CLINICAL AND MATERIALS SCIENCES
LETS Protein in Normal and Pathological Human Oral Epithelium B. R. ZETTER*, T. E. DANIELSt, C. QUADRA-WHITEt, and J. S. GREENSPANt
*Cancer Research Institute, and tDepartment of Oral Medicine and Hospital Dentistry, School of Dentistry, University of California, San Francisco, CA 94143 The distribution of LETS protein in human oral mucosa was studied by indirect immunofluorescence. Normal epithelium showed surface staining. Intracellular staining occurred in epithelial cell cytoplasm in lichen planus and pemphigoid. Surface staining was absent in discoid lupus erythematosus. In pemphigus, intercellular staining was seen near areas of acantholysis. J Dent Res 58(1):484488, January 1979
Introduction. Recently, a high molecular weight (220,000 dalton) glycoprotein has attracted attention as a major component of the cell surfaces and extracellular matrices of many tissues.1 Originally observed as a large external transformation-sensitive (LETS) protein present on normal fibroblasts but absent on cells transformed by many types of tumor virus,2 LETS protein has recently been found to be antigenically identical with a well-characterized plasma glycoprotein, the cold insoluble globulin (CIG).3 This protein is known by several terms: CIG for the form found in plasma; LETS or CSP (cell surface protein) for the form on cell surfaces; and fibronectin for the form in extracellular matrices and basement membrane. The term LETS protein is used in this report. Ample evidence indicates that one of the major functions of the LETS protein is to mediate the attachment of cells to other cells and to their substratum. When purified LETS protein is added to cultures of transformed cells that adhere poorly to a substratum, the strength of their attachment increases, and the morphology of the cells Received for publication July 10, 1978. Accepted for publication October 30, 1978. Requests for reprints should be addressed to Dr. John S. Greenspan, Department of Oral Medicine and Hospital Dentistry, School of Dentistry, University of California, San Francisco CA 94143 *Present address: Children's Hospital Medical Center, Harvard Medical School, Boston, Massachusetts
484
changes from round to flattened, with a well-spread cytoplasm indicative of increased contact with the substratum.46 LETS has also been shown to agglutinate sheep erythrocytes7 and has been implicated in the protease-induced aggregation of chick embryo fibroblasts.8 With many cell types, LETS protein is preferentially located in areas of cell-cell contact.9 In sparse cultures LETS protein is often first found on the intercellular processes between contacting cells, and in dense cultures its distribution follows the outline of the intercellular borders. These results suggest that LETS protein helps initiate and maintain cell-cell and cell-stratum contacts. Using indirect immunofluorescent staining for LETS protein, we have examined frozen sections of normal and diseased human oral mucosa. Our results indicate that production and distribution of LETS protein in the oral epithelium is altered in several pathological conditions, including lichen planus, discoid lupus erythematosus, mucous membrane pemphigoid, and pemphigus. These alterations may serve as markers for these conditions and may explain some of their histological or clinical features.
Materials and methods. In our institution, biopsy tissue from all patients with diffuse or chronic oral ulcerative disease is routinely examined by direct immunofluorescence as well as by conventional light microscopy. For this study, additional reactions with rabbit antihuman LETS antiserum were performed on 14 specimens of oral mucosa-five each from cases of lichen planus and mucous membrane pemphigoid, and two each from cases of pemphigus and discoid lupus erythematosus. As controls, two specimens of normal mucosa were obtained from patients who underwent oral surgical procedures yielding
Downloaded from jdr.sagepub.com at Univ of Connecticut / Health Center / Library on June 24, 2015 For personal use only. No other uses without permission.
Vol. 58 No. I
LETS IN HUMAN ORAL EPITIHELIUM
mucosa which was in excess of that needed for surgical primary closure and would normally have been discarded. The specimens were quick-frozen and stored in liquid nitrogen. Cryostat sections 5 pm thick were cut at 20 C and picked up on slides coated with formol-gelatin. Sections were washed twice for 15 min. in phosphate-buffered saline (PBS) and incubated for 30 min. at room temperature with rabbit anti-human LETS serum (donated by Dr. L. B. Chen),9'10 diluted 1:160 in PBS. After three more washes, sections were incubated with a 1:20 dilution of fluorescein-conjugated goat anti-rabbit IgG* (F/P molar ratio 2.8) for 30 min. more at room temperature, then washed again three times in PBS. The sections were mounted in PBS/ glycerine at pH 8.5 and observed by fluorescence microscopy with a dark field transmission fluorescence microscopet equipped with a high-pressure mercury vapor lamp (HBO-200). For excitation, FITC interferencet and BG 23 red-absorbing filters were used, while an FITC barrier was used above the specimen. In control experiments, the anti-LETS rabbit serum was replaced either with normal rabbit serum or with PBS alone. Previous control experiments with antiLETS serum absorbed with purified LETS protein (100 mg/ml) abolished staining on a variety of mammalian tissues.
485
Figures 1-5 All photomicrographs are of 5 gm cryostat sections of human oral mucosa, (B) indicates the location of the basement membrane.
Fig. lA-Normal oral mulcosa incubated with rabbit anti-human LETS serum followed by floorescein-conjulgated goat anti-rabbit IgG. X 156. Positive staining is seen in the connective tissue and in the superficial layer of the epithelium (S).
Results and discussion. Normal mucosa (Fig. 1)-Samples of normal nonkeratinized oral mucosa which were reacted for the presence of LETS protein showed positive immunofluorescent staining in two distinct locations: the connective tissue underlying the epithelium, and the superficial layer of the epithelium. No LETS staining was seen either within the cytoplasm or in the intercellular spaces of the epithelium. These results are consistent with previously reported data indicating that not all types of epithelial cells can express the LETS protein. Chen et a.l1 have reported that whereas primary cultures of kidney or liver cells produce LETS protein, epidermal basal epithelial cells do not. lHowever, connective tissue fibroblasts se-
Fig. B -Section, serial to IA, incuibated in normal rabbit serum followed by the fluorescein conjugate, X 156. This control, like the others used in this study, was negative.
crete a massive fibrillar network of LETS protein, found within the connective tissue of nearly all organs. Thus it is not surprising
tWild
to find LETS protein in the connective tissues but not throughout the epithelial layer of the normal oral mucosa. The presence of LETS in the superficial epithelium is, however, a new and somewhat unex-
LtZeiss
pected finding. This bright staining was
*Behring Diagnostics
Downloaded from jdr.sagepub.com at Univ of Connecticut / Health Center / Library on June 24, 2015 For personal use only. No other uses without permission.
486
J Dcii t Res Jaiiiiarj, 19 79 A L. ZETTER FRT
Fig. 2-Discoid lupus erythematosus. Sectioin prepared as in Fig. 1A, X 156. Staining was not found at the epithelial surface.
Fig. 3A -Pemphigoid. Section prepared as in Fig. IA, X 312. Marked intracellular staining of epitlhelial cells.
found only in sections stained with antiLETS serum and not in control sections incubated with normal rabbit serum or PBS. This surface-associated LETS protein may help to regulate loss of mutual adhesion and hence shedding of the superficial cells, or squames. It is also possible, but less likely, that it could represent surface absorption of LETS from saliva. Lichen planlus (Fig. 5)-Distribution of LETS protein in specimens of lichen planus was essentially similar to that observed in normal mucosa, except for selective intracellular staining in some, but not all, of the epithelial cell cytoplasm. In four cases the basal layer of epithelial cells lacked staining, whereas in the fifth case the negative zone included the deepest layers of the prickle zone. This intracellular fluorescence differs from the distribution of LETS protein in the connective tissue, where LETS is found primarily in an extracellular matrix or as a component of basement membranes. Our finding of LETS protein within oral epithelial cells suggests that the expression of LETS protein is a regulatable phenomenon. Chen et al. showed that the production of LETS protein by cultured fibroblasts could be modulated by the addition of certain peptide factors such as the epidermal growth factor (EGF) isolated from mouse submaxillary glands.12 Here we demonstrate that production of LETS protein by epithelial cells is altered in a pathological condition in vivo. This could be either a direct effect
Fig. 313-Section serial to 3A, prepared as in l B. Negative control.
of the disease process on the cells or anl indirect effect of exposure to increased concentrtations of inflammiiatory products, or even to EGF produced by damaged cells or in the saliva. Mucous membrane pemiphigoid (Fig. 3)-In pemphigoid, breakdown occurs betweein epithelium and connective tissue at the basement membrane. These specimens showed bright staining for LETS in epithelial cell cytoplasm, either throughout the epithelium or confined to the middle and upper epithelial cell layers. In addition, positive fluorescence was seen in the connective tissue and in the most superficial layers of the epithelium similar to normal mucosa. The LETS distribution here seems to be another example of the phenomenon, observed with
Downloaded from jdr.sagepub.com at Univ of Connecticut / Health Center / Library on June 24, 2015 For personal use only. No other uses without permission.
l TS INVI UMAN ORAA EPIlL Vol.o S8 No.NE I THL U 4487
l ! 7.245
io'W"'r !m MIT
wIllb" 4/1 Akk"
Fig. 4-Pemphigus. Section prepared as in Fig. IA, X 312. Intercellular staining of cells near an area of acantholysis (A). lichen planus, in which the epithelial cells produce LETS in response to a stimulus. In contrast to other cell types that primarily secrete the LETS protein that they synthesize, the epithelial cells in these two diseases seem to retain the LETS protein within their cytoplasm. Pemphigus (Fig. 4)-Pemnphigus is characterized by intercellular breakdown within the epithelium (acantholysis). The distribution of LETS protein in these specimens differed from that described in the other conditions. While there was little intracellular fluorescence in the epithelial cells, there was intercellular staining for LETS around certain cells, especially near the zones where acanltholysis had taken place. Since these are areas of marked disruption of cell-cell adhesions, perhaps the cells remaining in this area secrete LETS protein in order to promote intercellular adhesiveness. Alternatively, perhaps staining results from exposure of previously sequestered LETS. LETS staining in the surface epithelium and in the connective tissues was similar to normal. Discoid lupus erythe;natosus (DLI ") (Fig. 2)-DLE often effects changes in the oral mucosa, including hyperkeratosis, epithelial atrophy, liquefaction degeneration of the basal layer of the epithelium, and thickening of the basement membrane. When examined for the presence of LETS protein, specimens
1 ig. 5 -Lichen planus. Section prepared as in Fig. IA, X 200. IntracellulIar staining (I) in some epithelial cells and prominent staining in basement memnbrane zone.
from lesions of DLE showed one distinct difference from the normal: a complete lack of staining at the epithelial surface. Whereas the distribution of LETS protein in the epithelium and in the connective tissue was the same as in the normal mucosa, the absence of surface-associated LETS protein was striking and, to date, has been seen only in this condition. This absence might, therefore, be a useful marker for DLE. Furthermore, it may result from an attempt by cells of the epithelium to reduce excessive thickness by reducing surface-cell adhesiveness.
Conlclusion1s. LETS protein, intensely investigated since its rediscovery in 1973, has been implicated in phenomena as diverse as embryogenesis,13 hemostasis,3 and myogenesis,14,i 5 as well as
turn origenesis and metastasis.9'16 Hitherto, no reports of the distribution of LETS in human disease have appeared. In this report, we describe several instances in which the distribution of LETS protein is altered by
pathological conditions affecting the human oral mucosa. In discoid lupus erythematosus, the alteration involves a loss of the LETS protein normally present in the superficial layer of the epithelium. In all other conditions examined, mucosal disease seems to
Downloaded from jdr.sagepub.com at Univ of Connecticut / Health Center / Library on June 24, 2015 For personal use only. No other uses without permission.
488
ZETTER ETAL.
stimulate production of LETS protein by epithelial cells that do not normally produce it. Generally, the LETS protein detected in these cells remains intracellular. However, in the case of pemphigus, where acantholysis takes place, LETS protein is deposited in intercellular spaces, perhaps as an attempt by the cells to restore cell-cell adhesion. These preliminary results suggest that abnormal production of LETS protein by epithelial cells may be a general response to insult and could indicate stress or injury. Although this response is general, the amount and distribution of the LETS protein produced are different for the conditions described. Further work will be necessary to determine whether these patterns are useful as markers for these conditions, and may contribute to an understanding of their pathogenesis.
Acknowledgments. We would like to thank Dr. L. B. Chen
(Harvard Medical School) for his generous gift of monospecific antiserum to human LETS protein (CIG).
REFERENCES 1. STENMAN, S., and VAHERI, A.: Distribution of a Major Connective Tissue Protein, Fibronectin, in Normal Human Tissues, J. Exp Med 147:1054-1064, 1978. 2. HYNES, R. O.: Cell Surface Proteins and Malignant Transformation, Biochem Biophys Acta 458:73-107, 1976. 3. RUOSLAHTI, E., and VAHERI, A.: Interaction of Soluble Fibroblast Surface Antigen with Fibrinogen and Fibrin, J Exp Med 141: 497-501, 1975. 4. YAMADA, K. M.; YAMADA, S. S.; and PASTAN, I.: Cell Surface Protein Partially Restores Morphology, Adhesiveness, and Contact Inhibition of Movement to Transformed Fibroblasts, Proc Nat Acad Sci 73:12171221, 1976. 5. PEARLSTEIN, E.: Plasma Membrane Glycoprotein which Mediates Adhesion of Fibroblasts to Collagen, Nature (London) 262: 497-500, 1976.
J Dent Res January 19 79 6. ALI, I. U.; MAUTNER, V.; LANZA, R.; and HYNES, R. O.: Restoration of Normal Morphology, Adhesion and Cytoskeleton in Transformed Cells by Addition of a Transformation-Sensitive Surface Protein, Cell 11: 115-126, 1977. 7. YAMADA, K. M.; YAMADA, S. S.; and PASTAN, I.: The Major Cell Surface Glycoprotein of Chick Embryo Fibroblasts is an Agglutinin, Proc Nat Acad Sci 72:31583162, 1975. 8. ZETTER, B. R.; CHEN, L. B.; and BUCHANAN, J. M.: Effects of Protease Treatment on Growth, Morphology, Adhesion, and Cell Surface Proteins of Secondary Chick Embryo Fibroblasts, Cell 7:407412, 1976. 9. CHEN, L. B.; GALLIMORE, P. H.; and MCDOUGALL, J. H.: Correlation Between Tumor Induction and the Large External Transformation Sensitive Protein on the Cell Surface, Proc Nat Acad Sci 73:3570-3574, 1976. 10. BURRIDGE, K.: Changes in Cellular Glycoproteins After Transformation: Identification of Specific Glycoproteins and Antigens in Sodium Dodecyl Sulfate Gels, Proc Nat Acad Sci 73:44574461, 1976. 11. CHEN, L. B.; MAITLAND, N.; GALLIMORE, P. H.; and MCDOUGALL, J. H.: Detection of the Large External Transformation-Sensitive Protein on Some Epithelial Cells, Exp Cell Res 106:3946, 1977. 12. CHEN, L. B.; GUDOR, R. C.; SUN, T. T.; CHEN, A. B.; and MOSESSON, M. W.: Control of a Cell Surface Major Glycoprotein by Epidermal Growth Factor, Science 197:776778, 1977. 13. ZETTER, B. R.; and MARTIN, G. R.: Expression of a High Molecular Weight Cell Surface Glycoprotein (LETS Protein) by Preimplantation Mouse Embryos and Teratocarcinoma Stem Cells, Proc Nat Acad Sci 75:23242328, 1978. 14. HYNES, R. O.; MARTIN, G. S.; CUTCHLEY, D. R.; SHEARER, M.; and EPSTEIN, C. J.: Viral Transformation of Rat Myoblasts: Effects on Fusion and Surface Properties, Devel Biol 48:35-46, 1976. 15. CHEN, L. B.: Alteration in Cell Surface LETS Protein During Myogenesis, Cell 10:393400, 1977. 16. CHEN, L. B.; BURRIDGE, K.; MURRAY, A.; WALSH, M. L.; COPPLE, C. D.; BUSHNELL, A.; McDOUGALL, J. K.; and GALLIMORE, P. H.: Modulation of Cell Surface Glycocalyx Studies on Large, External, Transformation-Sensitive Protein, Ann NY Acad Sci 312:366-381, 1978.
Downloaded from jdr.sagepub.com at Univ of Connecticut / Health Center / Library on June 24, 2015 For personal use only. No other uses without permission.
LETS Protein in Normal and Pathological Human Oral Epithelium B. R. ZETTER*, T. E. DANIELSt, C. QUADRA-WHITEt, and J. S. GREENSPANt
*Cancer Research Institute, and tDepartment of Oral Medicine and Hospital Dentistry, School of Dentistry, University of California, San Francisco, CA 94143 The distribution of LETS protein in human oral mucosa was studied by indirect immunofluorescence. Normal epithelium showed surface staining. Intracellular staining occurred in epithelial cell cytoplasm in lichen planus and pemphigoid. Surface staining was absent in discoid lupus erythematosus. In pemphigus, intercellular staining was seen near areas of acantholysis. J Dent Res 58(1):484488, January 1979
Introduction. Recently, a high molecular weight (220,000 dalton) glycoprotein has attracted attention as a major component of the cell surfaces and extracellular matrices of many tissues.1 Originally observed as a large external transformation-sensitive (LETS) protein present on normal fibroblasts but absent on cells transformed by many types of tumor virus,2 LETS protein has recently been found to be antigenically identical with a well-characterized plasma glycoprotein, the cold insoluble globulin (CIG).3 This protein is known by several terms: CIG for the form found in plasma; LETS or CSP (cell surface protein) for the form on cell surfaces; and fibronectin for the form in extracellular matrices and basement membrane. The term LETS protein is used in this report. Ample evidence indicates that one of the major functions of the LETS protein is to mediate the attachment of cells to other cells and to their substratum. When purified LETS protein is added to cultures of transformed cells that adhere poorly to a substratum, the strength of their attachment increases, and the morphology of the cells Received for publication July 10, 1978. Accepted for publication October 30, 1978. Requests for reprints should be addressed to Dr. John S. Greenspan, Department of Oral Medicine and Hospital Dentistry, School of Dentistry, University of California, San Francisco CA 94143 *Present address: Children's Hospital Medical Center, Harvard Medical School, Boston, Massachusetts
484
changes from round to flattened, with a well-spread cytoplasm indicative of increased contact with the substratum.46 LETS has also been shown to agglutinate sheep erythrocytes7 and has been implicated in the protease-induced aggregation of chick embryo fibroblasts.8 With many cell types, LETS protein is preferentially located in areas of cell-cell contact.9 In sparse cultures LETS protein is often first found on the intercellular processes between contacting cells, and in dense cultures its distribution follows the outline of the intercellular borders. These results suggest that LETS protein helps initiate and maintain cell-cell and cell-stratum contacts. Using indirect immunofluorescent staining for LETS protein, we have examined frozen sections of normal and diseased human oral mucosa. Our results indicate that production and distribution of LETS protein in the oral epithelium is altered in several pathological conditions, including lichen planus, discoid lupus erythematosus, mucous membrane pemphigoid, and pemphigus. These alterations may serve as markers for these conditions and may explain some of their histological or clinical features.
Materials and methods. In our institution, biopsy tissue from all patients with diffuse or chronic oral ulcerative disease is routinely examined by direct immunofluorescence as well as by conventional light microscopy. For this study, additional reactions with rabbit antihuman LETS antiserum were performed on 14 specimens of oral mucosa-five each from cases of lichen planus and mucous membrane pemphigoid, and two each from cases of pemphigus and discoid lupus erythematosus. As controls, two specimens of normal mucosa were obtained from patients who underwent oral surgical procedures yielding
Downloaded from jdr.sagepub.com at Univ of Connecticut / Health Center / Library on June 24, 2015 For personal use only. No other uses without permission.
Vol. 58 No. I
LETS IN HUMAN ORAL EPITIHELIUM
mucosa which was in excess of that needed for surgical primary closure and would normally have been discarded. The specimens were quick-frozen and stored in liquid nitrogen. Cryostat sections 5 pm thick were cut at 20 C and picked up on slides coated with formol-gelatin. Sections were washed twice for 15 min. in phosphate-buffered saline (PBS) and incubated for 30 min. at room temperature with rabbit anti-human LETS serum (donated by Dr. L. B. Chen),9'10 diluted 1:160 in PBS. After three more washes, sections were incubated with a 1:20 dilution of fluorescein-conjugated goat anti-rabbit IgG* (F/P molar ratio 2.8) for 30 min. more at room temperature, then washed again three times in PBS. The sections were mounted in PBS/ glycerine at pH 8.5 and observed by fluorescence microscopy with a dark field transmission fluorescence microscopet equipped with a high-pressure mercury vapor lamp (HBO-200). For excitation, FITC interferencet and BG 23 red-absorbing filters were used, while an FITC barrier was used above the specimen. In control experiments, the anti-LETS rabbit serum was replaced either with normal rabbit serum or with PBS alone. Previous control experiments with antiLETS serum absorbed with purified LETS protein (100 mg/ml) abolished staining on a variety of mammalian tissues.
485
Figures 1-5 All photomicrographs are of 5 gm cryostat sections of human oral mucosa, (B) indicates the location of the basement membrane.
Fig. lA-Normal oral mulcosa incubated with rabbit anti-human LETS serum followed by floorescein-conjulgated goat anti-rabbit IgG. X 156. Positive staining is seen in the connective tissue and in the superficial layer of the epithelium (S).
Results and discussion. Normal mucosa (Fig. 1)-Samples of normal nonkeratinized oral mucosa which were reacted for the presence of LETS protein showed positive immunofluorescent staining in two distinct locations: the connective tissue underlying the epithelium, and the superficial layer of the epithelium. No LETS staining was seen either within the cytoplasm or in the intercellular spaces of the epithelium. These results are consistent with previously reported data indicating that not all types of epithelial cells can express the LETS protein. Chen et a.l1 have reported that whereas primary cultures of kidney or liver cells produce LETS protein, epidermal basal epithelial cells do not. lHowever, connective tissue fibroblasts se-
Fig. B -Section, serial to IA, incuibated in normal rabbit serum followed by the fluorescein conjugate, X 156. This control, like the others used in this study, was negative.
crete a massive fibrillar network of LETS protein, found within the connective tissue of nearly all organs. Thus it is not surprising
tWild
to find LETS protein in the connective tissues but not throughout the epithelial layer of the normal oral mucosa. The presence of LETS in the superficial epithelium is, however, a new and somewhat unex-
LtZeiss
pected finding. This bright staining was
*Behring Diagnostics
Downloaded from jdr.sagepub.com at Univ of Connecticut / Health Center / Library on June 24, 2015 For personal use only. No other uses without permission.
486
J Dcii t Res Jaiiiiarj, 19 79 A L. ZETTER FRT
Fig. 2-Discoid lupus erythematosus. Sectioin prepared as in Fig. 1A, X 156. Staining was not found at the epithelial surface.
Fig. 3A -Pemphigoid. Section prepared as in Fig. IA, X 312. Marked intracellular staining of epitlhelial cells.
found only in sections stained with antiLETS serum and not in control sections incubated with normal rabbit serum or PBS. This surface-associated LETS protein may help to regulate loss of mutual adhesion and hence shedding of the superficial cells, or squames. It is also possible, but less likely, that it could represent surface absorption of LETS from saliva. Lichen planlus (Fig. 5)-Distribution of LETS protein in specimens of lichen planus was essentially similar to that observed in normal mucosa, except for selective intracellular staining in some, but not all, of the epithelial cell cytoplasm. In four cases the basal layer of epithelial cells lacked staining, whereas in the fifth case the negative zone included the deepest layers of the prickle zone. This intracellular fluorescence differs from the distribution of LETS protein in the connective tissue, where LETS is found primarily in an extracellular matrix or as a component of basement membranes. Our finding of LETS protein within oral epithelial cells suggests that the expression of LETS protein is a regulatable phenomenon. Chen et al. showed that the production of LETS protein by cultured fibroblasts could be modulated by the addition of certain peptide factors such as the epidermal growth factor (EGF) isolated from mouse submaxillary glands.12 Here we demonstrate that production of LETS protein by epithelial cells is altered in a pathological condition in vivo. This could be either a direct effect
Fig. 313-Section serial to 3A, prepared as in l B. Negative control.
of the disease process on the cells or anl indirect effect of exposure to increased concentrtations of inflammiiatory products, or even to EGF produced by damaged cells or in the saliva. Mucous membrane pemiphigoid (Fig. 3)-In pemphigoid, breakdown occurs betweein epithelium and connective tissue at the basement membrane. These specimens showed bright staining for LETS in epithelial cell cytoplasm, either throughout the epithelium or confined to the middle and upper epithelial cell layers. In addition, positive fluorescence was seen in the connective tissue and in the most superficial layers of the epithelium similar to normal mucosa. The LETS distribution here seems to be another example of the phenomenon, observed with
Downloaded from jdr.sagepub.com at Univ of Connecticut / Health Center / Library on June 24, 2015 For personal use only. No other uses without permission.
l TS INVI UMAN ORAA EPIlL Vol.o S8 No.NE I THL U 4487
l ! 7.245
io'W"'r !m MIT
wIllb" 4/1 Akk"
Fig. 4-Pemphigus. Section prepared as in Fig. IA, X 312. Intercellular staining of cells near an area of acantholysis (A). lichen planus, in which the epithelial cells produce LETS in response to a stimulus. In contrast to other cell types that primarily secrete the LETS protein that they synthesize, the epithelial cells in these two diseases seem to retain the LETS protein within their cytoplasm. Pemphigus (Fig. 4)-Pemnphigus is characterized by intercellular breakdown within the epithelium (acantholysis). The distribution of LETS protein in these specimens differed from that described in the other conditions. While there was little intracellular fluorescence in the epithelial cells, there was intercellular staining for LETS around certain cells, especially near the zones where acanltholysis had taken place. Since these are areas of marked disruption of cell-cell adhesions, perhaps the cells remaining in this area secrete LETS protein in order to promote intercellular adhesiveness. Alternatively, perhaps staining results from exposure of previously sequestered LETS. LETS staining in the surface epithelium and in the connective tissues was similar to normal. Discoid lupus erythe;natosus (DLI ") (Fig. 2)-DLE often effects changes in the oral mucosa, including hyperkeratosis, epithelial atrophy, liquefaction degeneration of the basal layer of the epithelium, and thickening of the basement membrane. When examined for the presence of LETS protein, specimens
1 ig. 5 -Lichen planus. Section prepared as in Fig. IA, X 200. IntracellulIar staining (I) in some epithelial cells and prominent staining in basement memnbrane zone.
from lesions of DLE showed one distinct difference from the normal: a complete lack of staining at the epithelial surface. Whereas the distribution of LETS protein in the epithelium and in the connective tissue was the same as in the normal mucosa, the absence of surface-associated LETS protein was striking and, to date, has been seen only in this condition. This absence might, therefore, be a useful marker for DLE. Furthermore, it may result from an attempt by cells of the epithelium to reduce excessive thickness by reducing surface-cell adhesiveness.
Conlclusion1s. LETS protein, intensely investigated since its rediscovery in 1973, has been implicated in phenomena as diverse as embryogenesis,13 hemostasis,3 and myogenesis,14,i 5 as well as
turn origenesis and metastasis.9'16 Hitherto, no reports of the distribution of LETS in human disease have appeared. In this report, we describe several instances in which the distribution of LETS protein is altered by
pathological conditions affecting the human oral mucosa. In discoid lupus erythematosus, the alteration involves a loss of the LETS protein normally present in the superficial layer of the epithelium. In all other conditions examined, mucosal disease seems to
Downloaded from jdr.sagepub.com at Univ of Connecticut / Health Center / Library on June 24, 2015 For personal use only. No other uses without permission.
488
ZETTER ETAL.
stimulate production of LETS protein by epithelial cells that do not normally produce it. Generally, the LETS protein detected in these cells remains intracellular. However, in the case of pemphigus, where acantholysis takes place, LETS protein is deposited in intercellular spaces, perhaps as an attempt by the cells to restore cell-cell adhesion. These preliminary results suggest that abnormal production of LETS protein by epithelial cells may be a general response to insult and could indicate stress or injury. Although this response is general, the amount and distribution of the LETS protein produced are different for the conditions described. Further work will be necessary to determine whether these patterns are useful as markers for these conditions, and may contribute to an understanding of their pathogenesis.
Acknowledgments. We would like to thank Dr. L. B. Chen
(Harvard Medical School) for his generous gift of monospecific antiserum to human LETS protein (CIG).
REFERENCES 1. STENMAN, S., and VAHERI, A.: Distribution of a Major Connective Tissue Protein, Fibronectin, in Normal Human Tissues, J. Exp Med 147:1054-1064, 1978. 2. HYNES, R. O.: Cell Surface Proteins and Malignant Transformation, Biochem Biophys Acta 458:73-107, 1976. 3. RUOSLAHTI, E., and VAHERI, A.: Interaction of Soluble Fibroblast Surface Antigen with Fibrinogen and Fibrin, J Exp Med 141: 497-501, 1975. 4. YAMADA, K. M.; YAMADA, S. S.; and PASTAN, I.: Cell Surface Protein Partially Restores Morphology, Adhesiveness, and Contact Inhibition of Movement to Transformed Fibroblasts, Proc Nat Acad Sci 73:12171221, 1976. 5. PEARLSTEIN, E.: Plasma Membrane Glycoprotein which Mediates Adhesion of Fibroblasts to Collagen, Nature (London) 262: 497-500, 1976.
J Dent Res January 19 79 6. ALI, I. U.; MAUTNER, V.; LANZA, R.; and HYNES, R. O.: Restoration of Normal Morphology, Adhesion and Cytoskeleton in Transformed Cells by Addition of a Transformation-Sensitive Surface Protein, Cell 11: 115-126, 1977. 7. YAMADA, K. M.; YAMADA, S. S.; and PASTAN, I.: The Major Cell Surface Glycoprotein of Chick Embryo Fibroblasts is an Agglutinin, Proc Nat Acad Sci 72:31583162, 1975. 8. ZETTER, B. R.; CHEN, L. B.; and BUCHANAN, J. M.: Effects of Protease Treatment on Growth, Morphology, Adhesion, and Cell Surface Proteins of Secondary Chick Embryo Fibroblasts, Cell 7:407412, 1976. 9. CHEN, L. B.; GALLIMORE, P. H.; and MCDOUGALL, J. H.: Correlation Between Tumor Induction and the Large External Transformation Sensitive Protein on the Cell Surface, Proc Nat Acad Sci 73:3570-3574, 1976. 10. BURRIDGE, K.: Changes in Cellular Glycoproteins After Transformation: Identification of Specific Glycoproteins and Antigens in Sodium Dodecyl Sulfate Gels, Proc Nat Acad Sci 73:44574461, 1976. 11. CHEN, L. B.; MAITLAND, N.; GALLIMORE, P. H.; and MCDOUGALL, J. H.: Detection of the Large External Transformation-Sensitive Protein on Some Epithelial Cells, Exp Cell Res 106:3946, 1977. 12. CHEN, L. B.; GUDOR, R. C.; SUN, T. T.; CHEN, A. B.; and MOSESSON, M. W.: Control of a Cell Surface Major Glycoprotein by Epidermal Growth Factor, Science 197:776778, 1977. 13. ZETTER, B. R.; and MARTIN, G. R.: Expression of a High Molecular Weight Cell Surface Glycoprotein (LETS Protein) by Preimplantation Mouse Embryos and Teratocarcinoma Stem Cells, Proc Nat Acad Sci 75:23242328, 1978. 14. HYNES, R. O.; MARTIN, G. S.; CUTCHLEY, D. R.; SHEARER, M.; and EPSTEIN, C. J.: Viral Transformation of Rat Myoblasts: Effects on Fusion and Surface Properties, Devel Biol 48:35-46, 1976. 15. CHEN, L. B.: Alteration in Cell Surface LETS Protein During Myogenesis, Cell 10:393400, 1977. 16. CHEN, L. B.; BURRIDGE, K.; MURRAY, A.; WALSH, M. L.; COPPLE, C. D.; BUSHNELL, A.; McDOUGALL, J. K.; and GALLIMORE, P. H.: Modulation of Cell Surface Glycocalyx Studies on Large, External, Transformation-Sensitive Protein, Ann NY Acad Sci 312:366-381, 1978.
Downloaded from jdr.sagepub.com at Univ of Connecticut / Health Center / Library on June 24, 2015 For personal use only. No other uses without permission.
