Current Eye Research
ISSN: 0271-3683 (Print) 1460-2202 (Online) Journal homepage: http://www.tandfonline.com/loi/icey20
Ciliary Body in Experimental Autoimmune Uveitis: Tissue Repair and Immunoreactivity of Extracellular Matrix Substances Benjamin Peng & Hitoshi Shichi To cite this article: Benjamin Peng & Hitoshi Shichi (1992) Ciliary Body in Experimental Autoimmune Uveitis: Tissue Repair and Immunoreactivity of Extracellular Matrix Substances, Current Eye Research, 11:11, 1087-1097, DOI: 10.3109/02713689209015080 To link to this article: http://dx.doi.org/10.3109/02713689209015080
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Date: 29 March 2016, At: 17:49
Current Eye Research
Volume I 1 number 11 1992, 1087-1097
Ciliary body in experimental autoimmune uveitis: tissue repair and immunoreactivity of extracellular matrix substances Benjamin Peng and Hitoshi Shichi
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Kresge Eye Institute, Department of Ophthalmology, Wayne State University School of Medicine, Detroit, MI 48201, USA
ABSTRACT Experimental autoimmune uveoretinitis was induced in female Lewis rats with bovine retinal soluble antigen (S antigen). Tissue changes and immunoreactivitiesof transforming growth factor-p (TGF-p ), epidermal growth factor (EGF), and extracellular matrix compounds in the anterior segment (ciliary body) were investigated by immunocytochemical methods. Control animals received adjuvant only. The immunized animals were sacrificed at day 0, 3, 7, 14, 20, and 30 postimmunization. Tissue changes that occurred at the peak of inflammation (day 14) included destruction of the inner basement membrane, epithelial cell loss, distortion of the ciliary stroma, and loss of epithelial basal infoldings. Ciliary body architecture was regenerated almost completely by day 30. Basement membrane laminin and collagen type IV levels did not change much during the inflammatory process. Fibronectin labeling level peaked at day 14 postimmunization. Collagen type V level was low at day 14 and elevated at day 20 and day 30. TGF-p immunoreactivity peaked at day 14 and remained elevated thereafter. EGF labeling did not increase until day 20 and was maximal at day 30. Labeling of both growth factors was principally confinded to the stromal regions. The presence of TGF-p and EGF in the ciliary stroma at well defined intervals suggests a coordinated effect upon the synthesis and reorganization of the extracellular matrix and possibly upon the inflammatory cell population in the anterior tissue. INTRODUCTION A single immunization with the retinal soluble protein, S-antigen, causes a rapidly progressive and severe experimental autoimmune uveoretinitis (EAU) in Lewis rats (1, 2). Histological investigations of retinal pathology (3,4) have shown the development of vasculitis, cellular infiltration and subsequent destruction of the retinal photoreceptor layer at the peak of inflammation. The permanent loss of photoreceptors and other retinal cells marks the late stages in the affected retina. Compared to the tissue changes in the posterior segment, pathological changes in the anterior segment of the eye have been less well studied in this uveoretinitis (5). It is widely believed that the extracellularmatrix is instrumental in modulating
cellular behavior, as well as serving a role in the presentation of vital polypeptides such as growth factors and adhesion molecules, and their respective receptors (6, 7). In this work we have followed the course of disease in the ciliary body at the UltIaStNCtUld level. We have also examined several extracellular matrix substances and growth factors using immunocytochemicalmethods at several key time points following S-antigen immunization.
MATERIALS AND METHODS Female Lewis rats (Harlan Sprague-Dawley, Indianapolis, IN), 150 - 180 g., were immunized intradennally in the footpad with 100 - 200 p1 of a purified bovine S-antigen (100 pg) emulsified in complete Freund’s adjuvant (CFAXDifco, Detroit, MI) (1: 1 ratio) supplemented with H37 RA Mycobacterium tuberculosis (Difco, 7 mg per ml adjuvant). S-antigen was purified as described (8). Control animals were immunized with CFM Mycobacterium only. The Guiding Principles in the Care and Use of Animals (DHEWPubl. NIH 80-23) were followed to handle animals. The animals were sacrificed by rapid Co;! asphyxiation at 0, 3, 7, 14, 20, and 30 days postimmunization. The eyes were removed and carefully divided into the anterior hemisphere containing ciliary, iris, lens, cornea and peripheral retina, and the posterior cup possessing only retina and choroidal stmctures. The tissues were immediately immersed in fmtive. For histological survey, the ocular tissues were fured in a mixture of phosphate-bufferedsaline (PBS), 4% formaldehyde and 2% glutamldehyde for 2 hours, rinsed in buffer and then post-fixed in 1% aqueous osmium tetroxide for 1 hour. These tissues were hrther sectioned, serially dehydmted from buffer to 100% ethanol and 100% propylene oxide, and embedded in Epon-Araldite.
Received on July 8 , 1992; accepted on October 15, 1992
0 Oxford University Press
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Ultrathin sections (50-70 nm) were prepared and poststained with uranyl acetate imd lead citmte prior to
animals each were sacrificed at day 14, 20 and 30. The
examination with a Phillips EM 301 electron microscope. For immunocytochemistry, the tissues were fixed with PBV 4% formaldehyde/ 0.2% gkutaraldehyde for I hour,
I. The ciliary tissues exhibited few morphological changes until early signs of acute disease developed beginning at day 1 1 to 13. At day 3, mononuclear cells were noted rarely in the anterior tissues. The basement membranes were physiically intact, the ciliary epithelial cells did not exhibit obvious pathology and the vessels, endothelium and stroma were normal in appearance (Fig. IA). Day 7 postimmunization rats closely resembled day 3 animals. No increase in inflammatory cells was observed in the tissues from 4 animals at this time point. Severe destruction was obvious at day 14 (Fig. IB), with large numbers of polymorphonuclear macrophages (PMN's) in the massive cellular infiltrate. Epithelial cilia were eradicated and the basement membrane of the nonpigmented epithelium (ME) and the epithelial cell layers were disrupted. The ciliary stroma was distorted and vascular integrity compromised. At the peak period
serially dehydrated from buffer fo 80% ethanol, and embedded in LR m i t e (Polysciences, Warrington, PA). Ultrathin sections (50-70 nm) were incubated successively on phosphate-buffered droplets of 1% bovine serum albumin (BSA)/ 0.25% casein blocking agent (Sigma, St. Louis, MO) for 15 minutes, priniary antibody (Ab), PBS and 1% BSA for 20 minutes, 10 or 15 nm gold-conjugated secondary Abs JE-Y Laboratories, San Mateo, CA) diluted 1 :20 in 0.05 M Tr i~C 1(pH 7.4)-0.05% Tween 20 buffer
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for I hour, and finally rinsed in Tris-Tween and in distilled water. For control nonimmune sera or gamma-globulins (species-matched with primary antibodies) were used in place of primary antibody. Primary antibody concentration for anti-EGF (Sigma), polyclonal anti-
TGF-6 (R&D Systems, Minneapolis, MN), anti-fibronectin (Sigma), anti-laminin (E-Y Labs) and anti-collagens type N and t' (Southern Biotech. Assoc., Birmingham, AL) was at 1:20 dilution in PBS. 'The results of multiple trials showed that a 2-3 hour incubation in the growth factor antibody solutions produced optimal labeling, while 1- 1.5 hours were needed for sufficient labeling with antibodies directed toward collagens and fikronectin. The blocking agent used was selected for emcacy after comparison of combinations of BSA, nonfat dry milk, casein and nonimmune sera. Sections were contrasted with uranyl acetate only, and then viewed with a transmission electron microscope. Micrographs of immunocyttxhemically treated tissues were examined for immunogald labeling. Eyes from at teast 4 different animals at each time point were sectioned, labeled and compared. Iabeling levels were quantitated by counting gold particles per unit area (9 pm2) of photographs at the same magnification. RESIII, I'S Four control animals immunized only with adjuvant did not develop disease. Of 27 animals immunized with Santigen, 4 rats each were sacrificed at day 3 and day 7. Of 18 rats which developed hypracvte FAU at day 14-15, 6
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day of immunization was day 0.
of disease, hemorrhage was clinically observed in the anterior chamber. Retrnal changes (data not presented) were consistent with the published observations on hyperacute EAU (3, 4). These findings included an initial perivascular mononuclear infiltration extending into the photoreceptor layer, retinal detachment, disruption of the nuclear cell layers and pan retinal involvement. Remarkable recovery of the ciliary body architecture was seen by day 20 postimmunization (Fig. IC). Mononuclear cell infiltration was greatly reduced, and PMKs were scarce. Lymphocytes (cells with dark-outlined nuclei in Figs. 1B and C) were found in the epithelial layers and also in the perivascular extracellular matrix . The ciliary body stroma appeared edematous, varying in thickness about the vasculature several times that of normals. The blood vessels (postcapilllary venules) also appeared distorted, with capillary-sized vessels numerically less evident than in normal tissues. Lymphocyte infiltration at day 30 postimmunization (Fig. 1D)was scarce in ocular tissues. Ciliary epithelial reorganization such as restoration of cellular infoldings was well advanced, but the ordered features present in normal epithelium and stroma remained noticeably altered. The perivascular stroma was thickened and more complex. At day 30, the postinflammatory retina showed an atrophied remnant with
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Current Eye Research
Figure 1: Electron micrographs showing (A) normal ciliary body at day 3, (B) loss of basement membrane, ciliary infoldings and infiltration by neutrophils at day 14, (C) residual infiltrating cell, re-establishment of ciliary
infoldings, thickened perivascular stroma at day 20, (D) day 30. [orig. mag. A) 2800 B) 2200 C) 1300 D) 18001. Scale bars = 10 Bm.
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Figure 2: lmmunocytochemistry showing gold label for fibronectin. A) Label at day 3 in basal lamina. B) Heavy labeling of basal lamina at day 14. C) Label was present in lamina d e w and vessel lumen at day 20. D) Labeling at .. .
.
.
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~
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an absent photoreceptor layer and obvious cell loss in the nuclear layers. 2. Anti-fibronedin antibody labeling was found in the stroma and basement membme before and after the peak period of disease, similar to those in normals (day 0 and 3) (Fig. 2A). Antibody-conjugated gold particles were found at a high density specifically in the basement membranes of ciliary epithelium at the peak of inflammation (day 14). There was no label present intmcellularly at day 14 (Fig. 2B). There was a moderate level of intmvascular label not associated with specific structures at day 14 and 20 (Fig.
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day 30 was similar to' day 3 (A). [orig.mag. A) 7500 B,CP) 59001. Scale bars = 1.0 Fm, v = blood vessel lumen, r = red blood cell, n = epithelial nucleus, arrowheads = immunogold. ~
2C). It decreased at day 30 (Fig. 2D). Labeling levels at day 0, 3, I, 20 and 30 in the basement membranes were essentially the same. 3. Antibodies to TGF-0 showed a low level of labeling in normals and day 3 pastimmunization rats (Fig. 3A). A rise in labeling was most evident in the stroma at day 14 (Fig. 3B) but was not confined to that area. The increased label diminished at day 20 (Fig. 3C) but remained higher than normals even at day 30 (Fig. 3D). No label was noted intmvascularly and no intmcellular structures were labeled consistently. Areas of stroma that appeared enlarged and
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Current Eye Research
Figure 3: Immunocytochemistry for TGF-p. A) Gold labeling was seen in the basal lamina at day 3. B) Increased label was seen in basal lamina at day 14 . C) At day 20, label was increased over day 3 (A). D) At
day 30, label was similar to or slightly less than that at day 20. [orig. mag. A) 4300 B) 7500 C,D) 59001. Scale bars = 1.0 pm, v = blood vessel lumen, arrowheads = immunogold.
less ordered, consistently showed increased gold labeling at day 14, 20 and 30 postimmunization. 4. At day 3 (Fig. 4A), 7 (not shown) and 14 (Fig. 4B), EGF immunoreactivitywas expressed minimally. Labeling of epidermal growth factor was definitely elevated at day 20 (Fig. 4C). A comparative analysis of the data indicated that the level continued to increase further by day 30 (Fig. 4D). EGF immunoreactivitywas noted to be high in the stroma, but gold particles were also occasionally seen at high density in intracellularvesicles
(data not shown). There was no significant intravascular or intravitreal labeling. 5. Type V collagen labeling was low in EAU animals at postimmunization day 3 and day 14 (Figs. 5A,B) and increased in the basal lamina at day 20 and day 30 (Figs. 5C, D). No intracellular, intravascular or vitreal accumulationswere evident. 6. Antibodies against type IV collagen and laminin (Figs. 6 and 7) labeled specifically and strongly the l a m b densa of the basal lamina at each of the time points
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Figure 4 EGF immunoreactivity at A) day 3, B) day 14; pattern resembled day 3 (1 1 particled4 pm2), C) day 20; EGF labeling was increased in the basal lamina, D) day 30;
label was substantially higher (21 particled9 urn2). [orig. mag.A,B,C,D) 59001. Scale bars = 1.0 pm, v = blood vessel lumen, arrowheads = immunogold.
examined. No significant variations in the level of label coutd be discerned. No intracellular, intravascular or intmvitreal accumulations were noted. Fig. 8 summarizes changes in labeling intensities at
TGF-p immunoreactivity in the retinal vascular endothelium increaseid at day 14 and remained high for a long time.
different stages of EAU, It i s interesting to note that TGFp irnmunoreactivity reaches a maximum at the peak of inflammation (day 14) and decreases slightly but remains
DISCUSSION We have demonstrated in this study that the ciliary body quickly regeneiates its cellular architecture after the peak period of disease is over. We have also noted changes in extracellular matrix components which correlate with the progression of M U activity.
higher than normal thereafter. In contrast, EGF irnmunoreactivity slowly rises in the postinflammatory period. The TGF-p labeling pattern is similar to the result of oar previous experiment on the retina of EAU rat (9);
The acute onset olf disease is heralded by an initial
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Current Eye Research
Figure 5: Immunocytochemistry for type V collagen showing A) labeling of lamina densa at day 3, B) sparse label at day 14, C) and D) increased labeling of basal
lamina at day 20 and 30. [orig. mag. A,B,C,D) 75001. Scale bars = 1.0 pm, v = vessel lumen, arrowheads = immunogold.
vasculitis followed shortly by massive cellular infiltration and tissue destruction (5, 10, 11). The corona ciliaris is immediatelyvulnerable to the actions of inflammatory cells and any serum-borne immunogenic factors by virtue of its high vascularity. The degree to which it is affected can be attested to by the breach of vascular integrity and hemorrhage. Yet the ciliary process is apparently able to rapidly regenerate its architecture and specialized cellular processes while the retina sustains the permanent loss of the photoreceptor layer (3,4). The difference in the ability of the tissues to regenerate must have serious functional
implications, because of the importance that these stluctures have in the eye. At the peak phase of the disease, i.e., day 14 postimmunization, the WE basement membrane is destroyed, but reappears by day 20 along with the reestablished epithelial layers. Noting the severe disruption in the acute phase, the ability of the NPE to reconstruct the cell layer and ciliary infoldings, and to elaborate extracellular matrix is remarkable. Epithelial cell loss probably occurred during EAU,but was not quantitated in this study. Mitotic figures were not
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Current Eye Research
Figure 6: Type IV collagen shows strong labeling of basement membranes at day A) 3, B) 14, C ) 20 and D) 30. -
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[orig. mag. A,B,C,D) 75001. Scale bars = 1.0 pm, v = blood vessel lumen, arrowheads = immunogold. ---__. . . ~
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prominent in the ciliary tissues at any of the time points exanlined in this study. The mature ciliary tissues are a
relatively stable population in the mouse (12), and are expected to be stable in the rat also. Evidence for increased nuclear synthetic activity in the ciliary epithelia i n the course of EAlJ may be obtained by investigating the
Several authors have suggested interactions between growth factors in mcdels of development, cell proliferation and tissue repair (13, 14). TGF-fi and ECF are among the more commonly cited, and their models of interactions
-
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R
3
14
20
30
Time in days - E
m c5 Figure 8: Changes in immunolabelingintensities of TGFp, EGF and extracellular matrix components with time. Counting of gold particles per unit area (9 pm2) was made from photographs at the same magnification. F, fibronectin. T, TGF-p. E, EGF. C5, collagen type V.
vary depending on the model in question. Whether their presence in this model of EAU is a matter of synergism or coincidence is not known. A specific synergistic effect by TGF-0 and EGF on fibroblast cell culture gene transcription was reported, an observation suggestive of a possible physiological mechanism (15). A reciprocal action of the extracellular matrix on the activity of the growth factors has also been reported in vitro (16). We have noted that the POIYCIOMI TGF-p antibody labeling peaks before that of EGF in the ciliary stroma. Although the cellular concentration of TGF-p and EGF are generally very low, significant quantities of both factors are found in the extracellular matrix. As determined immunohistochemically,intracellular concentrations of these factors in the epithelium, fibroblasts or endothelium are not consistent. The large influx of inflammatorycells is a possible source, as TGF-0 is found at high concentrationsin platelets (17, 18) and macrophages (19). Changes in fibronectin and the constituent collagens,
such as type V, can reflect changes occurring in the extracellular matrix. Fibronectin in particular has been shown here to have a very high labeling intensity at day 14 postimmunization,which is localized to the basement membranes and stroma. Whether this is a function of the binding availability of antigenic sites a n d or increased free fibronectin binding or concentration may be answered with further studies. It is possible that more antigenic sites are available in a disrupted, edematous extracellular matrix. However, tissues at other time points which also have an edematous stroma do not demonstrate the same increase in labeling. Laminin and type IV collagen, which are also constituent basement membrane proteins, do not vary in the same fashion. The intraluminal fibronectin content at day 14 is suggestive of increased soluble fibronectin, which may induce the morphological tissue changes that are notable postuveitic sequelae, such as fibrosis, and alterations in the basal lamina (6, 20). Fibronectin has been shown to have a positive effect on corneal epithelial wound healing (21, 22) by stimulating epithelial migration in concert with EGF . It has also been demonstrated that TGF-0 and EGF are active in regulating the expression of fibronectin and collagens, by stimulating fibroblast chemotaxis, inducing increases in mRNA levels, and increasing incorporation into the extmcellular matrix (2329). TGF-p is known to increase cellular expression of the integrin family of adhesion protein receptors which mediate binding of cells to extracellular matrix proteins including collagen and fibronectin (28). TGF-0 has suppressive effects on the proliferation of T and B lymphocytes and monocytes (30). This may be an additional regulatory effect of TGF-p on the course of
EAU. We have demonstrated a remarkable pathophysiologicalprocess that occurs in the ciliary body more closely than before. We have shown a corresponding change in the detected levels of factors known to be involved in tissue repair. Further studies on the receptors to these factors and manipulation of factor levels in vivQ may be rewarding in deriving further answers in this useful model of autoimmune inflammation. ACKNOWLEDGEMENTS The authors thank Dr. Linda Hazlett for allowing us to use facilities supported by the core grant, Mark Heil for his
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Current Eye Research technical assistance in the purification of bovine S-antigen, Debra Jones for typing the manuscript, and Katherine Morehead for photographic service. Supported by EY03807, T32 EY07093, core grant P30 EY04068, and a grant from Research to Prevent Blindness.
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CORRESPONDING AUTHOR Dr. Hitoshi Shichi, Kresge Eye Institute, Wayne State University School of Medicine, Detroit, MI 48201, U.S.A. REFERENCES 1. Faure, J.-P. (1980) Autoimmunity and the retina. Curr. Topics Eye Res. 2, 215-302. 2. Gery, I., Mochizuki, M. and Nussenblatt, R.B. (1986) Retinal specific antigens and immunopathogenic processes they provoke. Prog. Retinal Res. 3, 75- 109. 3, De Kozak, Y., Thillaye, B., Renard, G. and Faure, J.Y. (1978) Hyperacute form of experimental autoimmune uveo-retinitis in Lewis rats; electron microscopic study. Graefe's Arch. Clin. Exp. Ophthalmol. 208, 135- 142. 4. Lin, W.-L., Essner, E. and Shichi, H. (1991) Breakdown of the blood-retinal barrier in S-antigeninduced uveoretinitis in rats. Graefe's Arch. Clin. Exp. Ophthalmol. 229, 457-463. 5 . Forrester, J.V., Liversidge, J., Dua, H.S., Towler, H. and McMenamin, P.G. (1990) Comparison of clinical and experimental uveitis. &IT. Eye Res. 9 ( S U , 75-84. 6. McDonald, J.A. (1988) Extracellular matrix assembly. Ann. Rev. C M . Biol. 4, 183-207. 7. Merwin, J.R., Anderson, J.M., Kocher, O., van Itallie, C.M. and Madri, J.A. (1990) Transforming growth factor beta 1 modulates extracellular matrix organization and cell-cell junctional complex formation during in vitro angiogenesis. J. Cell. Physiol. 142,117-128. 8. Das, N.D., Ulshafer, R.F., Tam, Z.S., Leverenz, V.R. and Shichi, H. (1984) Radioimmunocytochemical localization of retinal S-antigen with monoclonal antibodies. J. Histochem. Cytochem. 32, 834-838. 9. Mahalak, S.M., Lin, W.-L., Essner, E. and Shichi, H. (1991) hcreased immunoreactivity of collagen types I, KU and V, fibronectin and TGF-6 in retinal vessels of rats with experimental autoimmune uveoretinitis. Curr. Eye Res. lo, 1059-1063. 10. De Kozak, Y., Sakai, J. , $hillaye, B. and Faure, J.P. (1981) S antigen-induced experimental autoimmune uveoretinitis in rats. Curr. Eye Res. 1, 327-340. 11. Rao, N.A., Wacker, W.B. and Marak, G.E. (1979) Experimental allergic uveitis: Clinicopathologic features associated with varying doses of S-antigen. Arch. Ophthalmol. 97, 1954-1958. 12. McDonald, T.F. and Green, K. (1988) Cell turnover in ciliary epithelium compared to other slow renewing epithelia in the adult mouse. Curr.Eye Res. 1,247252. 13 Richter, K.H., Schnapke, R., Clauss, M., Fiistenberger, C., Him, 1). and Marks, F. (1990) Epidermal GI-chalone and transforming growth factor fr are two different endogenous inhibitors of epidermal cell proliferation. J. Cell. Physiol. 142,496 - 504.
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14. Tripathi, R.C., Borisuth, N.S.C. and Tripathi, B J . (199 1) Growth factors in the aqueous humor and their thempeutic impliciitions in glaucoma and anterior segment disorders of the human eye. Drug. Develop. R e s . 2 , 1-23. 15. Ranganathan, G. and Getz, M J . (1990) Cooperative stimulation of specific gene transcription by epidermal growth factor and transforming growth factor 61. J. Biol. Chem. 265, 3001-3004. 16. Schlumberger, W., Thie, M., Rauterberg, J. and Robenek, H. (199 I) Collagen synthesis in cultured aortic smooth muscle cells: modulation by collagen lattice culture, tmlsforming growth factor-61, and epidermal growth factor. Arterioscler. Thromb. It, 1660 - 1666. 17. Childs, BJ., Proper, J.A., Tucker, R.F. and Moses, H.L. (1982) Semm contains a platelet-derived transforming growth factor. Proc. Natl. Acad. Sci. USA,B, 5312-5316. 18. Assoian, R.K., Komoriya, A., Meyers, C.A., Miller, D.M. and Spom, M.B. (1983) Transforming growth factor-6 in human platelets: identification of a major storage site, purification and characterization. J. Biol. Chem. 258, 7155 - 7160. 19. Assoian , R.K., Fleurdelys, B.E., Stevenson, H.C., Miller, PJ., Madtes, D.K., Raines, E.W., Ross, R . and Spom, M.B. (1987) Expression and secretion of type 6 transforming growth factor by activated human macrophages. Proc. Natl. Acad. Sci. USA, &, 6020 6024. 20. Connor,T.B., Roberts,A.B., Sporn, M.B., Danielpour, D., ]Dart, L.L., Michels, R.G., de Bustros, S., Enger, C., Kato,H., Lansing, M., Hayashi, H. and Glaser, B.M. (1989) Correlation of fibrosis and translforming growth factor-6 type 2 levels in the eye. J. Clin. Invest. 83, 1661 - 1666. 21. Watanabe, K., Nakagawa, S. and Nishida, T. (1988) Chemotactic and haptotactic activities of fibronectin for cultured rabbit corneal epithelial cells. Invest. Ophthalmol. Vis. Sci. 29,572 - 577. 22. Nishida,T., Nakamura, M., Mishima, H.and Otori, T. (1990) Differential modes of action of fibronectin and epidermal growth factor on rabbit corneal epithelial migration. J. Cell. Physiol. 145,549 - 554. 23. Ignotz, R.A. and Massague, J. (1986) Transforming growth factor 0 stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J. Biol. Chem. 261,4337 4345. 24. Ignotz, R.A., Endo, T., Massague, J. (1987) Regulation of fiblronectin and type 1 collagen mRNA levels by transforming growth factor p. J. Biol. Chem. 262, 6443-6446. 25. Stlethwaite, A.E., Keski-Oja, J., Moses, H.L. and Kang, A.H. (19117) Stimulation of the chemotactic migration of h u n m fibroblasts by transforming growth factor p. .I. Exp. Med. 165,251 - 256. 26. Carpenter, G. and Cohen, S. (1975) Human epidermal growth factor and the proliferation of human fibroblasts. J. Cell. Physiol. 88,227 - 238. 27. Roberts, A.B., Spom, M.B., Assoian, R.K., Smith, J.M., Roche, N..S.,Wakefield, L.M., Heine, U.I., Liotta, L.A., Falanga, V., Kehrl, J.H. and Fauci, AS. (1986) Trainsforminggrowth factor type p: rapid induction of fibrosis and angiogenesis in vitro and stimulation of cclllagen formation in vitro. Proc. Natl. Acad. Sci. USA,83,4167 - 4171.
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28. Roberts, A.B., Heine, U.I., Flanders, K.C. and Spom, M.B. (1990) Transforming growth factor B: major role in regulation of extracellular matrix. Ann. New York A d . Sci. 593,225 - 232. 29. Peltonen, J., Hsiao, L.L., Jaakkola, S., Sollberg, S., Aumailley, M., Timpl, R., Chu, M.-L. and Uitto, J . (199 1) Activation of collagen gene expression in keloids: co-localition of type I and VI collagen and transforming growth factor-sl mRNA. J. Invest. Dermatol. 97, 240 - 248. 30. Massague, J. (1990) The transforming growth factor-0 family. Ann. Rev. Cell Biol. 6, 597-641.
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ISSN: 0271-3683 (Print) 1460-2202 (Online) Journal homepage: http://www.tandfonline.com/loi/icey20
Ciliary Body in Experimental Autoimmune Uveitis: Tissue Repair and Immunoreactivity of Extracellular Matrix Substances Benjamin Peng & Hitoshi Shichi To cite this article: Benjamin Peng & Hitoshi Shichi (1992) Ciliary Body in Experimental Autoimmune Uveitis: Tissue Repair and Immunoreactivity of Extracellular Matrix Substances, Current Eye Research, 11:11, 1087-1097, DOI: 10.3109/02713689209015080 To link to this article: http://dx.doi.org/10.3109/02713689209015080
Published online: 02 Jul 2009.
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Date: 29 March 2016, At: 17:49
Current Eye Research
Volume I 1 number 11 1992, 1087-1097
Ciliary body in experimental autoimmune uveitis: tissue repair and immunoreactivity of extracellular matrix substances Benjamin Peng and Hitoshi Shichi
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Kresge Eye Institute, Department of Ophthalmology, Wayne State University School of Medicine, Detroit, MI 48201, USA
ABSTRACT Experimental autoimmune uveoretinitis was induced in female Lewis rats with bovine retinal soluble antigen (S antigen). Tissue changes and immunoreactivitiesof transforming growth factor-p (TGF-p ), epidermal growth factor (EGF), and extracellular matrix compounds in the anterior segment (ciliary body) were investigated by immunocytochemical methods. Control animals received adjuvant only. The immunized animals were sacrificed at day 0, 3, 7, 14, 20, and 30 postimmunization. Tissue changes that occurred at the peak of inflammation (day 14) included destruction of the inner basement membrane, epithelial cell loss, distortion of the ciliary stroma, and loss of epithelial basal infoldings. Ciliary body architecture was regenerated almost completely by day 30. Basement membrane laminin and collagen type IV levels did not change much during the inflammatory process. Fibronectin labeling level peaked at day 14 postimmunization. Collagen type V level was low at day 14 and elevated at day 20 and day 30. TGF-p immunoreactivity peaked at day 14 and remained elevated thereafter. EGF labeling did not increase until day 20 and was maximal at day 30. Labeling of both growth factors was principally confinded to the stromal regions. The presence of TGF-p and EGF in the ciliary stroma at well defined intervals suggests a coordinated effect upon the synthesis and reorganization of the extracellular matrix and possibly upon the inflammatory cell population in the anterior tissue. INTRODUCTION A single immunization with the retinal soluble protein, S-antigen, causes a rapidly progressive and severe experimental autoimmune uveoretinitis (EAU) in Lewis rats (1, 2). Histological investigations of retinal pathology (3,4) have shown the development of vasculitis, cellular infiltration and subsequent destruction of the retinal photoreceptor layer at the peak of inflammation. The permanent loss of photoreceptors and other retinal cells marks the late stages in the affected retina. Compared to the tissue changes in the posterior segment, pathological changes in the anterior segment of the eye have been less well studied in this uveoretinitis (5). It is widely believed that the extracellularmatrix is instrumental in modulating
cellular behavior, as well as serving a role in the presentation of vital polypeptides such as growth factors and adhesion molecules, and their respective receptors (6, 7). In this work we have followed the course of disease in the ciliary body at the UltIaStNCtUld level. We have also examined several extracellular matrix substances and growth factors using immunocytochemicalmethods at several key time points following S-antigen immunization.
MATERIALS AND METHODS Female Lewis rats (Harlan Sprague-Dawley, Indianapolis, IN), 150 - 180 g., were immunized intradennally in the footpad with 100 - 200 p1 of a purified bovine S-antigen (100 pg) emulsified in complete Freund’s adjuvant (CFAXDifco, Detroit, MI) (1: 1 ratio) supplemented with H37 RA Mycobacterium tuberculosis (Difco, 7 mg per ml adjuvant). S-antigen was purified as described (8). Control animals were immunized with CFM Mycobacterium only. The Guiding Principles in the Care and Use of Animals (DHEWPubl. NIH 80-23) were followed to handle animals. The animals were sacrificed by rapid Co;! asphyxiation at 0, 3, 7, 14, 20, and 30 days postimmunization. The eyes were removed and carefully divided into the anterior hemisphere containing ciliary, iris, lens, cornea and peripheral retina, and the posterior cup possessing only retina and choroidal stmctures. The tissues were immediately immersed in fmtive. For histological survey, the ocular tissues were fured in a mixture of phosphate-bufferedsaline (PBS), 4% formaldehyde and 2% glutamldehyde for 2 hours, rinsed in buffer and then post-fixed in 1% aqueous osmium tetroxide for 1 hour. These tissues were hrther sectioned, serially dehydmted from buffer to 100% ethanol and 100% propylene oxide, and embedded in Epon-Araldite.
Received on July 8 , 1992; accepted on October 15, 1992
0 Oxford University Press
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Ultrathin sections (50-70 nm) were prepared and poststained with uranyl acetate imd lead citmte prior to
animals each were sacrificed at day 14, 20 and 30. The
examination with a Phillips EM 301 electron microscope. For immunocytochemistry, the tissues were fixed with PBV 4% formaldehyde/ 0.2% gkutaraldehyde for I hour,
I. The ciliary tissues exhibited few morphological changes until early signs of acute disease developed beginning at day 1 1 to 13. At day 3, mononuclear cells were noted rarely in the anterior tissues. The basement membranes were physiically intact, the ciliary epithelial cells did not exhibit obvious pathology and the vessels, endothelium and stroma were normal in appearance (Fig. IA). Day 7 postimmunization rats closely resembled day 3 animals. No increase in inflammatory cells was observed in the tissues from 4 animals at this time point. Severe destruction was obvious at day 14 (Fig. IB), with large numbers of polymorphonuclear macrophages (PMN's) in the massive cellular infiltrate. Epithelial cilia were eradicated and the basement membrane of the nonpigmented epithelium (ME) and the epithelial cell layers were disrupted. The ciliary stroma was distorted and vascular integrity compromised. At the peak period
serially dehydrated from buffer fo 80% ethanol, and embedded in LR m i t e (Polysciences, Warrington, PA). Ultrathin sections (50-70 nm) were incubated successively on phosphate-buffered droplets of 1% bovine serum albumin (BSA)/ 0.25% casein blocking agent (Sigma, St. Louis, MO) for 15 minutes, priniary antibody (Ab), PBS and 1% BSA for 20 minutes, 10 or 15 nm gold-conjugated secondary Abs JE-Y Laboratories, San Mateo, CA) diluted 1 :20 in 0.05 M Tr i~C 1(pH 7.4)-0.05% Tween 20 buffer
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for I hour, and finally rinsed in Tris-Tween and in distilled water. For control nonimmune sera or gamma-globulins (species-matched with primary antibodies) were used in place of primary antibody. Primary antibody concentration for anti-EGF (Sigma), polyclonal anti-
TGF-6 (R&D Systems, Minneapolis, MN), anti-fibronectin (Sigma), anti-laminin (E-Y Labs) and anti-collagens type N and t' (Southern Biotech. Assoc., Birmingham, AL) was at 1:20 dilution in PBS. 'The results of multiple trials showed that a 2-3 hour incubation in the growth factor antibody solutions produced optimal labeling, while 1- 1.5 hours were needed for sufficient labeling with antibodies directed toward collagens and fikronectin. The blocking agent used was selected for emcacy after comparison of combinations of BSA, nonfat dry milk, casein and nonimmune sera. Sections were contrasted with uranyl acetate only, and then viewed with a transmission electron microscope. Micrographs of immunocyttxhemically treated tissues were examined for immunogald labeling. Eyes from at teast 4 different animals at each time point were sectioned, labeled and compared. Iabeling levels were quantitated by counting gold particles per unit area (9 pm2) of photographs at the same magnification. RESIII, I'S Four control animals immunized only with adjuvant did not develop disease. Of 27 animals immunized with Santigen, 4 rats each were sacrificed at day 3 and day 7. Of 18 rats which developed hypracvte FAU at day 14-15, 6
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day of immunization was day 0.
of disease, hemorrhage was clinically observed in the anterior chamber. Retrnal changes (data not presented) were consistent with the published observations on hyperacute EAU (3, 4). These findings included an initial perivascular mononuclear infiltration extending into the photoreceptor layer, retinal detachment, disruption of the nuclear cell layers and pan retinal involvement. Remarkable recovery of the ciliary body architecture was seen by day 20 postimmunization (Fig. IC). Mononuclear cell infiltration was greatly reduced, and PMKs were scarce. Lymphocytes (cells with dark-outlined nuclei in Figs. 1B and C) were found in the epithelial layers and also in the perivascular extracellular matrix . The ciliary body stroma appeared edematous, varying in thickness about the vasculature several times that of normals. The blood vessels (postcapilllary venules) also appeared distorted, with capillary-sized vessels numerically less evident than in normal tissues. Lymphocyte infiltration at day 30 postimmunization (Fig. 1D)was scarce in ocular tissues. Ciliary epithelial reorganization such as restoration of cellular infoldings was well advanced, but the ordered features present in normal epithelium and stroma remained noticeably altered. The perivascular stroma was thickened and more complex. At day 30, the postinflammatory retina showed an atrophied remnant with
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Figure 1: Electron micrographs showing (A) normal ciliary body at day 3, (B) loss of basement membrane, ciliary infoldings and infiltration by neutrophils at day 14, (C) residual infiltrating cell, re-establishment of ciliary
infoldings, thickened perivascular stroma at day 20, (D) day 30. [orig. mag. A) 2800 B) 2200 C) 1300 D) 18001. Scale bars = 10 Bm.
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Figure 2: lmmunocytochemistry showing gold label for fibronectin. A) Label at day 3 in basal lamina. B) Heavy labeling of basal lamina at day 14. C) Label was present in lamina d e w and vessel lumen at day 20. D) Labeling at .. .
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an absent photoreceptor layer and obvious cell loss in the nuclear layers. 2. Anti-fibronedin antibody labeling was found in the stroma and basement membme before and after the peak period of disease, similar to those in normals (day 0 and 3) (Fig. 2A). Antibody-conjugated gold particles were found at a high density specifically in the basement membranes of ciliary epithelium at the peak of inflammation (day 14). There was no label present intmcellularly at day 14 (Fig. 2B). There was a moderate level of intmvascular label not associated with specific structures at day 14 and 20 (Fig.
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day 30 was similar to' day 3 (A). [orig.mag. A) 7500 B,CP) 59001. Scale bars = 1.0 Fm, v = blood vessel lumen, r = red blood cell, n = epithelial nucleus, arrowheads = immunogold. ~
2C). It decreased at day 30 (Fig. 2D). Labeling levels at day 0, 3, I, 20 and 30 in the basement membranes were essentially the same. 3. Antibodies to TGF-0 showed a low level of labeling in normals and day 3 pastimmunization rats (Fig. 3A). A rise in labeling was most evident in the stroma at day 14 (Fig. 3B) but was not confined to that area. The increased label diminished at day 20 (Fig. 3C) but remained higher than normals even at day 30 (Fig. 3D). No label was noted intmvascularly and no intmcellular structures were labeled consistently. Areas of stroma that appeared enlarged and
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Figure 3: Immunocytochemistry for TGF-p. A) Gold labeling was seen in the basal lamina at day 3. B) Increased label was seen in basal lamina at day 14 . C) At day 20, label was increased over day 3 (A). D) At
day 30, label was similar to or slightly less than that at day 20. [orig. mag. A) 4300 B) 7500 C,D) 59001. Scale bars = 1.0 pm, v = blood vessel lumen, arrowheads = immunogold.
less ordered, consistently showed increased gold labeling at day 14, 20 and 30 postimmunization. 4. At day 3 (Fig. 4A), 7 (not shown) and 14 (Fig. 4B), EGF immunoreactivitywas expressed minimally. Labeling of epidermal growth factor was definitely elevated at day 20 (Fig. 4C). A comparative analysis of the data indicated that the level continued to increase further by day 30 (Fig. 4D). EGF immunoreactivitywas noted to be high in the stroma, but gold particles were also occasionally seen at high density in intracellularvesicles
(data not shown). There was no significant intravascular or intravitreal labeling. 5. Type V collagen labeling was low in EAU animals at postimmunization day 3 and day 14 (Figs. 5A,B) and increased in the basal lamina at day 20 and day 30 (Figs. 5C, D). No intracellular, intravascular or vitreal accumulationswere evident. 6. Antibodies against type IV collagen and laminin (Figs. 6 and 7) labeled specifically and strongly the l a m b densa of the basal lamina at each of the time points
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Figure 4 EGF immunoreactivity at A) day 3, B) day 14; pattern resembled day 3 (1 1 particled4 pm2), C) day 20; EGF labeling was increased in the basal lamina, D) day 30;
label was substantially higher (21 particled9 urn2). [orig. mag.A,B,C,D) 59001. Scale bars = 1.0 pm, v = blood vessel lumen, arrowheads = immunogold.
examined. No significant variations in the level of label coutd be discerned. No intracellular, intravascular or intmvitreal accumulations were noted. Fig. 8 summarizes changes in labeling intensities at
TGF-p immunoreactivity in the retinal vascular endothelium increaseid at day 14 and remained high for a long time.
different stages of EAU, It i s interesting to note that TGFp irnmunoreactivity reaches a maximum at the peak of inflammation (day 14) and decreases slightly but remains
DISCUSSION We have demonstrated in this study that the ciliary body quickly regeneiates its cellular architecture after the peak period of disease is over. We have also noted changes in extracellular matrix components which correlate with the progression of M U activity.
higher than normal thereafter. In contrast, EGF irnmunoreactivity slowly rises in the postinflammatory period. The TGF-p labeling pattern is similar to the result of oar previous experiment on the retina of EAU rat (9);
The acute onset olf disease is heralded by an initial
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Figure 5: Immunocytochemistry for type V collagen showing A) labeling of lamina densa at day 3, B) sparse label at day 14, C) and D) increased labeling of basal
lamina at day 20 and 30. [orig. mag. A,B,C,D) 75001. Scale bars = 1.0 pm, v = vessel lumen, arrowheads = immunogold.
vasculitis followed shortly by massive cellular infiltration and tissue destruction (5, 10, 11). The corona ciliaris is immediatelyvulnerable to the actions of inflammatory cells and any serum-borne immunogenic factors by virtue of its high vascularity. The degree to which it is affected can be attested to by the breach of vascular integrity and hemorrhage. Yet the ciliary process is apparently able to rapidly regenerate its architecture and specialized cellular processes while the retina sustains the permanent loss of the photoreceptor layer (3,4). The difference in the ability of the tissues to regenerate must have serious functional
implications, because of the importance that these stluctures have in the eye. At the peak phase of the disease, i.e., day 14 postimmunization, the WE basement membrane is destroyed, but reappears by day 20 along with the reestablished epithelial layers. Noting the severe disruption in the acute phase, the ability of the NPE to reconstruct the cell layer and ciliary infoldings, and to elaborate extracellular matrix is remarkable. Epithelial cell loss probably occurred during EAU,but was not quantitated in this study. Mitotic figures were not
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Figure 6: Type IV collagen shows strong labeling of basement membranes at day A) 3, B) 14, C ) 20 and D) 30. -
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[orig. mag. A,B,C,D) 75001. Scale bars = 1.0 pm, v = blood vessel lumen, arrowheads = immunogold. ---__. . . ~
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prominent in the ciliary tissues at any of the time points exanlined in this study. The mature ciliary tissues are a
relatively stable population in the mouse (12), and are expected to be stable in the rat also. Evidence for increased nuclear synthetic activity in the ciliary epithelia i n the course of EAlJ may be obtained by investigating the
Several authors have suggested interactions between growth factors in mcdels of development, cell proliferation and tissue repair (13, 14). TGF-fi and ECF are among the more commonly cited, and their models of interactions
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3
14
20
30
Time in days - E
m c5 Figure 8: Changes in immunolabelingintensities of TGFp, EGF and extracellular matrix components with time. Counting of gold particles per unit area (9 pm2) was made from photographs at the same magnification. F, fibronectin. T, TGF-p. E, EGF. C5, collagen type V.
vary depending on the model in question. Whether their presence in this model of EAU is a matter of synergism or coincidence is not known. A specific synergistic effect by TGF-0 and EGF on fibroblast cell culture gene transcription was reported, an observation suggestive of a possible physiological mechanism (15). A reciprocal action of the extracellular matrix on the activity of the growth factors has also been reported in vitro (16). We have noted that the POIYCIOMI TGF-p antibody labeling peaks before that of EGF in the ciliary stroma. Although the cellular concentration of TGF-p and EGF are generally very low, significant quantities of both factors are found in the extracellular matrix. As determined immunohistochemically,intracellular concentrations of these factors in the epithelium, fibroblasts or endothelium are not consistent. The large influx of inflammatorycells is a possible source, as TGF-0 is found at high concentrationsin platelets (17, 18) and macrophages (19). Changes in fibronectin and the constituent collagens,
such as type V, can reflect changes occurring in the extracellular matrix. Fibronectin in particular has been shown here to have a very high labeling intensity at day 14 postimmunization,which is localized to the basement membranes and stroma. Whether this is a function of the binding availability of antigenic sites a n d or increased free fibronectin binding or concentration may be answered with further studies. It is possible that more antigenic sites are available in a disrupted, edematous extracellular matrix. However, tissues at other time points which also have an edematous stroma do not demonstrate the same increase in labeling. Laminin and type IV collagen, which are also constituent basement membrane proteins, do not vary in the same fashion. The intraluminal fibronectin content at day 14 is suggestive of increased soluble fibronectin, which may induce the morphological tissue changes that are notable postuveitic sequelae, such as fibrosis, and alterations in the basal lamina (6, 20). Fibronectin has been shown to have a positive effect on corneal epithelial wound healing (21, 22) by stimulating epithelial migration in concert with EGF . It has also been demonstrated that TGF-0 and EGF are active in regulating the expression of fibronectin and collagens, by stimulating fibroblast chemotaxis, inducing increases in mRNA levels, and increasing incorporation into the extmcellular matrix (2329). TGF-p is known to increase cellular expression of the integrin family of adhesion protein receptors which mediate binding of cells to extracellular matrix proteins including collagen and fibronectin (28). TGF-0 has suppressive effects on the proliferation of T and B lymphocytes and monocytes (30). This may be an additional regulatory effect of TGF-p on the course of
EAU. We have demonstrated a remarkable pathophysiologicalprocess that occurs in the ciliary body more closely than before. We have shown a corresponding change in the detected levels of factors known to be involved in tissue repair. Further studies on the receptors to these factors and manipulation of factor levels in vivQ may be rewarding in deriving further answers in this useful model of autoimmune inflammation. ACKNOWLEDGEMENTS The authors thank Dr. Linda Hazlett for allowing us to use facilities supported by the core grant, Mark Heil for his
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CORRESPONDING AUTHOR Dr. Hitoshi Shichi, Kresge Eye Institute, Wayne State University School of Medicine, Detroit, MI 48201, U.S.A. REFERENCES 1. Faure, J.-P. (1980) Autoimmunity and the retina. Curr. Topics Eye Res. 2, 215-302. 2. Gery, I., Mochizuki, M. and Nussenblatt, R.B. (1986) Retinal specific antigens and immunopathogenic processes they provoke. Prog. Retinal Res. 3, 75- 109. 3, De Kozak, Y., Thillaye, B., Renard, G. and Faure, J.Y. (1978) Hyperacute form of experimental autoimmune uveo-retinitis in Lewis rats; electron microscopic study. Graefe's Arch. Clin. Exp. Ophthalmol. 208, 135- 142. 4. Lin, W.-L., Essner, E. and Shichi, H. (1991) Breakdown of the blood-retinal barrier in S-antigeninduced uveoretinitis in rats. Graefe's Arch. Clin. Exp. Ophthalmol. 229, 457-463. 5 . Forrester, J.V., Liversidge, J., Dua, H.S., Towler, H. and McMenamin, P.G. (1990) Comparison of clinical and experimental uveitis. &IT. Eye Res. 9 ( S U , 75-84. 6. McDonald, J.A. (1988) Extracellular matrix assembly. Ann. Rev. C M . Biol. 4, 183-207. 7. Merwin, J.R., Anderson, J.M., Kocher, O., van Itallie, C.M. and Madri, J.A. (1990) Transforming growth factor beta 1 modulates extracellular matrix organization and cell-cell junctional complex formation during in vitro angiogenesis. J. Cell. Physiol. 142,117-128. 8. Das, N.D., Ulshafer, R.F., Tam, Z.S., Leverenz, V.R. and Shichi, H. (1984) Radioimmunocytochemical localization of retinal S-antigen with monoclonal antibodies. J. Histochem. Cytochem. 32, 834-838. 9. Mahalak, S.M., Lin, W.-L., Essner, E. and Shichi, H. (1991) hcreased immunoreactivity of collagen types I, KU and V, fibronectin and TGF-6 in retinal vessels of rats with experimental autoimmune uveoretinitis. Curr. Eye Res. lo, 1059-1063. 10. De Kozak, Y., Sakai, J. , $hillaye, B. and Faure, J.P. (1981) S antigen-induced experimental autoimmune uveoretinitis in rats. Curr. Eye Res. 1, 327-340. 11. Rao, N.A., Wacker, W.B. and Marak, G.E. (1979) Experimental allergic uveitis: Clinicopathologic features associated with varying doses of S-antigen. Arch. Ophthalmol. 97, 1954-1958. 12. McDonald, T.F. and Green, K. (1988) Cell turnover in ciliary epithelium compared to other slow renewing epithelia in the adult mouse. Curr.Eye Res. 1,247252. 13 Richter, K.H., Schnapke, R., Clauss, M., Fiistenberger, C., Him, 1). and Marks, F. (1990) Epidermal GI-chalone and transforming growth factor fr are two different endogenous inhibitors of epidermal cell proliferation. J. Cell. Physiol. 142,496 - 504.
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14. Tripathi, R.C., Borisuth, N.S.C. and Tripathi, B J . (199 1) Growth factors in the aqueous humor and their thempeutic impliciitions in glaucoma and anterior segment disorders of the human eye. Drug. Develop. R e s . 2 , 1-23. 15. Ranganathan, G. and Getz, M J . (1990) Cooperative stimulation of specific gene transcription by epidermal growth factor and transforming growth factor 61. J. Biol. Chem. 265, 3001-3004. 16. Schlumberger, W., Thie, M., Rauterberg, J. and Robenek, H. (199 I) Collagen synthesis in cultured aortic smooth muscle cells: modulation by collagen lattice culture, tmlsforming growth factor-61, and epidermal growth factor. Arterioscler. Thromb. It, 1660 - 1666. 17. Childs, BJ., Proper, J.A., Tucker, R.F. and Moses, H.L. (1982) Semm contains a platelet-derived transforming growth factor. Proc. Natl. Acad. Sci. USA,B, 5312-5316. 18. Assoian, R.K., Komoriya, A., Meyers, C.A., Miller, D.M. and Spom, M.B. (1983) Transforming growth factor-6 in human platelets: identification of a major storage site, purification and characterization. J. Biol. Chem. 258, 7155 - 7160. 19. Assoian , R.K., Fleurdelys, B.E., Stevenson, H.C., Miller, PJ., Madtes, D.K., Raines, E.W., Ross, R . and Spom, M.B. (1987) Expression and secretion of type 6 transforming growth factor by activated human macrophages. Proc. Natl. Acad. Sci. USA, &, 6020 6024. 20. Connor,T.B., Roberts,A.B., Sporn, M.B., Danielpour, D., ]Dart, L.L., Michels, R.G., de Bustros, S., Enger, C., Kato,H., Lansing, M., Hayashi, H. and Glaser, B.M. (1989) Correlation of fibrosis and translforming growth factor-6 type 2 levels in the eye. J. Clin. Invest. 83, 1661 - 1666. 21. Watanabe, K., Nakagawa, S. and Nishida, T. (1988) Chemotactic and haptotactic activities of fibronectin for cultured rabbit corneal epithelial cells. Invest. Ophthalmol. Vis. Sci. 29,572 - 577. 22. Nishida,T., Nakamura, M., Mishima, H.and Otori, T. (1990) Differential modes of action of fibronectin and epidermal growth factor on rabbit corneal epithelial migration. J. Cell. Physiol. 145,549 - 554. 23. Ignotz, R.A. and Massague, J. (1986) Transforming growth factor 0 stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J. Biol. Chem. 261,4337 4345. 24. Ignotz, R.A., Endo, T., Massague, J. (1987) Regulation of fiblronectin and type 1 collagen mRNA levels by transforming growth factor p. J. Biol. Chem. 262, 6443-6446. 25. Stlethwaite, A.E., Keski-Oja, J., Moses, H.L. and Kang, A.H. (19117) Stimulation of the chemotactic migration of h u n m fibroblasts by transforming growth factor p. .I. Exp. Med. 165,251 - 256. 26. Carpenter, G. and Cohen, S. (1975) Human epidermal growth factor and the proliferation of human fibroblasts. J. Cell. Physiol. 88,227 - 238. 27. Roberts, A.B., Spom, M.B., Assoian, R.K., Smith, J.M., Roche, N..S.,Wakefield, L.M., Heine, U.I., Liotta, L.A., Falanga, V., Kehrl, J.H. and Fauci, AS. (1986) Trainsforminggrowth factor type p: rapid induction of fibrosis and angiogenesis in vitro and stimulation of cclllagen formation in vitro. Proc. Natl. Acad. Sci. USA,83,4167 - 4171.
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28. Roberts, A.B., Heine, U.I., Flanders, K.C. and Spom, M.B. (1990) Transforming growth factor B: major role in regulation of extracellular matrix. Ann. New York A d . Sci. 593,225 - 232. 29. Peltonen, J., Hsiao, L.L., Jaakkola, S., Sollberg, S., Aumailley, M., Timpl, R., Chu, M.-L. and Uitto, J . (199 1) Activation of collagen gene expression in keloids: co-localition of type I and VI collagen and transforming growth factor-sl mRNA. J. Invest. Dermatol. 97, 240 - 248. 30. Massague, J. (1990) The transforming growth factor-0 family. Ann. Rev. Cell Biol. 6, 597-641.
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