C57BL/6小鼠性别差异对髓鞘少突胶质糖蛋白诱导的实验性自身脑脊髓炎疾病的影响

多发性硬化是人类常见的中枢神经系统自身免疫性炎症致脱髓鞘疾病．流行病学研究发现,女性患者多于男性,其平均发病时间早于男性．实验性自身免疫性脑脊髓炎(EAE)与多发性硬化症有相似的临床症状和病理特征,是被广泛应用于人类疾病研究的动物模型．本实验利用髓鞘少突胶质糖蛋白MOG_33-35免疫C57BL/6小鼠建立EAE模型,观察29天．通过疾病评分发现雌雄小鼠在发病率、起病时间上均无明显差别,但雄鼠的发病症状明显比雌鼠严重．在其病理切片HE染色中观察到雄性小鼠中枢浸润的炎性细胞多于雌性小鼠,并且在LFB染色中同样观察到雄鼠脱髓鞘区域明显增大．对其发病高峰期中枢浸润细胞的染色分析时,可以发现雄性小鼠中浸润的CD4 T细胞及其亚群T_H-1和T_H-17细胞均有明显增加．这些都表明MOG_33-35免疫C57BL/6小鼠建立的EAE模型存在着性别差异的影响,这一发现为今后建立多发性硬化症的动物模型中动物性别的选择提供了一定的参考依据．     

C57BL/6J小鼠实验性自身免疫性脑脊髓炎模型的建立及其病理特征

目的探讨C57BL/6J小鼠建立实验性自身免疫性脑脊髓炎（EAE）模型的可能性及其发病特点。方法使用PLP139-151抗原及其C57BL/6J小鼠自制脑脊髓匀浆（spinal cord homogenate,SCH）免疫C57BL/6J小鼠,使用完全福（氏）免疫佐剂为免疫佐剂,并在尾静脉注射百日咳杆菌,建立EAE模型,与经典的PLP139-151免疫的SJL/J小鼠EAE模型进行对比。结果PLP139-151免疫C57BL/6J小鼠仅有一只小鼠表现为尾部张力明显降低;自制SCH免疫C57BL/6J小鼠可见明显脱髓鞘改变。与PLP139-151免疫SJL/J小鼠组相比发病率较低（P〈0.05）,神经功能评分比较没有明显差异（P〉0.05）,但发病时间长于PLP139-151免疫SJL/J小鼠组（P〈0.05）。结论SCH免疫C57BL/6J小鼠的EAE动物模型,主要表现为急性单相病程,从临床表现和病理学特点来看符合人类MS的病理特点,值得在以后的研究中进一步研究探讨。     

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG_35-55 is suspended in complete Freund''s adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.     

De novo central nervous system processing of myelin antigen is required for the initiation of experimental autoimmune encephalomyelitis

We demonstrate the absolute requirement for a functioning class II-restricted Ag processing pathway in the CNS for the initiation of experimental autoimmune encephalomyelitis (EAE). C57BL/6 (B6) mice deficient for the class II transactivator, which have defects in MHC class II, invariant chain (Ii), and H-2M (DM) expression, are resistant to initiation of myelin oligodendrocyte protein (MOG) peptide, MOG(35-55)-specific EAE by both priming and adoptive transfer of encephalitogenic T cells. However, class II transactivator-deficient mice can prime a suboptimal myelin-specific CD4(+) Th1 response. Further, B6 mice individually deficient for Ii and DM are also resistant to initiation of both active and adoptive EAE. Although both Ii-deficient and DM-deficient APCs can present MOG peptide to CD4(+) T cells, neither is capable of processing and presenting the encephalitogenic peptide of intact MOG protein. This phenotype is not Ag-specific, as DM- and Ii-deficient mice are also resistant to initiation of EAE by proteolipid protein peptide PLP(178-191). Remarkably, DM-deficient mice can prime a potent peripheral Th1 response to MOG(35-55), comparable to the response seen in wild-type mice, yet maintain resistance to EAE initiation. Most striking is the demonstration that T cells from MOG(35-55)-primed DM knockout mice can adoptively transfer EAE to wild-type, but not DM-deficient, mice. Together, these data demonstrate that the inability to process antigenic peptide from intact myelin protein results in resistance to EAE and that de novo processing and presentation of myelin Ags in the CNS is absolutely required for the initiation of autoimmune demyelinating disease.     

Effects of progesterone in the spinal cord of a mouse model of multiple sclerosis

The spinal cord is a target of progesterone (PROG), as demonstrated by the expression of intracellular and membrane PROG receptors and by its myelinating and neuroprotective effects in trauma and neurodegeneration. Here we studied PROG effects in mice with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis characterized by demyelination and immune cell infiltration in the spinal cord. Female C57BL/6 mice were immunized with a myelin oligodendrocyte glycoprotein peptide (MOG_40–54). One week before EAE induction, mice received single pellets of PROG weighing either 20 or 100 mg or remained free of steroid treatment. On average, mice developed clinical signs of EAE 9–10 days following MOG administration. The spinal cord white matter of EAE mice showed inflammatory cell infiltration and circumscribed demyelinating areas, demonstrated by reductions of luxol fast blue (LFB) staining, myelin basic protein (MBP) and proteolipid protein (PLP) immunoreactivity (IR) and PLP mRNA expression. In motoneurons, EAE reduced the expression of the alpha 3 subunit of Na,K-ATPase mRNA. In contrast, EAE mice receiving PROG showed less inflammatory cell infiltration, recovery of myelin proteins and normal grain density of neuronal Na,K-ATPase mRNA. Clinically, PROG produced a moderate delay of disease onset and reduced the clinical scores. Thus, PROG attenuated disease severity, and reduced the inflammatory response and the occurrence of demyelination in the spinal cord during the acute phase of EAE.     

E- and P-selectin are not required for the development of experimental autoimmune encephalomyelitis in C57BL/6 and SJL mice

In multiple sclerosis and in its animal model experimental autoimmune encephalomyelitis (EAE), inflammatory cells migrate across the endothelial blood-brain barrier (BBB) and gain access to the CNS. It is well-established that alpha4 integrins are actively involved in leukocyte recruitment across the BBB during EAE. In contrast, the role of endothelial E- and P-selectin in this process has been a controversial issue. In this study, we demonstrate that P-selectin protein can be detected in meningeal blood vessel endothelial cells in healthy SJL and C57BL/6 mice and on rare parenchymal CNS blood vessels in C57BL/6, but not SJL, mice. During EAE, expression of P-selectin but not E-selectin was found up-regulated on inflamed CNS microvessels surrounded by inflammatory infiltrates irrespective of their meningeal or parenchymal localization with a more prominent immunostaining detected in C57BL/6 as compared with SJL mice. P-selectin immunostaining could be localized to CNS endothelial cells and to CD41-positive platelets adhering to the vessel wall. Despite the presence of P-selectin in wild-type mice, E/P-selectin-deficient SJL and C57BL/6 mice developed clinical EAE indistinguishable from wild-type mice. Absence of E- and P-selectin did neither influence the activation of myelin-specific T cells nor the composition of the cellular infiltrates in the CNS during EAE. Finally, endothelial-specific tetracycline-inducible expression of E-selectin at the BBB in transgenic C57BL/6 mice did not alter the development of EAE. Thus, E- and P-selectin are not required for leukocyte recruitment across the BBB and the development of EAE in C57BL/6 and in SJL mice.     

Overcoming unresponsiveness in experimental autoimmune encephalomyelitis (EAE) resistant mouse strains by adoptive transfer and antigenic challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2(b)) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated. The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 μg/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 μg/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.     

不同剂量MOG抗原免疫诱导小鼠自身免疫性脑脊髓炎模型的比较

目的比较不同剂量髓鞘少突胶质细胞糖蛋白(myelin oligodendrocyte glycoprotein,MOG35-55)免疫诱导C57BL/6小鼠实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)的作用。方法将C57BL/6小鼠分为正常组和三组不同剂量MOG35-55诱导的EAE模型组,共4组。模型组分别以每只200、100、50μg的MOG35-55与完全弗氏佐剂(complete Freund s adjuvant,CFA)混合的乳化抗原皮下注射免疫诱导EAE模型,正常组以生理盐水替代。观察不同剂量MOG35-55对C57BL/6小鼠体重、发病率以及神经功能评分等影响,同时取小鼠脑和脊髓,利用光镜和透射电镜观察小鼠病理组织学改变。结果三组不同剂量MOG35-55均能诱导EAE模型,发病率为100%,呈慢性单相病程,病理学观察发现小鼠脑和脊髓有炎性细胞浸润、脱髓鞘及轴突损伤等改变。但小剂量组在体重减轻、临床症状评分及病理学改变等方面均较中、大剂量组明显。结论用MOG35-5550μg剂量免疫诱导的C57BL/6小鼠EAE模型稳定,可在今后的研究中应用。     

A transgenic model of central nervous system autoimmunity mediated by CD4+ and CD8+ T and B cells

Experimental autoimmune encephalomyelitis (EAE) is a widely used model of multiple sclerosis. In NOD mice, EAE develops as a relapsing-remitting disease that transitions to a chronic progressive disease, making the NOD model the only mouse model that recapitulates the full clinical disease course observed in most multiple sclerosis patients. We have generated a TCR transgenic mouse that expresses the α- and β-chains of a myelin oligodendrocyte glycoprotein (MOG) 35-55-reactive TCR (1C6) on the NOD background. 1C6 TCR transgenic mice spontaneously generate both CD4(+) and CD8(+) T cells that recognize MOG and produce proinflammatory cytokines, allowing for the first time to our knowledge the simultaneous examination of myelin-reactive CD4(+) and CD8(+) T cells in the same host. 1C6 CD8(+) T cells alone can induce optic neuritis and mild EAE with delayed onset; however, 1C6 CD4(+) T cells alone induce severe EAE and predominate in driving disease when both cell types are present. When 1C6 mice are crossed with mice bearing an IgH specific for MOG, the mice develop spontaneous EAE with high incidence, but surprisingly the disease pattern does not resemble the neuromyelitis optica-like disease observed in mice bearing CD4(+) T cells and B cells reactive to MOG on the C57BL/6 background. Collectively, our data show that although myelin-reactive CD8(+) T cells contribute to disease, disease is primarily driven by myelin-reactive CD4(+) T cells and that the coexistence of myelin-reactive T and B cells does not necessarily result in a distinct pathological phenotype.     

Separation of T and B lymphocytes from various mouse strains by density gradient electrophoresis

T and B mouse spleen lymphocytes were separated by density gradient electrophoresis on the basis of their surface charge. In all strains examined, the T lymphocytes were found in the high mobility fractions and the B in the low. The T and B cells were separated completely in most fractions, with some overlapping in the middle. Significant differences were found in the electrophoretic distribution profiles between the strains: C57BL/6j, C57BL/10j, (BALB/cXC57BL/6j)F₁, and all the following: B₆·C-H-2^d/cBy (congenic to C57BL/6j), BALB/c, CBA/H/T6j, C57BL/10Sn, and C3H. The C57BL/6j and the (BALB/cXC57BL/6j)F₁ cells appear more heterogeneous as far as electrophoretic mobility is concerned. Almost all the other strains give two major peaks. Moreover, the high mobility areas are less populated in the C57BL/6j and the (BALB/cXC57BL/6j)F₁ animals than in all the others. The above differences were found consistently when cells prepared by different methods were electrophoresed. It is concluded that the surface charge of lymphocytes may be genetically determined. Possible dependency on the H-2 complex or non-H-2 areas is discussed.     

Deletion of the complement anaphylatoxin C3a receptor attenuates, whereas ectopic expression of C3a in the brain exacerbates, experimental autoimmune encephalomyelitis

The C3aR is expressed throughout the CNS and is increased in expression on glial cells during CNS inflammation. However, the role that C3a and the C3aR play in chronic inflammation, such as in the demyelinating disease experimental autoimmune encephalomyelitis (EAE), remains unclear. We show in this study that deletion of the C3aR is protective in myelin oligodendrocyte glycoprotein-induced EAE in C57BL/6 mice. C3aR-deficient (C3aR(-/-)) mice had a significantly attenuated course of EAE compared with control mice during the chronic phase of the disease. Immunohistochemical analysis demonstrated modestly reduced macrophage and T cell infiltration in the spinal cords of C3aR(-/-) mice. To examine the role of C3a in EAE, we developed a transgenic mouse that expresses C3a exclusively in the CNS using the glial fibrillary acidic protein (GFAP) promoter. We observed that C3a/GFAP mice had exacerbated EAE during the chronic phase of the disease, with significant mortality compared with nontransgenic littermates. C3a/GFAP mice had massive meningeal and perivascular infiltration of macrophages and CD4(+) T cells. These studies indicate that C3a may contribute to the pathogenesis of demyelinating disease by directly or indirectly chemoattracting encephalitogenic cells to the CNS.     

Genetic Background Can Result in a Marked or Minimal Effect of Gene Knockout (GPR55 and CB2 Receptor) in Experimental Autoimmune Encephalomyelitis Models of Multiple Sclerosis

Endocannabinoids and some phytocannabinoids bind to CB₁ and CB₂ cannabinoid receptors, transient receptor potential vanilloid one (TRPV1) receptor and the orphan G protein receptor fifty-five (GPR55). Studies using C57BL/10 and C57BL/6 (Cnr2 ^tm1Zim) CB₂ cannabinoid receptor knockout mice have demonstrated an immune-augmenting effect in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis. However, other EAE studies in Biozzi ABH mice often failed to show any treatment effect of either CB₂ receptor agonism or antagonism on inhibition of T cell autoimmunity. The influence of genetic background on the induction of EAE in endocannabinoid system-related gene knockout mice was examined. It was found that C57BL/6.GPR55 knockout mice developed less severe disease, notably in female mice, following active induction with myelin oligodendrocyte glycoprotein 35-55 peptide. In contrast C57BL/6.CB₂ (Cnr2 ^Dgen) receptor knockout mice developed augmented severity of disease consistent with the genetically and pharmacologically-distinct, Cnr2 ^tm1Zim mice. However, when the knockout gene was bred into the ABH mouse background and EAE induced with spinal cord autoantigens the immune-enhancing effect of CB₂ receptor deletion was lost. Likewise CB₁ receptor and transient receptor potential vanilloid one knockout mice on the ABH background demonstrated no alteration in immune-susceptibility, in terms of disease incidence and severity of EAE, in contrast to that reported in some C57BL/6 mouse studies. Furthermore the immune-modulating influence of GPR55 was marginal on the ABH mouse background. Whilst sedative doses of tetrahydrocannabinol could induce immunosuppression, this was associated with a CB₁ receptor rather than a CB₂ receptor-mediated effect. These data support the fact that non-psychoactive doses of medicinal cannabis have a marginal influence on the immune response in MS. Importantly, it adds a note of caution for the translational value of some transgenic/gene knockout and other studies on low-EAE susceptibility backgrounds with inconsistent disease course and susceptibility.     

Role of the humoral immune response in resistance to Theiler''s virus infection.

Theiler's virus, a murine picornavirus, persists in the central nervous system of susceptible strains of mice, causing chronic inflammation and demyelination in the white matter of the spinal cord. Resistant strains, however, clear the virus and do not develop late disease. In this study, we compared the characteristics of T and B lymphocytes in C57BL/6 (resistant) and SJL/J (susceptible) mice 1 week after intracerebral infection. We detected a marked increase of the number of immunoglobulin M (IgM)-secreting cells in the spleens of C57BL/6 detected a marked increase of the number of immunoglobulin M (IgM)-secreting cells in the spleens of C57BL/6 mice (but not in those of SJL/J mice), which correlated with higher levels of serum IgM antiviral antibodies. The role of the humoral response in virus clearance and resistance was demonstrated by a marked decrease in the number of infected spinal cord cells in SJL/J mice after passive transfer of serum from infected C57BL/6 donors. The B-cell response was found to be partly T cell independent. These results suggest an important role of the early humoral immune response in resistance to Theiler's virus-induced disease.     

Separation of T and B lymphocytes from various mouse strains by density gradient electrophoresis.

T and B mouse spleen lymphocytes were separated by density gradient electrophoresis on the basis of their surface charge. In all strains examined, the T lymphocytes were found in the high mobility fractions and the B in the low. The T and B cells were separated completely in most fractions, with some overlapping in the middle. Significant differences were found in the electrophoretic distribution profiles between the strains: C57BL/6j, C57BL/10j, (BALB/cXC57BL/6j)F1, and all the following: B6.C-H-2d/cBy (congenic to C57BL/6j), BALB/c, CBA/H/T6j, C57BL/10Sn, and C3H. The C57BL/6j and the (BALB/cXC57BL/6j)F1 cells appear more heterogeneous as far as electrophoretic mobility is concerned. Almost all the other strains give two major peaks. Moreover, the high mobility areas are less populated in the C57BL/6j and the (BALB/cXC57BL/6j)F1 animals than in all the others. The above differences were found consistently when cells prepared by different methods were electrophoresed. It is concluded that the surface charge of lymphocytes may be genetically determined. Possible dependency on the H-2 complex or non-H-2 areas is discussed.     

Passive induction of experimental allergic encephalomyelitis

Experimental allergic encephalomyelitis (EAE) is a widely used animal model of the human demyelinating disease multiple sclerosis. EAE is initiated by immunization with myelin antigens in adjuvant or by adoptive transfer of myelin-specific T cells, resulting in inflammatory infiltrates and demyelination in the central nervous system. Induction of EAE in rodents typically results in ascending flaccid paralysis with inflammation primarily targeting the spinal cord. This protocol describes passive induction of EAE by adoptive transfer of T cells isolated from mice primed with myelin antigens into na?ve mice. The advantages of using this method versus active induction of EAE are discussed.     

Time-Dependent Progression of Demyelination and Axonal Pathology in MP4-Induced Experimental Autoimmune Encephalomyelitis

BackgroundMultiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) characterized by inflammation, demyelination and axonal pathology. Myelin basic protein/proteolipid protein (MBP-PLP) fusion protein MP4 is capable of inducing chronic experimental autoimmune encephalomyelitis (EAE) in susceptible mouse strains mirroring diverse histopathological and immunological hallmarks of MS. Limited availability of human tissue underscores the importance of animal models to study the pathology of MS.MethodsTwenty-two female C57BL/6 (B6) mice were immunized with MP4 and the clinical development of experimental autoimmune encephalomyelitis (EAE) was observed. Methylene blue-stained semi-thin and ultra-thin sections of the lumbar spinal cord were assessed at the peak of acute EAE, three months (chronic EAE) and six months after onset of EAE (long-term EAE). The extent of lesional area and inflammation were analyzed in semi-thin sections on a light microscopic level. The magnitude of demyelination and axonal damage were determined using electron microscopy. Emphasis was put on the ventrolateral tract (VLT) of the spinal cord.ResultsB6 mice demonstrated increasing demyelination and severe axonal pathology in the course of MP4-induced EAE. In addition, mitochondrial swelling and a decrease in the nearest neighbor neurofilament distance (NNND) as early signs of axonal damage were evident with the onset of EAE. In semi-thin sections we observed the maximum of lesional area in the chronic state of EAE while inflammation was found to a similar extent in acute and chronic EAE. In contrast to the well-established myelin oligodendrocyte glycoprotein (MOG) model, disease stages of MP4-induced EAE could not be distinguished by assessing the extent of parenchymal edema or the grade of inflammation.ConclusionsOur results complement our previous ultrastructural studies of B6 EAE models and suggest that B6 mice immunized with different antigens constitute useful instruments to study the diverse histopathological aspects of MS.     

Cutting edge: CD8 T cell-mediated demyelination is IFN-gamma dependent in mice infected with a neurotropic coronavirus

Mice infected with the murine coronavirus, mouse hepatitis virus, strain JHM (MHV) develop an immune-mediated demyelinating encephalomyelitis. We showed previously that adoptive transfer of MHV-immune splenocytes depleted of either CD4 or CD8 T cells to infected RAG1(-/-) recipients (mice deficient in recombination activation gene 1) resulted in demyelination. Herein we show that transfer of CD8 T cell-enriched splenocytes from MHV-immune IFN-gamma(-/-) donors resulted in a substantial decrease in demyelination (4.8% of the white matter of the spinal cord compared with 26.3% in those receiving cells from C57BL/6 donors). Similar numbers of lymphocytes were present in the CNS of recipients of either C57BL/6 or IFN-gamma(-/-) CD8 T cells, suggesting that IFN-gamma was not crucial for lymphocyte entry into the CNS. Rather, IFN-gamma was critical for optimal activation or migration of macrophages or microglia into the white matter in the context of CD8 T cell-mediated demyelination.     

T cell autoimmunity in Ig transgenic mice.

Autoantibodies directed at a diverse group of proteins of the U1/Sm ribonucleoprotein (snRNP) are characteristic of systemic lupus erythematosus and are found in the MRL murine model of this disease. This study examines the role of transgenic B lymphocytes in the regulation of autoreactive T cells to the snRNP autoantigen. Transgenic mice were developed bearing an Ig heavy chain gene specific for the D protein component of murine snRNP. B lymphocytes in these mice are neither deleted nor anergic and are of an immature (heat-stable Aghigh) phenotype. T lymphocytes from anti-snRNP transgenic mice were examined using a recombinant form of the D protein of the murine snRNP complex. Our results revealed that transgenic anti-snRNP B cell APCs stimulated CD4 T cells from wild-type C57BL/6 and MRL lpr/lpr mice, while nonspecific APCs failed to stimulate CD4 T cells. This study demonstrates that autoreactive T cells are not deleted from wild-type mice, although their activation is facilitated by autoantigen-specific APCs. The snRNP-reactive T cells in C57BL/6 transgenic mice are tolerized, in contrast to those T cells from MRL lpr/lpr transgenic mice. These studies implicate a role for autoreactive B lymphocytes in the in vivo activation and/or diversification of autoreactive T cells.     

Over-expression of Bcl-2 provides protection in septic mice by a trans effect

Transgenic mice that over-express B cell leukemia/lymphomas (Bcl)-2 in myeloid cells under control of the human MRP8 promoter (hMRP8-Bcl-2) or in T lymphocytes under the E micro promoter (E micro -Bcl-2) were compared with C57BL/6 control mice following cecal ligation and puncture (CLP). There was a significant difference in outcome between the hMRP8-Bcl-2 and control mice with 100% survival in the hMRP8-Bcl-2 mice vs 25% survival in the control mice. In separate experiments there was a significant difference between E micro -Bcl-2 and control mice with 87.5 and 22.2% survival, respectively. Adoptive transfer of CD11b-positive bone marrow cells from hMRP8-Bcl-2 or C57BL/6 mice to C57BL/6 mice subjected to CLP resulted in 100 and 0% survival, respectively. Adoptive transfer of CD11b-positive cells from either hMRP8-Bcl-2 or C57BL/6 mice to Rag-1(-/-) mice (no mature T or B cells) subjected to CLP resulted in survival of 87.5 and 12.5%, respectively. The hMRP8-Bcl-2 mice had significantly more neutrophils and fewer bacteria in the peritoneum compared with C57BL/6 mice 24 h after CLP. These experiments show that Bcl-2 over-expression is protective in CLP and that protection is independent of lymphocytes. We propose that over-expression of Bcl-2 in T cells or myeloid cells induce release of a molecule(s) that protects against death following CLP.