The juxtamembrane wedge negatively regulates CD45 function in B cells

CD45 is a receptor-like protein tyrosine phosphatase highly expressed on all nucleated hematopoietic cells. We previously generated mice containing a point mutation in the juxtamembrane wedge of CD45. Demonstrating the critical negative regulatory function of the wedge, the CD45 E613R mutation led to a lymphoproliferative disorder (LPD) and a lupus-like autoimmune syndrome. Here we show the central role of B cells in this phenotype. Genetic elimination of B cells, but not T cells, ablates the LPD. In contrast to CD45-deficient B cells, the E613R mutation generates hyperresponsive B cells. Comparison of CD45-deficient and CD45 E613R mice reveals dichotomous effects of these mutations on B cell development. Together, the results support a role for CD45 as a rheostat, with both positive and negative regulatory functions, that fine-tunes the signal transduction threshold at multiple checkpoints in B cell development.     

CD4 and CD45 regulate qualitatively distinct patterns of calcium mobilization in individual CD4+ T cells

An early consequence of T cell activation is an increase in intracellular calcium concentration. Recent advances in video laser microscopic techniques enable the examination of individual cells over time following stimulation. Such studies have revealed that cells can undergo qualitatively distinct patterns of calcium mobilization, suggesting that different patterns of calcium flux may be associated with different signaling pathways and may differentially affect late events in cell activation. In this report, we identify distinct patterns of calcium mobilization in CD4⁺ T cells following the antibody-mediated cross-linking of either CD3 or CD4, or following the cross-linking of both CD3 and CD4 simultaneously. These effects can be further modified by the cross-linking of CD45. We find that antibody cross-linking of CD3 alone induces a single spike in the vast majority of cells shortly after the addition of the cross-linking antibody. In contrast, cross-linking CD4 alone induces a delayed pattern of repetitive calcium spikes which are decreased in amplitude compared to CD3 cross-linking. Simultaneous cross-linking of CD3 and CD4 induces a sustained increase in intracellular calcium mobilization which is dependent on the presence of extracellular calcium. This sustained increase in intracellular calcium concentration is also seen following physiologic cross-linking of CD3 and CD4 after T cell interaction with specific antigen and antigen-presenting cells. Finally, the simultaneous cross-linking of CD45, CD3 and CD4 abrogates the sustained increase in calcium seen following CD3 and CD4 cross-linking. These results suggest that the qualitative nature of T cell receptor signaling can be modulated by the molecular association of other signaling molecules, which may be part of the T cell receptor complex or not.     

The CD45 tyrosine phosphatase regulates Campath-1H (CD52)-induced TCR-dependent signal transduction in human T cells

Campath-1H, a humanized mAb undergoing clinical trials for treatment of leukemia, transplantation and autoimmune diseases, produces substantial lymphocyte depletion in vivo.The antibody binds to CD52, a highly glycosylated molecule attached to the membrane by a glycosylphosphatidylinositol anchor. Cross-linked Campath-1H is known to activate T cells in vitro. We have investigated the molecular basis for these effects by comparing the protein tyrosine phosphorylation signals induced by Campath-1H and the CD3 mAb OKT3 in primary T cells, and in CD45(+)TCR(+), CD45(-)TCR(+) and CD45(+)TCR(-) Jurkat subclones transfected with CD52. Our results show that Campath-1H triggers similar tyrosine phosphorylation events as OKT3 in both primary T cells and in the CD45(+)TCR(+) Jurkat sub-clone, albeit at quantitatively lower levels. However, no phospholipase C gamma 1 activation nor calcium signals were detected in response to CD52 ligation. The CD52-mediated induction of protein tyrosine phosphorylation was absolutely dependent upon the expression of both the TCR and the CD45 phosphotyrosine phosphatase at the cell surface. Cross-linking of Campath-1H was essential for signal transduction in all cells investigated. Fluorescence resonance energy transfer was used to demonstrate CD52 homo-association at the cell surface in Jurkat T cells in a TCR- and CD45-independent manner, and CD52-TCR association in CD45(+)TCR(+) cells. We propose a model to explain the activating effects of Campath-1H in which CD52 mAb cross-linking causes the trapping of TCR polypeptides within molecular complexes at the cell surface, thereby inducing signals via the TCR by a process which depends on the CD45-mediated regulation of the p56(lck) and p59(fyn) tyrosine kinases.     

CD45 expression by murine B cells and T cells: Alteration of CD45 isoforms in subpopulations of activated B cells

The CD45 family of high molecular weight cell surface glycoproteins is abundantly expressed by virtually all hematopoietic cells. CD45 molecules exist as multiple isoforms whose extracellular portions vary in protein structure and carbohydrate content but whose intracellular portions are highly conserved and possess tyrosine phosphatase activity. In this review we summarize current studies describing CD45 isoform expression on peripheral and thymic lymphocytes. Further, we analyze changes in CD45 isoform expression by selective populations of activated B cells.     

Receptor editing in CD45-deficient immature B cells

B cell receptor signaling threshold regulates negative selection of autoreactive B cells and determines the mechanism of B cell tolerance. Using mice carrying immunoglobulin transgene specific for MHC class I antigen K(k) (3-83 Tg mice), and IL-7-driven bone marrow (BM) culture system, we have previously shown that receptor editing is a major mechanism in B cell tolerance. To test the role of BCR signaling competence on the induction of tolerance-mediated receptor editing, we crossed the 3-83 Tg mice with mice deficient in CD45, a protein tyrosine phosphatase that functions asa positive regulator of the BCR signaling. We found that in the absence of self-antigen allelic exclusion is efficiently imposed in 3-83 Tg CD45(-/-) mice, although numbers of peripheral B cells are reduced. Using our BM culture system, we show here that immature 3-83 Tg CD45(-/-) B cells encountering self-antigen are developmentally arrested and undergo secondary light chain recombination and receptor editing, not different than CD45-sufficient cells. Thus, lack of CD45 does not abolish the receptor editing competence in immature B cells encountering high avidity membrane-bound antigen.     

Abnormal CD45RC expression and elevated CD45 protein tyrosine phosphatase activity in LEC rat peripheral CD4+ T cells

LEG rats are known to show a maturational arrest in the development of CD4⁺8⁺ to CD4⁺8^? cells in the thymus. Despite the blockade of maturation of CD4⁺8^? thymocytes, CD4⁺ T cells were observed in peripheral lymphoid organs, and these cells exhibit a defect in interleukin-2 (IL-2) production upon concanavalin A (Con A) stimulation. Although peripheral CD4⁺ cells in normal rat highly expressed CD45RC (CD45RC^high), the level of CD45RC expression was low (CD45RC^low) in LEC rat peripheral CD4⁺ cells. However, CD4⁺ cells from both strains highly expressed CD45 when those cells were stained by pan-CD45 mAb, suggesting that LEC rat CD4⁺ cells are deficient in expression of the CD45RC isoform, but not of CD45 molecules. When backcross rats from (F344 × LEC)F₁ × LEC were examined, the phenotype for CD45 expression pattern in CD4⁺ cells was clearly correlated with IL-2 production level in response to Con A stimulation. Thus, CD45RC^low cells exhibit a defect in IL-2 production, while CD45RC^high cells show normal IL-2 production. Protein tyrosine phosphatase (PTPase) activity in the membrane fraction of LEC rat CD4⁺ cells was threefold higher than that of normal rat CD4⁺ cells. Con A stimulation led to an increase in tyrosine phosphorylation levels, especially 100- and 40-kDa proteins, in normal rat CD4⁺ cells. In LEC rat CD4⁺ cells, however, the level of tyrosine phosphorylation in those proteins were very low. These results suggest that an elevated CD45 PTPase activity is responsive for a defect in IL-2 production in LEC rat peripheral CD4⁺ T cells.     

The Role of the Protein Tyrosine Phosphatase CD45 in Regulation of B Lymphocyte Activation

The transmembrane protein tyrosine phosphatase CD45 is expressed throughout B cell development and differentiation, with the exception of terminally differentiated plasma cells on which its expression is down regulated. Numerous studies using CD45-deficient B cell lines and CD45-deficient mice have clearly demonstrated that CD45 plays an important role in modulating the signal that is transduced via the B cell antigen receptor by regulating the phosphorylation state of Src family kinases. Spatial and temporal controls enable CD45 to promote B cell antigen receptor signal transduction by constitutively maintaining Src family kinases in a partially active state, such that the B cell is able to effectively respond to an antigenic challenge. Moreover, CD45 is required for optimal activation of Ca²⁺-dependent and MAP kinase-dependent signal transduction pathways in the B cell. The net result is that CD45 affects the B cell response by controlling the relative threshold of sensitivity to a given antigenic stimulus. Thus, CD45 expression and function is required for normal B cell development, tolerance induction, and responsiveness to antigen.     

CD45-mediated regulation of LFA1 function in human natural killer cells. Anti-CD45 monoclonal antibodies inhibit the calcium mobilization induced via LFA1 molecules

The TA218 and T205 monoclonal antibodies (mAb) were selected on the basis of their ability to inhibit the non-major histocompatibility complex-restricted lysis of the murine mastocytoma P815 cell line mediated by CD3^?CD16⁺ natural killer (NK) cells. Both mAb were found to react with CD45 molecules, as demonstrated by immunoprecipitation after surface iodination and western blot analysis. A panel of tumor target cells susceptible to lysis by polyclonal or clonal CD3^?CD16⁺ NK cells was used to study the mAb-mediated inhibitory effect. The inhibition of cytolysis mediated by TA218 and T205 mAb was found to consistently parralel the inhibition mediated (with the same tumor target cells) by the anti-LFAlα mAb TS.1.22 or by the anti-LFA1β mAb TS.1.18. However, different from the anti-LFAl mAb, T205 or TA218 mAb did not inhibit the binding of activated CD3^?CD16⁺ effector NK cells to the same tumor target cells. This finding supported the concept that the anti-CD45 mAb-mediated inhibition could occur at a post-binding stage. In polyclonal or clonal CD3-CD16⁺ NK cellsT205 orTA218 mAb were found to reduce by 50–70 % the intracellular Ca⁺⁺ ([Ca⁺⁺]i) mobilization induced by anti-LFAlα or anti-LFA1β mAb. On the other hand, TA218 and T205 mAb did not inhibit the Ca⁺⁺ mobilization induced by anti-CD 16 mAb or phytohemagglutinin, thus suggesting that, in NK cells, CD45 molecules may exert a selective inhibitory effect on the signal transduction mediated by LFA1 molecules. In line with this hypothesis, the cytolytic activity of human NK clones was triggered in the presence of the hybridoma cells secreting either anti-CD16 or anti-LFAla mAb (as “triggering targets”). This effect of anti-LFAlα, but not of anti-CD16 hybridoma was susceptible to inhibition by the anti-CD45 mAb T205 or TA218. Further, experiments on cloned NK cells indicated that T205 or TA218 mAb induced a strong decrease in the constitutive phosphorylation of the LFAlα chain (but not of HLA class I antigens). Taken together, these studies suggest that in human NK lymphocytes, CD45 molecule may regulate both the activation state and the function of the LFA1 molecule.     

CD45 regulates apoptosis in peripheral T lymphocytes

Programmed cell death (apoptosis) is a key mechanism for regulating lymphocyte numbers. Murine lymph node lymphocytes cultured in vitro without added stimuli show significant levels of apoptosis over 24 h, detectable by staining with Annexin V. CD4 and CD8 T lymphocytes from transgenic (Tg) mice expressing single CD45RABC or CD45RO isoforms show increased apoptosis and the extent of apoptosis is inversely correlated with the level of CD45 expression. CD45 Tg cells exhibit phosphatidyl serine translocation and DNA oligonucleosome formation, and can be partially rescued from apoptosis by culture in caspase inhibitors or common gamma-chain-binding cytokines. We conclude that CD45 is an important regulator of spontaneous apoptosis in T lymphocytes and this mechanism may contribute to the disease associations reported for individuals expressing CD45 variant alleles.     

Effect of activation of protein kinase C on CD45 isoform expression and CD45 protein tyrosine phosphatase activity in T cells

The T200/leukocyte common antigen (CD45) is a family of at least five large-molecular weight glycoproteins, which are differentially expressed on T cell subsets. The CD45 antigen consists of a variable heavily glycosylated exterior domain, a single membrane-spanning region, and a large cytoplasmic domain that has protein tyrosine phosphatase (PTPase) activity. In this study, we examined the effects of activation of protein kinase C (PKC) on the phosphorylation and expression of CD45 isoforms and PTPase activity in human T cells. After activation of PKC by phorbol 12-myristate 13-acetate (PMA), CD45RA expression rapidly increased within the first 24 h, whereas CD45R0 expression did not change within this time. However by 48 h, expression of CD45R0 also began to increase. Metabolic labeling showed that the rapid increment in CD45RA expression observed after PMA stimulation is primarily due to increased de novo synthesis of the 205-kDa and not the 220-kDa molecule. PMA treatment resulted in the phosphorylation of each CD45 isoform to a degree corresponding to its relative surface expression. Significantly, we found that the phosphorylation of CD45 by PKC activation down-regulated CD45 PTPase activity.     

The role of the protein tyrosine phosphatase CD45 in regulation of B lymphocyte activation

The transmembrane protein tyrosine phosphatase CD45 is expressed throughout B cell development and differentiation, with the exception of terminally differentiated plasma cells on which its expression is down regulated. Numerous studies using CD45-deficient B cell lines and CD45-deficient mice have clearly demonstrated that CD45 plays an important role in modulating the signal that is transduced via the B cell antigen receptor by regulating the phosphorylation state of Src family kinases. Spatial and temporal controls enable CD45 to promote B cell antigen receptor signal transduction by constitutively maintaining Src family kinases in a partially active state, such that the B cell is able to effectively respond to an antigenic challenge. Moreover, CD45 is required for optimal activation of Ca2+-dependent and MAP kinase-dependent signal transduction pathways in the B cell. The net result is that CD45 affects the B cell response by controlling the relative threshold of sensitivity to a given antigenic stimulus. Thus, CD45 expression and function is required for normal B cell development, tolerance induction, and responsiveness to antigen.     

Characterization of the protein tyrosine phosphatase CD45 on human epidermal Langerhans cells

Human Langerhans cells (LC) express CD45, but clear data about the isoform(s) and their function(s) are lacking. In the present study, double labeling experiments reveal that freshly isolated LC from normal skin are CD45RO⁺/RA^?/RB^?. However, after isolation and short-time culture where LC undergo an in vitro maturation resembling that to lymphoid dendritic cells, CD45RB emerges whereas CD45RO expression decreases. This evolution results from dynamic alternative RNA splicing. Addition of granulocyte-macrophage colony-stimulating factor or tumor necrosis factor-α to short-time cultures has no significant effect on CD45RB, but both cytokines accelerate the loss of CD45RO. LC isolated from lesional skin of atopic eczema highly express CD45RO and CD45RB. Cross-linking of CD45 on LC isolated from atopic individuals inhibits the calcium mobilization in response to activation via Fcε receptor type I(FcεRI). Hence, the protein tyrosine phosphatase CD45 from human LC is subjected to a splicing phenomenon related to the differentiation and activation stage of these cells and regulates their FcεRI-mediated activation.     

Evidence that the CD45 phosphatase regulates the activity of the phospholipase C in mouse T lymphocytes.

The importance of the tyrosine phosphatase CD45 in the regulation of lymphocyte activation was first demonstrated using antibodies against the extracellular domain of CD45 in functional assays. More recently it was reported that CD45-negative mutants were nonresponsive to stimulation through the T cell receptor-CD3 complex. We have studied the effect of CD45 cross-linking on the early signals induced by CD3 in mouse T cells. We show that CD45 cross-linking inhibits the increase in inositol phosphates and cytoplasmic Ca2+ induced by cross-linking of CD3. This indicates that CD45 is involved in the regulation of phospholipase C.     

CD45 negatively regulates tumour necrosis factor and interleukin-6 production in dendritic cells

CD45 is known to regulate signalling through many different surface receptors in diverse haemopoietic cell types. Here we report for the first time that CD45-/- bone marrow dendritic cells (BMDC) are more activated than CD45+/+ cells and that tumour necrosis factor (TNF) and interleukin-6 (IL-6) production by BMDC and splenic dendritic cells (sDC), is increased following stimulation via Toll-like receptor (TLR)3 and TLR9. Nuclear factor-kappaB activation, an important downstream consequence of TLR3 and TLR9 signalling, is also increased in CD45-/- BMDC. BMDC of CD45-/- mice also produce more TNF and IL-6 following stimulation with the cytokines TNF and interferon-alpha. These results show that TLR signalling is increased in CD45-/- dendritic cells and imply that CD45 is a negative regulator of TLR and cytokine receptor signalling in dendritic cells.     

Antigen-independent adhesion of CD45RA (naive) and CD45RO (memory) CD4 T cells to B cells.

We found that naive (CD45RA+) CD4 T cells have a lower capacity of adhesion to Epstein-Barr virus (EBV) immortalized B cells than memory (CD45RO+) CD4 T cells, as judged by conjugate formation. This would appear to be due to differences in the expression of adhesion molecules [lymphocyte function-associated antigen (LFA)-1, CD2]. However, kinetic studies showed that the degree of adhesion of naive T cells to B cells was stable over 60 min while that of memory T cells, like that of unseparated CD4 T cells, was characterized by a rapid formation and rapid dissociation of conjugates. This could be explained by a difference in the sensitivity of naive and memory CD4 T cells to down-regulation of antigen-independent adhesion by CD4-MHC class II interaction. Indeed, memory T cells also adhered stably to MHC class II(-) B cells. The adhesion of memory T cells, but not naive T cells, to MHC class II(+) B cells was sensitive to inhibition by OKT4a an anti-CD4 antibody, human immunodeficiency (HIV) gp160 (env) protein and a 12-mer peptide encompassing the 35-46 sequence of the HLA, DR beta 1 domain and previously shown to inhibit activation of HLA class II-restricted CD4 T cell responses. Since MHC class II expression did not influence the degree of conjugate formation by naive or memory CD4 T cells with B cells, CD4-MHC class II interaction does not appear to be involved in binding itself, but may down-regulate the adhesion of memory but not naive CD4 T cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Negative regulation of apoptotic death in immature B cells by CD45

Cross-linking of membrane IgM receptor on B cells induces tyroslnephosphorylation within 1 mln. This biochemical alteration triggersa cascade of signaling events which ultimately leads to activationin mature B cells but growth arrest and cell death by apoptosisin immature B cells. To study the mechanisms underlying thebifurcation of signals, we chose to examine the role of receptor-typeprotein tyroslne phosphatase (PTP) CD45 using CD45^– clonesisolated from an immature B cell line WEHI-231. Here we reportthat in CD45^– clones, tyroslne phosphorylation was constltutlvelyinduced but not enhanced by antl-IgM stimulation and anti-lgM-lnducedCa²⁺ flux was slightly delayed but evidently prolonged. Further,the degree of growth arrest and DNA fragmentation induced byantl-IgM antibody was more evident in CD45^– clones thanthe parental cells. These results indicate that initial alterationsin signaling are effectively transduced into effector signalsand that IgM receptor-mediated growth arrest and apoptosis inimmature B cells are negatively regulated by CD45.     

B淋巴细胞在抗CD45RB抗体诱导的移植免疫耐受中的作用

 目的: 探讨B淋巴细胞在抗CD45RB抗体诱导的移植免疫耐受中的作用。方法: 抗CD45RB抗体对BALB/c裸鼠进行预处理后制备脾脏单细胞悬液,与BALB/c小鼠T淋巴细胞和C57BL/6小鼠脾细胞混合培养,流式细胞术分析Th1、Th2、Treg和Tm淋巴细胞。以B6.μMT^-/-小鼠为受体、BALB/c小鼠为供体建立皮肤移植模型,移植后向受体鼠腹腔注射抗CD45RB单抗,监测脾淋巴细胞CD3⁺CD45RB^hi细胞比例。在混合淋巴培养过程中加入抗CD45RB单抗,分离B细胞,建立以BALB/c小鼠为供体、B6.μMT^-/-小鼠为受体的心脏移植模型,通过尾静脉注射B细胞给B6.μMT^-/-小鼠,观察受体鼠生存期和B细胞分布。结果: 在裸鼠体内用抗CD45RB抗体处理过的B淋巴细胞,与T淋巴细胞混合培养时,可使Treg和Th2淋巴细胞比例明显升高,Th1淋巴细胞的比例明显下降,Tm细胞无明显变化。在体内B淋巴细胞缺失的情况下,抗CD45RB抗体依然能够降低T细胞表面CD45RB的表达,与对照组B淋巴细胞存在组相比,抗CD45RB抗体对T淋巴细胞表面CD45RB下调更为快速,但最终CD3⁺CD45RB^hi T细胞比例无明显变化。体外抗CD45RB抗体处理过的B淋巴细胞可以延长受体鼠的生存时间。B6.μMT^-/-鼠在接受抗CD45RB抗体处理的B细胞并进行同种异体心脏移植后,B细胞可向胸腺迁移。结论: 在抗CD45RB抗体诱导的免疫耐受中,B淋巴细胞可能通过介导各T淋巴细胞亚群比例发挥着重要作用,且在中枢耐受中也起到一定作用,但是仅靠B淋巴细胞无法形成完全耐受。     

Modulation of CD45 tyrosine phosphatase activity by antigen

CD45 is a widely distributed phosphatase which modulates the activity of Lck by controlling the phosphorylation status of two tyrosine residues localized in the catalytic activation loop and in the negative regulatory domain. Little is known about the regulation of CD45 activity upon T cell activation. In the present study, we found that, in resting lymphocytes, an enzymatically active fraction of CD45 molecules is associated to the CD4 coreceptor. TCR engagement by an agonist ligand markedly inhibited this pool of CD45 phosphatase without affecting the CD4 / CD45 association. These results reveal that the modulation of the CD4-associated CD45 phosphatase activity is a very early biochemical event triggered by TCR stimulation. Since the recruitment of CD4 is an initial step in the activation process, the inhibition of this pool of CD45 molecules would be crucial to prevent dephosphorylation of relevant substrates which promote the activation process.     

Detection of restricted isoform expression and tyrosine phosphatase activity of CD45 in murine dendritic cells

CD45 is a cell surface transmembrane tyrosine phosphatase. It is expressed as distinct protein isoforms via alternative splicing of exons 4, 5 and 6. In T and B lymphocytes, CD45 is thought to play a critical role in antigen-dependent signaling through their respective antigen receptor complexes. However, the isoform expression and enzymatic activity of CD45 in other leukocytes remains largely unknown. Here, we examine the isoform expression and phosphatase activity of CD45 in murine dendritic cells (DC). Flow cytometric double-labeling analysis and biochemical analysis of purified splenic DC CD45 demonstrate that DC express both the CD45RB and CD45RO isoforms. Flow cytometric analyses of freshly isolated splenic DC and thymic DC also indicate the expression of CD45RB and CD45RO on these DC populations. In addition, we find that purified splenic DC CD45 possesses a high level of intrinsic tyrosine phosphatase activity. These data therefore establish the restricted isoform expression pattern of CD45 in murine DC and demonstrate that cells lacking specific antigen receptor complexes have active tyrosine phosphatase activity associated with CD45.     

Protection against hydrogen peroxide-induced injury by Z-ligustilide in PC12 cells

Z-ligustilide (Z-LIG) is the primary lipophilic compound of the Chinese medicine Danggui (Radix Angelica sinensis). Previous studies demonstrated that Z-LIG had significant neuroprotective potential in both transient and permanent cerebral ischemia, possibly through antioxidant and anti-apoptotic mechanisms. The present study examined the mechanisms of Z-LIG on hydrogen peroxide (H₂O₂)-induced injury in PC12 cells. Following exposure of the cells to H₂O₂ (500 μM), a significant reduction in cell survival and total antioxidant capacity (TAC), as well as increased intracellular reactive oxygen species (ROS), were observed. In addition, H₂O₂ treatment significantly upregulated Bax expression, cleaved-caspase 3, and cytosolic cytochrome-c, and decreased Bcl-2 protein levels. Pretreatment of the cells with Z-LIG (0.1, 1.0, 2.5, or 5.0 μg/ml) significantly attenuated H₂O₂-induced cell death, attenuated increased intracellular ROS levels, and decreased Bax expression, cleaved-caspase 3, and cytochrome-c. Further, Z-LIG improved cellular TAC and concentration-dependently upregulated Bcl-2 expression. These results demonstrate that Z-LIG has a pronounced protective effect against H₂O₂-induced cytotoxicity, at least partly through improving cellular antioxidant defense and inhibiting the mitochondrial apoptotic pathway. These findings suggest that Z-LIG may be useful in the treatment of neurodegenerative disorders in which oxidative stress and apoptosis are mainly implicated.