Anti-acetylcholine-receptor antibody concentrations after thymectomy in patients with myasthenia gravis.

Serum concentrations of anti-acetylcholine-receptor (anti-AChR) antibody were measured in patients with myasthenia gravis. In those patients undergoing thymectomy concentrations were measured before and after the operation to see whether there might be a connection between the thymus and antibody production. We found no correlation between antibody concentration and either thymectomy or duration and severity of the disease before the operation. Our results suggest that if anti-AChR antibodies are the principal pathogenic factor in myasthenia gravis then immunological and neurophysical variables other than the total serum anti-AChR antibody concentration contribute to the severity of the disease.     

Antibodies to acetylcholine receptor and tetanus toxoid: in vitro synthesis by thymic lymphocytes

Thymic lymphocytes (TL) of patients with myasthenia gravis (MG) have been reported to synthesize antibodies to acetylcholine receptors (anti-AChR). Incubation of TL with pokeweed mitogen (PWM), a polyclonal T cell- and monocyte-dependent activator of B cell differentiation, was reported to inhibit TL in vitro synthesis of anti-AChR. We studied the TL of 16 patients with MG without thymoma. TL of 10 of 16 patients synthesized anti-AChR in vitro without stimulation. In the presence of PWM, the amount of anti-AChR synthesized by the TL of these 10 patients increased in five, decreased in three, and was unaffected in two. There was a correlation between serum anti-AChR titer and PWM-stimulated synthesis (r = 0.87), but not with unstimulated synthesis (r = 0.33) of anti-AChR by TL. There was no correlation between the amount of synthesized anti-AChR and the amount of secreted IgG or with the percentage of B cells (surface Ig+) in the TL suspensions. TL of three age-matched cardiac surgery controls failed to synthesize detectable anti-AChR, although two of three synthesized IgG. Four patients with MG were booster immunized with tetanus toxoid (TT) 3 to 4 wk prior to thymectomy. TL of three synthesized anti-TT in vitro, whereas TL of three nonboosted MG patients failed to synthesize anti-TT. Thus, we have shown that TL of some patients with MG are capable of anti-AChR synthesis, with evidence of heterogeneity of the in vitro response, and the B cell repertoire in the thymus may, in part, reflect recent systemic immune events of the host.     

Characterization of peripheral blood acetylcholine receptor-binding B cells in experimental myasthenia gravis

In myasthenia gravis (MG), the neuromuscular transmission is impaired by antibodies (Abs) specific for muscle acetylcholine receptor (AChR). Anti-AChR Abs can be detected in the serum of MG patients, although their levels do not correlate with disease severity. In this study, we developed a flow cytometric assay for the detection of peripheral blood AChR-specific B cells to characterize B cell phenotypes associated with experimental autoimmune myasthenia gravis (EAMG). Alexa-conjugated AChR was used as a probe for AChR-specific B cells (B220+Ig+). Mice with EAMG had significantly elevated frequencies of AChR-specific IgG2+ and IgM+ B cells. While the frequencies of IgG2+ B cells and plasma anti-AChR IgG2 levels significantly correlated with the clinical grades of EAMG, the frequencies of IgM+ B cells and plasma anti-AChR IgM levels did not. These results indicate that the frequency of AChR-specific and IgG1+ (mouse IgG2 equivalent) peripheral blood B cells and anti-AChR IgG1 levels could be potential biomarkers for MG disease severity.     

Induction of peripheral tolerance to experimental autoimmune myasthenia gravis by acetylcholine receptor-pulsed dendritic cells

Dendritic cells (DC) are usually regarded as antigen-presenting cells involved in T cell activation, but DC also directly and indirectly affect B cell activation, antibody synthesis, and isotype switch. In the present study, bone marrow (BM)-derived DC from healthy rats were pulsed in vitro with acetylcholine receptor (AChR) and injected subcutaneously into healthy Lewis rats. No clinical signs of the first phase of experimental autoimmune myasthenia gravis (EAMG) were observed during 3 weeks of observation. Upon immunization with AChR and complete Freund's adjuvant, the rats that had received AChR-pulsed DC did not develop clinical EAMG. This tolerance of rats injected with AChR-pulsed DC was associated with reduced expression of B cell-activating factor (BAFF) and by reduced numbers of B cells among splenic mononuclear cells (MNC) compared to rats injected with medium or unpulsed DC. Anti-AChR IgG antibody-secreting cells were decreased, while the ratio of IgG1:IgG2b isotypes was enhanced in rats treated with AChR-pulsed DC compared to control EAMG rats. These results demonstrate that AChR-pulsed DC induce peripheral tolerance to EAMG by possibly inhibiting the expression of BAFF and production of anti-AChR antibodies, providing a possible potential for immunotherapy of antibody-mediated autoimmune diseases.     

Mice with IFN-gamma receptor deficiency are less susceptible to experimental autoimmune myasthenia gravis

IFN-gamma can either adversely or beneficially affect certain experimental autoimmune diseases. To study the role of IFN-gamma in the autoantibody-mediated experimental autoimmune myasthenia gravis (EAMG), an animal model of myasthenia gravis in humans, IFN-gammaR-deficient (IFN-gammaR-/-) mutant C57BL/6 mice and congenic wild-type mice were immunized with Torpedo acetylcholine receptor (AChR) plus CFA. IFN-gammaR-/- mice exhibited significantly lower incidence and severity of muscle weakness, lower anti-AChR IgG Ab levels, and lower Ab affinity to AChR compared with wild-type mice. Passive transfer of serum from IFN-gammaR-/- mice induced less muscular weakness compared with serum from wild-type mice. In contrast, numbers of lymph node cells secreting IFN-gamma and of those expressing IFN-gamma mRNA were strongly augmented in the IFN-gammaR-/- mice, reflecting a failure of negative feedback circuits. Cytokine studies by in situ hybridization revealed lower levels of lymphoid cells expressing AChR-reactive IL-1beta and TNF-alpha mRNA in AChR + CFA-immunized IFN-gammaR-/- mice compared with wild-type mice. No differences were found for AChR-reactive cells expressing IL-4, IL-10, or TGF-beta mRNA. These results indicate that IFN-gamma promotes systemic humoral responses in EAMG by up-regulating the production and the affinity of anti-AChR autoantibodies, thereby contributing to susceptibility to EAMG in C57BL/6-type mice.     

Interferon acts directly on human B lymphocytes to modulate immunoglobulin synthesis

At different times of exposure, interferon (IFN) enhanced and suppressed pokeweed mitogen- (PWM) induced IgG synthesis by human peripheral blood lymphocytes (PBL). Pretreatment of PBL and IFN frequently increased antibody production by more than 100% when compared with that by untreated PBL. Results of experiments in which PBL were separated into T and B subpopulations indicated that IFN preparations acted directly on B cells. Thus, mixtures of IFN-treated B cells and untreated T cells from 5 of 7 persons tested produced 81% to 500% more IgG than untreated, matched control cells. However, IFN-treated monocytes mixed with untreated B and T cells or IFN-treated T cells mixed with untreated B cells failed to enhance IgG production significantly in similar assays. In contrast to the pretreatment protocol, when IFN was present in the incubation mixture throughout the PWM assay, IgG production decreased. Sephadex chromatography of the IFN and tests of the resulting fractions indicated that the IgG production-enhancing activity was located in the fraction carrying the antiviral activity.     

Proteasome inhibition with bortezomib depletes plasma cells and autoantibodies in experimental autoimmune myasthenia gravis

Bortezomib, an inhibitor of proteasomes, has been reported to reduce autoantibody titers and to improve clinical condition in mice suffering from lupus-like disease. Bortezomib depletes both short- and long-lived plasma cells; the latter normally survive the standard immunosuppressant treatments targeting T and B cells. These findings encouraged us to test whether bortezomib is effective for alleviating the symptoms in the experimental autoimmune myasthenia gravis (EAMG) model for myasthenia gravis, a disease that is characterized by autoantibodies against the acetylcholine receptor (AChR) of skeletal muscle. Lewis rats were immunized with saline (control, n = 36) or Torpedo AChR (EAMG, n = 54) in CFA in the first week of an experimental period of 8 wk. After immunization, rats received twice a week s.c. injections of bortezomib (0.2 mg/kg in saline) or saline injections. Bortezomib induced apoptosis in bone marrow cells and reduced the amount of plasma cells in the bone marrow by up to 81%. In the EAMG animals, bortezomib efficiently reduced the rise of anti-AChR autoantibody titers, prevented ultrastructural damage of the postsynaptic membrane, improved neuromuscular transmission, and decreased myasthenic symptoms. This study thus underscores the potential of the therapeutic use of proteasome inhibitors to target plasma cells in Ab-mediated autoimmune diseases.     

The role of the thymus in maturational development of phytohemagglutinin and pokeweed mitogen responsiveness

A sensitive culture system for measuring lymphocyte transformation under physiological conditions by thymidine incorporation into DNA has been developed to study mouse and chick cell responses to mitogens. Both phytohemagglutinin (PHA) and pokeweed mitogen (PWM) stimulated thymus and spleen lymphocytes. Reduced but definite responses were obtained with lymph nodes, but negligible response with bone marrow cells.Thymocytes of newborn mice did not respond to PHA, but responded well to PWM. PHA responsiveness of thymocytes increased with aging until 12 weeks of postnatal life and then decreased in older animals. The level of background thymidine incorporation increased with advancing age. Spleen cells of 2-week-old mice were transformed by PHA and PWM, but in contrast to mouse thymus there was no decrease in older animals.Neonatal thymectomy of mice reduced the response of spleen cells to both PHA and PWM, especially in younger animals. The reduction was almost complete in the case of the PHA response, but only partial with the PWM response. Spleen cells from bursectomised chickens, checked for absence of B cell function, still responded well to both PWM and PHA.The results suggest PHA is a marker for T-lymphocytes in a certain “mature” stage of differentiation. PWM appears to stimulate a wider spectrum of cells.     

Experimental autoimmune myasthenia gravis may occur in the context of a polarized Th1- or Th2-type immune response in rats.

Experimental autoimmune myasthenia gravis (EAMG) is a T cell-dependent, Ab-mediated autoimmune disease induced in rats by a single immunization with acetylcholine receptor (AChR). Although polarized Th1 responses have been shown to be crucial for the development of mouse EAMG, the role of Th cell subsets in rat EAMG is not well established. In the present work we show that while the incidence and severity of EAMG are similar in Lewis (LEW) and Brown-Norway (BN) rats, strong differences are revealed in the immune response generated. Ag-specific lymph node cells from LEW rats produced higher amounts of IL-2 and IFN-gamma than BN lymph node cells, but expressed less IL-4 mRNA. IgG1 and IgG2b anti-AChR isotype predominated in BN and LEW rats, respectively, confirming the dichotomy of the immune response observed between the two strains. Furthermore, although IL-12 administration or IFN-gamma neutralization strongly influenced the Th1/Th2 balance in BN rats, it did not affect the disease outcome. These data demonstrate that a Th1-dominated immune response is not necessarily associated with disease severity in EAMG, not only in rats with disparate MHC haplotype but also in the same rat strain, and suggest that in a situation where complement-fixing Ab can be generated as a consequence of either Th1- or Th2-mediated T cell help, deviation of the immune response will not be an adequate strategy to prevent this Ab-mediated autoimmune disease.     

Amelioration of experimental autoimmune myasthenia gravis in rats by neonatal FcR blockade

The neonatal FcR (FcRn) plays a critical role in IgG homeostasis by protecting it from a lysosomal degradation pathway. It has been shown that IgG has an abnormally short half-life in FcRn-deficient mice and that FcRn blockade significantly increases the catabolism of serum IgG in mice. Therefore, reduction of serum IgG half-life may have therapeutic benefits in Ab-mediated autoimmune diseases. We have studied the therapeutic effects of an anti-rat FcRn mAb, 1G3, in two rat models of myasthenia gravis, a prototypical Ab-mediated autoimmune disease. Passive experimental autoimmune myasthenia gravis was induced by administration of an anti-acetylcholine receptor (AChR) mAb, and it was shown that treatment with 1G3 resulted in dose-dependent amelioration of the disease symptoms. In addition, the concentration of pathogenic Ab in the serum was reduced significantly. The effect of 1G3 was also studied in an active model of experimental autoimmune myasthenia gravis in which rats were immunized with AChR. Treatment with 1G3 significantly reduced the severity of the disease symptoms as well as the levels of total IgG and anti-AChR IgG relative to untreated animals. These data suggest that FcRn blockade may be an effective way to treat Ab-mediated autoimmune diseases.     

The effect of colony stimulating factor on the synthesis of ribonucleic acid by mouse bone marrow cells in vitro.

The effect of granulocyte-macrophage colony stimulating factor (GM-CSF) on the synthesis of RNA in liquid cultures of mouse bone marrow, spleen, thymus, peritoneal, peripheral blood leukocytes and lymph node cells was investigated. GM-CSF appeared to stimulate RNA-synthesis in syngeneic bone marrow cells within ten minutes of adding it to the culture. In the presence of GM-CSF bone marrow cultures maintained their initial rate of RNA synthesis for approximately ten hours. GM-CSF had no apparent effect on the uptake of 3H-uridine into bone marrow cells. This stimulation was still observed in the presence of puromycin and cycloheximide, but was abrogated by actinomycin D. The magnitude of the stimulation was not affected by the density of cells between 1 and 20 x 10(6) cells/ml but was slightly smaller at 0.1 and 40 x 10(6) cells/ml. Increasing concentration of GM-CSF (up to 2 X 105 units per ml) led to increased stimulation of RNA synthesis in bone marrow cells, but a significant stimulation could be detected at concentrations as low as 800 units/ml. GM-CSF did not significantly stimulate RNA synthesis in spleen, thymus, mesenteric or subcutaneous lymph node cells. However a small stimulation was observed in peripheral blood leukocytes and peritoneal cells. Autoradiographic studies showed that GM-CSF stimulated RNA synthesis in blast cells, myelocytes, metamyelocytes and polymorphs. Nucleated erythroid cells showed no increased labeling with GM-CFS. Labeling in lymphoid-like cells was highly variable but the level of labeling did not appear to be influenced by GM-CSF.     

Phenotypic markers for the feline monocyte: rosette formation with human erythrocytes and a monoclonal antibody which binds myeloid cells

A small population of cells with the ability to form rosettes with human erythrocytes was found in feline peripheral blood leukocytes (PBL) (10%) and bone marrow (9%), but not in purified granulocyte preparations, thymus, and lymph node tissues. The morphologic appearance and ability to phagocytize latex beads indicated these cells were monocytes. A monoclonal antibody, CM277, with a binding specificity for feline peripheral blood phagocytes was also characterized. Immunofluorescent microscopy revealed CM277 to bind specifically to monocytes and polymorphonuclear neutrophils. The binding of CM277 to monocytes was also shown by human erythrocyte-rosette formation wherein there was a high degree of correlation between these two phenotypic markers for cells ingesting latex beads. Monocytes, polymorphonuclear neutrophils, and T lymphocytes of the cat rosette with guinea pig erythrocytes (GPE) and using CM277 we were able to determine the contribution of the former two cell types to the GPE-rosetting population. Monocytes and polymorphonuclear neutrophils comprised the majority of the GPE-rosetting cells in fresh PBL (greater than 60%), but after culturing overnight, there was a substantial decrease in these cells (less than 35%). In contrast, GPE-rosetting T lymphocytes comprised approximately 10% of the cells in fresh PBL, and after in vitro culture for 1 day they constituted 35-45% of all cells. The removal of monocytes by human erythrocyte-rosetting did not affect the pokeweed mitogen-induced synthesis of Ig, but did lead to an increased production of interleukin 2. Removal of the GPE-rosetting population from PBL resulted in a marked decrease in interleukin 2 production, pointing to a positive contribution of GPE-rosetting T lymphocytes to the synthesis of this lymphokine.     

种间胚胎植入对母体免疫系统T细胞比例的影响

目的:研究种间胚胎植入期母体外周血、外周免疫器官(淋巴结、脾脏)、中枢免疫器官(胸腺、骨髓)中总T细胞的百分比变化,并探讨这种变化对种间胚胎植入的影响.方法:利用荧光标记的单克隆抗体染色结合流式细胞术,检测种间、同种胚胎移植以及同期假孕母体外周血、淋巴结、脾脏、胸腺、骨髓中T淋巴细胞的百分率.结果:种间胚胎植入时其外周血T细胞计数极显著低于同种和同期假孕小鼠(P＜0.01),而淋巴结、胸腺、骨髓中的T细胞计数则极显著高于同期假孕小鼠(P＜0.01).脾脏中同种胚胎植入母体则极显著高于种间和同期假孕小鼠(P＜0.01),两后者之间无显著性差异(P＞0.05).结论:种间妊娠时早在植入期开始,母体全身免疫系统就开始发生不利于种间妊娠的反应.     

The role of the thymus in the ontogeny of the immune system

The proper development of the organs of the immune system is dependent on at least three factors: (1) the development of anlagen with the capacity to trap antigens and support the proliferation of lymphoid and plasma cell precursors; (2) the production by the bone marrow of lymphoid and plasma cell precursors which seed in the lymphoid organs; and (3) the thymus, which seeds reactive cells to the lymphoid organs and produces a humoral factor stimulating antigen-triggered proliferation of primitive lymphoid and plasma cells. Studies on cell population changes in the lymph nodes following thymectomy in mice confirm earlier evidence that most cells produced in the thymus do not seed to the lymphoid organs, but die locally in the thymus.     

Guinea pig and gerbil erythrocytes rosette with different cells in the blood, bone marrow, and thymus of the cat

Cat thymocytes, bone marrow cells, and peripheral blood leukocytes (PBL) formed rosettes with guinea pig (GP) and gerbil (G) erythrocytes (E). In PBL from adult cats the frequency of rosettes was 27% with GPE and GE, while an average of 33% bone marrow cells formed rosettes with GPE and only 4% with GE. Thymocytes from kittens showed a high percentage of rosettes with both GPE and GE (35 to 81%), with the frequency of each type varying with the thymus tested. Fluorescein isothiocyanate labeling of one of the erythrocyte species revealed these cells to be rosetting with different nucleated cells; i.e., a low percentage (3-5%) of the rosettes formed with PBL and bone marrow had both labeled and unlabeled erythrocytes. In contrast, "mixed" rosettes were observed with a significant number of thymocytes, averaging 33% of thymocytes from six animals. A further distinction between the GE- and GPE-rosetting cells was revealed by a monoclonal antibody which blocked GE rosette formation without interfering with the binding of GPE to PBL and thymocytes. PBL could be depleted of either GPE- or GE-rosetting cells, with retention of IgG+ cells and cells capable of rosetting with the second erythrocyte species in the nonrosetting fractions. Stimulation of the latter nonrosetting fractions with pokeweed mitogen for induction of Ig synthesis revealed a T-lymphocyte specificity of the GE- and GPE-rosetting cells. PBL depleted of GE-rosetting cells yielded an increased Ig production, two- to threefold above the control; in contrast, depletion of GPE-rosetting cells from PBL resulted in a failure of the remaining cells to respond. These results suggest that T-suppressor cells of the cat are contained in the GE-rosetting fraction and T-helper cells are rosetted with GPE.     

Selective in vitro inhibition of an antibody response to purified acetylcholine receptor by using antigen-ricin A chain immunotoxin

Purified acetylcholine receptor (AChR) covalently coupled to the catalytically toxic A chain of ricin has been used to selectively eliminate rat lymph node cells involved in in vitro anti-AChR antibody responses. The resulting inhibition was specific in view of the lack of such inhibition of anti-Keyhole limpet hemocyanin antibody responses. Furthermore, when fractionated B cell or T cell populations were treated with AChR-A chain, both populations were found to be sensitive to the specific cytotoxicity. However, T cell cytotoxicity required higher concentrations of the immunotoxin. Furthermore, when AChR-immune lymphocytes were treated with AChR-A chain in vitro, they became unable to mediate secondary adoptive transfer responses in vivo. The abrogation of the anti-AChR adoptive response correlated with the lack of muscle weakness characteristic of experimental autoimmune myasthenia gravis. Thus, it is possible, in principle, to eliminate clones of antigen-reactive lymphocytes with antigen-ricin A chain immunotoxins. This lets open the possibility of using such agents in immunotherapeutic approaches to autoimmune disease.     

Effect of thymectomy on human peripheral blood T cell pools in myasthenia gravis

The human thymus is required for establishment of the T cell pool in fetal life, but postnatal thymectomy does not lead to immunodeficiency in humans. Because thymectomy in humans is performed for treatment of myasthenia gravis (MG), we have studied patients with MG for effects of thymectomy on peripheral blood (PB) naive (CD45RA(+), CD62L(+)) and memory (CD45RO(+)) T cells. We have also determined the effect of thymectomy on levels of PB cells containing signal joint TCR delta excision circles (TRECs), a molecular marker of thymus emigrants that have divided few times after leaving the thymus. In 17 nonthymectomized and 26 thymectomized MG patients studied at varying times after thymectomy (1 day to 41 years), we found no significant mean difference in PB T cell TREC levels between ages 40 and 80 years. However, both thymectomized and nonthymectomized MG patients had lower PB T cell TREC levels than did age-matched normal subjects (p < 0.0001 for both). These data demonstrated that MG itself or treatment for MG decreased thymopoiesis independent of thymectomy. Next, to control for disease activity and treatment, we prospectively studied 10 MG patients before and from 27 to 517 days after thymectomy. We found that thymectomy decreased CD4 or CD8 T cell TREC concentrations most when thymopoiesis was active before thymectomy (six of six patients), but had little effect in patients when thymopoiesis was minimal (four of four patients). In contrast, there was no significant effect of thymectomy on absolute numbers of naive PB T cells. Thus, in MG, removal of a thymus with active thymopoiesis resulted in a significant fall in PB TREC(+) T cells postthymectomy.     

Changes in the H-alloantigen-recognizing function of mouse lymphoid cells following hydrocortisone administration]

The capacity of spleen, thymus, and bone marrow cells of intact (control) and of hydrocortisone-treated mice CBA to induce the lymph node type of graft-v-host reaction (GVHR) in hybrids F1 (CBA X c57bl) was studied. After hydrocortisone injection (2.5 mg per mouse) the donor spleen cells became more active in GVHR, considering the value of lymph node indices and immunoblast content in the regional lymph node as compared with a control group. Following transplantation of thymus cells taken from the hydrocortison-treated donors the immunoblast count was higher, although the lymph node weight remained the same as in the control group. On the contrary, following the transfer of the bone marrow cells from the hydrocortisone-treated mice the lymph nodes enlarged, while the immunoblast count remained as low as in control. Consequently, exogenously conditioned increase in the hydrocortisone level was accompanied by an enrichment of the spleen and thymus cell populations with T-lymphocytes, proliferating in response to H-alloantigens.     

"Lymph node" form of graft-versus-host reaction in mice]

Local type of the graft-versus-host reaction was induced in adult hybrid mice (CBA X C57BL) F1 by the transfer of splenic, thymus and bone marrow CBA cells subcutaneously into the right hind footpad.The weight gain of the regional knee lymph node and blast accumulation in it 7 days later were used as indices of the graft-versus-host activity. After the transfer of 5 and 20 x 10(6) splenic cells the absolute weight of the regional lymph node was 8-10 times higher than that of the contralateral control; it was also significantly greater in comparison with controls which received live syngeneic or semiallogeneic dead cells from the same source. Contrary to controls, in case of the live cells a direct dose-effect dependency could be seen. The lymph node weight gain was accompanied by a regular immunoblast accumulation. The effect of the thymus and the bone marrow cells was less pronounced than that of the splenic cells.     

Lymphocyte subpopulations in sensitization and specific immunotherapy in an experiment. Lymphocyte subpopulations in sensitization to staphylococci, Artemisia pollen and their combinations

The distribution of T- and B-lymphocytes in the body of guinea pigs was studied in different groups of the animals. As shown in this study, in delayed hypersensitivity to staphylococci the number of PE- and E-rosette-forming cells increased in the blood, the spleen, and the lymph nodes and decreased in the thymus; the number of EA- and EAC-rosette-forming cells decreased in the bone marrow and the spleen, the number of T gamma-suppressors decreased in the bone marrow and the distant lymph node. Immediate hypersensitivity to tarragon pollen induced the general increase of the content of T- and B-lymphocytes; the number of T gamma-cells decreased in the thymus, the bone marrow, and the lymph nodes and increased in the spleen. The characteristic features of combined microbial-pollen sensitization were the high content of B-cells in all lymphoid organs (except the thymus), a low level of T-lymphocytes in the blood and the peripheral lymphoid organs, the decreased number of T gamma-cells in most of the immunogenetic organs.