Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair.

Dysferlin and calpain are important mediators of the emergency response to repair plasma membrane injury. Our previous research revealed that membrane injury induces cleavage of dysferlin to release a synaptotagmin-like C-terminal module we termed mini-dysferlinC72. Here we show that injury-activated cleavage of dysferlin is mediated by the ubiquitous calpains via a cleavage motif encoded by alternately spliced exon 40a. An exon 40a-specific antibody recognizing cleaved mini-dysferlinC72 intensely labels the circumference of injury sites, supporting a key role for dysferlinExon40a isoforms in membrane repair and consistent with our evidence suggesting that the calpain-cleaved C-terminal module is the form specifically recruited to injury sites. Calpain cleavage of dysferlin is a ubiquitous response to membrane injury in multiple cell lineages and occurs independently of the membrane repair protein MG53. Our study links calpain and dysferlin in the calcium-activated vesicle fusion of membrane repair, placing calpains as upstream mediators of a membrane repair cascade that elicits cleaved dysferlin as an effector. Of importance, we reveal that myoferlin and otoferlin are also cleaved enzymatically to release similar C-terminal modules, bearing two C2 domains and a transmembrane domain. Evolutionary preservation of this feature highlights its functional importance and suggests that this highly conserved C-terminal region of ferlins represents a functionally specialized vesicle fusion module.

Gamma tropomyosin gene products are required for embryonic development.

The actin filament system is essential for many cellular functions, including shape, motility, cytokinesis, intracellular trafficking, and tissue organization. Tropomyosins (Tms) are rod-like components of most actin filaments that differentially affect their stability and flexibility. The Tm gene family consists of four genes, alphaTm, betaTm, gammaTm (Tm5 NM, where "NM" indicates "nonmuscle"), and deltaTm (Tm4). Multiple isoforms of the Tm family are generated by alternative splicing of three of these genes, and their expression is highly regulated. Extensive spatial and temporal sorting of Tm isoforms into different cellular compartments has been shown to occur in several cell types. We have addressed the function of the low-molecular-weight Tms encoded by the gammaTm gene by eliminating the corresponding amino-terminal coding sequences from this gene. Heterozygous mice were generated, and subsequent intercrossing of the F1 pups did not result in any viable homozygous knockouts. Genotype analysis of day 2.5 morulae also failed to detect any homozygous knockouts. We have failed in our attempts to delete the second allele and generate in vitro double-knockout cells, although 51 clones displayed homologous recombination back into the originally targeted locus. We therefore conclude that low-molecular-weight products from the gammaTm gene are essential for both embryonic development and cell survival.

Lymphotoxin controls alphaEbeta7-integrin expression by peripheral CD8+ T cells.

Lymphotoxin (LT)-alpha, a member of the TNF family, is recognized as an important mediator in different aspects of lymphoid organ development. Targeted disruption of this molecule resulted in a substantial reduction in the proportion of alphaEbeta7-integrin(high) CD8+ T cells detectable in peripheral lymphoid organs. This defect, however, was not observed on mature CD4-CD8+ thymocytes. To determine whether this was due to downregulation of beta7-integrin expression by peripheral CD8+ T cells or a failure of thymic emigration of CD8+ beta7-integrin(high) T cells, beta7-integrin was examined on recent thymic emigrants (RTE). When analysed within 16 h after leaving the thymus CD4-CD8+ RTE in both LT-alpha-/- and wild type (wt) mice remained beta7-integrin(high) and were indistinguishable. However, within 3-5 days, emigration loss of beta7-integrin became evident in LT-alpha-/- mice. Despite this loss, the proportion of thymically derived alphabetaTCR+ T-cell populations in the intestinal epithelium, an important target tissue of CD8+ alphaEbeta7-integrin(high) T cells, was increased in the absence of LT-alpha. In contrast, B cells were detectable only rarely in the intestinal tissue of LT-alpha-/- mice. The expression of E-Cadherin remained unchanged. These results indicate that a LT-alpha-dependent process maintains a high level of alphaEbeta7-integrin expression by peripheral CD8+ T cells, and with this control mechanism LT-alpha may help to regulate CD8+ T-cell numbers in the tissues.

Generation of splenic follicular structure and B cell movement in tumor necrosis factor-deficient mice.

Secondary lymphoid tissue organogenesis requires tumor necrosis factor (TNF) and lymphotoxin alpha (LTalpha). The role of TNF in B cell positioning and formation of follicular structure was studied by comparing the location of newly produced naive recirculating and antigen-stimulated B cells in TNF-/- and TNF/LTalpha-/- mice. By creating radiation bone marrow chimeras from wild-type and TNF-/- mice, formation of normal splenic B cell follicles was shown to depend on TNF production by radiation-sensitive cells of hemopoietic origin. Reciprocal adoptive transfers of mature B cells between wild-type and knockout mice indicated that normal follicular tropism of recirculating naive B cells occurs independently of TNF derived from the recipient spleen. Moreover, soluble TNF receptor-IgG fusion protein administered in vivo failed to prevent B cell localization to the follicle or the germinal center reaction. Normal T zone tropism was observed when antigen-stimulated B cells were transferred into TNF-/- recipients, but not into TNF/LTalpha-/- recipients. This result appeared to account for the defect in isotype switching observed in intact TNF/LTalpha-/- mice because TNF/LTalpha-/- B cells, when stimulated in vitro, switched isotypes normally. Thus, TNF is necessary for creating the permissive environment for B cell movement and function, but is not itself responsible for these processes.

Challenging cytokine redundancy: inflammatory cell movement and clinical course of experimental autoimmune encephalomyelitis are normal in lymphotoxin-deficient, but not tumor necrosis factor-deficient, mice.

Lymphotoxin (LT) is widely regarded as a proinflammatory cytokine with activities equivalent to tumor necrosis factor (TNF). The contribution of LT to experimental autoimmune encephalomyelitis (EAE) was examined using TNF/LTalpha-/- mice, TNF-/- mice, and a new LTalpha-/- line described here. All mice were generated directly in the C57BL/6 strain and used for the preparation of radiation bone marrow chimeras to reconstitute peripheral lymphoid organs and restore immunocompetence. This approach overcame the problems related to the lack of lymph nodes that results from LTalpha gene targeting. We show here that when LT is absent but TNF is present, EAE progresses normally. In contrast, when TNF is absent but LT is present, EAE is delayed in onset and inflammatory leukocytes fail to move normally into the central nervous system parenchyma, even at the peak of disease. In the absence of both cytokines, the clinical and histological picture is identical to that seen when TNF alone is deficient, including demyelination. Furthermore, the therapeutic inhibition of TNF and LTalpha with soluble TNF receptor in unmanipulated wild-type or TNF-/- mice exactly reproduces these outcomes. We conclude from these studies that TNF and LT are functionally distinct cytokines in vivo, and despite sharing common receptors, show no redundancy of function nor mutual compensation.

Critical points of tumor necrosis factor action in central nervous system autoimmune inflammation defined by gene targeting.

Tumor necrosis factor (TNF)-dependent sites of action in the generation of autoimmune inflammation have been defined by targeted disruption of TNF in the C57BL/6 mouse strain. C57BL/6 mice are susceptible to an inflammatory, demyelinating form of experimental autoimmune encephalomyelitis (EAE) induced by the 35-55 peptide of myelin oligodendrocyte glycoprotein. Direct targeting of a strain in which EAE was inducible was necessary, as the location of the TNF gene renders segregation of the mutated allele from the original major histocompatibility complex by backcrossing virtually impossible. In this way a single gene effect was studied. We show here that TNF is obligatory for normal initiation of the neurological deficit, as demonstrated by a significant (6 d) delay in disease in its absence relative to wild-type (WT) mice. During this delay, comparable numbers of leukocytes were isolated from the perfused central nervous system (CNS) of WT and TNF-/- mice. However, in the TNF-/- mice, immunohistological analysis of CNS tissue indicated that leukocytes failed to form the typical mature perivascular cuffs observed in WT mice at this same time point. Severe EAE, including paralysis and widespread CNS perivascular inflammation, eventually developed without TNF. TNF-/- and WT mice recovered from the acute illness at the same time, such that the overall disease course in TNF-/- mice was only 60% of the course in control mice. Primary demyelination occurred in both WT and TNF-/- mice, although it was of variable magnitude. These results are consistent with the TNF dependence of processes controlling initial leukocyte movement within the CNS. Nevertheless, potent alternative mechanisms exist to mediate all other phases of EAE.

Distinct roles for lymphotoxin-alpha and tumor necrosis factor in organogenesis and spatial organization of lymphoid tissue.

Specialized roles for the pro-inflammatory cytokines tumor necrosis factor (TNF) and lymphotoxin (LT) were characterized in TNF/LT alpha -/- and TNF -/- mice established by direct gene targeting of C57BL/6 ES cells. The requirement for LT early in lymphoid tissue organogenesis is shown to be distinct from the more subtle and varied role of TNF in promoting correct microarchitectural organization of leukocytes in LN and spleen. Development of normal Peyer's patch (PP) structure, in contrast, is substantially dependent on TNF. Only mice lacking LT exhibit retarded B cell maturation in vivo and serum immunoglobulin deficiencies. A temporal hierarchy in lymphoid tissue development can now be defined, with LT being an essential participant in general lymphoid tissue organogenesis, developmentally preceeding TNF that has a more varied and subtle role in promotion of correct spatial organization of leukocytes in LN and spleen PP development in TNF -/- mice is unusual, indicating that TNF is a more critical participant for this structure than it is for other lymphoid tissues.

Tumor necrosis factor blockade in actively induced experimental autoimmune encephalomyelitis prevents clinical disease despite activated T cell infiltration to the central nervous system.

Recently, we demonstrated that experimental autoimmune encephalomyelitis (EAE) in the rat, passively transferred using myelin basic protein (MBP)-reactive encephalitogenic CD4+ T cells, was preventable by administration of a p55-tumor necrosis factor-IgG fusion protein (TNFR-IgG). This was despite quantitatively and qualitatively normal movement of these MBP-specific T cells to the central nervous system (CNS). To extend these findings, the effect of TNFR-IgG on EAE actively induced by injection of MBP in complete Freund's adjuvant was examined. This form of EAE in the rat typically involves an acute, self-limiting neurological deficit, substantial CNS inflammation, but minimal demyelination. Here we show that administration of TNFR-IgG prior to onset of disease signs completely prevented the neurological deficit or markedly reduced its severity. This blockade of clinical disease was dissociated from weight loss which occurred at the same tempo and magnitude as in control rats exhibiting neurological signs of disease such as paralysis. The timing of TNF blockade was critical as established clinical disease was relatively refractory to TNFR-IgG treatment. Activated CD4+ T cells expressing normal or elevated levels of VLA4, major histocompatibility complex class II, MRC OX40 and CD25 were isolated from or immunohistochemically localized in the CNS of clinically healthy rats treated before disease onset. There was a reduction of the amount of other inflammatory leukocytes in the CNS of these treated animals but, more importantly, the activation state of inflammatory leukocytes, as well as that of microglia isolated from treated animals, was reduced. Thus, TNFR-IgG, when administered before disease onset, appears to act by inhibiting an effector function of activated T cells and possibly other inflammatory leukocytes necessary to bring about the neurological deficit. However, while TNF is a critically important cytokine for the early events leading to initiation of EAE, it is not a necessary factor in the acute neurological deficit characteristic of this form of EAE, once disease onset has occurred.

Gene targeting in C57BL/6 ES cells. Successful germ line transmission using recipient BALB/c blastocysts developmentally matured in vitro.

There are significant advantages to the production of gene-knockout mice directly in mouse strains other than 129. The availability now of ES cells derived from the C57BL/6 mouse strain presents workers with a valuable alternative. A major difficulty, however, is the requirement for BALB/c blastocysts as recipients for ES cell injection. Using standard procedures, few BALB/c blastocysts can be obtained. This limitation has now been resolved by harvesting BALB/c embryos at the early morula stage and maturing these to blastocysts by in vitro culture. Of early morulae harvested and cultured, over 70% were recovered as fully expanded and injectable blastocysts. C57BL/6 ES cell injection of these blastocysts has enabled the production of a number of gene-knockout mice with a success rate similar to that reported for ES cells derived from the 129 mouse strains.

Microglia induce CD4 T lymphocyte final effector function and death.

Microglia, a type of tissue macrophage, are the only cells in the central nervous system (CNS) parenchyma to express some major histocompatibility complex (MHC) class II constitutively or to upregulate expression readily. They are thought to play a role in CD4 T cell activation in autoimmune diseases such as multiple sclerosis, as well as in neurodegenerative conditions, Alzheimer's disease in particular. We show here that highly purified MHC class II+ microglia when tested directly ex vivo do indeed support an effector response by an encephalitogenic myelin basic protein-reactive CD4 T cell line from which production of the proinflammatory cytokines, interferon gamma and tumor necrosis factor, is elicited, but not interleukin (IL)-2 secretion or proliferation. After this interaction, the T cells die by apoptosis. Other nonmicroglial but CNS-associated macrophages isolated in parallel stimulate full T cell activation, including IL-2 production, proliferation, and support T cell survival. Neither CNS-derived population expresses B7.1/B7.2. Resident macrophages that terminate effector T cells in tissues constitute a novel and broadly applicable regulatory measure of particular relevance to processes of self-tolerance against sequestered antigens.

Unimpaired autoreactive T-cell traffic within the central nervous system during tumor necrosis factor receptor-mediated inhibition of experimental autoimmune encephalomyelitis.

The critical role of tumor necrosis factor (TNF) as a mediator in autoimmune inflammatory processes is evident from in vivo studies with TNF-blocking agents. However, the mechanisms by which TNF, and possibly also its homologue lymphotoxin alpha, contributes to development of pathology in rheumatoid arthritis and Crohn disease and in animal models like experimental autoimmune encephalomyelitis is unclear. Possibilities include regulation of vascular adhesion molecules enabling leukocyte movement into tissues or direct cytokine-mediated effector functions such as mediation of tissue damage. Here we show that administration of a TNF receptor (55 kDa)-IgG fusion protein prevented clinical signs of actively induced experimental autoimmune encephalomyelitis. Significantly, the total number of CD4+ T lymphocytes isolated from the central nervous system of clinically healthy treated versus diseased control animals was comparable. By using a CD45 congenic model of passively transferred experimental autoimmune encephalomyelitis to enable tracking of myelin basic protein-specific effector T lymphocytes, prevention of clinical signs of disease was again demonstrated in treated animals but without quantitative or qualitative impediment to the movement of autoreactive T lymphocytes to and within the central nervous system. Thus, despite the uninterrupted movement of specific T lymphocytes into the target tissue, subsequent disease development was blocked. This provides compelling evidence for a direct effector role of TNF/lymphotoxin alpha in autoimmune tissue damage.