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CD22/Siglec-2 is a B cell membrane-bound lectin that recognizes glycan ligands containing alpha2,6-linked sialic acid, and negatively regulates signaling through the B cell antigen receptor (BCR). Previous studies demonstrated that synthetic sialosides that bind to CD22 augment BCR signaling by inhibiting CD22-mediated BCR regulation. Here we demonstrate that, after antigen stimulation, CD22 forms a cap together with BCR, and translocates to lipid rafts. Both co-capping of CD22 with BCR and translocation of CD22 to lipid rafts were markedly blocked by a synthetic alpha2,6-linked sialic acid, Neu5Gcalpha2-6GalbetaSE. These results strongly suggest that synthetic glycan ligand excludes CD22 from BCR-containing lipid rafts. Because CD22-mediated signal regulation requires phosphorylation of CD22 by Lyn that localizes in lipid rafts and is activated by BCR, synthetic glycan ligand regulates localization of CD22 crucial for signal regulation.

Calpain represents a family of Ca(2+)-dependent cytosolic cysteine proteases found in almost all eukaryotes and some bacteria, and is involved in a variety of biological phenomena, including brain function. Several substrates of calpain are aggressively proteolyzed under pathological conditions, e.g., in neurodegenerating processes, fodrin is proteolyzed by calpain. Because very small amounts of substrate are proteolyzed by calpain under normal biological conditions, the molecular identities of calpain substrates are largely unknown. In this study, an extensive survey of the substrates of p94/calpain 3 in COS7 cells was executed using iTRAQ(TM) labeling and 2-D LC-MALDI analysis. p94 was used because: (i) several p94 splicing variants are expressed in brain tissue even though p94 itself is a skeletal-muscle-specific calpain, and (ii) it exhibits Ca(2+)-independent activity in COS cells, which makes it useful for evaluating the effects of p94 protease activity on proteins without perturbing the cells. Our approach revealed several novel protein substrates for p94, including the substrates of conventional calpains, components of the protein synthesis system, and enzymes of the glycolytic pathway. The results demonstrate the usefulness and sensitivity of this approach for mining calpain substrates. A combination of this method with other analytical methods would contribute to elucidation of the biological relevance of the calpain family.

The B cell membrane molecules CD22 and CD72 contain ITIMs in their cytoplasmic portion, and negatively regulate signaling through BCR. Various lines of evidence suggest that ligation of BCR containing IgG (IgG-BCR) transmits augmented signaling due to lack of CD22-mediated signal regulation. However, the signaling capacities of BCR containing IgA and IgE remain largely undefined. In this study, we demonstrate that both IgE-BCR and IgG-BCR, but not IgA-BCR, transmit augmented signaling compared with IgM-BCR. Ligation of IgE-BCR does not induce signaling events required for CD22-mediated signal inhibition, and restoration of these signaling events by coligation of CD22 with BCR abrogates signal augmentation. Furthermore, the cytoplasmic portion of IgE but not that of IgA is sufficient for suppressing CD22-mediated signal inhibition. These findings strongly suggest that the cytoplasmic portion of IgE but not that of IgA reverses CD22-mediated signal inhibition, leading to augmentation of signaling through IgE-BCR but not IgA-BCR. Augmented IgE-BCR signaling appears to play a role in production of large amounts of IgE during helminth infection, whereas regulated signaling through IgA-BCR may be crucial for constitutive production of IgA for mucosal immunity.

4-Hydroxy-2-nonenal (HNE), a racemic mixture of 4R- and 4S-enantiomers, is a major product of lipid peroxidation and is believed to be largely responsible for the cytopathological effects observed during oxidative stress. HNE reacts with histidine to form a stable HNE-histidine Michael addition-type adduct possessing three chiral centers in the cyclic hemiacetal structure. We have previously raised the mAbs, anti-R mAb 310 and anti-S mAb S412, that enantioselectively recognized the R-HNE-histidine and R-HNE-histidine adducts, respectively, and demonstrated the presence of both epitopes in vivo. In the present study, to further investigate the anti-HNE immune response, we analyzed the variable genes and primary structure of these Abs and found that the sequence of R310 was highly homologous to anti-DNA autoantibodies, the hallmark of systemic lupus erythematosus. An x-ray crystallographic analysis of the R310 Fab fragment showed that the R-HNE-histidine adduct binds to a hydrophobic pocket in the antigen-binding site. Despite the structural identity to the anti-DNA autoantibodies, however, R310 showed only a slight crossreactivity with the native double-stranded DNA, whereas the Ab immunoreactivity was dramatically enhanced by the treatment of the DNA with 4-oxo-2-nonenal (ONE), an analog of HNE. Moreover, the 7-(2-oxo-heptyl)-substituted 1,N2-etheno-type ONE-2'-deoxynucleoside adducts were identified as alternative epitopes of R310. Molecular mimicry between the R-HNE-histidine configurational isomers and the ONE-DNA base adducts is proposed for the dual crossreactivity.

Various abnormalities have been described in B cells from patients with systemic autoimmune diseases such as systemic lupus erythematosus (SLE) and lupus-prone mice. Many of the abnormalities do not appear to be connected with the pathogenesis of the disease. However, various animal models developing lupus-like disease including both spontaneous mutans such as (NZB x NZW)F1 and MRL/lpr and mice generated by transgenic or knockout technology such as Bim-deficient and CD40L-transgenic mice show defect in apoptosis of mature B cells induced by ligation of the B cell antigen receptor (BCR). BCR-mediated apoptosis appears to be involved in deletion of self-reactive B cells. Thus, defect in BCR-mediated apoptosis is a widely observed B cell abnormality in lupus-prone mice and may play a role in the pathogenesis of systemic autoimmune diseases by abrogating deletion of self-reactive B cells.

High-resolution the Mn Kalpha1 X-ray fluorescence spectra (HRXRF) were measured for a variety of manganese compounds, the oxidation number (valence) of which was from II to VII. Plots of the valence against the full width at half maximum (FWHM) and the chemical shift of the Kalpha1 X-ray fluorescence spectra give a curve and a liniar relation, respectively. The coefficient of correlation (R2) for the latter plot was 0.989 between valency II and VII. More excellent linearlity from III to IV was obtained with R2 = 0.995, which enabled us to determine the oxidation number of manganese atom in a nickel ion doped spinel structure, LiMn2O4 (LiNixMn(2-x)O4, with 0.2 < x < 0.42). From the valence of manganese ion obtained from HRXRF, the oxidation number of nickel ion is concluded to be divalent.

The inhibitory coreceptors CD22 and CD72 downmodulate B-cell receptor (BCR) signaling and function as a molecular switch, determining whether antigen-stimulated B cells undergo apoptosis or proliferation. These coreceptors carry an intrinsic property for associating with the BCR, and this association is crucial for the initiation of signal inhibition through phosphorylation of these coreceptors by BCR-associated kinases. Recent findings have demonstrated that signal inhibition by these coreceptors is regulated by ligands for the coreceptors and by molecules binding to the coreceptors or the BCR. Moreover, signal inhibition by CD22 depends on the BCR isotype. These findings suggest a dynamic regulation of these coreceptors through molecular interactions on the B-cell surface.

Several studies indicate that CD4(+) T cells, macrophages, and dendritic cells initially mediate intestinal inflammation in murine models of human inflammatory bowel disease. However, the initial role of B cells in the development of intestinal inflammation remains unclear. In this study we present evidence that B cells can trigger intestinal inflammation using transgenic (Tg) mice expressing CD40 ligand (CD40L) ectopically on B cells (CD40L/B Tg). We demonstrated that CD40L/B Tg mice spontaneously developed severe transmural intestinal inflammation in both colon and ileum at 8-15 wk of age. In contrast, CD40L/B TgxCD40(-/-) double-mutant mice did not develop colitis, indicating the direct involvement of CD40-CD40L interaction in the development of intestinal inflammation. The inflammatory infiltrates consisted predominantly of massive aggregated, IgM-positive B cells. These mice were also characterized by the presence of anti-colon autoantibodies and elevated IFN-gamma production. Furthermore, although mice transferred with CD4(+) T cells alone or with both CD4(+) T and B220(+) B cells, but not B220(+) cells alone, from diseased CD40L/B Tg mice, develop colitis, mice transferred with B220(+) B cells from diseased CD40L/B Tg mice and CD4(+) T cells from wild-type mice also develop colitis, indicating that the Tg B cells should be a trigger for this colitis model, whereas T cells are involved as effectors. As it has been demonstrated that CD40L is ectopically expressed on B cells in some autoimmune diseases, the present study suggests the possible contribution of B cells in triggering intestinal inflammation in human inflammatory bowel disease.

The CD40 molecule transmits a signal that abrogates apoptosis induced by ligation of the antigen receptor (BCR) in both primary B cells and B-cell lines such as WEHI-231. Expression of Bcl-xL and A1, antiapoptotic members of the Bcl-2 family, is enhanced by CD40 ligation, and is suggested to mediate CD40-induced B-cell survival. CD40 ligation also promotes cell cycle progression by increasing the levels of cyclin-dependent kinases (CDKs) required for cell cycle progression, and reducing expression of the CDK inhibitor p27(kip1). Here we demonstrate that cell cycle inhibition by retrovirus-mediated p27(kip1) expression does not modulate the levels of Bcl-xL or A1, but significantly reduces the survival of BCR-ligated WEHI-231 cells by CD40 ligation. This indicates that cell cycle progression is crucial for CD40-mediated survival of B cells.

Ag-induced B cell proliferation in vivo requires a costimulatory signal through CD40, whereas B cell Ag receptor (BCR) ligation by anti-Ig H chain Abs, such as anti-Ig micro H chain Ab and anti-Ig delta H chain Ab, alone induces proliferation of B cells in vitro, even in the absence of CD40 ligation. In this study, we demonstrate that CD40 signaling is required for survival and proliferation of B cells stimulated by protein Ags in vitro as well as in vivo. This indicates that the in vitro system represents B cell activation in vivo, and that protein Ags generate BCR signaling distinct from that by anti-Ig H chain Abs. Indeed, BCR ligation by Ags, but not by anti-Ig H chain Abs, efficiently phosphorylates the inhibitory coreceptors CD22 and CD72. When these coreceptors are activated, anti-Ig H chain Ab-stimulated B cells can survive and proliferate only in the presence of CD40 signaling. Conversely, treatment of Ag-stimulated B cells with anti-CD72 mAb blocks CD72 phosphorylation and induces proliferation, even in the absence of CD40 signaling. These results strongly suggest that activation of B cells by anti-Ig H chain Abs involves their ability to silence the inhibitory coreceptors, and that the inhibitory coreceptors install requirement of CD40 signaling for survival and proliferation of Ag-stimulated B cells.

Pten is a tumor suppressor gene mutated in human cancers. We used the Cre-loxP system to generate a B cell-specific mutation of Pten in mice (bPten(flox/flox)mice). bPten(flox/flox) mice showed elevated numbers of B1a cells and increased serum autoantibodies. Among B2 cells in bPten(flox/flox) spleens, numbers of marginal zone B (MZB) cells were significantly increased while those of follicular B (FOB) cells were correspondingly decreased. Pten-deficient B cells hyperproliferated, were resistant to apoptotic stimuli, and showed enhanced migration. The survival kinase PKB/Akt was highly activated in Pten-deficient splenic B cells. In addition, immunoglobulin class switch recombination was defective and induction of activation-induced cytidine deaminase (AID) was impaired. Thus, Pten plays a role in developmental fate determination of B cells and is an indispensable regulator of B cell homeostasis.

The immunoglobulin G (IgG)-containing B lymphocyte antigen receptor (IgG-BCR) transmits a signal distinct from that of IgM-BCR or IgD-BCR, although all three use the same signal-transducing component, Igalpha/Igbeta. Here we demonstrate that the inhibitory coreceptor CD22 down-modulates signaling through IgM-BCR and IgD-BCR, but not that through IgG-BCR, because of the IgG cytoplasmic tail, which prevents CD22 phosphorylation. These results suggest that the cytoplasmic tail of IgG specifically enhances IgG-BCR signaling by preventing CD22-mediated signal inhibition. Enhanced signaling through IgG-BCR may be involved in efficient IgG production, which is crucial for immunity to pathogens.

CD40 ligand (CD40L) is ectopically expressed on B cells in patients with systemic lupus erythematosus (SLE) and lupus-prone BXSB mice. To assess the role of the ectopic CD40L expression in development of SLE, we have established transgenic mice expressing CD40L on B cells. Some of the 12- to 14-mo-old CD40L-transgenic mice spontaneously produced autoantibodies such as antinuclear Abs, anti-DNA Abs, and antihistone Abs. Moreover, approximately half of the transgenic mice developed glomerulonephritis with immune-complex deposition, whereas the kidneys of the normal littermates showed either no pathological findings or only mild histological changes. These results indicate that CD40L on B cells causes lupus-like disease in the presence of yet unknown environmental factors that by themselves do not induce the disease. Thus, ectopic CD40L expression on B cells may play a crucial role in development of SLE.

Signaling through the B cell antigen receptor (BCR) is negatively regulated by the SH2 domain-containing protein-tyrosine phosphatase SHP-1, which requires association with tyrosine-phosphorylated proteins for activation. Upon BCR ligation, SHP-1 has been shown to associate with the BCR, the cytoplasmic protein-tyrosine kinases Lyn and Syk, and the inhibitory co-receptors CD22 and CD72. How SHP-1 is activated by BCR ligation and regulates BCR signaling is, however, not fully understood. Here we demonstrate that, in the BCR-expressing myeloma line J558L mu 3, CD72 expression reduces the BCR ligation-induced phosphorylation of the BCR component Ig alpha/Ig beta and its cytoplasmic effectors Syk and SLP-65. Substrate phosphorylation was restored by expression of dominant negative mutants of SHP-1, whereas the SHP-1 mutants failed to enhance phosphorylation of the cellular substrates in the absence of CD72. This indicates that SHP-1 is efficiently activated by CD72 but not by other pathways in J558L mu m3 cells and that inhibition of SHP-1 specifically activated by CD72 reverses CD72-induced dephosphorylation of cellular substrates in these cells. Taken together, BCR-induced SHP-1 activation is likely to require inhibitory co-receptors such as CD72, and SHP-1 appears to mediate the negative regulatory effect of CD72 on BCR signaling by dephosphorylating Ig alpha/Ig beta and its downstream signaling molecules Syk and SLP-65.

PTEN, a tumor suppressor gene, is essential for embryogenesis. We used the Cre-loxP system to generate a T cell-specific deletion of the Pten gene (Pten(flox/-) mice). All Pten(flox/-) mice develop CD4+ T cell lymphomas by 17 weeks. Pten(flox/-) mice show increased thymic cellularity due in part to a defect in thymic negative selection. Pten(flox/-) mice exhibit elevated levels of B cells and CD4+ T cells in the periphery, spontaneous activation of CD4+ T cells, autoantibody production, and hypergammaglobulinemia. Pten(flox/-) T cells hyperproliferate, are autoreactive, secrete increased levels of Th1/Th2 cytokines, resist apoptosis, and show increased phosphorylation of PKB/Akt and ERK. Peripheral tolerance to SEB is also impaired in Pten(flox/-) mice. PTEN is thus an important regulator of T cell homeostasis and self-tolerance.

The B cell antigen receptor (BCR) is composed of the membrane form of the immunoglobulin (Ig) and the Ig-alpha/Ig-beta heterodimer, which function as the antigen recognition component and the signaling component, respectively. A signal transmitted by BCR modulates gene expression, adhesion or survival, thereby determining the fate of antigen-encountered B cells. BCR proximal signaling occurs within cholesterol- and sphingolipid-rich plasma membrane microdomains termed lipid rafts, and involves tyrosine kinases such as Lyn, Syk and Btk and the adapter molecule SLP65/BLNK. Although the distal signaling cascades via BCR are not yet fully elucidated, various components are already identified, such as lipid kinases and small G-proteins. BCR signaling is regulated by various membrane molecules termed co-receptors such as CD19 and CD22. The BCR co-receptors appear to be required for normal immune functions. Viral proteins such as LMP2 also regulate BCR signaling to maintain viral latency. Various aspects of BCR signaling and its regulatory mechanisms are discussed in this issue.

Although the B cell antigen receptor (BCR) transmits survival and activation signals, BCR ligation can induce apoptosis in both immature and mature B cells. BCR-mediated apoptosis is suggested to play a role in self-tolerance by deleting self-reactive B cells. Generation of an apoptotic signal through BCR appears to depend on the composition of the higher order BCR complex and is suggested to occur outside the plasma membrane microdomains, termed lipid rafts. During BCR-mediated apoptosis, mitochondrial dysfunction is induced and is essential for apoptosis, probably by activating both caspases, cysteine proteases that play a central role in apoptosis, and caspase-independent effectors for apoptosis. Although signaling pathways for apoptosis are not yet fully defined in BCR-mediated apoptosis, expression of the proto-oncogene product c-Myc is enhanced upon BCR ligation, and c-Myc appears to mediate BCR ligation-induced apoptosis by causing mitochondrial dysfunction, suggesting that BCR-mediated apoptosis is a form of Myc-induced apoptosis.

Ras is essential for the transition from early B cell precursors to the pro-B stage, and is considered to be involved in the signal cascade mediated by pre-B cell antigen receptors. To examine the role of p21(ras) in the late stage of B cell differentiation, we established transgenic mice (TG) expressing a dominant-inhibitory mutant of Ha-ras (Asn-17 Ha-ras) in B lineage cells at high levels after the early B cell precursor stage. Expression of p21(Asn-17) (Ha-ras) was associated with a prominent reduction in the number of late pre-B cells, but had little effect on proliferation of early pre-B cells. Inhibition of p21(ras) activity markedly reduced the life span of pre-B cells, due, at least in part, to downregulation of the expression of an antiapoptotic protein, Bcl-xL. Thus, the apparent role for p21(ras) activity in pre-B cell survival may explain the decreased numbers of late pre-B cells in Asn-17 Ha-ras TG. Consistent with this possibility, overexpression of Bcl-2 in Asn-17 Ha-ras TG reversed the reduction in the number of late pre-B cells undergoing immunoglobulin light chain gene (IgL) rearrangement and progressing to immature B cells. These results suggest that p21(ras) mediates effector pathways responsible for pre-B cell survival, which is essential for progression to the late pre-B and immature B stages.

Antigen receptor ligation-induced apoptosis is thought to play a role in self-tolerance by deleting autoreactive lymphocytes. Antigen receptor ligation-induced apoptosis of mature T cells and T cell lines requires autocrine or paracrine activation of Fas (CD95/APO-1). Whether B cell antigen receptor (BCR)-mediated apoptosis requires Fas or related molecules is unclear. Here we demonstrate that expression of either CrmA, the cowpox virus serpin, or an inhibitor of the adapter protein FADD/MORT1 blocks Fas-mediated apoptosis but has no effect on BCR ligation-induced apoptosis of the B cell line WEHI-231. In contrast, expression of Bcl-2 blocks BCR-mediated but not Fas-induced apoptosis in WEHI-231 cells. These results indicate that BCR ligation activates an apoptotic signaling pathway distinct from Fas-mediated apoptosis in WEHI-231 cells, and that BCR-mediated apoptosis of WEHI-231 cells does not require Fas or related molecules such as DR3, DR4 and DR5, as all of these death receptors require FADD/MORT1 and/or CrmA-sensitive caspases for induction of apoptosis. Moreover, extensive BCR ligation induces death of mature B cells from C57BL/6-lpr/lpr mice as efficiently as those from C57BL/6 mice, indicating that Fas is not essential for BCR-mediated apoptosis of mature B cells. In contrast, BCR ligation-induced apoptosis is reduced in mature B cells from MRL mice and this is not affected by the lpr mutation. Since MRL-lpr/lpr mice but not C57BL/6-lpr/lpr mice develop severe autoimmune disease, defects in BCR-mediated apoptosis in the MRL background, together with lpr mutation, may contribute to the development of severe autoimmune disease in MRL-lpr/lpr mice by allowing survival of self-reactive B cells.

The immunoreceptor tyrosine-based inhibition motif (ITIM) is found in various membrane molecules such as CD22 and the low-affinity Fc receptor for IgG in B cells and the killer cell-inhibitory receptor and Ly-49 in NK cells. Upon tyrosine phosphorylation at the ITIMs, these molecules recruit SH2 domain-containing phosphatases such as SH2-containing tyrosine phosphatase-1 and negatively regulate cell activity. The B cell surface molecule CD72 carries an ITIM and an ITIM-like sequence. We have previously shown that CD72 is phosphorylated and recruits SH2-containing tyrosine phosphatase-1 upon cross-linking of the Ag receptor of B cells (BCR). However, whether CD72 modulates BCR signaling has not yet been elucidated. In this paper we demonstrate that expression of CD72 down-modulates both extracellular signal-related kinase (ERK) activation and Ca2+ mobilization induced by BCR ligation in the mouse B lymphoma line K46micromlambda, whereas BCR-mediated ERK activation was not reduced by the ITIM-mutated form of CD72. Moreover, coligation with CD72 with BCR reduces BCR-mediated ERK activation in spleen B cells of normal mice. These results indicate that CD72 negatively regulates BCR signaling. CD72 may play a regulatory role in B cell activation, probably by setting a threshold for BCR signaling.

Self-tolerance is induced in B cells at various maturational stages by diverse self-antigens B cell tolerance involves multiple mechanisms, ie. clonal deletion, clonal anergy, receptor editing and maturation arrest. The mechanism utilized for self-tolerance depends on both the maturational stage of B cells and the molecular nature of the self-antigens. B cell tolerance is abrogated by various mechanisms such as defects in inhibitory co-receptors, overexpression of CD19, T cell help and defects in the death receptor Fas (CD95). Since all of these molecules regulate B cell apoptosis mediated by either the antigen receptor or Fas, B cell apoptosis may play a role in the induction and maintenance of B cell tolerance. Moreover, environmental factors such as intestinal lipopolysaccharide also play a role in the breakdown of B cell tolerance.

Deregulated expression of c-Myc has been shown to induce or enhance apoptosis in various different cell types. c-Myc requires p53 for apoptosis in some but not all the cell types, indicating heterogeneous mechanisms for c-Myc-induced apoptosis. In B lymphoma line WEHI-231, stable expression of c-Myc has been demonstrated to protect cells from BCR-mediated apoptosis. However, stable expression of c-Myc carrying pro-apoptotic functions may generate variant cells resistant to apoptosis. By utilizing an inducible system for c-Myc, we demonstrated here that deregulated expression of c-Myc induced apoptosis of WEHI-231 by itself, indicating that c-Myc induces apoptosis in WEHI-231 as is the case for other cell types. When transactivation of p53 was inactivated, WEHI-231 cells overexpressing c-Myc no longer underwent apoptosis in the absence of other stimuli, but showed markedly enhanced apoptosis in the presence of BCR ligation. These results indicate that deregulated c-Myc expression enhances apoptosis by a p53-independent pathway in the presence of BCR signaling but requires p53 for apoptosis in the absence of BCR crosslinking in WEHI-231. BCR ligation may thus activate a p53-independent pathway of c-Myc-induced apoptosis.

Co-receptors have been shown to regulate the antigen-receptor signaling threshold for B cell responses by modulating the activation of signaling molecules that are essential for transmitting a signal through the antigen-receptor. Co-receptors appear to modulate the signaling threshold for B cell tolerance distinctly from that for B cell activation.

Antigen receptor (BCR) transduces either pro-apoptotic or anti-apoptotic signals of mature B cells depending on the nature of stimuli. Mature B cells also undergo apoptosis by signaling through CD95. Those apoptotic signals through BCR or CD95 are blocked by various transmembrane signaling such as those via CD40, BCR, CD21 and IL-4 receptor, presumably generated by interaction with T helper cells or the components of innate immunity such as complements. Induction of B cell apoptosis and its regulation are likely to play important roles in humoral immunity. Indeed, spontaneous models as well as patients of systemic autoimmune diseases show defects in apoptosis of mature B cells mediated by BCR or CD95.

Cross-linking of surface immunoglobulin (sIg) has been shown to induce either activation or apoptosis of mature B cells presumably depending on the nature of antigens. However, the nature of antigens for induction of mature B-cell apoptosis is not yet fully understood. We cross-linked sIg of mature B cells with various amounts of either anti-Ig antibodies in the soluble form or anti-Ig coupled to erythrocytes or myeloma cells as surrogate membrane-bound antigens. Anti-Ig antibodies coupled to cell surface membrane induced rapid and extensive apoptosis of normal spleen B cells even in the absence of signalling via the Fc receptor. In contrast, soluble anti-Ig induced proliferation or apoptosis of mature B cells depending on the concentration of anti-Ig. The extent of apoptosis induced by soluble anti-Ig was limited compared to that induced by membrane-bound anti-Ig. These results suggest that mature B cells undergo apoptosis or proliferation depending on whether antigens are soluble or membrane-bound and on antigen doses.

Activation signals of lymphocytes are negatively regulated by the membrane molecules carrying the immunoreceptor tyrosine-based inhibition motif (ITIM). Upon tyrosine phosphorylation, ITIMs recruit SH2-containing phosphatases such as SHP-1, resulting in down-modulation of cell activation. We showed that the cytoplasmic domain of the CD72 molecule carries an ITIM and is associated in vitro with SHP-1 upon tyrosine phosphorylation. Moreover, cross-linking of B cell Ag receptor (BCR) enhances both tyrosine phosphorylation of CD72 and association of CD72 with SHP-1 in B cell line WEHI-231. These results indicate that CD72 recruits SHP-1 upon tyrosine phosphorylation induced by BCR signaling, suggesting that CD72 is a negative regulator of BCR signaling.

Lines of evidence suggest that self-reactive B cells are deleted or functionally inactivated at the several different steps of maturation from immature B cells to antibody producing cells. These self-tolerance mechanisms appear to involve B cell apoptosis induced by signaling via the antigen receptor (surface immunoglobulin) or Fas. In mice prone to systemic autoimmune diseases such as bcl-2 transgenic, NZB or (NZB x NZW) F1 mice, antigen receptor-mediated B cell apoptosis is defective. In another autoimmunity-prone mice MRL/lpr, autoantibody production requires defects of Fas in B cells. These findings strongly suggest that the defects in B cell tolerance play an important role in the pathogenesis of systemic autoimmune diseases.

Several lines of evidence suggest that interaction with antigens generates a negative signal via the antigen receptor of B lymphocytes (cell surface immunoglobulin; sIg), resulting in apoptosis, growth arrest or functional inactivation, and that activation of B cells requires an additional co-stimulatory signal such as a T cell-derived signal through the B cell membrane molecule CD40. In the B cell line WEHI-231, sIg crosslinking induces apoptosis and cell cycle arrest at the late G1 phase, both of which are reversed by CD40 signaling. Crosslinking of sIg reduces the activity of cyclin dependent kinase (Cdk)2 required for cell cycle progression in the late G1 phase by induction of a Cdk inhibitor (CKI) p27Kip1, but the induction of p27Kip1 is abrogated by CD40 signaling. These results strongly suggest that p27Kip1 plays some role in negative signaling via sIg, resulting in growth arrest of antigen-stimulated B cells.

Alymphoplasia (aly) mice are thought to provide a new model for systemic Sjögren's syndrome (SS), since they reveal remarkable infiltration of mononuclear cells into salivary glands, lachrymal glands and kidneys, and show histological findings similar to those in patients with SS. Cell transfer experiments demonstrate that T cells induce the infiltration of mononuclear cells into several tissues in aly mice. To analyse the pathogenesis of cell infiltration in various tissues, we examined T-cell receptor (TCR) V beta usage of T cells in salivary glands, lachrymal glands and kidneys from aly mice, using family-polymerase chain reaction (PCR) and PCR-single-strand conformation polymorphism (SSCP) methods. The results of SSCP demonstrated that the infiltrating T cells in the three organs expanded clonally, suggesting that they proliferate by antigen-driven stimulation. Some TCR V beta genes (V beta 1, 3, 6, 11, 12, 16) were commonly used in salivary glands, lachrymal glands and kidneys, while the V beta 7 gene was specifically expressed in kidneys. SSCP also showed that there were a few shared T-cell clones (V beta 3- and V beta 6-positive cells) among the three tissues. Indeed, sequence analysis of accumulated T cells showed that a conserved amino acid (leucine) at position 98 in the TCR V beta complementary determining region (CDR) 3 was detected in all organs at high frequency (41-57%) and the amino acid sequence motif (LG) was specifically conserved at a frequency of 32% in the three organs. In conclusion, T cells that infiltrate into lachrymal glands, salivary glands and kidneys of aly mutant mice might recognize shared common epitopes in all three organs and a kidney-specific antigen.

Mice homozygous for an autosomal recessive mutation aly (alymphoplasia) lack both lymph nodes and Peyer's patches, and show defects in both humoral and cellular immunity. Histopathological analysis revealed chronic inflammatory changes in exocrine organs such as the salivary gland, lacrimal gland, and pancreas of the homozygotes (aly/aly), but not the heterozygotes (aly/+). In these exocrine organs, mononuclear cells consisting mainly of CD4+ T cells infiltrate periductal areas, and, in some cases, the cell infiltration extended to lobules. The inflammatory changes in exocrine organs were transferred by a T cell-enriched fraction of spleen cells from homozygous animals. These results suggest that autoimmune mechanisms mediated by self-reactive T cells may be involved in the inflammatory lesions of various exocrine organs in the homozygous mice, although these mice show immunodeficiency. Inflammatory changes were also observed in the lung of the homozygotes. Since Sjögren's syndrome is characterized by diffuse lymphocyte infiltration in the periductal areas of the lacrimal and salivary glands and is occasionally associated with pulmonary disease, aly/aly mice may serve as a unique spontaneous model of Sjögren's syndrome.

The cross-linking of surface immunoglobulins (sIg) of B cells can transmit a negative signal, resulting in cell cycle arrest, apoptosis or both. Signaling via the B cell antigen CD40 reverses the sIg-mediated negative signaling and induces activation and proliferation of B cells. We investigated the molecular mechanism for cell cycle regulation by negative and positive signaling via sIg and CD40, respectively, by using the B cell line WEHI-231. Cross-linking of sIg almost completely reduced the activity of cyclin-dependent kinase (Cdk) 2, essential for cell cycle progression in the late G1 phase, although the level of Cdk2 was not reduced. Among the factors that regulate Cdk2 activation, the activity of the Cdk-activating kinase (CAK) appeared intact and cyclin E was reduced only partially in sIg-cross-linked WEHI-231. In contrast, sIg cross-linking induced a significant Cdk inhibitor (CKI) activity. Since a 27-kDa protein was co-precipitated with Cdk2 in anti-Ig-treated, but not untreated WEHI-231, and the CKI activity in anti-Ig-treated WEHI-231 was neutralized by anti-p27Kip1 antibodies, it is most likely that p27Kip1 is responsible for the CKI activity induced by sIg cross-linking. p27Kip1 may thus play a role in growth inhibition of B cells by negative signaling via sIg. In contrast, CD40 signaling enhanced Cdk2 activity and reduced the p27Kip1 level in anti-Ig-treated WEHI-231, suggesting that the reduction of p27Kip1 plays an important role in the abrogation of sIg-mediated growth arrest by CD40 signaling. Taken together, p27Kip1 is likely to be a crucial target molecule of the negative signaling via sIg and the positive signaling via CD40 essential for T cell-dependent immune responses.

The alymphoplasia (aly) mutation of mice causes the systemic absence of lymph nodes, Peyer's patches and well-defined lymphoid follicles in the spleen. We found that antibody responses are elicited, albeit weakly, to either T cell-dependent or T cell-independent antigen by aly/aly mutants. However, isotype switching was defective. The T cell-dependent immune response was not elicited in splenectomized aly/aly mice. Neither hypermutation nor germinal center formation was observed in aly/aly mice. These results suggest that T-B collaboration requires either lymph nodes or spleen, and that hypermutation and affinity maturation depend on germinal center formation.

Mature B cells undergo programmed cell death when surface (s) Ig is extensively multimerized. A signal that blocks death of B cells is thus required for activation of B cells in response to antigen stimulation. Here we show that only a few diverse transmembrane signals capable of inducing activation and proliferation of B cells blocked sig-mediated death of normal mature B cells, and that there is no correlation between mitogenic activity and the ability to rescue B cells from death. The results suggest that a specific signal is required for abrogating B cell death induced by sig cross-linking. Signaling via IL-4 receptor and CD40, both of which are derived from activated T cells, blocked sig-mediated death, as described previously. Signaling through a B cell antigen CD72, a counter-receptor of the pan-T antigen CD5, also blocked death of anti-Ig-treated mouse spleen B cells. CD72 signal may play a role in survival of B cells at the initial step of T-B interaction, where resting T cells recognize antigens presented by B cells. Moreover, B cell death by anti-Ig was blocked by T cell-independent antigens such as lipopolysaccharide and dextran sulfate, and spleen B cells from New Zealand mice, which are prone to autoantibody-dependent autoimmune diseases, were resistant to sig-mediated death. Mechanisms for blocking sig-mediated death may therefore be required in antibody response to foreign antigens regardless of T independence or T dependence and in autoantibody production.

A mAb J43 has been produced against the product of the mouse PD-1 gene, a member of the Ig gene superfamily, which was previously isolated from an apoptosis-induced T cell hybridoma (2B4.11) by using subtractive hybridization. Analyses by flow cytometry and immunoprecipitation using the J43 mAb revealed that the PD-1 gene product is a 50-55 kDa membrane protein expressed on the cell surface of several PD-1 cDNA transfectants and 2B4.11 cells. Since the molecular weight calculated from the amino acid sequence is 29, 310, the PD-1 protein appears to be heavily glycosylated. Normal murine lymphoid tissues such as thymus, spleen, lymph node and bone marrow contained very small numbers of PD-1(+) cells. However, a significant PD-1(+) population appeared in the thymocytes as well as T cells in spleen and lymph nodes by the in vivo anti-CD3 mAb treatment. Furthermore, the PD-1 antigen expression was strongly induced in distinct subsets of thymocytes and spleen T cells by in vitro stimulation with either anti-CD3 mAb or concanavalin A (Con A) which could lead T cells to both activation and cell death. Similarly, PD-1 expression was induced on spleen B cells by in vitro stimulation with anti-IgM antibody. By contrast, PD-1 was not significantly expressed on lymphocytes by treatment with growth factor deprivation, dexamethasone or lipopolysaccharide. These results suggest that the expression of the PD-1 antigen is tightly regulated and induced by signal transduction through the antigen receptor and do not exclude the possibility that the PD-1 antigen may play a role in clonal selection of lymphocytes although PD-1 expression is not required for the common pathway of apoptosis.

Activation mechanisms of B-1 (Ly-1 B) cells have been suggested to be different from those of conventional B cells. To assess the role of various interleukins (IL) in the activation of B-1 cells, we injected IL-4, IL-5 or IL-10 into nonanemic anti-red blood cells (RBC) autoantibody-transgenic mice, in which conventional B cells are clonally deleted but peritoneal B-1 cells persist without secreting Ig. Intraperitoneal or intramuscular injection of IL-5 or IL-10, but not IL-4, increased the number of antibody-producing peritoneal B-1 cells by four- to five-fold, resulting in increased anti-RBC serum autoantibody and induction of hemolytic anemia. These results suggest that IL-5 or IL-10 may play an important role in the terminal differentiation of B-1 cells into antibody-producing cells in vivo.

Our recent studies on an autoantibody-transgenic mouse line demonstrated that peritoneal B-1 cells are responsible for autoimmune symptoms. However, whether B-1 cells in the peritoneum are generally involved in the pathogenesis of autoimmune disease remains controversial. To test the possible involvement of peritoneal B-1 cells in autoimmune symptoms of autoimmune-prone NZB mice, we eliminated the peritoneal cells by hypotonic shock with repeated i.p. injection of distilled water every 7 days into neonatal or 8-week-old NZB mice. By this treatment, B-1 cells, which self-renew within the peritoneal cavity, are expected to be preferentially eliminated, while other peritoneal cells can be easily supplied from bone marrows after this treatment. Indeed, in distilled water-treated old NZB mice, the number of B-1 cells decreased in spleen as well as in lamina propria of the gut but the numbers of conventional B cells and T cells did not change. Moreover, the production of autoantibodies against erythrocytes significantly decreased and the occurrence of autoimmune hemolytic anemia was reduced in 12-month-old treated NZB mice. Similarly, the elimination of peritoneal cells of NZB/NZW (NZB/W) F1 mice by water injection decreased anti-DNA IgG antibodies in the sera and reduced the pathological changes of the kidney. These results suggest that peritoneal B-1 cells may be a source of autoantibody-producing cells in autoimmune diseases of NZB and NZB/W F1 mice.

In autoimmune diseases, IgG class autoantibodies are generally considered to be more pathognomonic than IgM class ones. Although Epstein-Barr virus (EBV)-transformation of lymphocytes is a useful method to obtain human monoclonal autoantibodies, it tends to result predominantly in IgM-producing cells. We depleted IgM+ cells before EBV-transformation with a Magnetic Cell Separator (MACS) in order to increase the chance of acquisition of cells producing IgG class anti-thyrotropin (TSH) receptor antibodies (TRAb). As a result, we obtained four independent B cell clones producing IgG class monoclonal thyroid-stimulating antibodies (TSAb) from three patients with Graves' disease. None of these clones showed any TSH binding inhibitor immunoglobulin (TBII) activity, suggesting independence of TSAb-producing lymphocytes from those producing TBII.

Apoptosis (programmed cell death) has been suggested to be involved in clonal elimination of self-reactive lymphocytes for the normal function of the immune system. By crosslinking the antigen receptor (surface immunoglobulin; sIg) on the peritoneal B cells of normal mice, we found that strong crosslinking of sIg induces apoptosis of mature B cells, suggesting that interaction with membrane-bound self-antigens may eliminate self-reactive mature B cells by apoptosis. Antigen-receptor-mediated B cell apoptosis is blocked when a signal is transduced via the CD40 molecule on the B cell surface. Because the ligand of CD40 (CD40L) is expressed on activated T helper cells, B cells may escape from apoptosis and are activated when the immune system interacts with foreign antigens, which are normally able to activate T helper cells. Moreover, sIg crosslinking fails to induce apoptosis of both bcl-2-transgenic mice and autoimmune-disease-prone New Zealand mice. In these mice, the defect in sIg-mediated apoptosis of mature B cells may allow generation of self-reactive B cells, resulting in pathogenic consequences.

About a half of the antierythrocyte autoantibody transgenic (autoAb Tg) mice, in which almost all B cells are detected in the spleen, lymph nodes, and Peyer's patches, but not in the peritoneal cavity, suffer from autoimmune hemolytic anemia. The occurrence of this disease is strongly linked to production of autoAb by activated peritoneal B-1 cells in the Tg mice. In this study, we have shown that oral administration of lipopolysaccharides (LPS) activated B-1 cells in the lamina propria of the gut as well as the peritoneal cavity in the healthy Tg mice and induced the autoimmune symptoms in all the Tg mice. The activation of peritoneal and lamina propria B-1 cells by enteric LPS is found not only in the anti-RBC autoAb Tg mice and normal mice but also in the aly mice which congenitally lack lymph nodes and Peyer's patches. These results suggest that B-1 cells in the two locations may form a common pool independent of Peyer's patches and lymph nodes, and can be activated by enteric thymus-independent antigens or polyclonal activators such as LPS. The induction of autoimmune hemolytic anemia in the Tg mice by enteric LPS through the activation of B-1 cells in the lamina propria of gut and in the peritoneal cavity suggests that B-1 cells and bacterial infection may play a pathogenic role in the onset of autoimmune diseases.

We induced differentiation of mouse embryonic stem (ES) cells into lymphoid cells by culturing in methylcellulose, followed by the co-culture with a bone marrow stromal cell line ST2 in the presence of IL-7. These lymphoid cells expressed transcripts of the recombination activating genes, RAG-1 and RAG-2, as well as the C mu gene, although the lymphoid cells did not express surface antigens specific to T or B lymphocytes such as T200, CD4, CD8 and B220, or transcripts of B lymphocyte-specific genes such as lambda 5 and mb-1. D-J rearrangement was detectable in the lymphoid cells differentiated from ES cells in vitro and a sizeable number of both B and T lymphocytes were generated in vivo when the ES-derived lymphoid cells were transferred into RAG-2-deficient mice, which contain no B or T lymphocytes. The results indicate that in the in vitro co-culture system, ES cells give rise to immature lymphocyte precursors which have potentials to differentiate into both mature B and T lymphocytes in vivo. The ES-derived lineage marker-negative lymphocyte precursors would thus provide useful materials for studying early events of lymphopoiesis.

Background: Programmed cell death (apoptosis) is an essential process in the development of various tissues and seems to be involved in the elimination of self-reactive immature T and B lymphocytes when they interact with self antigens. Indeed, signaling through the antigen receptor of immature T cells induces their apoptotic cell death. Immature B cells have also been shown to be eliminated when they interact with antigens, although the involvement of apoptosis has yet to be demonstrated. In contrast, little is known about the elimination of mature lymphocytes upon interaction with antigens. We have previously demonstrated that Ly1 B cells in the peritoneal cavity of transgenic mice undergo apoptotic cell death upon interaction with antigens. As Ly1 B cells constitute a B-cell lineage distinct from conventional B cells, it is important to know whether conventional B cells also undergo apoptosis upon antigen-receptor cross-linking., Results: Our experiments show that, in vivo, strong cross-linking of cell-surface immunoglobulins induced apoptotic death of normal, mature B cells in the peritoneal cavity, regardless of whether they were conventional or Ly1 B cells. The same treatment did not kill, but rather activated, B cells in bcl-2-transgenic, apoptosis-resistant mice. Peritoneal B cells from autoimmune-disease-prone New Zealand mouse strains were also found to be resistant to cell death induced by surface immunoglobulin cross-linking., Conclusion: Self-reactive B cells are eliminated by the binding of antigen at both mature and immature stages. B-cell activation appears to require, in addition to antigen binding, a second signal that induces expression of rescue molecules such as the bcl-2 gene product. Resistance to B-cell apoptosis induced by antigen receptor cross-linking may play a crucial role in the production of autoantibodies and in the pathogenesis of the autoimmune diseases found in the strains of mice used here.

To test whether the product of the bcl-2 proto-oncogene blocks clonal deletion of self-reactive B cells, we have generated transgenic mice carrying the bcl-2 gene and the immunoglobulin genes for the anti-erythrocyte 4C8 antibody. In these transgenic mice, clonal deletion of self-reactive immature B cells in the bone marrow was not inhibited in spite of expression of the bcl-2 gene. In contrast, self-antigen-induced clonal deletion of mature self-reactive Ly-1 B (B1) cells in the peritoneal cavity was inhibited in the transgenic mice. These results indicate that the mechanism for clonal deletion of immature self-reactive B cells in the bone marrow differs from that of mature self-reactive B cells in the periphery.

In mice transgenic for an autoantibody, self-reactive B cells have been shown to be eliminated upon interaction with membrane-bound self-antigens in the periphery as well as in the bone marrow, suggesting that both immature and mature B cells are eliminated by multimerization of surface immunoglobulins (sIg). Activation of mature B cells by antigens may thus require a second signal that inhibits sIg-mediated apoptosis. Such a second signal is likely to be provided by T helper cells, because B-cell tolerance is more easily induced in the absence of T helper cells. To assess the molecular nature of the signal that inhibits sIg-mediated apoptosis, we used anti-IgM-induced apoptotic death of WEHI-231 B lymphoma cells as a model system. Here we report that the signal for abrogating sIg-mediated apoptosis is generated by association of the CD40L molecule on T cells with the CD40 molecule on WEHI-231 cells. T-cell help through CD40 may thus determine whether B cells are eliminated or activated upon interaction with antigens.

We previously established Epstein-Barr virus (EBV)-transformed bullous pemphigoid (BP) patient lymphoblastoid cell lines, which produced human monoclonal anti-basement membrane zone antibodies. In the present study, we established two independent human-human hybridomas by fusion of these EBV transformants with a human B-cell line. These hybridomas, designated 5E-HY-4B and 10D-HY-8B, were very stable and showed a high yield of monoclonal antibody (MoAb) secretion. Each cell line was tetraploid and showed combined rearranged segments of immunoglobulin heavy-chain gene derived from both an EBV transformant and a parent cell. Immunoblot analysis showed that the 5E-HY-4B MoAb recognized the 230-kDa BP antigen but that the 10D-HY-8B MoAb did not show any reactivity. In contrast, both MoAbs precipitated the 230-kDa BP antigen with immunoprecipitation. These results indicate that the two MoAbs reacted with different epitopes on the 230-kDa BP antigen: a continuous epitope for the 5E-HY-4B MoAb and a conformation-dependent epitope for the 10D-HY-8B MoAb. This speculation was confirmed at the molecular level by the result that the fusion protein produced by a partial cDNA for the 230-kDa mouse BP antigen reacted with the 5E-HY-4B MoAb but not with the 10D-HY-8B MoAb. Furthermore, the study of the reactivity with fusion proteins of a series of deleted clones restricted the epitope for the 5E-HY-4B MoAb within the region with 114 amino acid residues in the C-terminal domain of the 230-kDa BP antigen.

We constructed an expression vector encoding a truncated Ig mu chain that lacks both VH and CH1 domains (mu delta m chain) and introduced the mu delta m vector into the Ig negative Abelson pre-B cell line P17-27. The transfectants expressed a large amount of the mu delta m chain on their surface, which was not complexed with the lambda 5 and VpreB surrogate light chain molecules. While P17-27 transfected with a vector for the intact micron chain (P17-27 micron) shows V kappa rearrangements in culture, V kappa rearrangements were not detected in P17-27 mu delta m cells. When the mu delta m chains on the cell surface were crosslinked by anti-mu antibodies, V kappa gene rearrangements were induced in P17-27 mu delta m. These results strongly suggest that crosslinking of the micron-lambda 5-VpreB complex on the pre-B cell surface generates a signal that activates V kappa gene rearrangement, and that the lambda 5 and VpreB molecules are necessary for the spontaneous crosslinking of surface Ig on pre-B cells.

Studies on transgenic mice expressing immunoglobulins against self-antigens have shown that self-tolerance is maintained by active elimination (clonal deletion), functional inactivation (clonal anergy) of self-reactive B cells, or a combination of both. We have established and characterized a transgenic mouse line expressing an anti-erythrocyte autoantibody. In contrast to other autoantibody transgenic lines, about 50% of the animals of this transgenic line suffer from autoimmune disease, indicating a loss of self-tolerance. Here we show that peritoneal Ly-1 B cells (also known as B-1 cells) are responsible for this autoimmune disease in our transgenic mice. A few self-reactive Ly-1 B cells that have somehow escaped the deletion mechanism expand in the peritoneum because of the absence of self-antigen. These Ly-1 B cells are eliminated in vivo by apoptosis once exposed to self-antigen. On the basis of these results we propose a novel autoantibody production mechanism whereby self-reactive B cells sequestered in compartments free of self-antigens may survive, proliferate and be activated for generation of pathogenic autoantibodies in autoimmune diseases.