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Immunohistochemical localization of calpains and calpastatin in the rabbit eye
272
Brain Research, 611 (1993) 272-278 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.0(/
BRES 18813
Immunohistochemical localization of calpains and calpastatin in the rabbit eye H~.kan Persson a, Seiichi Kawashima b and Jan-Olof Karlsson
a
a Department of Anatomy and Cell Biology, Unit~ersityof G6teborg, Giiteborg (Sweden) and h Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo (Japan) (Accepted 8 December 1992)
Key words: Calpain; Calpastatin; Eye; lmmunohistochemistry; Rabbit
The localization of the two Ca-activated extralysosomal proteases m-calpain and #-calpain in the eye of the adult rabbit was examined by immunohistochemistry,using poly- and monoclonal antibodies against the corresponding rabbit antigens. Immunoreactivity against the two forms of calpains was observed in the epithelial cells on the external and internal surfaces of the cornea as well as in the epithelial cells covering the iris and ciliary body. The sclera and choroid layers showed a relatively weak immunoreactivity. Using anti m-calpain antibodies, the pigment epithelium in the retina was heavily labelled as well as the outer and inner plexiform layers. The outer and inner borders of the Miiller cells were clearly labelled. The outer segments of the receptor cells showed a strong immunoreactivityfor both/x-calpain and m-calpain. Labelling was also observed in the retinal ganglion cells and in the nerve fiber layer. The immunohistochemical localization of calpastatin, an endogenous inhibitor of both m- and /x-calpain was also examined. A high level of calpastatin immunoreactivity was observed in the outer segments of the receptor cells. The results may be compatible with a role for calpains, especially m-calpain, in the secretory/phagocytic process and as modulators of the cytoskeleton in cell processes.
INTRODUCTION
f u n c t i o n has b e e n unequivocally coupled to the activity of calpain. T h e cellular localization a n d tissue distribu-
Calpains ( E C 3.4.22.17) are Ca-activated n e u t r a l
tion of these p r o t e i n s may give i m p o r t a n t clues for
SH-proteases which are f o u n d in most m a m m a l i a n
e l u c i d a t i o n of their functions. O n e a p p r o a c h is to es-
cells together with a specific e n d o g e n o u s inhibitor, calpastatin. T h e r e are two types of calpains in the cell; /x-calpain a n d m-calpain, which r e q u i r e /xM and m M c o n c e n t r a t i o n s of Ca, respectively, for initiation of activity. T h e c a l p a i n - c a l p a s t a t i n system may be involved in various aspects of the cell function, such as degradation of cytoskeletal 3'4"15'23 a n d myelin p r o t e i n s 5'14'~2'2~,
tablish this i n f o r m a t i o n in a single o r g a n with m a n y morphologically a n d functionally different cell types such as the eye. To our knowledge there are no studies on the histochemical localization of calpains a n d calpastatin in the eye.
modification a n d activation of enzymes, particularily p r o t e i n kinases 26, and r e g u l a t i o n of v o l t a g e - d e p e n d e n t calcium c h a n n e l s z. For a r e c e n t a n d c o m p r e h e n s i v e review of calpains a n d calpastatin see Croall a n d DeM a r t i n i 7. Histochemical studies have indicated that both /z-calpain a n d m - c a l p a i n are p r e s e n t in all parts of the n e u r o n , including the nerve terminals, a n d that they may be associated with cytoskeletal e l e m e n t s l'z°. Most investigations on calpain a n d calpastatin have b e e n devoted to in vitro e x p e r i m e n t s a n d no specific
MATERIALS AND METHODS Animals Healthy adult New Zealand White rabbits (body weight 3.5-5.5 kg) were used for immunohistochemical procedures. Antibodies Chicken polyclonal antibodies against /z-calpain and m-calpain were produced against the corresponding rabbit antigens (Karlsson et al., in preparation). Briefly, gel slices containing the 80 kDa subunit of m-calpain from rabbit lung, purified as previously describedz8 were mixed with incomplete Freunds adjuvant and used as immunogen. The IgY fraction was isolated from the yolk via precipitation with PEG and gel filtration on Sephacryl S-300 HR (antibody
Correspondence: J.-O. Karlsson, Department of Anatomy and Cell Biology, University of G6teborg, Medicinaregatan 5, S-413 90 G6teborg, Sweden. Fax: (46) (31) 829690.
273 c2a). For /z-calpain, the whole enzyme (80+30 kDa subunits) was used as immunogen. The isolated IgY fraction was passed through a column with m-calpain coupled to a Sepharose column to absorb cross-reacting antibodies to give an antibody (c3x) specific for /zcalpain. On Western blots these antibodies reacted only with the corresponding antigen. In a direct ELISA assay the m-calpain antibody (c2a) showed a 3% cross-reactivity with /x-calpain. The antibody against /z-calpain (c3x) showed 6% cross-reactivity against m-calpain (Karlsson et al., in preparation). Mouse monoclonal antibodies against the 80 kDa subunits of rabbit skeletal muscle/z-calpain (3CllB10) and m-calpain (2G4C9) were produced using standard procedures 12'13. A purified mouse polyclonal antibody preparation against a 40 kDa truncated fragment of the C-terminal half of calpastatin was produced as described previously t7.
Tissue preparation Rabbits were anesthetized with sodium pentobarbital (40 mg/kg body weight), exsanguinated with one liter ice-cold Tyrode's solution administered via the left ventricle and perfusion fixed with 4%
paraformaldehyde in phosphate buffer for 5 min. The animals were then enucleated bilaterally and the eyes opened by a small cut through the sclera posteriorly to the attachments of the external ocular muscles. The vitreous body was carefully rinsed out with fixation solution using a syringe and the eyes immersion fixed for another 24 h in perfusion fixation fluid. The eyes were then cut in half along the mid-sagittal plane and the halves transferred to 10% sucrose in PBS for 10 h, embedded in O.C.T mounting medium (Miles Inc., Elkhart, IN, USA), frozen in liquid N2 and 8-~m-thick cryostat sections were collected on poly-L-lysine coated glass slides.
Immunohistochemistry All incubations were done at +4°C. Cryostat sections were preincubated for 20 min in a blocking solution consisting of 5% bovine serum albumin (BSA), 1% normal rabbit serum and 0.5% Tween 20 in phosphate-buffered saline, pH 7.2 (PBS). After a brief rinse in PBS, the sections were incubated overnight with primary antibody; a 1:20 dilution of polyclonal anti /z-calpain antibody preparation, a 1:10 dilution of anti m-calpain antibody preparation
Fig. 1. Immunofluorescence distribution on 8-/zm-thick sections of cornea, ciliary body and iris. FITC optics, a: cornea stained with polyclonal antibodies against m-calpain. The arrow indicates the endothelium, b: cornea stained with polyclonal antibodies against m-calpain. The asterisk indicates the epithelium, c: cornea stained with monoclonal antibodies against m-calpain. The asterisk indicates the epithelium, d: anterior epithelium of cornea stained with polyclonal antibodies against /z-calpain. e: cornea stained with monoclonal antibodies against /~-calpain. f: cornea stained with mouse antibodies against calpastatin. The arrow indicates the endothelium. Asterisk indicates the epithelium, g: iris stained with polyclonal antibodies against m-calpain. The arrowhead indicates the anterior part. Arrow indicates the posterior part. h: the ciliary body stained with polyclonal antibodies against m-calpain. Arrows indicate packed blood cells in ciliary capillaries, i: the ciliary body stained with mouse antibodies against calpastatin. The arrow indicates the endothelium. Scale bars; 10/zm (a,c,d,e), 50 ~m (b,f,g,h,i).
274 or a 1 : 100 dilution of monoclonal anti /z-calpain, m-calpain antibodies or anti calpastatin antibodies. The sections were then rinsed 3 x 15 min in PBS, incubated lh with a 1:30 dilution of fluorescein isothiocyanate(FITC)-conjugated rabbit anti-chicken ( D A K O A / S , Copenhagen, Denmark) or anti-mouse (Sigma Inc. St. Louis, MO, USA) antibodies, again rinsed 3 × 15 min in PBS and mounted in D A B C O anti-fading agent (2.5 g 1,4 diazo-bicyclo(2,2)octane dissolved in 90 ml glycerin at 45°C on a water bath and 10 ml PBS pH 8.2 added. Stored in aliquots at -70°).
l"Tuorescence microscopy Cryostat sections were viewed in a Nikon Microphot-FXA microscope fitted with FITC epifluorescence optics. Photomicrographs were taken with Kodak Tri-X Pan black and white 400 A S A film.
Control procedures For cryostat sections, control incubations were performed where primary antibody was either omitted or replaced with parent myeloma cell culture supernatant (for monoclonal antibodies). A 10% rabbit eye homogenate was prepared by mincing fixed eyes in a Waring blendor followed by homogenization in a teflon-glass homogenizer. Large, remaining connective tissue pieces were then removed and the homogenate was washed three times in PBS with intervening centrifugations at 12,000 x g. After the last centrifugation, the pellet was suspended in PBS to give a 10% (v/v) suspension. All secondary antibodies were preabsorbed for 2 h against the rabbit eye homogenate and then filtered through 0.22 micrometer syringe filters with luer adapters (Microgon Inc., Laguna Hills, CA). This filtering also effectively removed FITC dust from the conjugated secondary antibodies. Before blocking nonspecific antibody binding, the sections were treated with 20 m M glycine in PBS for 10 min, and then rinsed for 5 min in PBS to eliminate tissue autofluorescence. These precautions were crucial and very effective, giving a very feeble, even background on the cryostat sections.
RESULTS Cornea
The polyclonal antibody against m-calpain gave an intensely positive reaction against the endothelium of the anterior chamber, covering the posterior side of the cornea (Fig. la). The underlying Descemets membrane was negative as well as the bundles of collagen fibers in the cornea. Immunoreactive flattened structures, probably representing the cytoplasmic extensions of fibroblasts, were noted between the collagen bundles in the corneal stroma. In addition, a diffuse staining was noted in the parts of the stroma underlying Descemets membrane (Fig. lb, see also Fig. la). The corneal epithelum was distinctly stained with a more intense
reaction present in a gradient towards the basal membrane (Fig. lb). The monoclonal antibodies against m-calpain, gave a distribution of reactivity very similar to that obtained with the polyclonal antibodies. The anterior epithelium showed a distinct positive reaction and the basal cells appeared to have a stronger immunoreactivity. As with the polyclonal antibody preparation the fibroblasts between the unstained collagen bundles in the corneal stroma were stained by these antibodies (Fig. ic). Incubation of the sections with polyclonal antibodies against ~-calpain revealed a tissue distribution with a general similarity to that observed using the antibodies against m-calpain. The epithelium and endothelium covering the anterior (Fig. ld) and posterior aspects of the cornea respectively, were distinctly and homogenously stained. A weak staining of the stromal fibroblasts was noted but, as compared to m-calpain, less staining was generally found in association with the corneal stroma. The monoclonal antibody against #-calpain stained the corneal epithelum intensely and uniformly whereas the corneal stroma was essentially negative (Fig. le). Less staining was observed over the cell nuclei as compared with the polyclonal antiserum. Incubation of the tissue sections with the antibodies against calpastatin showed a distribution similar to that observed with the calpain antibodies. The epithelium and especially the endothelium were distinctly stained (Fig. lf) and, again, antibody reactivity associated with structures probably representing fibroblast cytoplasmic extensions between the stromal collagen bundles was also seen. In general, the reaction was weaker than with the anti-calpain antibodies. Iris
The polyclonal antibodies against m-calpain stained the fibroblasts and the pigmented cells of the anterior part of the iris very strongly. The epithelium covering the posterior surface was also clearly positive. The central connective tissue was essentially negative, re-
Fig. 2. Immunofluorescense distribution on 8-~m-thick sections of retina. FITC optics, a: the retina stained with polyclonal antibodies against m-calpain. The arrow indicates pigment epithelium. Abbreviations: ILM, internal limiting membrane; NFL, nerve fibre layer; GC, Ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; O N L outer nuclear layer; ELM, external limiting membrane; RCL, receptor cell layer; PE, pigment epithelium; Ch, choroidea, b: the retina stained with polyclonal antibodies against m-calpain. The asterisk indicates the nerve fiber layer. Abbreviations: ILM, internal limiting membrane; ELM, external limiting membrane, c: the retina stained with monoclonal antibodies against m-calpain. The arrow indicates the inner limiting membrane. The black arrowhead indicates the outer limiting membrane. The black asterisk indicates the outer segments of the receptor cells. The white arrowhead indicates the choriocapillary layer of the choroid. The white asterisk indicates the sclera, d: the retina stained with polyclonal antibodies against ~-calpain. The arrowhead indicates retinal ganglion cells. The arrow shows the inner nuclear layer. The asterisk indicates the receptor cell layer, e: the retina stained with mouse polyclonal antibodies against calpastatin. Asterisk shows the receptor cell layer. The arrow indicates the previously described heavily stained structures in the choriocapillary layer. Scale bars: 30 # m .
276 vealing the course of stained radially and concentrically running fibers of the dilatator and sphincter muscles, respectively (Fig. lg). Incubation of sections of the iris with the polyclonal antibodies against /~-calpain showed that the muscle fibers were heavily stained as well as the posterior epithelium layers and scattered cells in the central connective tissue.
Ciliary body The polyclonal antibodies against m-calpain labelled the two layers of columnar cells covering the ciliary processes. The cells in the basal layer showed a stronger reactivity (Fig. lh). The fibers of the ciliary muscle were stained by the antibodies, while the surrounding loose connective tissue was negative. The endothelium of the ciliary capillaries was stained by the antibodies. Positive erythrocytes were occasionally seen within the capillaries. Incubation with the polyclonal antibody against /zcalpain revealed a uniform and weak labelling of the epithelial cell layers compared to the staining pattern of the antibody against m-calpain. The central connective tissue and ciliary muscle fibers, as well as erythrocytes within the ciliary capillaries, were heavily labelled. The calpastatin antibody revealed a clear and equally strong staining of the two cell layers in the epithelium. Very little staining of the connective tissue was found. The endothelium in the blood vessels showed a positive reaction with the antibody (Fig. li).
Retina Compared with the choroid and sclera the retina showed a generally stronger reaction with the polyclonal antibody against m-calpain (Fig. 2a and b). The pigment epithelium was densely stained. The inner segments of the cones showed a clearly positive reaction (Fig. 2a). In addition the outer segments of the receptor cells showed a relatively faint reaction. A distinct staining of the outer and inner plexiform layers was also observed. The cell nuclei in both the outer and inner nuclear layer stained very faintly or not at all, but were surrounded by strongly stained cytoplasm in the cell body region (Fig. 2a). A positive reaction was seen also in the nerve fiber layer and in the cell bodies of the retinal ganglion cells. The resolution did not allow a distinction between the axons in the nerve fiber layer and processes from the Miiller cells close to the vitreous body. A marked positive reaction was found at the location of the outer and inner parts of the Miiller cells corresponding to the outer and inner limiting membranes (Fig. 2b).
In the choriocapillary layer beneath the pigment epithelium conspicious, densely stained cell-like structures were observed (Fig. 2a and b). Most of these structures had the size and shape of erythrocytes whereas others were larger, somewhat elongated and showed a fine granulation in the cytoplasm (see also Fig. 2e). The staining pattern of the monoclonal antibodies against m-calpain was very similar to that obtained with the polyclonal antibodies. The inner and outer parts of the Miiller ceils (inner and outer limiting membrane) were markedly positive as well as parts of the inner segments of the cones (Fig. 2c). The retinal staining with the polyclonal antibody against/x-calpain was relatively weak compared to the staining of the inner parts of the choroid. The retinal distribution of immunoreactivity was more homogenously distributed than that of the polyclonal antibodies against m-calpain and also considerably weaker except for the staining of the choroid which was stronger (Fig. 2d). A dense staining of blood cells in the choroid was observed. Compared to the results obtained using antibodies against m-calpain the pigment epithelium was more weakly stained. In the receptor cell layer, structures that might correspond to the outer and inner segments of the receptor cells were labelled. The cell bodies in the retinal ganglion cell layer but also in the inner nuclear layer were weakly but distinctly labelled by the antibody (Fig. 2d). With the monoclonal antibodies against ~-calpain, a w~ak staining of the retinal layers was observed (data not shown). The general pattern was very similar to that obtained using the polyclonal antibodies. The cell bodies of the retinal ganglion cells showed a relatively high immunoreactivity as compared to the rest of the retina. Heavily stained blood cells were present in the choroidal vessels. The previously described finely granulated cells beneath the pigment cell layer were also seen. The antibodies against calpastatin gave a weak labelling of most parts of the retina (Fig. 2e). However the outer and inner segments of the receptor cells were densely stained, as well as the outer limiting membrane. Some staining was also observed in the outer plexiform layer. The previously described cells beneath the pigment epithelium were also labelled. Notably, a very weak labelling of the pigment epithelium was seen. DISCUSSION An earlier study, using western blotting and ELISA together with estimation of Ca-activated proteolytic
277 activity, has presented biochemical evidence for the presence of calpain and calpastatin in extracts from corneal epithelium of the rabbit 25. In line with this, it was demonstrated in the present immunohistochemical study that the epithelia covering both sides of the cornea were heavily labelled by poly- and monoclonal antibodies against /z-calpain and m-calpain. For comparison, in normal human epidermis it has been shown that /z-calpain primarily is localized in the granular layer and to a lesser degree in the basal layer 16. Our data indicate a more homogenous labelling of all cells in the anterior epithelium with slightly more m-calpain in the basal cell layers. Some reactivity was also observed in the keratocytes (fibroblasts) in the corneal stroma especially with the antibodies against m-calpain. Less labelling of the connective tissue was observed with the antibodies against /z-calpain. The immunoreactivity for calpastatin was very similar to that observed for/z-calpain. The general impression of a high level of m-calpain in the epithelial cells, on both sides of the cornea, compared to that of the fibroblasts in the connective tissue may imply that m-calpain is involved in a specific function in these cells. It is highly probable that the proliferation rate is higher in the epithelium and a possibility is that m-calpain is involved in the mitotic process 24. The corneal epithelium also has an extensive capacity for repair and m-calpain may be involved in modifications of the well developed cytoskeleton which occur in association with changes in cell shape and migratory activity. Another possibility is that m-calpain may be important for secretory processes in epithelial tissues l~. The endothelium covering the posterior part of the cornea is highly active in this regard, continously pumping fluid and ions from the stroma into the aqueous. The epithelium covering the ciliary processes is responsible for the formation of aquous humor. Active secretion by the inner (non-pigmented) cells probably plays a major role in this process 6. Relatively more immunoreactivity in the two layers of epithelial cells compared to the stroma was observed with the antibody against m-calpain. The epithelial layers were less stained with the antibodies against /x-calpain. These findings are, in line with the findings in the cornea, compatible with a role for m-calpain in the secretory process. Earlier investigations on the ultrastructural localization of calpains in the central nervous system have shown /~-calpain and m-calpain to be present in neuronal perikarya as well as in dendritic and axonal processes 9'1°'2°'21. Glial cell bodies and processes were also stained in these studies. In many cases /x-calpain was found deposited along the cytoskeleton2°. In the
present investigation, m-calpain immunoreactivity was generally much stronger in the retina than in the connective tissue of the choroid and sclera. All types of neuronal cells, including the receptors, as well as the (astrocytic) Miiller ceils were labelled. Particularly dense labelling was observed in the pigment epithelium. These cells are characterized by a high content of melanin granules (in pigmented animals) and a very high phagocytotic activity towards the outer segments of rods and cones. As discussed in several reports, there is evidence for a role of calpains in the endocytotic/exocytotic process 11'24'27. These cells also play an important role in controlling both fluid volume and the ionic milieu of the subretinal space. In the present study the pigment epithelium showed a lower immunoreactivity for p.-calpain as compared to m-calpain. Both /z-calpain and m-calpain immunoreactivity was observed in the outer segments of the receptor cells. Ca-activated proteolytic activity has earlier been described in photoreceptor cells of invertebrates s'~9. The functional significance of a hypothetical calpain-induced proteolytic activity in the outer segment of the receptor ceils secondary to a Ca influx from light sensitive cGMP-gated channels is not known. The shedding of packets of outer segment disk membranes or the secretion of interphotoreceptor matrix proteins may also be regulated by calpains. A rather surprising finding was the conspicious immunoreactivity for calpastatin in the outer segments of the photoreceptors. The high level of calpastatin at this site may imply a strict control of calpain activity in the outer segments. The inner and outer limiting membranes consisting of processes from the Mfiller cells, considered as a type of astrocytes, were strongly positive for the antibodies against m-calpain, in contrast to the rather faint reactivity for /x-calpain. One possibility is that m-calpain has a specialized role during modification of tight junctions a n d / o r rearrangement of the cytoskeleton. This suggestion is also supported by the finding of a relatively higher labelling of the outer and inner plexiform layers by the antibodies to m-calpain as compared to the/z-calpain labelling. Acknowledgements. This work was supported by the Swedish Medical Research Council (Projects 3157 and 5932), Axel Ax:son Johnsons Stiftelse, Magn. Bergvalls Stiftelse, Stiftelsen Handlanden Hj. Svenssons Forskningsfond, Lundbergs Stiftelse, O.E. and Edla Johanssons stiftelse, GLS and SLS.
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278 2 Belles, B., Hescheler, J., Trautwein, W., Blomgren, K. and Karlsson, J.-O., A possible physiological role of the Ca-dependent protease calpain and its inhibitor calpastatin on the Ca current in guinea pig myocytes, Eur. J. Physiol., 412 (1988), 554-556. 3 Billger, M., Wallin, M. and Karlsson, J.-O., Proteolysis of tubulin and microtubule-associated proteins 1 and 2 by calpain I and II. Difference in sensitivity of assembled and disassembled microtubules, Cell Calcium, 9,(1988) 33-44. 4 Burgoyne, R.D. and Cumming, R., Evidence for the presence of high-M(r) microtubule-associated proteins and their Ca-dependent proteolysis in synaptosomal cytosol, FEBS Lett., 146 (1982) 273-277. 5 Chakrabarti, A.K., Yoshida, Y., Powers, J.M., Singh, I., Hogan, E.L. and Banik, N.L., Calcium-activated neutral protease in rat brain myelin and subcellular fractions. J. Neurosci. Res., 20 (1988) 351-358. 6 Cole, D.F., Ocular fluids. In H. Davson (Ed), The Eye, Academic Press, New York, 1984, pp. 269-390. 7 Croall, D.E. and DeMartino, G.N., Calcium-activated neutral protease (calpain) system: Structure, function and regulation, Physiol. Retd., 71 (1991) 813-847. 8 DeCouet, H.G., Stowe, S. and Blest, A.D., Membrane-associated actin in the rhabdomeral microvilli of crayfish photoreceptors, J. Cell Biol., 98, (1984) 834-846. 9 Fukuda, T., Adachi, E., Kawashima, S., Yoshiya, I. and Hashimoto, P., Immunohistochemical distribution of calciumactivated neutral proteinases and endogenous CANP inhibitor in the rabbit hippocampus, J. Comp. NeuroL,. 302 (1990) 100-109. l0 Hamakubo, T., Kannagi, R., Murachi, T. and Matus, A., Distribution of calpain I and II in rat brain, J. Neurosci., 6 (1986) 3103-3111. 11 Hayashi, M., Kasai, Y. and Kawashima, S., Preferential localization of calcium-activated neutral protease in epithelial tissues, Biochem. Biophys. Res. Commun., 148 (1987) 567-574. 12 Inomata, M., Kasai, Y., Nakamura, M. and Kawashima, S., Activation mechanism of calcium-activated neutral protease, J. Biol. Chem., 263 (1988) 19783-19787. 13 Kasai, Y., lnomata, M., Hayashi, M., Imahori, K. and Kawashima, S., Isolation and characterization of monoclonal antibodies against calcium-activated neutral protease with low calcium sensitivity, J. Biochem., 100 (1986) 183-190. 14 Kerlero de Rosbo, N., Carnegie, P., and Bernard, C., Quantitative electroimmunoblotting study of the calcium activated neutral protease in human myelin, Z Neurochem., 47 (1986) 1007-1012. 15 Malik, M.N., Fenko, M.D., Iqbal, K. and Wisniewski, H.M., Purification and characterization of two forms of Ca-activated
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neutral protease from calf brain, Z Biol. Chem., 258 (1983) 8955-8962. Miyachi, Y., Yoshimura, N., Suzuki, S., Hamakubo, T., Kannagi, R., Imamura, S. and Murachi, T., Biochemical demonstration and immunohistochemical localization of calpain in human skin, Z Invest. Dermatol., 86 (1986) 346-349. Nakamura, M., Inomata, M., Imajoh, S., Suzuki, K. and Kawashima, S., Fragmentation of an endogenous inhibitor upon complex formation with high- and low-Cae+-requiring forms of calcium-activated neutral proteases, Biochemistry, 28 (1989) 449455. Nilsson, E. and Karlsson, J.-O., Slow anterograde axonal transport of calpain I and II, Neurochem. Int., 17 (1990) 487-494. Oldenburg, K. and Hubbell, W.L., Invertebrate rhodopsin cleavage by an endogenous calcium activated protease, Exp. Eye Res., 51 (1990) 463-472. Perlmutter, L., Siman, R., Gall, C., Seubert, P., Baudry, M. and Lynch, G., The ultrastructural localization of calcium-activated protease "calpain" in rat brain, Synapse, 2 (1988) 79-88. Perlmutter, L.S., Gall, C., Baudry, M. and Lynch, G., Distribution of calcium-activated protease calpain in the rat brain, J. Comp. Neurol., 296 (1990) 269-276. Persson, H. and Karlsson, J.-O., Calpain activity in a subcellular fraction enriched in partially degraded CNS myelin fragments compared with myelin, Neurosci. Lett., 130 (1991) 81-84. Reville, W.J., Goll, D.E., Stromer, M.H., Robson, R.M. and Dayton, W.R., A Ca-activated protease possibly involved in myofibrillar protein turnover, J. Cell. Biol., 70 (1976) 1-8. Schollmeyer, J.E., Calpain II involvement in mitosis, Science, 240 (1988) 911-913. Shearer, T.R., Azuma, M., David, L.L., Yamagata, Y. and Murachi, T., Calpain and calpastatin in rabbit corneal epithelium, Curr. Eye Res., 9 (1990) 39-44. Suzuki, K. and Ohno, S., Calcium activated neutral protease: Structure-function relationship and functional implications, Cell Struct. Funct., 15 (1990) 1-6. Verhallen, P.F.J., Bevers, E.M., Comfuruis, P. and Zwaal, R.F.A., Fluoride-dependent calcium-induced platelet procoagulant activity shows that calpain is involved in increased phospholipid transbilayer movement, Biochim. Biophys. Acta, 942 (1988) 150158. Yanagisawa, K., Sato, S., Amaya, N. and Miyatake, T., Degradation of myelin basic protein by calcium-activated neutral protease (CANP) in human brain and inhibition by E-64 analogue, Neurochem. Res., 8 (1983) 1285-1293.
Brain Research, 611 (1993) 272-278 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.0(/
BRES 18813
Immunohistochemical localization of calpains and calpastatin in the rabbit eye H~.kan Persson a, Seiichi Kawashima b and Jan-Olof Karlsson
a
a Department of Anatomy and Cell Biology, Unit~ersityof G6teborg, Giiteborg (Sweden) and h Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo (Japan) (Accepted 8 December 1992)
Key words: Calpain; Calpastatin; Eye; lmmunohistochemistry; Rabbit
The localization of the two Ca-activated extralysosomal proteases m-calpain and #-calpain in the eye of the adult rabbit was examined by immunohistochemistry,using poly- and monoclonal antibodies against the corresponding rabbit antigens. Immunoreactivity against the two forms of calpains was observed in the epithelial cells on the external and internal surfaces of the cornea as well as in the epithelial cells covering the iris and ciliary body. The sclera and choroid layers showed a relatively weak immunoreactivity. Using anti m-calpain antibodies, the pigment epithelium in the retina was heavily labelled as well as the outer and inner plexiform layers. The outer and inner borders of the Miiller cells were clearly labelled. The outer segments of the receptor cells showed a strong immunoreactivityfor both/x-calpain and m-calpain. Labelling was also observed in the retinal ganglion cells and in the nerve fiber layer. The immunohistochemical localization of calpastatin, an endogenous inhibitor of both m- and /x-calpain was also examined. A high level of calpastatin immunoreactivity was observed in the outer segments of the receptor cells. The results may be compatible with a role for calpains, especially m-calpain, in the secretory/phagocytic process and as modulators of the cytoskeleton in cell processes.
INTRODUCTION
f u n c t i o n has b e e n unequivocally coupled to the activity of calpain. T h e cellular localization a n d tissue distribu-
Calpains ( E C 3.4.22.17) are Ca-activated n e u t r a l
tion of these p r o t e i n s may give i m p o r t a n t clues for
SH-proteases which are f o u n d in most m a m m a l i a n
e l u c i d a t i o n of their functions. O n e a p p r o a c h is to es-
cells together with a specific e n d o g e n o u s inhibitor, calpastatin. T h e r e are two types of calpains in the cell; /x-calpain a n d m-calpain, which r e q u i r e /xM and m M c o n c e n t r a t i o n s of Ca, respectively, for initiation of activity. T h e c a l p a i n - c a l p a s t a t i n system may be involved in various aspects of the cell function, such as degradation of cytoskeletal 3'4"15'23 a n d myelin p r o t e i n s 5'14'~2'2~,
tablish this i n f o r m a t i o n in a single o r g a n with m a n y morphologically a n d functionally different cell types such as the eye. To our knowledge there are no studies on the histochemical localization of calpains a n d calpastatin in the eye.
modification a n d activation of enzymes, particularily p r o t e i n kinases 26, and r e g u l a t i o n of v o l t a g e - d e p e n d e n t calcium c h a n n e l s z. For a r e c e n t a n d c o m p r e h e n s i v e review of calpains a n d calpastatin see Croall a n d DeM a r t i n i 7. Histochemical studies have indicated that both /z-calpain a n d m - c a l p a i n are p r e s e n t in all parts of the n e u r o n , including the nerve terminals, a n d that they may be associated with cytoskeletal e l e m e n t s l'z°. Most investigations on calpain a n d calpastatin have b e e n devoted to in vitro e x p e r i m e n t s a n d no specific
MATERIALS AND METHODS Animals Healthy adult New Zealand White rabbits (body weight 3.5-5.5 kg) were used for immunohistochemical procedures. Antibodies Chicken polyclonal antibodies against /z-calpain and m-calpain were produced against the corresponding rabbit antigens (Karlsson et al., in preparation). Briefly, gel slices containing the 80 kDa subunit of m-calpain from rabbit lung, purified as previously describedz8 were mixed with incomplete Freunds adjuvant and used as immunogen. The IgY fraction was isolated from the yolk via precipitation with PEG and gel filtration on Sephacryl S-300 HR (antibody
Correspondence: J.-O. Karlsson, Department of Anatomy and Cell Biology, University of G6teborg, Medicinaregatan 5, S-413 90 G6teborg, Sweden. Fax: (46) (31) 829690.
273 c2a). For /z-calpain, the whole enzyme (80+30 kDa subunits) was used as immunogen. The isolated IgY fraction was passed through a column with m-calpain coupled to a Sepharose column to absorb cross-reacting antibodies to give an antibody (c3x) specific for /zcalpain. On Western blots these antibodies reacted only with the corresponding antigen. In a direct ELISA assay the m-calpain antibody (c2a) showed a 3% cross-reactivity with /x-calpain. The antibody against /z-calpain (c3x) showed 6% cross-reactivity against m-calpain (Karlsson et al., in preparation). Mouse monoclonal antibodies against the 80 kDa subunits of rabbit skeletal muscle/z-calpain (3CllB10) and m-calpain (2G4C9) were produced using standard procedures 12'13. A purified mouse polyclonal antibody preparation against a 40 kDa truncated fragment of the C-terminal half of calpastatin was produced as described previously t7.
Tissue preparation Rabbits were anesthetized with sodium pentobarbital (40 mg/kg body weight), exsanguinated with one liter ice-cold Tyrode's solution administered via the left ventricle and perfusion fixed with 4%
paraformaldehyde in phosphate buffer for 5 min. The animals were then enucleated bilaterally and the eyes opened by a small cut through the sclera posteriorly to the attachments of the external ocular muscles. The vitreous body was carefully rinsed out with fixation solution using a syringe and the eyes immersion fixed for another 24 h in perfusion fixation fluid. The eyes were then cut in half along the mid-sagittal plane and the halves transferred to 10% sucrose in PBS for 10 h, embedded in O.C.T mounting medium (Miles Inc., Elkhart, IN, USA), frozen in liquid N2 and 8-~m-thick cryostat sections were collected on poly-L-lysine coated glass slides.
Immunohistochemistry All incubations were done at +4°C. Cryostat sections were preincubated for 20 min in a blocking solution consisting of 5% bovine serum albumin (BSA), 1% normal rabbit serum and 0.5% Tween 20 in phosphate-buffered saline, pH 7.2 (PBS). After a brief rinse in PBS, the sections were incubated overnight with primary antibody; a 1:20 dilution of polyclonal anti /z-calpain antibody preparation, a 1:10 dilution of anti m-calpain antibody preparation
Fig. 1. Immunofluorescence distribution on 8-/zm-thick sections of cornea, ciliary body and iris. FITC optics, a: cornea stained with polyclonal antibodies against m-calpain. The arrow indicates the endothelium, b: cornea stained with polyclonal antibodies against m-calpain. The asterisk indicates the epithelium, c: cornea stained with monoclonal antibodies against m-calpain. The asterisk indicates the epithelium, d: anterior epithelium of cornea stained with polyclonal antibodies against /z-calpain. e: cornea stained with monoclonal antibodies against /~-calpain. f: cornea stained with mouse antibodies against calpastatin. The arrow indicates the endothelium. Asterisk indicates the epithelium, g: iris stained with polyclonal antibodies against m-calpain. The arrowhead indicates the anterior part. Arrow indicates the posterior part. h: the ciliary body stained with polyclonal antibodies against m-calpain. Arrows indicate packed blood cells in ciliary capillaries, i: the ciliary body stained with mouse antibodies against calpastatin. The arrow indicates the endothelium. Scale bars; 10/zm (a,c,d,e), 50 ~m (b,f,g,h,i).
274 or a 1 : 100 dilution of monoclonal anti /z-calpain, m-calpain antibodies or anti calpastatin antibodies. The sections were then rinsed 3 x 15 min in PBS, incubated lh with a 1:30 dilution of fluorescein isothiocyanate(FITC)-conjugated rabbit anti-chicken ( D A K O A / S , Copenhagen, Denmark) or anti-mouse (Sigma Inc. St. Louis, MO, USA) antibodies, again rinsed 3 × 15 min in PBS and mounted in D A B C O anti-fading agent (2.5 g 1,4 diazo-bicyclo(2,2)octane dissolved in 90 ml glycerin at 45°C on a water bath and 10 ml PBS pH 8.2 added. Stored in aliquots at -70°).
l"Tuorescence microscopy Cryostat sections were viewed in a Nikon Microphot-FXA microscope fitted with FITC epifluorescence optics. Photomicrographs were taken with Kodak Tri-X Pan black and white 400 A S A film.
Control procedures For cryostat sections, control incubations were performed where primary antibody was either omitted or replaced with parent myeloma cell culture supernatant (for monoclonal antibodies). A 10% rabbit eye homogenate was prepared by mincing fixed eyes in a Waring blendor followed by homogenization in a teflon-glass homogenizer. Large, remaining connective tissue pieces were then removed and the homogenate was washed three times in PBS with intervening centrifugations at 12,000 x g. After the last centrifugation, the pellet was suspended in PBS to give a 10% (v/v) suspension. All secondary antibodies were preabsorbed for 2 h against the rabbit eye homogenate and then filtered through 0.22 micrometer syringe filters with luer adapters (Microgon Inc., Laguna Hills, CA). This filtering also effectively removed FITC dust from the conjugated secondary antibodies. Before blocking nonspecific antibody binding, the sections were treated with 20 m M glycine in PBS for 10 min, and then rinsed for 5 min in PBS to eliminate tissue autofluorescence. These precautions were crucial and very effective, giving a very feeble, even background on the cryostat sections.
RESULTS Cornea
The polyclonal antibody against m-calpain gave an intensely positive reaction against the endothelium of the anterior chamber, covering the posterior side of the cornea (Fig. la). The underlying Descemets membrane was negative as well as the bundles of collagen fibers in the cornea. Immunoreactive flattened structures, probably representing the cytoplasmic extensions of fibroblasts, were noted between the collagen bundles in the corneal stroma. In addition, a diffuse staining was noted in the parts of the stroma underlying Descemets membrane (Fig. lb, see also Fig. la). The corneal epithelum was distinctly stained with a more intense
reaction present in a gradient towards the basal membrane (Fig. lb). The monoclonal antibodies against m-calpain, gave a distribution of reactivity very similar to that obtained with the polyclonal antibodies. The anterior epithelium showed a distinct positive reaction and the basal cells appeared to have a stronger immunoreactivity. As with the polyclonal antibody preparation the fibroblasts between the unstained collagen bundles in the corneal stroma were stained by these antibodies (Fig. ic). Incubation of the sections with polyclonal antibodies against ~-calpain revealed a tissue distribution with a general similarity to that observed using the antibodies against m-calpain. The epithelium and endothelium covering the anterior (Fig. ld) and posterior aspects of the cornea respectively, were distinctly and homogenously stained. A weak staining of the stromal fibroblasts was noted but, as compared to m-calpain, less staining was generally found in association with the corneal stroma. The monoclonal antibody against #-calpain stained the corneal epithelum intensely and uniformly whereas the corneal stroma was essentially negative (Fig. le). Less staining was observed over the cell nuclei as compared with the polyclonal antiserum. Incubation of the tissue sections with the antibodies against calpastatin showed a distribution similar to that observed with the calpain antibodies. The epithelium and especially the endothelium were distinctly stained (Fig. lf) and, again, antibody reactivity associated with structures probably representing fibroblast cytoplasmic extensions between the stromal collagen bundles was also seen. In general, the reaction was weaker than with the anti-calpain antibodies. Iris
The polyclonal antibodies against m-calpain stained the fibroblasts and the pigmented cells of the anterior part of the iris very strongly. The epithelium covering the posterior surface was also clearly positive. The central connective tissue was essentially negative, re-
Fig. 2. Immunofluorescense distribution on 8-~m-thick sections of retina. FITC optics, a: the retina stained with polyclonal antibodies against m-calpain. The arrow indicates pigment epithelium. Abbreviations: ILM, internal limiting membrane; NFL, nerve fibre layer; GC, Ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; O N L outer nuclear layer; ELM, external limiting membrane; RCL, receptor cell layer; PE, pigment epithelium; Ch, choroidea, b: the retina stained with polyclonal antibodies against m-calpain. The asterisk indicates the nerve fiber layer. Abbreviations: ILM, internal limiting membrane; ELM, external limiting membrane, c: the retina stained with monoclonal antibodies against m-calpain. The arrow indicates the inner limiting membrane. The black arrowhead indicates the outer limiting membrane. The black asterisk indicates the outer segments of the receptor cells. The white arrowhead indicates the choriocapillary layer of the choroid. The white asterisk indicates the sclera, d: the retina stained with polyclonal antibodies against ~-calpain. The arrowhead indicates retinal ganglion cells. The arrow shows the inner nuclear layer. The asterisk indicates the receptor cell layer, e: the retina stained with mouse polyclonal antibodies against calpastatin. Asterisk shows the receptor cell layer. The arrow indicates the previously described heavily stained structures in the choriocapillary layer. Scale bars: 30 # m .
276 vealing the course of stained radially and concentrically running fibers of the dilatator and sphincter muscles, respectively (Fig. lg). Incubation of sections of the iris with the polyclonal antibodies against /~-calpain showed that the muscle fibers were heavily stained as well as the posterior epithelium layers and scattered cells in the central connective tissue.
Ciliary body The polyclonal antibodies against m-calpain labelled the two layers of columnar cells covering the ciliary processes. The cells in the basal layer showed a stronger reactivity (Fig. lh). The fibers of the ciliary muscle were stained by the antibodies, while the surrounding loose connective tissue was negative. The endothelium of the ciliary capillaries was stained by the antibodies. Positive erythrocytes were occasionally seen within the capillaries. Incubation with the polyclonal antibody against /zcalpain revealed a uniform and weak labelling of the epithelial cell layers compared to the staining pattern of the antibody against m-calpain. The central connective tissue and ciliary muscle fibers, as well as erythrocytes within the ciliary capillaries, were heavily labelled. The calpastatin antibody revealed a clear and equally strong staining of the two cell layers in the epithelium. Very little staining of the connective tissue was found. The endothelium in the blood vessels showed a positive reaction with the antibody (Fig. li).
Retina Compared with the choroid and sclera the retina showed a generally stronger reaction with the polyclonal antibody against m-calpain (Fig. 2a and b). The pigment epithelium was densely stained. The inner segments of the cones showed a clearly positive reaction (Fig. 2a). In addition the outer segments of the receptor cells showed a relatively faint reaction. A distinct staining of the outer and inner plexiform layers was also observed. The cell nuclei in both the outer and inner nuclear layer stained very faintly or not at all, but were surrounded by strongly stained cytoplasm in the cell body region (Fig. 2a). A positive reaction was seen also in the nerve fiber layer and in the cell bodies of the retinal ganglion cells. The resolution did not allow a distinction between the axons in the nerve fiber layer and processes from the Miiller cells close to the vitreous body. A marked positive reaction was found at the location of the outer and inner parts of the Miiller cells corresponding to the outer and inner limiting membranes (Fig. 2b).
In the choriocapillary layer beneath the pigment epithelium conspicious, densely stained cell-like structures were observed (Fig. 2a and b). Most of these structures had the size and shape of erythrocytes whereas others were larger, somewhat elongated and showed a fine granulation in the cytoplasm (see also Fig. 2e). The staining pattern of the monoclonal antibodies against m-calpain was very similar to that obtained with the polyclonal antibodies. The inner and outer parts of the Miiller ceils (inner and outer limiting membrane) were markedly positive as well as parts of the inner segments of the cones (Fig. 2c). The retinal staining with the polyclonal antibody against/x-calpain was relatively weak compared to the staining of the inner parts of the choroid. The retinal distribution of immunoreactivity was more homogenously distributed than that of the polyclonal antibodies against m-calpain and also considerably weaker except for the staining of the choroid which was stronger (Fig. 2d). A dense staining of blood cells in the choroid was observed. Compared to the results obtained using antibodies against m-calpain the pigment epithelium was more weakly stained. In the receptor cell layer, structures that might correspond to the outer and inner segments of the receptor cells were labelled. The cell bodies in the retinal ganglion cell layer but also in the inner nuclear layer were weakly but distinctly labelled by the antibody (Fig. 2d). With the monoclonal antibodies against ~-calpain, a w~ak staining of the retinal layers was observed (data not shown). The general pattern was very similar to that obtained using the polyclonal antibodies. The cell bodies of the retinal ganglion cells showed a relatively high immunoreactivity as compared to the rest of the retina. Heavily stained blood cells were present in the choroidal vessels. The previously described finely granulated cells beneath the pigment cell layer were also seen. The antibodies against calpastatin gave a weak labelling of most parts of the retina (Fig. 2e). However the outer and inner segments of the receptor cells were densely stained, as well as the outer limiting membrane. Some staining was also observed in the outer plexiform layer. The previously described cells beneath the pigment epithelium were also labelled. Notably, a very weak labelling of the pigment epithelium was seen. DISCUSSION An earlier study, using western blotting and ELISA together with estimation of Ca-activated proteolytic
277 activity, has presented biochemical evidence for the presence of calpain and calpastatin in extracts from corneal epithelium of the rabbit 25. In line with this, it was demonstrated in the present immunohistochemical study that the epithelia covering both sides of the cornea were heavily labelled by poly- and monoclonal antibodies against /z-calpain and m-calpain. For comparison, in normal human epidermis it has been shown that /z-calpain primarily is localized in the granular layer and to a lesser degree in the basal layer 16. Our data indicate a more homogenous labelling of all cells in the anterior epithelium with slightly more m-calpain in the basal cell layers. Some reactivity was also observed in the keratocytes (fibroblasts) in the corneal stroma especially with the antibodies against m-calpain. Less labelling of the connective tissue was observed with the antibodies against /z-calpain. The immunoreactivity for calpastatin was very similar to that observed for/z-calpain. The general impression of a high level of m-calpain in the epithelial cells, on both sides of the cornea, compared to that of the fibroblasts in the connective tissue may imply that m-calpain is involved in a specific function in these cells. It is highly probable that the proliferation rate is higher in the epithelium and a possibility is that m-calpain is involved in the mitotic process 24. The corneal epithelium also has an extensive capacity for repair and m-calpain may be involved in modifications of the well developed cytoskeleton which occur in association with changes in cell shape and migratory activity. Another possibility is that m-calpain may be important for secretory processes in epithelial tissues l~. The endothelium covering the posterior part of the cornea is highly active in this regard, continously pumping fluid and ions from the stroma into the aqueous. The epithelium covering the ciliary processes is responsible for the formation of aquous humor. Active secretion by the inner (non-pigmented) cells probably plays a major role in this process 6. Relatively more immunoreactivity in the two layers of epithelial cells compared to the stroma was observed with the antibody against m-calpain. The epithelial layers were less stained with the antibodies against /x-calpain. These findings are, in line with the findings in the cornea, compatible with a role for m-calpain in the secretory process. Earlier investigations on the ultrastructural localization of calpains in the central nervous system have shown /~-calpain and m-calpain to be present in neuronal perikarya as well as in dendritic and axonal processes 9'1°'2°'21. Glial cell bodies and processes were also stained in these studies. In many cases /x-calpain was found deposited along the cytoskeleton2°. In the
present investigation, m-calpain immunoreactivity was generally much stronger in the retina than in the connective tissue of the choroid and sclera. All types of neuronal cells, including the receptors, as well as the (astrocytic) Miiller ceils were labelled. Particularly dense labelling was observed in the pigment epithelium. These cells are characterized by a high content of melanin granules (in pigmented animals) and a very high phagocytotic activity towards the outer segments of rods and cones. As discussed in several reports, there is evidence for a role of calpains in the endocytotic/exocytotic process 11'24'27. These cells also play an important role in controlling both fluid volume and the ionic milieu of the subretinal space. In the present study the pigment epithelium showed a lower immunoreactivity for p.-calpain as compared to m-calpain. Both /z-calpain and m-calpain immunoreactivity was observed in the outer segments of the receptor cells. Ca-activated proteolytic activity has earlier been described in photoreceptor cells of invertebrates s'~9. The functional significance of a hypothetical calpain-induced proteolytic activity in the outer segment of the receptor ceils secondary to a Ca influx from light sensitive cGMP-gated channels is not known. The shedding of packets of outer segment disk membranes or the secretion of interphotoreceptor matrix proteins may also be regulated by calpains. A rather surprising finding was the conspicious immunoreactivity for calpastatin in the outer segments of the photoreceptors. The high level of calpastatin at this site may imply a strict control of calpain activity in the outer segments. The inner and outer limiting membranes consisting of processes from the Mfiller cells, considered as a type of astrocytes, were strongly positive for the antibodies against m-calpain, in contrast to the rather faint reactivity for /x-calpain. One possibility is that m-calpain has a specialized role during modification of tight junctions a n d / o r rearrangement of the cytoskeleton. This suggestion is also supported by the finding of a relatively higher labelling of the outer and inner plexiform layers by the antibodies to m-calpain as compared to the/z-calpain labelling. Acknowledgements. This work was supported by the Swedish Medical Research Council (Projects 3157 and 5932), Axel Ax:son Johnsons Stiftelse, Magn. Bergvalls Stiftelse, Stiftelsen Handlanden Hj. Svenssons Forskningsfond, Lundbergs Stiftelse, O.E. and Edla Johanssons stiftelse, GLS and SLS.
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