Physiological cell death in B lymphocytes: I. Differential susceptibility of WEHI-231 sublines to anti-lg inducedphysiological cell death and lack of correlation with bcl-2 expression

WEHI-231 is a murine lymphoma generally considered to representan Immature B cell. Cross-linking of slg on WEHI-231 leads togrowth arrest and eventually physiological cell death (PCD).We characterized three sublines of WEHI-231 by flow cytometryand compared their responses with slg cross-linking. All sublineshad Identical expression of a series of common B cell surfacemarkers (IgM, IgD, FCR, ICAM-1, and CD45), but one was I-A^–.Despite the phenotyplc similarities between these sublines,antl-IgM caused aptotosls in only two sublines, although itinhibited growth in all three. The growth arrest induced byantl-IgM was reversible by lipopolysaccharide and T_h2 clonesand independent of FcR engagement. Antl-lgD, unlike antl-IgM,Induced neither growth arrest nor apoptosls. To further comparethe sublines' susceptibility to PCD, we investigated their responsesto antl-IgM by ultrastructural morphology, [³H]thymldlne release,propidium Iodide exclusion, and incorporation into DNA. By allthese experimental criteria, two of the WEHI-231 sublines weresusceptible to PCD while the third demonstrated remarkable resistanceto antl-IgM, but not irradiation or T_h1-induced PCD. This differentialsusceptibility to PCD did not correlate with either bcl-2 levelsin the resting cells or to the decrease in bcl-2 expressionfollowing slg engagement. We discuss the Implications of thesefindings for our understanding of PCD in B cells.     

B7 2 (CD86) is essential for the development of IL-4-producing T cells

The CD28/CTLA-4 ligands, B7–1 (CD80) and B7–2 (CD86),provide a co-stimulatory signal necessary for optimal T cellactivation. We have examined the effect of blocking B7–1and B7–2 in an in vitro system using ovalbumin-specificT cells from ß TCR-transgenic mice. This system allowedus to examine the interaction of B7 co-stimulators on physiologicantigen-presenting cells (APC) with antigen-specific T helperprecursor (T_hp) cells. We report that blocking T_hp/B7–1or B7–2 interactions in a primary response differentiallyaffects the cytokine profile observed in a secondary stimulation,even in the absence of additional anti-B7 antibody. Engagementof B7–2 in the primary stimulation was found to be essentialfor production of the T_h2 cytokine, IL-4, but not the T_h1 cytokines,IL-2 and IFN-, in a secondary stimulation. Conversely, inclusionof the anti-B7–1 mAb in cultures using highly purifiednaive T cells increased levels of IL-4 and significantly depressedlevels of IFN-, upon re-stimulation. The effect of the anti-B7–2mAb in reducing IL-4 production could be overcome by the additionof recombinant IL-4 in the primary stimulation. The effectsof the anti-B7–2 mAb appear to be due to blocking andnot cross-linking, as F(ab) fragments mimicked the intact antibody.Taken together, our data demonstrate that the interaction betweenThp and B7–2 favors the development of T_h2 cells.     

Differential regulation of surface immunoglobulin and Lyb2 mediated B cell activation: II. cAMP dependent (prostaglandin E2) and independent (IFN-{gamma}) mechanisms of regulation of B lymphocyte activation

We have recently demonstrated that pharmacological agents thatelevated cAMP inhibited sigM but not Lyb2 mediated activationof murine B lymphocytes. In this report we show evidence fordifferential regulation of prostagiandin E₂ (PGE₂), a physiologicalagent that elevated cAMP and IFN- on sigM and Lyb2 mediatedB cell activation. PGE₂ inhibited anti-lgM but not anti-Lyb2induced DNA synthesis in a dose-dependent manner. Interestingly,rlFN- also inhibited anti-lgM but not anti-Lyb2 induced DNAsynthesis. rlFN- exerted its effects directly on B cells sincedepletion of T cells and G-10 Sephadex adherent cells did notalter effects of IFN- on anti-lgM and anti-Lyb2 induced DNAsynthesis. Pretreatment of B cells with IL-4 and/or IL-5 didnotprevent the IFN- mediated inhibition of the anti-lgM response.The inhibitory effect of IFN- was observed during early stagesof B cell activation. Thus IFN- inhibited anti-µ inducedblast transformation and subsequent progression into the G₁phase of the cell cycle. The differential effects exerted byPGE₂ and rIFN- appeared to be mediated by distinct mechanisms.ThusPGE₂ but not rIFN-, at concentrations inhibitory to the slgMresponse, induced elevation of intracellular cAMP levels. Theseresults demonstrate that physiologically relevant immunomodulatorssuch as PGE₂ and IFN- can differentially regulate murine B cellresponses mediated through the antigen receptor and Lyb2 moleculesby cAMP dependent and independent mechanisms. Relevance of thisregulation for the induction of antibody synthesis by T_h1 andT_h2 types of helper T cells is discussed.     

Role of macrophages and {alpha}{beta} T lymphocytes in early interleukin 10 production during Listeria monocytogenes infection

Immunity to intracellular bacteria including Listeria monocytogenesis determined by T_h1 cells and CD8 T cells which produce interferon_h.Here we show that high levels of IL-10 are released by splenocytesfrom mice infected with L. monocytogenes. IL-10 was detectedon day 1 after infection, peaked on day 4, and subsequentlydeclined. Cell separation studies and experiments with RAG-1-deficientmice, which do not possess mature B cells or T cells, revealedthat the macrophage Is the major cellular source of early IL-10production. Elevated IL-10 production in RAG-1 mutants and TCRßmutants, but not in TCR mutants, Is consistent with an inhibitionof macrophage IL-10 release by ß T cells. High IL-10production was also seen after infection with another intracellularbacterium, Mycobacterlum bovis. Since IL-10 Inhibits T_h1 cellresponses, certain pathogens might use induction of this cytokineas an evasion mechanism from the protective Immune responseof the host. However, our findings showing high levels of IL-10production in infectious models which are dominated by T_h1 cellresponses suggest that IL-10 alone is insufficient for directingT_h0 differentiation into the T_h2 cell pathway. These findingstherefore challenge the view of IL-10 as a unique and decisivedetermlnator of the T_h2 cell pathway.     

Differential production of IL-12 in BALB/c and DBA/2 mice controls IL-4 versus IFN-{gamma} synthesis in primed CD4 lymphocytes

The profile of cytokines produced by CD4 T cells is profoundlyinfluenced by the presence of IL-12. Here we demonstrate thatduring re-stimulation of antigen-specific immune responses invitro, antigen-primed lymph node cells from DBA/2 mice produced3- to 30-fold more IL-12 than did cells from BALB/c mice, whichare identical at the major histocompatibility locus. The straindifferences in IL-12 production were observed only in antigen-drivenresponses (and not in responses induced by bacterial products),and were dependent upon an interaction between CD4 T cells andlymph node adherent cells. In addition, differences in the quantityof IL-12 produced by DBA/2 and BALB/c antigen-presenting cells(APC) was not dependent on differential production of IFN- byT cells, since APC from DBA/2 mice still produced much greaterquantities of IL-12 than did BALB/c APC when each was culturedwith the same H-2^d-restricted T_h2 clones, in the complete absenceof IFN, or when each was cultured with primed (BALB/c x DBA/2)F₁T cells. The level of IL-12 produced in the cultures criticallyaffected cytokine production in CD4 T cells, since neutralizationof endogenous IL-12 in DBA/2 cultures, which are predisposedtowards developing T_h1 responses, reduced IFN- production andenhanced IL-4 synthesis to levels normally seen in BALB/c cultures,which are predisposed toward developing Th2 responses. We proposetherefore that differential production of antigen-driven IL-12is a mechanism by which the genetic background in DBA/2 andBALB/c mice can affect the pattern of cytokine synthesis byT cells during the development of adaptive immune responses.     

Defective IL-5-receptor-mediated signaling in B cells of X-linked immunodeficient mice

Murine (m) IL-5 induces proliferation and differentiation ofboth Ly-1⁺; B cells and activated conventional B cells. X-linkedimmunodeficient (XID) mice do not respond to thymus-independenttype II antigens, and have an abnormal response to a varietyof activation signals through Ig receptors, CD40 and cytokinereceptors. Furthermore, XID mice show a B cell specific defect,reflected in decreased numbers of IL-5R⁺ B cells and reducedresponsiveness of IL-5R⁺ B cells to mIL-5. We generated IL-5Rtransgenic (5R-Tg) mice in which B cells expressed recombinantIL-5R. We crossed male 5R-Tg mice with female XID mice and usedtheir offspring to determine the IL-5 responsiveness of theseB cells. All B cells of F₁ male mice carrying the xid gene togetherwith the transgene expressed the recombinant IL-5R. However,those mice lacked Ly-1 B cells and their B cells acquired responsivenessto mIL-5. Interestingly, XID-5R-Tg B cells, but not XID B cells,acquired mIL-5 proliferatlve and Ig-secretory responsivenessonly in the presence of suboptimal doses of Ilpopolysaccharide.Stimulation of these B cells with mIL-5 plus phorbol myristateacetate induced proliferation, but not Ig secretion. These resultsindicate that the impaired mIL-5 responsiveness of B cells inXID mice is due to an abnormality of IL-5R-mediated signalingwhich may correlate with the xid gene mutation, alteration ofa single amino acid of Bruton's tyroslne kinase.     

Selective activation of VH3A10+ rheumatoid factor producing B cells by staphylococcal enterotoxin D

Staphylococcal enterotoxin D (SED) is a T cell superantigenwhich selectively targets ß TCRs bearing particularV^ß elements. A second function of SED relates to thepreferential activation of a B cell subset characterized bya high frequency of rheumatoid factor (RF) producing B cells.To define the molecular basis of the SED-induced B cell repertoireshift, we have analyzed Ig heavy chain genes in B cell clonesexpanded after SED stimulation and compared them with B cellclones established in the presence of anti-CD3 stimulated helpercells. Gene segments of the V_H3 family were most frequentlyutilized under both stimulation conditions (42% anti-CD3; 47%SED). Sequence analysis of V_H3 gene segments demonstrated thatthe repertoire of V_H3 elements in B cell clones from SED drivenand anti-CD3 driven cultures were distinct (P=0.01). RF activitywas closely associated with the expression of selected V_H3 elements.B cell clones stimulated with SED preferentially expressed V_H3A10,whereas V_H26 was the gene segment dominantly used in B cellclones expanded with anti-CD3 stimulated helper cells. The usageof J_H and D_H elements was indistinguishable in SED and anti-CD3driven B cell clones, suggesting that SED targets V_H3⁺ B cellsthrough a V_H-specific mechanism. Comparison of the closely relatedsequences of the SED responsive V_H3A10 and the SED non-responsiveV_H26 element suggested a role of a sequence polymorphism inthe CDR2 reminiscent of B cell reactivity to conventional antigens.In contrast to conventional antigens, SED can induce differentiationof a high frequency of naive B cells. Thus, this staphylococcalenterotoxin combines selective activation of T cells with selectiveactivation of B cells and might be able to direct T cell helpto RF producing B cells.     

Sharing of the IL-2 receptor {gamma} chain with the functional IL-9 receptor complex

The third subunit, the so-called common (_c) chain, of the IL-2receptor is shared among the receptors for IL-2, IL-4, IL-7and IL-15, and dysfunction of the _c chain is thought to causeX-linked severe combined immunodeficiency (XSCID) ascribed toimpairment of early T cell development. However, cytokines linkedto XSCID are as yet unidentified. A mAb specific for the _c chain,TUGm2, profoundly inhibited cell proliferation in response toIL-9. Another mAb, TUGm3, immunoprecipltated [¹²⁵I]IL-9 cross-linkedwith either the IL-9 receptor or the _c chain. These resultsdemonstrate that the _c chain is included in the functional receptorcomplex for IL-9, which was initially characterized as a T cellgrowth factor and is essential for IL-9-dependent growth signaltransductlon.     

Co-transfer of B cells converts resistance into susceptibility in T cell-reconstituted, Leishmania major-resistant C.B-17 scid mice by a non-congnate mechanism

Resistance to infection of mice with Leishmania major parasitesis dependent on the production of IFN- by CD4⁺ T helper cells.C.B-17 scid mice, lacking both T and B cells, succumb very quicklyto the infection, but develop resistance if reconstituted withappropriate numbers of T cells from BALB/c mice. In this model,we studied the role of B cells with regard to their abilityto influence disease outcome and to function as antigen-presentingcells for T cells. For this purpose, we reconstituted scid mice(H-2^d) with either T cells or with T and B cells obtained from(BALB/c x BALB.B)F₁ mice (H-2^d x ^b), and infected them withL. major parasites 1 day after reconstitution. Mice reconstitutedwith T cells alone cured the disease, whereas additional B cellreconstitution led to susceptibility. Healing was associatedwith a predominant T_h1-type response. In all mice, L. mayor-specificT cell proliferation was restricted to the MHC phenotype ofthe recipient (H-2^d) but not to that of the donor (H-2^d x ^b),indicating that there was no detectable contribution of donorB cells in the priming of a T cell response. Furthermore, Bcells, when purified from infected BALB/c mice, were unableto stimulate a L. mayor-specific CD4⁺ T cell clone (L1/1) withoutaddition of exogenous antigen, in contrast to macrophages fromthe same animal. These data suggest that B cells, in vivo, donot carry L. major antigen in a form capable of activating specificCD4⁺ T cells. Therefore, B cells promote disease by means otherthan cognate interaction with CD4⁺ T cells.     

Signal transduction in human B cells initiated via Ig{beta} ligation

Ig and Igß heterodimers are non-covalently associatedwith Ig to compose the antigen receptor complexes on B cells.The demonstration that different sets of tyrosine kinases bindto the cytoplasmic tails of Ig and Igß suggests thatIg and Igß may activate distinct second messengerpathways. In this study, we examined the effects of mAbs againstan exposed epitope of human Igß on pre-B and B celltriggering. Cross-linkage of Igß on B cells leadsto activation of tyrosine kinases, hydrolysis of phosphatidylinositides,and elevation of intracellular Ca²⁺, effects qualitatively identicalto those of anti-_µ mAbs. Our observations thus indicatethat cross-linking of Igß does not segregate signaltransduction pathways connected with the cytoplasmic talls ofIg and Igß. Ig ligation has been reported to be moreeffective in triggering pre-B than B cells, whereas our resultsindicated that Igß ligation is more efficient in triggeringB than pre-B cells. In addition to their activation properties,the anti-Igß mAbs effectively modulated B cell receptorcomplexes and blocked terminal differentiation of all plasmacell isotypes. The findings support the idea that anti-Igßcould serve as a universal B cell immunosuppressant.     

Differential regulation of early and late stages of B lymphocyte development by the {micro} and {delta} membrane heavy chains of Ig

Regulation of B lymphocyte development by the µ-membrane(µ_m) and -membrane (_m) heavy chains of Ig was examinedin an Ig transgenic mouse model. Mice were bred on a commonC57BL/6 (B6) background, and expressed rearranged and hypermutatedheavy and light chain transgenes encoding high-affinity receptorsfor the foreign antigen hen egg rysozyme (HEL). At no stagewere they exposed to HEL. variation of the Ig heavy chain constructyielded four different types of Ig transgenlc mice in whichdeveloping B lineage cells either expressed µ_m and _m inthe normal physiological sequence (µ_m then µ_m+_m),or produced µ_m alone, µ_m alone or µ_m +_m fromthe onset of heavy chain expression in the bone marrow. ImmatureB220^low, HSA^high and mature B220^high, HSA^low B cells were producedin all mice regardless of their developmental pattern of µ_mand _m expression. However, production of immature B cells wasmost efficient when µ_m heavy chain was expressed aloneduring early B cell development Thus expression of _m duringthis period either in the presence or absence of µ_m resultedin a 2- to 3-fold reduction in the numbers of immature B cellsin the spleen as well as altered levels of surface B220 andHSA on these cells in spleen and bone marrow respectively. Bycontrast, normal maturationally regulated expression of _m ledto the presence of increased numbers of mature B cells in thespleen and lengthened the average lifespan of these cells asdetermined by in vivo incorporation of 5-bromo-2'-deoxyurtdlne.These results pointed to selective effects of µ_m and _mheavy chains on regulation of the early and late stages of Bcell development respectively, and provided a rational basisfor co-expression of µ_m and _m as well as the delayed expressionof _m during normal B cell development     

Skewed T cell receptor V{alpha} repertoire among superantigen reactive murine T cells

Reactivity of murine T cells with viral or bacterial superantigensis clearly correlated with the expression of TCR V_ßdomains. Thus, T cells responding to the minor lymphocyte stimulatorylocus (Mls-1^a) or staphylococcal enterotoxin B (SEB) expresspredominantly TCR V_ß6 or V_ß8.2 respectively.We have investigated the involvement of the other major variableelement of the TCR, the V domain, in these superantigen responses.Using a panel of anti-TCR V mAbs, It is demonstrated that theTCR V repertoire among superantigen stimulated V_ß6⁺or V_ß8.2⁺ blasts (responding to Mls-1^a or SEB respectivelyin vitro) is altered in comparison with anti-CD3 stimulatedcells expressing the same V domains. Furthermore, the TCR Vrepertoire is strongly skewed in TCR V_ß8.2 transgenicmice that have undergone extensive peripheral clonal deletionafter SEB injection. These data imply that the V domain influencessuperantigen recognition by sthe TCR.     

IL-2 receptor {alpha} chain (CD25JAC) expression defines a crucial stage in pre-B cell development

The analysis of the expression of the a chain of the IL-2 receptor(CD25.TAC) on the surface of B lineage cells In mouse bone marrowreveals that it is a useful marker to distinguish pre-B-I frompre-B-II cells. CD25 Is not expressed on CD45R(B220)⁺ c-kit⁺CD43⁺ TdT⁺ ₅⁺ C_µ^– slg^– lgH chain locus DJ_H-rearrangedpre-B-I cells of mouse bone marrow. It is expressed on largecycling CD45R(B220)⁺ c-kit⁺ CD43⁺ TdT⁺ ₅⁺ C_µ^– sig^–and on small resting CD45R(B220)⁺ c-kit⁺ CD43^– TdT⁺ ₅⁺C_µ^– sig^– sig- IgH chain locus VHDJH-rearrangedpre-B-II cells. Therefore, the transition from pre-B-I to largepre-B-II cells is marked by the downregulation of c-kit andterminal deoxynucleotldyl transferase (TdT), and by the upregulattonof CD25. SCID, RAG-2T, _µMT and ₆T mutant mice do havenormal, If not elevated numbers of pre-B-I cells but lack allCD25⁺ pre-B-II cells in their bone marrow. The expression ofa transgenic H chain under control of the _µH chain enhancerin RAG-2T bone marrow B lineage precursors allows the developmentof large and small CD25⁺ pre-B-II cells. The results suggestthat the differentiation of pre-B-I to pre-B-II cells in mousebone marrow requires the expression of _µH chains and surrogateL chains in membranes, probably on the surface of precursorB cells.     

Intercellular adhesion molecule-1 and leukocyte function-associated antigen-1 are involved in protection mediated by CD3+TCR{alpha}{beta} T cells at the early stage after infection with Listeria monocytogenes in rats

To Investigate the significance of Intercellular adhesion molecule-1(ICAM-1) and leukocyte function-associated antlgen-1 (LFA-1)In host defense against infection with Intracellular parasites,we examined the effects of In vivo pretreatment with mAbs toICAM-1 (1A29) and LFA-1 (WT-1) on the protection against Infectionwith Listeria monocytogenesIn Fisher F344/N rats. Expressionof ICAM-1 and LFA-1 molecules on T cells In spleen, liver andperitoneal cavity of rats was down-regulated after i.p. administrationwith daily doses of 300 µg of either 1A29 or WT-1 for10 days. The survival rate of rats inoculated with viable Listeriawas significantly reduced byIn vivo pretreatment with 1A29 togetherwith WT-1 for 10 days but not by In vivo pretreatment with controlmAb. The numbers of bacteria In the spleen In rats pretreatedwith both 1A29 and WT-1 were significantly increased on day3 and day 6 after Infection with 1 x 10⁷ of viable Listeriacorresponding to 1/30 of LD₅₀ to normal rats. Thus, the resistanceagainst llsterial Infection was severely Impaired by combinationalpretreatment with mAbs In ICAM-1 and LFA-1. As shown In ourprevious report, the early appearance of CD3⁺TCRß^–T cells, presumably TCR cells, was evident In the peritonealcavity and liver of control rats at the early stage after llsterialInfection, while this was suppressed at this stage in rats pretreatedwith both 1A29 and WT1. These results suggest that the ICAM-1and LFA-1 adhesion pathway may be critically involved in protectlveroles of CD3⁺TCR_ß– T cells at the early stageof rat listeriosis.     

Identification, in mouse macrophages and in serum, of a soluble receptor for the Fc portion of IgG (Fc{gamma}R) encoded by an alternatively spliced transcript of the Fc{gamma}RII gene

Low affinity FcR are a heterogeneous group of glycoproteinswhich exist in transmembrane (TM) as well as in soluble forms.Two membrane isoforms of the murine type II FcR, FcRilb1 andFc;Rilb2, have been described. They result from the translationof alternatively spliced premRNA, FcRilb2 lacking sequencesof the first intracytoplasmic domain (IC1). Soluble forms ofFcR (sFcR) have previously been shown to result from proteolysisof membrane receptors. We report here the identification, inmacrophages, of a mRNA derived from the FCRll gene by splicingexons encoding the TM and IC1 domains, i.e. corresponding toa TM-deleted FcRllb2 mRNA. A soluble protein possibly encodedby this mRNA was identified in macrophage supernatants. In accordancewith FcR nomenclature, we propose to name this new FcRll IsoformFcRllb3. It is the most abundant 8FcR present in serum, as comparedwith 8FcR resulting from cleavage of membrane FcR.     

Rearrangement and expression of {chi} light chain genes can occur without {micro} heavy chain expression during differentiation of pre-B cells

The kinetics of light (L) chain gene rearrangement and expressionon mRNA and protein level has been studied with four stromalcell/IL-7 reactive, long-term in vitro proliferating pre-B celllines and clones, two from fetal liver of normal mice and twofrom fetal liver of E_µH-bcl-2 transgenic (bcl-2-tg) mice.These pre-B cell lines and clones are DJ_H-rearranged on bothH chain alleles. Two of the clones harbor H chain rearrangementswhich do not allow the expression of V_HDJ_H rearranged H chaingenes as _µH chain proteins. Upon removal of IL-7 fromthe pre-B cell cultures all four cell lines rearrange V_H-DJ_Hand V_L-J_L gene segments, loose the surface expression of c-kit,CD43, and surrogate light chain, as well as the capacity tobe clonable on stromal cells in the presence of IL-7. Pre-Bcells from normal mice die by apoptosis during differentiation,while those from bcl-2-tg mice do not. All four lines and clonesexpress comparable levels of mRNA for _µH and _µLchains with the same time kinetics during 3 days of differentiation.However, only two of the four pre-B cell lines and clones express_µH chain protein, whereas all four pre-B cell lines andclones express _µL chain protein at comparable levels between2x10⁵ and 1.40x10⁶ _µL chain molecules per cell. Theseresults suggest that _µH chain expression is not mandatoryfor rearrangement and normal expression of _µL chain geneswhen pre-B cells differentiate to B cells.     

IL-5 receptor positive B cells, but not eosinophils, are functionally and numerically influenced in mice carrying the X-linked immune defect

Mouse IL-5 (mIL-5) acts on B cells and eosinophils to inducegrowth and differentiation through the mIL-5 specific receptor(mIL-5R). The functional high-affinity mIL-5R is a heterodimercomposed of and ß chains. We investigated the expressionof mIL-5R and the responsiveness of B cells and eosinophilsto mIL-5 In X-linked immunodeficient (xld) mice. mIL-5R expressionanalyzed by using mAbs specific for and ß chainsrevealed that xld B cells had fewer mIL-5R⁺mIL-5Rß⁺than BALB/c B cells. In particular, a decrease in the numberof peritoneal mIL-5R⁺ B cells among Ly-1 B cells (known as B-1cells) was remarkable. Furthermore, the frequency of precursorsof mIL-5 responsive B cells in xld mice was 100-fold lower thanthat of BALB/c mice. Interestingly, sorted mIL-5R+ peritonealB cells from xld mice displayed a low response to mIL-5. Intraperltonealinjection of mIL-5 into BALB/c mice induced polyclonal IgM productionand an increase in the number of eosinophils. The same regimenfailed to induce an increase in the same parameters in xld mice.However, xld mice showed mIL-5-induced eoslnophilla in peripheralblood to a similar extent as BALB/c mice. Eosinophils from mIL-5-injectedxld mice expressed both and ß chains of mIL-5, andresponded to mlL-5 with prolonged in vitro survival.