重症肌无力患者胸腺内microRNA-29表达水平的探究

目的 检测重症肌无力(myasthenia gravis,MG)患者胸腺组织中microRNA-29的表达水平并探讨其与MG发病的相关性.方法 22例经手术治疗的MG患者胸腺组织作为实验组,22例经手术治疗的非MG伴胸腺异常患者胸腺组织作为对照组.通过实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)的方法检测实验组及对照组胸腺组织中microRNA-29表达水平,采用秩和检验(Mann-Whitney U test)分析实验组与对照组中microRNA-29表达水平.采用Spearman秩相关分析实验组胸腺中microRNA-29表达水平与定量重症肌无力(quantitative myasthenia gravis score,QMG)之间的相关性.结果 MG患者胸腺组织中microRNA-29表达水平较对照组显著增高(P=0.032);依据MG患者性别、年龄、临床特点、胸腺病理结果将实验组分成不同临床亚组,microRNA-29在各组间表达差异无统计学意义(P值分别为0.614、0.471、0.267、0.329);microRNA-29表达水平与QMG呈显著正相关性(r=0.689,P=0.000a).结论 microRNA-29在MG患者胸腺组织中表达增高,其表达水平与肌无力严重程度呈正相关,而与患者性别、年龄、临床类型、胸腺病理结果无明显关联.     

Engagement of CD20 suppresses apoptosis in germinal center B cells

In a screen of 67 monoclonal antibodies (mAb) included in the Blind Panel of B cell antibodies for the 5th International Workshop on Human Leukocyte Differentiation Antigens, only the CD20 mAb – included as a positive control for immunophenotyping studies – was found to suppress the spontaneous apoptosis which occurs in human germinal center (GC) B cells when placed in tissue culture at 37°C. Further detailed study using the 1F5 mAb confirmed this observation, showing that rescue from apoptosis via CD20, while not as efficient as that obtained on ligating CD40, was of similar magnitude to that achieved on engagement of surface immunoglobulin (sIg) by immobilized antibody. Also similar to anti-Ig, the CD20 mAb rescued from apoptosis without priming for the proliferation of GC B cells: this was quite different to its action on resting, non-GC B cells, where it provides a potent priming signal for cell cycle progression in response to IL-4 or anti-CD40. Unlike the survival signal engendered via sIg, CD20 engagement neither mobilized Ca²⁺ from intracellular stores or opening of a Ca²⁺ channel with 1F5, nor did it affect the ability of anti-Ig to open a Ca²⁺ gate in GC B cells. An unexpected feature of CD20-mediated rescue of GC B cells from apoptosis was a failure to turn on Bcl-2 expression.     

重症肌无力患者CD5+B细胞的变化

目的：研究ＣＤ＾＋Ｂ细胞在重症肌无力（ＭＣ）发病中的作用。方法Ｌ：采用免疫荧光双标记技术和流式细胞仪对３９例ＭＧ患者和１８例健康对照者周围血中ＣＤ５＾＋细胞（ＣＤ５＾＋ＣＤ１９＾＋）的百分率进行测定,同时用ＥＬＩＳＡ间接法检测ＭＣ患者血清中ＡｃｈＲａｂ。结果：在ＭＧ患者周围血ＣＤ５＾＋Ｂ细胞百分率显著高于对照组,而且在血清ＡｃｈＲａｂ阳性和阴性ＭＧ中ＣＤ５＾＋Ｂ细胞均明显高于对照组;但在血清Ａｃｈ     

Basement membrane proteins and reticulin in a normal thymus and the thymus in myasthenia gravis

Summary The distribution of basement membrane (BM) proteins, laminin and type IV collagen were studied immunohistochemically in a series of 12 normal thymuses representing different age groups (0–52 years) and in 10 cases of myasthenia gravis (age 7–53 years). The staining pattern was compared with that of conventional reticulin staining. BM proteins were present at the capsule-parenchyma interface and scantily distributed in the medullary stroma, where they were closely associated with reticulin fibres. The extrathymic perivascular space was effectively vizualized by the staining of the BM's marginal to it. The fiber network present in this space stained with reticulin stain and, less continuously, in BM stainings. Lymph node like tissue with germinal centers was occasionally present in the perivascular spaces in normal thymuses and commonly in the myasthenia gravis cases, where the perivascular spaces were often dilated. The BM's of the perivascular space were mostly continuous in normal cases, but discontinuities were observed in cases of myasthenia gravis, especially in the spaces which were widely dilated. Immunohistochemical detection of BM proteins seems to be useful in the study of thymic structure, particularly in the demonstration of the characteristic changes of the perivascular space in myasthenia gravis. It is suggested that the reticulin fibres present in the medulla and in the perivascular space contain laminin and type IV collagen.     

Antigen receptor-induced apoptosis of human germinal center B cells is targeted to a centrocytic subset

The outcome of the signals transduced through the B cell antigen receptor (BCR) depends both on their maturational stage and on the extent of receptor cross-linking. It is established that the BCR-mediated apoptosis of immature B cells represents an important mechanism for tolerance induction in the pre-immune B cell compartment. We show here that mature germinal center (GC) B cells can re-acquire sensitivity to BCR-induced cell death following CD40 ligation. In contrast, neither virgin nor memory B cells become susceptible to antigen receptor-triggered apoptosis upon CD40 stimulation, suggesting that this phenomenon may play a role in the shaping of the mature B cell repertoire in GC. Our data reveal that the death signal evoked through the BCR does not involve the Fcγ receptors, does not operate through the Fas/Fas ligand system, and can be blocked by interleukin-4. Finally, we found that the acquisition of sensitivity to the death-promoting effect of anti-Ig antibodies in CD40-stimulated GC B cell cultures correlates with the induction of a centrocytic phenotype. We propose that negative regulatory signals via the BCR may delete somatically mutated centrocytes with self-reactivity.     

Clonal expansions of CD4+ B helper T cells in autoimmune myasthenia gravis

The weakness in myasthenia gravis (MG) is mediated by T helper cell (Th)-dependent autoantibodies against neuromuscular epitopes. So far, analyzing Th phenotypes or antigen specificities has yielded very few clues to pathogenesis. Here we adopt an alternative antigen-independent approach, analyzing T cell receptor (TCR) Vbeta usage/expansions in blood from 118 MG patients. We found major expansions (>or= five standard deviations above the mean of 118 healthy, individually age- and sex-matched controls) in diverse Vbeta in 21 patients (17.6%, p<0.001) among CD4+ T cells, and in 45 patients (38.1%, p<0.001) among CD8+ T cells. In informative probands, the expanded CD4+ cells consistently showed a Th cell phenotype (CD57+CXCR5+) and expressed Th1 cytokines. Furthermore, their expression of markers for activation, lymphocyte trafficking and B cell-activating ability persisted for >or=3 years. Surprisingly, we noted a selective decline in the expansions/their CD57 positivity while the probands' MG was improving. CDR3 spectratyping suggested mono- or oligoclonal origins, which were confirmed by the prevalent TCR Vbeta CDR3 sequences of Th cells cloned from repeat bleeds. Thus, our data provide evidence for persistent clonally expanded CD4+ B helper T cell populations in the blood of MG patients. These unexpected CD4+ expansions might hold valuable clues to MG immunopathogenesis.     

CD38 signaling by agonistic monoclonal antibody prevents apoptosis of human germinal center B cells

The present study demonstrates that an agonistic anti-CD38 monoclonal antibody (mAb) (IB4) is capable of preventing apoptosis of human tonsillar germinal center (GC) B cells as measured by either morphological methods on Giemsa-stained cytospin preparations or flow cytometry on propidium iodidestained cells. Two other anti-CD38 mAb (Leu-17 and OKT10) consistently failed to prevent apoptosis in the same cells, even when tested over a wide range of concentrations. Furthermore, exposure of GC B cells to IB4 mAb up-regulates the bcl-2 proto-oncogene product in a manner similar to that observed with CD40 ligand (CD40L). The ability of IB4 mAb to prevent apoptosis of GC B cells was inferior to that of both anti-CD40 mAb and CD40L. No synergistic or additive effects were observed when IB4 mAb was used together with CD40L. Unlike anti-CD40 mAb or CD40L, IB4 mAb neither induced a proliferation of GC B cells nor increased their proliferative response to anti-CD40, CD40L or recombinant interleukin-4, used alone or in combination. The present results are consistent with the recent findings on either the feature of the CD38 molecules to deliver activation signals and on the mechanisms of selection of B cells that operates in the GC.     

Identification of a tonsil IgD+ B cell subset with phenotypical and functional characteristics of germinal center B cells

We have identified and isolated a subpopulation of IgD⁺ B cells (IgD⁺ CD38⁺ B cells) from human tonsils which expresses the germinal center (GC)-associated surface markers CD10, CD38, CD75, CD77 and CD95/Fas. The heterogeneity of expression of several markers on IgD⁺ CD38⁺ B cells suggests that this population can be further subdivided into two discrete subtypes. On a functional basis, IgD⁺ CD38⁺ B cells behave as GC B cells as they rapidly and spontaneously undergo apoptosis in vitro and cannot be stimulated to synthesize DNA upon cross-linking of the antigen receptor. However, in contrast with most GC B cells, IgD⁺ CD38⁺ B cells have not completed Ig class switching since they predominantly secrete IgM following stimulation in vitro and lack surface expression of secondary isotypes. Immunoenzymatic staining performed on tonsil tissue sections revealed that IgD⁺ CD38⁺ B cells are located in two distinct histological structures: within the GC of a few classical secondary follicles, in which they appear as scattered cells, and within rare atypical GC, homogeneously constituted of IgD⁺ B cells. Taken together, our findings indicate that IgD⁺ CD38⁺ B cells constitute a novel subset of GC B cells. The possibility that these cells could represent an early stage of the follicular reaction or be generated in response to certain bacterial carbohydrate antigens is discussed.     

IL-2和IL-4联合诱导B细胞死亡受体CCR3的表达及其功能研究

目的　研究在IL 2和IL 4作用下 ,趋化性细胞因子受体CCR3在人生发中心 (germinalcenter,GC)B细胞上的表达及其功能特性。方法　采用流式细胞术检测人GCB细胞上CCR3表达和在CCR3配体eotaxin作用下B细胞的凋亡 ,实时定量RT PCR和Northernblot法检测GCB细胞内CCR3mRNA的表达 ,淋巴细胞趋化和黏附试验检测B细胞的趋化和黏附能力。结果　人GCB细胞极低表达趋化性细胞因子受体CCR3,经IL 2和IL 4作用后 ,GCB细胞高表达CCR3,但此时CCR3不能在其配体作用下诱导GCB细胞的趋化和黏附功能 ,而是诱导GCB细胞凋亡。结论　IL 2和IL 4联合诱导人GCB细胞CCR3表达 ,CCR3可能具有死亡受体的功能。     

Thymic myoid cell turnover in myasthenia gravis patients and in normal controls

 Thymic myoid cells (TMC) are sparse muscle-like cells in the thymic medulla, which are believed to trigger the autoimmune response in myasthenia gravis (MG). Ultrastructural investigations have revealed mature, degenerating, and immature TMC, but the number of TMC in MG patients does not differ from that in controls. We examined the turnover of TMC at the subcellular level, performing an immunocytochemical study with muscle-specific anti-desmin labelling of 10 thymuses derived from MG patients with lymphofollicular hyperplasia and from 8 normal controls. All thymuses examined revealed mature, immature, and degenerating TMC. Mature TMC contained desmin filaments in between Z-discs provided the sarcomeres were arranged in register. Morphological features of degenerating TMC included hypercontracted sarcomeres, cytoplasmic granular debris, chromatin clumping and, occasionally, membrane-bound bodies. Macrophages were not involved in the process. Immature TMC were of small diameter and contained myofilaments not arranged in myofibrils. In an MG thymus, small immature TMC were found clustered with dying TMC. It may be that degeneration of TMC is a stimulus for the generation of new TMC with faster turnover. This mechanism may mean that more antigen is available in MG patients than in normal controls, despite constant TMC numbers Received: 2 July 1997 / Accepted: 20 November 1997     

Innate immunity in myasthenia gravis thymus: Pathogenic effects of Toll-like receptor 4 signaling on autoimmunity

The thymus is the main site of immune sensitization to AChR in myasthenia gravis (MG). In our previous studies we demonstrated that Toll-like receptor (TLR) 4 is over-expressed in MG thymuses, suggesting its involvement in altering the thymic microenvironment and favoring autosensitization and autoimmunity maintenance processes, via an effect on local chemokine/cytokine network. Here, we investigated whether TLR4 signaling may favor abnormal cell recruitment in MG thymus via CCL17 and CCL22, two chemokines known to dictate immune cell trafficking in inflamed organs by binding CCR4. We also investigated whether TLR4 activation may contribute to immunodysregulation, via the production of Th17-related cytokines, known to alter effector T cell (Teff)/regulatory T cell (Treg) balance. We found that CCL17, CCL22 and CCR4 were expressed at higher levels in MG compared to normal thymuses. The two chemokines were mainly detected around medullary Hassall's corpuscles (HCs), co-localizing with TLR4⁺ thymic epithelial cells (TECs) and CCR4⁺ dendritic cells (DCs), that were present in higher number in MG thymuses compared to controls. TLR4 stimulation in MG TECs increased CCL17 and CCL22 expression and induced the production of Th17-related cytokines. Then, to study the effect of TLR4-stimulated TECs on immune cell interactions and Teff activation, we generated an in-vitro imaging model by co-culturing CD4⁺ Th1/Th17 AChR-specific T cells, naïve CD4⁺CD25⁺ Tregs, DCs and TECs from Lewis rats. We observed that TLR4 stimulation led to a more pronounced Teff activatory status, suggesting that TLR4 signaling in MG thymic milieu may affect cell-to-cell interactions, favoring autoreactive T-cell activation. Altogether our findings suggest a role for TLR4 signaling in driving DC recruitment in MG thymus via CCL17 and CCL22, and in generating an inflammatory response that might compromise Treg function, favoring autoreactive T-cell pathogenic responses.     

Suppression of apoptosis in normal and neoplastic human B lymphocytes by CD40 ligand is independent of Bcl-2 induction

The tendency of isolated germinal center (GC) B cells to undergo apoptosis was suppressed by recombinant cell-bound CD40 ligand (CD40L): after 2 days at 37°C, > 80 % of cells remained viable in the presence of CD40L as compared to < 1 % in control cultures. CD40L sustained a high rate of DNA synthesis in GC cells and was more effective than monoclonal antibody to CD40 in this regard. Group I Burkitt lymphoma (BL) cell lines induced to undergo apoptosis with anti-immunoglobulin or calcium ionophore were also protected by CD40L. In BL cells, this route of rescue was not accompanied by induction of Bcl-2 protein, the expression of which has been linked to hemopoietic cell survival. Bcl-2 was induced in GC cells responding to CD40L, but its appearance was a relatively late event not reaching significant levels over controls until day 2 of culture. Thus induction of Bcl-2 appears to be secondary to the survival signal imparted by CD40L. These findings are discussed in relation to a potential role for CD40L in supporting B cell tumors in vivo and the discovery that the molecular defect in the X-linked Hyper-IgM syndrome is targeted to the CD40L gene.     

Re-expression of RAG-1 and RAG-2 genes and evidence for secondary rearrangements in human germinal center B lymphocytes

V(D)J recombination occurs in immature B cells within primary lymphoid organs. However, recent evidence demonstrated that the recombination activating genes RAG-1 and RAG-2 can also be expressed in murine germinal centers (GC) where they can mediate secondary rearrangements. This finding raises a number of interesting questions, the most important of which is what is the physiological role, if any, of secondary immunoglobulin (Ig) gene rearrangements. In the present report, we provide evidence that human GC B cells that have lost surface immunoglobulin re-express RAG-1 and RAG-2, suggesting that they may be able to undergo Ig rearrangement. Furthermore, we describe two mature B cell clones in which secondary rearrangements have possibly occurred, resulting in light chain replacement. The two clones carry both κ and λ light chains productively rearranged, but fail to express the κ chain on the cell surface due to a stop codon acquired by somatic mutation. Interestingly, the analysis of the extent of somatic mutations accumulated by the two light chains might suggest that the λ chain could have been acquired through a secondary rearrangement. Taken together, these data suggest that secondary Ig gene rearrangements leading to replacement may occur in human GC and may contribute to the peripheral B cell repertoire.     

Identification of a novel subpopulation of germinal center B cells characterized by expression of IgD and CD70

CD27, which belongs to the tumor necrosis-factor receptor family, is expressed on germinal center (GC) but not on naive B cells, suggesting an important function of this molecule in the regulation of the GC reaction. We described here the expression of CD70, which is the ligand for CD27. We observed that in most tonsils, CD70 is only expressed on part of the IgD^?, CD38^? B cell population, which have been described as memory B cells. However, in 10 % of the tonsils tested, CD70⁺ IgD⁺ GC were found. The CD70⁺ GC B cells were small cells that also expressed CD44 and CD39, but were CD10^? and CD38^?, suggesting that they represent very recent immigrants that are in the process of forming a GC. The concordant expression of CD27 and its ligand CD70 on this primordial subset of GC B cells suggests an important role for CD27/CD70 interaction at this stage of GC formation.     

未成熟树突状细胞诱导实验性自身免疫性重症肌无力耐受的试验研究

目的 探讨未成熟树突状细胞(iDC)对实验性自身免疫性重症肌无力(EAMG)的影响。方法 用小剂量粒-巨噬细胞集落刺激因子(GM-CSF)体外诱导骨髓前体细胞，获得的iDC用乙酰胆碱受体(ACAR)负载后皮下接种同基因大鼠，3周后通过体外试验观察脾细胞对AChR的增殖反应，同时用AChR和完全弗氏佐剂(CFA)免疫大鼠，观察免疫7周后MG相关指标的改变。结果 小剂量GMCSF诱导所获得的iDC与成熟DC(mDC)相比，其表面MHCⅡ和共刺激分子CD80、CD86的表达低，摄取dextran-FITC的能力强但刺激同种T细胞增殖的能力弱。接种iDC、mDC、AChR负载的mDC的大鼠和对照组一样，体外脾细胞对AChR的刺激均强烈增殖，用AChR和CFA免疫后，产生明显的MG症状，重复电刺激出现明显衰减，血清AChRAb滴度明显增高，神经肌肉接头呈现典型的MG样改变；而接种AChR负载的iDC的大鼠脾细胞对AChR的刺激呈现明显的增殖抑制但对卵清蛋白的刺激仍强烈增殖，MG相关指标均未见明显改变。结论 骨髓前体细胞经小剂量GM-CSF诱导产生了iDC，AChR负载的iDC可诱导EAMG耐受。     

A subset of anti-CD21 antibodies promote the rescue of germinal center B cells from apoptosis

Germinal center cells (GCC) are programmed to die by apoptosis unless they receive a positive signal for rescue. The primary signal in vivo is believed to be dependent on interaction with antigen held as immune complexes on follicular dendritic cells (FDC), a subset of which express large amounts of CD23, a low-affinity receptor for IgE. Recombinant soluble CD23 (sCD23) and interleu-kin-1 have been found to potentiate the survival of GCC in vitro. Recently, CD23 was shown to interact specifically with a ligand other than IgE, namely CD21 (CR2/Epstein-Barr virus receptor). In the present study, we show that a subset of anti-CD21 monoclonal antibodies behave similarly to soluble CD23 in their effect on GCC inasmuch as they: (i) diminish the occurrence of apoptosis; (ii) promote a plasmacytoid appearance in rescued cells; (in) up-regulate expression of the Bcl-2 proto-oncogene. These findings indicate that FDC-derived CD23 exerts its effect on GCC via CD21.     

IL-10 interrupts memory B cell expansion in the germinal center by inducing differentiation into plasma cells

Germinal center (GC) B cells undergo proliferation, somatic hypermutation and isotype switching in the course of differentiation into plasma cells to produce high-affinity antibodies. To understand the molecular mechanism regulating the expansion of memory B cells and the termination of expansion by differentiation into plasma cells, we investigated the effect of interleukin-2 (IL-2), IL-4, IL-10 and CD40 ligand (CD40L) on the differentiation of GC B cells in the defined culture system containing a follicular dendritic cell (FDC)-like cell line. IL-2, IL-4 and CD40L are required for the optimum proliferation and differentiation of GC B cells. When IL-10 was added to this culture condition, CD20⁺ CD38⁺ GC B cells sequentially differentiated into CD20⁺ CD38⁻ memory B cells and then CD20⁻ CD38⁺ plasma cells. In the absence of IL-10, the resulting CD20⁺ CD38⁻ memory B cells continued to proliferate and retained its phenotype. The proliferation of memory B cells was interrupted by addition of IL-10 which induced the differentiation into plasma cells. The expression of CD80 and CD86 was up-regulated in the memory B cells, compared to naive B cells and plasma cells. The identity of memory B cells generated in vitro from GC B cells was further substantiated since memory B cells generated in vivo displayed the identical pattern of proliferation and differentiation under the same culture condition. These results highlight the potent role of GCT helper cells in the expansion and differentiation of memory B cells by regulating different cytokine production.     

Recirculating and germinal center B cells differentiate into cells responsive to polysaccharide antigens

Antibodies against bacterial capsular polysaccharides play a critical protective role. Responses to these antigens can occur without the help or control of T cells and are associated with marginal zone (MZ) B cells. Capsular antigens are diverse and some cross-react with self-carbohydrate epitopes. This diversity may explain the recruitment of non-autoreactive recirculating B cells and memory B cells to the MZ in addition to other B cells, some of which are weakly autoreactive cells, that are recruited to the MZ without entering the recirculating pool. To test whether memory B cells respond to polysaccharide-based antigens, mice with hapten-specific memory B cells were challenged with hapten-polysaccharide. Hapten-specific plasma cells producing high affinity antibody with Ig V-region mutations were induced. To test whether naive recirculating B cells can form MZ cells that respond to polysaccharide, recirculating B cells from lymph nodes were transferred into Rag-1-deficient mice. MZ cells differentiated from the donor cells without proliferation or T cell help and responded to polysaccharide-based antigen. The differentiation of B cells both from germinal centers and the recirculating pool to the MZ phenotype is likely to make an important contribution to the repertoire of B cells that respond to polysaccharide antigens.     

Protein tyrosine phosphorylation is mandatory for CD40-mediated rescue of germinal center B cells from apoptosis

Spontaneous apoptosis in germinal center (GC) B cells can be arrested either by engaging cell surface immunoglobulin (Ig) with immobilized ligand or, more effectively, by treatment with soluble monoclonal antibody (mAb) directed against CD40. The present study examines the intracellular signal transduction pathways through which rescue from spontaneous apoptosis is engendered in GC B cells following ligation of surface CD40. Cross-linking the surface CD40 of GC B cells with mAb consistently resulted in enhanced tyrosine phosphorylation on a number of distinct substrates: this process could be blocked, in a dose-dependent fashion, by pre-treating GC B cells with the selective protein tyrosine kinase(s) (PTK) inhibitor, herbimycin A. Moreover, the pattern of phosphorylation on tyrosine observed following treatment with anti-CD40 was remarkably similar to that triggered by polyvalent anti-Ig. By contrast, anti-CD40 failed to stimulate the increase in inositol 1,4,5-trisphosphate and cytosolic free calcium observed in both GC B cells and resting B lymphocytes following ligation of surface Ig. The involvement of the signaling pathways generated in the rescue of GC B cells from apoptosis was studied by using selective inhibitors of PTK and of extracellular and intracellular Ca²⁺. Pre-incubation with the PTK inhibitor herbimycin A (5 μM) abrogated anti-CD40-mediated rescue of GC B cells from apoptosis, while genistein (40 μM) and the tyrphostins AG490 (10 μ M) and AG814 (25 μ M) significantly inhibited this process. Consistent with these results, herbimycin A (5 μM) abolished the expression of the 26 kDa bcl-2 protooncogene product, which confers resistance to apoptosis, normally observed following culture with anti-CD40. The Ca²⁺ chelators BAPTA and EGTA did not significantly affect CD40-promoted rescue. Taken together, these results indicate that CD40 of GC B cells is coupled to functional PTK but not to the phosphatidylinositol signaling pathway and that tyrosine phosphorylation is mandatory for CD40-mediated rescue of GC B cells from apoptosis.