Report |
Address correspondence to Norma W. Andrews, Section of Microbial Pathogenesis, Boyer Center for Molecular Medicine, Yale University School of Medicine, 295 Congress Ave., New Haven, CT 06510. Tel.: (203) 737-2410. Fax: (203) 737-2630. email: norma.andrews{at}yale.edu
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Abstract |
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Key Words: exocytosis; repair; lysosome; inflammatory myopathy; knockout mouse
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Introduction |
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Ca2+-regulated exocytosis of lysosomes was recently recognized as a ubiquitous process, which is not restricted to specialized secretory cells. Several cell types, such as fibroblasts and epithelial cells, respond to Ca2+ elevations by exposing lysosomal proteins on the plasma membrane and by releasing lysosomal contents (Rodriguez et al., 1997). Among several fluorescently tagged intracellular compartments analyzed by total internal reflection fluorescence microscopy, membrane-proximal lysosomes were found to be the major Ca2+-regulated exocytotic population (Jaiswal et al., 2002). In addition to other markers, these membrane-proximal lysosomes also contain Syt VII (Jaiswal et al., 2002). These findings are consistent with previous studies implicating Syt VII in the regulation of lysosomal exocytosis (Martinez et al., 2000), in the lysosome-mediated cell invasion mechanism of Trypanosoma cruzi (Caler et al., 2001), and in the process by which cells repair plasma membrane wounds (Reddy et al., 2001).
Ca2+ influx at the site of membrane injury triggers exocytosis, a process thought to be essential for cell resealing (McNeil and Steinhardt, 1997). Lysosomes are likely candidates for the vesicular population involved in this process, because inhibition of lysosomal exocytosis by introducing recombinant Syt VII C2A, anti-Syt VII C2A, or antiLamp-1 antibodies greatly reduced the efficiency of plasma membrane resealing in wounded fibroblasts (Reddy et al., 2001). Thus, a major role of Ca2+-triggered lysosomal exocytosis might be the maintenance of plasma membrane integrity. However, very limited information is available on the physiological consequences of defective membrane repair. A role for the sarcolemma protein dysferlin in a muscle-specific resealing mechanism was recently suggested, although the nature of the putative exocytotic vesicles involved in that process is still unknown (Bansal et al., 2003). To investigate the in vivo consequences of disrupting Ca2+-dependent exocytosis mediated by the ubiquitously expressed Syt VII, we performed targeted gene disruption in mice by homologous recombination.
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Results and discussion |
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The NH2-terminal, membrane-proximal region of synaptotagmins has been reported to mediate Ca2+-independent clustering (Bai et al., 2000; Fukuda et al., 2001a), so a putative dominant negative effect mediated by this domain cannot be ruled out. Immunofluorescence results with antibodies against the membrane-proximal Syt VII spacer domain, however, show that both the wild-type and mutated forms of Syt VII are detected in a normal lysosomal localization pattern, with no evidence of aggregation or mistargeting (Fig. 2 A). Therefore, as previously reported for the Golgi-targeted synaptotagmin isoform Syt IV (Fukuda et al., 2001b), our results indicate that the targeting signal of Syt VII is located within the membrane proximal spacer region (unpublished data).
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To investigate the consequences of impaired resealing in the mouse tissues, histological examination of various organs was performed in 14-wk-old wild-type and Syt VIIdeficient littermates. No obvious abnormalities were detected in the brain, liver, heart, exocrine pancreas, spleen, or kidneys of the mutant mice (unpublished data). However, sections of the skin and skeletal muscle showed an enhanced accumulation of connective tissue elements, indicative of fibrosis (Fig. 3 A). Examination of the skeletal muscle of younger animals revealed an extensive endomysial cellular infiltration in 4-wk-old mutant mice, and scattered foci of inflammation and fiber degeneration at 8 wk old (Fig. 3 B, left and center). At this stage, several muscle fibers were completely surrounded by inflammatory cells, and invasion of degenerating fibers by macrophages and eosinophils was apparent on light (Fig. 3 B, center) and electron microscopy analysis (Fig. 4 A). Mast cells and bundles of collagen fibers were frequently observed in the endomysial space (Fig. 4 A). The presence of inflammatory cells infiltrating the skeletal muscle of 8-wk-old Syt VII -/- mice was confirmed by immunocytochemistry with specific antibodies against markers of T cells (CD3), macrophages and NK cells (CD11b), and neutrophils and eosinophils (Ly-6G) (Fig. 4 B). Inflammatory cells were less abundant in the skeletal muscle of older Syt VII -/- mice but fibrosis remained detectable until 44 wk old (Fig. 3 B; Fig. 5 A).
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Conditions leading to autoimmunity are still poorly understood. It is known that self-reactive T cells can be deleted or suppressed in the periphery after the uptake and presentation of self-antigens by antigen presenting cells (Green and Flavell, 1999; Mellman and Steinman, 2001). However, most of the evidence available points to apoptotic cells generated during tissue turnover as the most common source of self-antigens acquired by antigen presenting cells. This steady-state antigen presentation process is thought to lead to tolerance when occurring in the absence of an inflammatory stimulus (Green and Flavell, 1999; Mellman and Steinman, 2001; Steinman and Nussenzweig, 2002). The autoimmune response associated with defective membrane repair in Syt VIIdeficient mice suggests that tolerance to antigens released from wounded cells may not be so readily achieved. Such release of intracellular contents mimics necrosis, a process that is proinflammatory and can lead to autoimmunity (Gallucci et al., 1999; Li et al., 2001). In this context, it is significant that the pathology observed in the Syt VII mutant mice occurs largely in tissues under mechanical stress, the skin and skeletal muscle. A similar pattern is observed in human polymyositis (affecting predominantly skeletal muscle) and dermatomyositis (affecting skeletal muscle and skin), suggesting that mechanical injury may represent an important element in the etiology of these serious tissue-specific autoimmune disorders.
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Materials and methods |
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Cells and antibodies
MEF were prepared from day 13.5 embryos (Tournier et al., 2000). Bone marrowderived macrophages were prepared by flushing tibia and femur bones with Dulbecco's minimal essential medium (DMEM) (Life Technologies) followed by culture in DMEM 10% FBS, and 30% L629 supernatant containing macrophage stimulating factor for 5 d. Syt VII-specific antibodies were generated by immunizing a rabbit with a recombinant GST-fusion protein containing amino acids 46133 of the Syt VII spacer region (Sugita et al., 2001), followed by affinity purification on Affigel (Bio-Rad Laboratories)-immobilized peptide. mAbs specific for CD3 (553239), CD11b (557395), and Ly-6G (553124) were obtained from BD Biosciences. AntiLamp-1 mAbs (1D4B) were obtained from the Developmental Studies Hybridoma Bank. Immunofluorescent localization of Lamp-1 and Syt VII was performed on MEFs fixed with 4% PFA in the presence of 0.1% saponin.
Histological analysis
Killed mice were perfusion fixed with 0.1 M cacodylate buffer, pH 7.4, containing 4% PFA, and 1% glutaraldehyde followed by organ removal, embedding in Epon resin, sectioning, and staining with toluidine blue. Thin sections of Epon-embedded muscle tissue were processed for transmission EM and examined in an electron microscope (Tecnai 12; Philips). H&E or Masson's trichrome staining was performed in paraffin-embedded blocks of tissues previously fixed in PBS containing 4% PFA. Immunocytochemistry was performed on 5-µm transverse cryosections of skeletal muscle using biotinylated mAbs followed by streptavidin-HRP (BD Biosciences) and DAB color development. Sections were examined in a microscope (model Axiovert 135; Carl Zeiss MicroImaging, Inc.) equipped with Metamorph software (Universal Imaging Corp.).
Hydroxyproline, creatine kinase, and T. cruzi invasion assays
Hydroxyproline content in mouse tissues was measured colorimetrically as described previously (Hao et al., 2000). Organs were weighed immediately after removal from killed mice, and the results were expressed as micrograms of hydroxyproline per milligrams of tissue. Quantitative, kinetic determination of creatine kinase in serum of control and Syt VII -/- mice was performed as described previously (Bogdanovich et al., 2002). T. cruzi invasion of wild-type (WT) and Syt VIIdeficient (KO) MEFs was assayed as described previously (Tardieux et al., 1992). At least 200 host cells from randomly chosen microscopic fields were analyzed for each experiment point.
Grip strength measurement
A grip strength meter (Columbus Instruments) was used to quantify the strength of wild-type and Syt VII -/- mice according to the manufacturer's instructions. Male mice were allowed to grab a triangular ring connected to a tension digital force transducer, and then gently pulled by the tail, away from the bar that measures the maximum tension produced. Three trials were done consecutively on each mouse, with no more than 30 s between each trial. The three values were averaged and correlated with the weight of the animal to generate data expressed as Newton of tension per kilogram of weight of the mouse.
Wound repair assays using fibroblast-collagen matrices
Suspensions of neutralized Vitrogen 100 collagen (Cohesion Corp.) at 1.5 mg/ml in serum-free DMEM containing 5 x 106 MEFs/ml were prepared as described previously (Lin et al., 1997; Reddy et al., 2001). The anchored matrices were lifted to initiate cell contraction and wounding by inserting a thin spatula between the collagen matrix and the dish surface. Samples of the supernatant were taken at 2-min intervals, always with replacement of an equal volume of 0.5 ml of fresh media. Background levels of LDH and ß-hexosaminidase activity (as determined in Reddy et al., 2001) detected pre-wounding were subtracted from each value. Total ß-hexosaminidase and LDH activity was determined in extracts of polymerized matrices treated with 1% Triton X-100 for 30 min.
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Acknowledgments |
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K. Kobayashi is a recipient of Inflammatory Bowel Disease grants from the Eli and Edythe L. Broad Foundation; R.A. Flavell is an Investigator of the Howard Hughes Medical Institute; and work in F. Gorelick's laboratory is supported by a merit award from the Veterans Administration. This work was supported by a National Research Service Award postdoctoral fellowship to D.R. Liston; a Research Supplements for Underrepresented Minorities award to K. Fowler; by the National Institutes of Health (NIH) and Burroughs Wellcome Scholar awards to N.W. Andrews; and G. Cline, R. LePine, and NIH mouse center grant U24DK-59635.
Submitted: 28 May 2003
Accepted: 7 July 2003
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