ig isotype switching directly at the level of the murine B cell has not been previously reported. In this report we show that IL-10 selectively up-regulated IgM to IgG3 class switching in lipopolysaccharide (LPS)-activated cultures through a direct effect on membrane (m) IgM+IgG3(-)B cells in vitro. IL-10 stimulated a 3- to 4-fold enhancement (from 6-8 to 20-30%) in membrane mIgG3(+) cells and a significant increase in Smu-Sgamma3 DNA rearrangement events as measured by digestion-circularization PCR (DC-PCR) over that observed with LPS alone. IL-10 induction of switching to IgG3 was not accompanied by a corresponding increase in the steady-state levels of germline CHgamma3 RNA. By contrast, IL-10 strongly inhibited the transforming growth factor-beta-mediated generation of mIgA+ cells and Smu-Salpha DNA rearrangement events in LPS-, but not CD40 ligand (CD40L)-activated B cells. This effect was not accompanied by changes in the steady-state levels of germline CHalpha RNA. IL-10 had no effect on IL-4-mediated switching to either IgG1 or IgE in either LPS- or CD40L-activated B cells. Thus, IL-10 can either enhance or suppress switching to particular murine Ig isotypes but it differs from most other murine cytokines in that its effects on switching do not appear to be associated with changes in the corresponding steady-state levels of germline CH RNA.

Animals↗

PubMed1996

IFN-gamma is a potent inducer of Ig secretion by sort-purified murine B cells activated through the mIg, but not the CD40, signaling pathway.

IFN-gamma has been shown to either stimulate or inhibit Ig secretion. No studies have yet addressed the basis for these seemingly conflicting properties nor whether IFN-gamma acted directly at the level of the B cell to mediate its effects. Thus, we studied the ability of IFN-gamma to regulate Ig secretion in sort-purified, resting murine B cells that were >99% Ig+, activated either through membrane Ig using unconjugated or dextran-conjugated anti-IgD antibodies (alphadelta-dex) or through CD40 using soluble or membrane CD40 ligand (CD40L). B cells activated with alphadelta-dex proliferated but do not secrete Ig, even in the presence of IL-1 + IL-2. We demonstrate that IFN-gamma only when added subsequent to B cell stimulation with alphadelta-dex, but not unconjugated anti-IfD antibody, plus IL-1 + IL-2 induces up to 100-fold enhancements in Ig secretion and in the numbers of Ig-secreting cells. The predominant Ig isotype secreted is IgM, with IgG3 and IgG2a comprising the majority of non-IgM antibody. IFN-gamma must act in concert with IL-2 for stimulation of Ig secretion. Further, IFN-gamma synergizes with IL-3 + granulocyte-macrophage colony stimulating factor for induction of Ig synthesis. IFN-gamma also enhances IgA syntheses by transforming growth factor-beta-induced membrane IgA+ cells. By contrast, 125IIFN-gamma fails to stimulate Ig secretion in B cells activated with CD40L in the presence or absence of IL-1 + IL-2 or IL-4. However, the combination of CD40L and alphabeta-dex is strongly synergistic for IFN-gamma-induced Ig secretion. Thus, these data establish that IFN-gamma can act directly on the B cell to induce Ig synthesis without the participation of any other cell and demonstrates that the mode of activation of the B cell plays an important role in directing the action of IFN-gamma.

Animals↗

PubMed1995

Bacterial lipoproteins may substitute for cytokines in the humoral immune response to T cell-independent type II antigens.

Bacterial lipoproteins share a common structural motif that has been shown to stimulate proliferation and Ig secretion of murine B cells, in a manner distinct from that mediated by LPSs. Studies of lipoprotein-mediated B cell activation utilized heterogeneous populations of lymphoid cells, leaving unresolved their ability to directly activate resting B cells, as well as their ability to interact with other B cell stimuli. Using highly enriched and/or sort-purified resting murine B cells, we demonstrate that, in contrast to previous reports, lipoproteins (lipoprotein-D, lipoprotein-OspA, and/or the synthetic analogue Pam3Cys) stimulate little, if any, proliferation or Ig secretion in resting B cells. However, when combined with a multivalent membrane (m)Ig-mediated cross-linking signal, dextran-conjugated anti-IgD Abs (alpha delta-dex), lipoproteins mediate up to 10,000-fold inductions in IgM secretion and up to 25-fold enhancements in cellular proliferation relative to that observed with alpha delta-dex alone, in the absence of added cytokines. This mIg-mediated enhancement of Ig secretion was not observed when B cells were stimulated with bivalent, unconjugated anti-Ig. CD40 ligand (CD40L), shows a similar, although somewhat more moderate, synergy with lipoproteins for induction of proliferation and IgM secretion. By contrast, lipoproteins by themselves are relatively ineffective at costimulating Ig secretion in the presence of various combinations of cytokines. These data suggest that bacteria may induce Ag-specific humoral immunity through the action of bacterial polysaccharides that mediate an Ag-specific multivalent mIg signal, in concert with bacterial lipoproteins that deliver ancillary signals, without a requirement for recruitment of non-B cell types.

Animals↗

PubMed1995

IL-3 and granulocyte-macrophage colony-stimulating factor strongly induce Ig secretion by sort-purified murine B cell activated through the membrane Ig, but not the CD40, signaling pathway.

Sort-purified resting murine B cells proliferate in response to dextran-conjugated anti-IgD Abs (alpha delta-dex) but fail to secrete significant amounts of Ig even after the addition of IL-1 + IL-2. We show that either IL-3 or granulocyte-macrophage CSF (GM-CSF) stimulates 10- to 50-fold enhancements in IgM secretion by sort-purified B cells treated with alpha delta-dex + IL-1 + IL-2, and that the combined actions of IL-3 and GM-CSF are typically greater than additive. Both IL-3 and GM-CSF act primarily as B cell differentiation factors, although IL-3 induces a modest enhancement in cellular outgrowth. The enhancing effects of IL-3 and GM-CSF require multivalent Ag receptor cross-linkage, mediated by alpha delta-dex, as neither cytokine induces IgM secretion in the presence of unconjugated anti-IgD Abs. Although both alpha delta-dex and IL-1 + IL-2 are required for optimal IL-3- and GM-CSF-mediated IgM secretion, both IL-3 and GM-CSF stimulate a modest IgM secretory response by cells activated with alpha delta-dex alone. In this regard, supernatant from either an activated CD4+ Th1 or Th2 clone potently induces IgM secretion by alpha delta-dex + IL-1 + IL-2-activated B cells and this is due, in large part, to the presence in these supernatants of either IL-3 and/or GM-CSF. Neither IL-3 nor GM-CSF stimulates significant IgM secretion by B cells activated through the CD40 signaling pathway alone, although the combination of CD40 and membrane Ig signaling leads to a strong enhancement of the IL-3 + GM-CSF-mediated IgM synthesis above that obtained with membrane Ig signaling alone. The demonstration that IL-3 and GM-CSF act directly as differentiation factors for B cells activated through their Ag receptor establishes a novel cytokine pathway for induction of humoral immunity.

Animals↗

PubMed1995

Novel in vitro model for high-rate IgA class switching.

The parameters necessary for induction of high-rate IgA class switching are unknown. Thus, although TGF-beta is switch factor for the IgA class, the percentage of membrane (m)IgA+ cells generated in vitro in response to TGF-beta and various individual modes of B cell activation is limited to 1 to 2% of the total B cell population, a percentage far below that observed within Peyer's patches. In this report we determined a set of parameters that act synergistically to generate up to 15 to 20% mIgA+ cells in vitro. A dual mode of B cell activation is required whereby signaling through CD40 or in response to LPS stimulation must occur in concert with multivalent Ag receptor crosslinking. A complex cytokine requirement is also revealed in that both IL-4 and IL-5 must be present with TGF-beta for high-rate IgA class switching to occur. By contrast, IFN-gamma, a known antagonist of IL-4, strongly suppresses the induction of mIgA+ cells in response to these stimuli. This novel cellular system should serve as a powerful tool for studying the molecular mechanisms that underly the IgA class switch and may provide insight into the physiologic parameters that induce it.

Animals↗

PubMed1995

Multivalent, but not divalent, antigen receptor cross-linkers synergize with CD40 ligand for induction of Ig synthesis and class switching in normal murine B cells. A redefinition of the TI-2 vs T cell-dependent antigen dichotomy.

A number of previous studies have suggested that cross-linkage of the B cell Ag receptor may be critical for induction of humoral immune responses to T cell-dependent (TD) Ags in vivo. Previous work also indicated a critical role, in these responses, for CD40-mediated signaling mediated by binding of the inducible T cell membrane protein, CD40 ligand (CD40L). Data in this manuscript demonstrate that concentrations of bivalent anti-IgD or anti-IgM Ab as high as 30 micrograms/ml induced little if any enhancement of CD40-dependent Ig secretion by resting murine B cells. In contrast, concentrations as low as 3 pg/ml of multivalent, dextran-conjugated, anti-IgD (alpha delta-dex) or anti-IgM (alpha mu-dex) were strongly synergistic with CD40L for induction of B cell proliferation, viable cell outgrowth, Ig isotype switching, and maturation to Ig secretion. As many as 30% of the B cells became membrane IgG1+ after stimulation with CD40L, anti-Ig-dextran, and IL-4 + IL-5, with a concomitant three- to fivefold increase in numbers of viable cells as compared with control cultures. High Ig secretory responses were obtained in response to the combined actions of CD40L and alpha delta-dex or alpha mu-dex, utilizing concentrations of B cell activator that when acting alone induced only modest Ig secretion. Surprisingly, although we previously demonstrated that alpha delta-dex selectively and strongly suppressed IgE production by T cell-activated B cells, it strikingly augmented IgE expression by CD40L-activated B cells. These data suggest 1) a key role for Ag receptor cross-linkage in CD40-dependent induction of humoral immune responses, 2) that to achieve a membrane Ig-dependent enhancing effect in the presence of activated T cells, TD Ags must be displayed to the B cell as a multivalent array of epitopes, 3) that picomolar concentrations of Ag can mediate this effect, and 4) that at least for induction of IgE responses, B cell stimulation via CD40L or via activated T cells may lead to a qualitatively different pathway of activation.

Animals↗

PubMed1995

Antigen receptor cross-linking differentially regulates germ-line CH ribonucleic acid expression in murine B cells.

Cytokines are believed to regulate Ig class switching, in part, through selective modulation of germ-line constant heavy (CH) gene transcription. B cell activators such as LPS or activated T cell membranes also influence germ-line CH RNA expression in the absence of exogenous cytokines. In this report we determined whether multivalent Ag receptor cross-linking, utilizing dextran-conjugated anti-IgD Abs (alpha delta-dex), could also regulate germ-line CH RNA expression. We demonstrated that alpha delta-dex markedly inhibited germ-line epsilon RNA expression, but strongly augmented germ-line gamma 1 RNA, in LPS + IL-4-stimulated cultures. This was correlated with > 90% alpha delta-dex-mediated suppression in the secretion of IgE and generation of membrane (m)IgE+ cells, and a more modest 50% reduction in IgG1 synthesis and mIgG1+ cells. Furthermore, alpha delta-dex inhibited the LPS induction of both gamma 3 and gamma 2b germ-line RNA and the associated secretion of IgG3 and IgG2b. A similar alpha delta-dex-mediated suppression of germ-line gamma 2a RNA and IgG2a secretion in LPS + IFN-gamma-stimulated cultures was observed. By contrast, activation of resting B cells with alpha delta-dex alone led to induction of germ-line gamma 3, gamma 1, and gamma 2b RNA but did not stimulate detectable expression of RNA specific for gamma 2a or epsilon. These studies demonstrate that: 1) germ-line gamma 1 gene expression is regulated uniquely, 2) germ-line transcription and switch recombination can be dissociated, 3) the germ-line transcription of each IgG isotype has an independent pattern of regulation, and 4) cross-linking of the Ag receptor, by itself, can stimulate small amounts of germ-line CH RNA.

Animals↗

PubMed1995

T cell independent antigens.

T cell independent antigens type 2 (TI-2), which are represented predominantly by polysaccharide antigens, stimulate humoral antibody responses in the absence of T-cell help. We and others have recently reported that natural killer cells and/or natural killer cell derived lymphokines may provide a form of 'help' that is necessary for the induction and maintenance of TI-2 responses. Two natural killer cell derived lymphokines, interferon-gamma and granulocyte-macrophage colony-stimulating factor, show synergistic stimulatory activity in inducing Ig secretion in B cells stimulated by a multivalent ligand that mimics TI-2 antigens. The recent finding that natural killer cells have receptors for various classes of polysaccharides supports a role for these cells in regulating responses to TI-2 antigens.

Antigens, Differentiation↗

PubMed1995

T cell-independent antigens type 2.

In this review we have attempted to define the characteristics of TI-2 antigens that enable them to stimulate antibody production in the absence of T cell help. One of the most critical properties of this group of antigens is their ability to deliver prolonged and persistent signaling to the B cell. This by itself is not however sufficient to stimulate Ig synthesis, and they must therefore stimulate non-T cells to interact with the B cells either directly or indirectly via cytokine production. There is evidence implicating the NK cell and T cell as playing this important role in response to TI antigens. Furthermore, we discuss the importance of cytokines such as IL-3, GMCSF, and IFN-gamma, which significantly enhance antibody production by these antigens. Finally, we present evidence demonstrating that B cell activation via TI stimuli does not play merely a permissive role in allowing for cell cycle entry and enhanced responsiveness to other stimuli. Rather, the nature of the B cell activating signal is critical in determining the quantitative and qualitative profile of Ig isotype production.

Animals↗

PubMed1994

Synergy of IL-1 and stem cell factor in radioprotection of mice is associated with IL-1 up-regulation of mRNA and protein expression for c-kit on bone marrow cells.

Administration of IL-1 and stem cell factor (SCF) to mice 18 h before lethal 60Co whole-body irradiation resulted in synergistic radioprotection, as evidenced by increased numbers of mice surviving 1,200 to 1,300 cGy doses of radiation and the recovery of increased numbers of c-kit+ bone marrow cells at 1 and 4 days after the lethal dose of 950 cGy. Anti-SCF Ab inhibited IL-1-induced radioprotection, indicating that endogenous production of SCF is necessary for radioprotection by IL-1. Conversely, radioprotection induced by SCF was reduced by anti-IL-1R Ab, indicating that endogenous IL-1 contributes to SCF radioprotection. SCF, unlike IL-1 does not induce hemopoietic CSFs and IL-6 or gene expression of a scavenging mitochondrial enzyme manganese superoxide dismutase in the bone marrow, suggesting that SCF and IL-1 radioprotect by distinct pathways. The mRNA expression for c-kit (by Northern blot analysis) and 125I-SCF binding on bone marrow cells was elevated within 2 and 4 h of IL-1 administration respectively. Four days after LD 100/30 radiation the recovery of c-kit+ bone marrow cells was increased sixfold in IL-1-treated mice, almost 20-fold in SCF-treated mice, and 40-fold in mice treated with the combination of the two cytokines. Thus, endogenous production of both IL-1 and SCF is required for resistance to lethal irradiation and the synergistic radioprotective effect of the two cytokines may, in part, depend on IL-1 and SCF-induced increases in numbers of c-kit+ hemopoietic stem and progenitors cells that survive lethal irradiation.

Adrenalectomy↗

PubMed1994

An in vitro model for T cell-independent induction of humoral immunity. A requirement for NK cells.

We previously established an in vitro polyclonal model for membrane Ig-mediated Ig secretion by B cells in response to T cell-independent type 2 (TI-2) Ags, by using dextran-conjugated anti-IgD Abs (alpha delta-dex). We demonstrated that resting B cells activated with alpha delta-dex plus IL-2 secreted large amounts of Ig only in the presence of NK cells. In this report we show that, in contrast to small B cell-enriched spleen cells that require activation with the combination of alpha delta-dex and IL-2 for induction of Ig secretion, large B cells were induced to secrete Ig in response to alpha delta-dex, alone. These responses were inhibited by previous depletion of asialo-Gm-1+ cells from the B cell-enriched population, and restored both by the addition of freshly explanted NK cells and by in vitro-activated NK cells, suggesting a requirement for NK cells. Small alpha delta-dex-activated B cells could, however, also be stimulated to secrete Ig in the absence of added cytokines but only after the addition of in vitro-activated NK cells and not freshly explanted splenic NK cells. When highly purified large B cells, obtained by electronic cell sorting, were cultured with in vitro-activated NK cells, Ig secretion was induced even in the absence of any other added B cell stimulus. Because the splenic marginal zone has been implicated in humoral immune responses to TI-2 Ags, we further fractionated large B cells into marginal zone (MZB) and follicular (FB) B cells by electronic cell sorting. In vitro-activated NK cells stimulated large MZB, but not FB, cells to secrete Ig in the absence of exogenous stimuli. These data establish a T cell-independent model for induction of Ig synthesis in the absence of any added cytokine and demonstrate a role for freshly explanted NK cells in stimulating Ab production in response to TI-2 Ags. Further, they show that pre-activated NK cells can induce Ig secretion from pre-activated B cells even in the absence of any added stimuli. These data also underscore a special role for the MZB cell in these responses.

Animals↗

PubMed1993

Natural killer cells induce activated murine B cells to secrete Ig.

We previously demonstrated that dextran-conjugated anti-IgD antibodies (alpha delta-dex) induce proliferation of small, B cell-enriched murine spleen cells (Be cells), and in the presence of IL-2, stimulate Ig secretion in vitro. We have shown that alpha delta-dex-stimulated B cells provide an in vitro model for studying B cell activation by T cell-independent type 2 (TI-2) Ag, as exemplified by the bacterial polysaccharides. We now show that highly purified resting B cells, obtained by electronic cell sorting (Bsp cells), fail to secrete Ig in the presence of alpha delta-dex + IL-2. The alpha delta-dex + IL-2-induced Ig secretory response of Bsp cells is restored upon addition of splenic non-B, non-T cells or a pure population of in vitro-generated NK cells. Similarly, pretreatment of Be cells with anti-AsGm-1 plus complement inhibits Ig secretion in response to alpha delta-dex + IL-2. An IL-2-induced NK cell supernatant (NKSN) is equally potent at stimulating Ig secretion by alpha delta-dex-activated Bsp cells, indicating that cell contact between Bsp and activated NK cells is not required for this effect. IL-2 stimulates not only NK cells, but B cells as well, since addition of anti-IL-2 + anti-IL-2R antibodies to Bsp cell cultures, in the presence of alpha delta-dex + NKSN, inhibits Ig secretion. These data describe a novel animal model for NK cell-induced B cell maturation to Ig secretion and suggest a pathway for Ig production in response to T1-2 Ag.

Animals↗

PubMed1993

Interleukin 5 induces S mu-S gamma 1 DNA rearrangement in B cells activated with dextran-anti-IgD antibodies and interleukin 4: a three component model for Ig class switching.

The cellular signals required for induction of immunoglobulin (Ig) class switching are only partially understood. Two processes that are considered to be necessary for such induction are DNA synthesis and germline constant heavy (CH) gene transcription. We now show that an additional signal, as mediated by interleukin 5 (IL-5), is also required. To induce proliferation of resting B cells, but not Ig secretion, we utilized anti-IgD antibodies conjugated to dextran (alpha delta-dex). The addition of IL-4, a well-established switch factor for the IgG1 subclass, to alpha delta-dex-activated cell cultures failed to induce IgG1 secretion or mIgG1+ cells unless IL-5 was also present. While IL-4 stimulated an increase in germline gamma 1 RNA in alpha delta-dex-activated cells, this effect could neither be induced nor enhanced by IL-5. By contrast, IL-5 strongly enhanced steady-state levels of productive gamma 1 RNA induced by alpha delta-dex and IL-4, suggesting that IL-5 stimulated IgG1 switch rearrangement. To test this possibility we measured switch (S) mu-S gamma 1 DNA recombination events using a newly developed assay, digestion circularization polymerase chain reaction (DC-PCR). We demonstrated that IL-5 was necessary for induction of S mu-S gamma 1 DNA rearrangement in alpha delta-dex plus IL-4-activated cells but that it had little effect on rearrangement in the absence of IL-4. Our data strongly suggest, therefore, a three-component model for induction of Ig class switching. This model includes germline CH gene transcription, DNA synthesis, and a third component that is necessary for recombination.

Animals↗

PubMed1993

Transforming growth factor-beta 1 is required for secretion of IgG of all subclasses by LPS-activated murine B cells in vitro.

Addition of transforming growth factor-beta 1 (TGF-beta 1) to in vitro cultures of murine B cells activated with bacterial LPS selectively stimulates IgG2b and IgA class switching and decreases cellular proliferation. To assess a possible role for endogenous TGF-beta in modulating the Ig isotypes produced by LPS-activated cells, we utilized a neutralizing anti-TGF-beta mAb to abrogate endogenous TGF-beta activity. Anti-TGF-beta antibody, over a range of relatively low cell densities, strikingly inhibited both IgG3 and IgG2b production in response to LPS, with little or no change in the concentrations of secreted IgM. This effect of anti-TGF-beta antibody was specific, since it did not occur with an isotype-matched control mAb and was completely reversed with exogenous TGF-beta 1. Optimal IgG3 secretion occurred at concentrations of TGF-beta that were approximately eightfold lower than that necessary for maximal synthesis of IgG2b. Neutralization of endogenous TGF-beta in LPS-activated cultures was associated with an approximately twofold increase in proliferation and viable cell yields, a modest decrease in the percentage of membrane (m)IgG2b+ cells, and a modest increase in the percentage of mIgG3+ cells. This latter finding indicated that TGF-beta was not required for IgG3 class switching, but for maturation of mIgG3+ cells into Ig secretors. Highly purified B cells, obtained by electronic cell sorting, released active TGF-beta in response to LPS and showed a similar marked reduction in LPS-mediated IgG3 and IgG2b secretion in the presence of anti-TGF-beta antibody. Abrogation of endogenous TGF-beta activity in LPS-activated cultures also resulted in a striking reduction in IFN-gamma-mediated IgG2a production, and a more modest decrease in the synthesis of IgG1 and IgE in the presence of IL-4. These data indicate that relatively low concentrations of TGF-beta are essential for stimulating optimal IgG secretion by LPS-activated B cells, in an Ig isotype-nonspecific manner, and may regulate these responses in an autocrine fashion.

Animals↗