British Journal of Rheumatology 1992;31:231-234
INTEGRIN EXPRESSION BY HUMAN ARTICULAR CHONDROCYTES BY D. M. SALTER, D. E. HUGHES, R. SIMPSON AND D. L. GARDNER Department of Pathology, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG SUMMARY Expression of integrins, a family of cell adhesion proteins, by human articular cartilage chondrocytes in vivo has been studied by immunohistological techniques using cryostat sections and a panel of anti-integrin monoclonal antibodies. Chondrocytes strongly expressed (31 and a5 integrin subunits, known to form the classical fibronectin receptor, VLA-5, strongly, a l was expressed more weakly and variably; a3 was expressed by occasional cells only. Chondrocytes did not express P2, p3 or other integrin a subunits tested. KEY WORDS:
Cartilage, Immunohistology, Monoclonal antibodies, Cell ahesion molecules.
theatre or autopsy room, the joints opened and disarticulated. A coronal block was obtained by sawing through the articular surface and underlying bone. A 5 mm wide strip of full thickness articular cartilage was removed using a scalpel, placed in OCT and snapfrozen in liquid nitrogen. Cartilage was not taken from joints with macroscopic evidence of osteoarthrosis. Immunohistology Four pirn frozen sections were cut with a Bright cryostat and mounted on poly-L-lysine coated glass slides, allowed to come to room temperature and fixed in acetone. Cases were stained with a panel of well characterized monoclonal antibodies (MCA) (Table I) against pi (CD29) [6], |J2 (CD18) [7], P3 (CD61) [8] and cd-5 integrin subunits (CD49a-e) [6, 9-12] by an avidin-biotin-complex (ABC) technique [13,14] using a Shandon sequenza. Positive controls were sections of human tonsil. Negative controls were provided by omitting the primary antibody. TS2/16, TS2/7, HP2/1 were gifts from F Sanchez-Madrid, University of Madrid, Gi9 and Gil4 were gifts from S Santoso, Institute of Clinical Immunology and Transfusion Medicine, Giessen. AO43 and BIIG2 were gifts from S Dedhar, University of British Columbia, MHM23 and Y2/51 were obtained from Dako Ltd. RESULTS The results are summarized in Table I. Cartilage from all cases and joint surfaces gave similar results.
MATERIALS AND METHODS
P Chains Positive staining for pi integrin (CD29) was seen in TABLE I
Tissues Fresh human articular cartilage was obtained from medial femoral condyles (11 cases), medial tibial plateaux (13 cases), distal tibia (four cases) or taluses (eight cases) of below (seven cases) or above (four cases) knee amputations for peripheral vascular disease; at autopsy from sudden deaths (nine cases), or from the femoral head (one case) removed at hemiarthroplasty for osteoporotic neck fracture. Fourteen cases were male (age range 16-80, mean 48 years), seven were female (age range 35-89, mean 70.9 years). Cartilage was sampled in the following way. Resections and intact joints were received directly from
INTEGRIN EXPRESSION BY ARTICULAR CARTILAGE CHONDROCYTES
Submitted 17 January; revised version accepted 16 May 1991. Correspondence to Dr D. Salter.
MCA
Specificity Dilution
TS2/16 MHM23 Y2/51 TS2/7 Gi 14 Gi9 AO43 HP2/1 BIIG2
P1/CD29 P2/CD18 P3/CD61 cd/CD49a a2/CD49b «2/CD49b «3/CD49c a4/CD49d «5/CD49e
1/40 1/100 1/400 1/10 1/100 1/100 1/400 1/10 1/400
Superficial Mid Deep Calcified zone zone zone zone
±1-
±/-
±/-
±/-
+ , All cells positive; ±, positive and negative cells, ±_, occasional positive cells/most cells negative, - , all cells negative. 231
Downloaded from http://rheumatology.oxfordjournals.org/ at New York University on July 7, 2015
ARTICULAR cartilage consists of cells, chondrocytes, embedded in a proteoglycan- and collagen-rich matrix. Normal adult cartilage shows zonal variation with differences in chondrocyte morphology, biochemical composition and metabolism [1]. Control of chondrocyte function is central to the maintenance and integrity of structure and organization of this tissue. In a relatively paucicellular tissue such as cartilage, cellmatrix interactions are potentially important in the regulation of chondrocyte metabolism. Anchorin CII has been shown to be expressed by chondrocytes and to bind type II collagen in vitro [2], but other molecules are likely to be involved in chondrocyte-matrix adhesion. A number of cell adhesion receptors have now been recognized which may regulate cell-matrix and cell-cell adhesion, but it is unclear which, if any, of these participate in chondrocytematrix binding. One potentially important group is the integrins [3,4], a family of heterodimeric transmembrane glycoproteins. Integrins are distributed widely in many tissues; through interactions with matrix components including collagen, fibronectin and vitronectin, they may modulate cell function [3-5]. This study was undertaken to determine the expression of integrins by human articular chondrocytes in vivo and so increase our understanding of the molecular basis of chondrocyte-matrix interactions.
232
BRITISH JOURNAL OF RHEUMATOLOGY VOL. XXXI NO. 4
all cases, chondrocytes in all anatomical zones showing uniform cytoplasmic membrane positivity using the ABC immunostaining technique (Fig. la). Initial studies with the less sensitive indirect immunoperoxidase technique demonstrated positive and negative cells at a lower titre (1/40) of primary antibody and strong and weak staining of the majority of cells when the primary antibody was used at a higher titre (1/10) suggesting variable expression of this molecule (results not shown). There was no reactivity with anti-p*2 and anti-p3 MCA.
DISCUSSION This study shows that chondrocytes of adult human articular cartilage express pi integrin (CD29) and a5 (CD49e) in a similar distribution. This suggests that the majority of articular chondrocytes express the pia5 complex, VLA-5, which constitutes the classical fibronectin receptor [15]. Expression of al and a3 is less consistent and weaker. We were not able to test other integrin subunits such as a6 (VLA-6) and aV which are known to associate with (31 [16,17] and therefore
\
a
*
b
FIG. 1.—Human articular cartilage reacted with (a) TS2/16, anti-pi integrin, (b) BII G2, anti-a5, (c) negative control. There is a positive reaction product at the cytoplasmic membrane of chondrocytes in cartilage stained with TS2/16 and BII G2 (arrows). ABC-immunoperoxidase. haematoxylin counterstain: (a) x500. (b) X500, (c) x500.
Downloaded from http://rheumatology.oxfordjournals.org/ at New York University on July 7, 2015
a Chains Positive cytoplasmic membrane staining of chondrocytes for a5 was seen in all cases (Fig. lb) in a similar distribution to that observed with the anti-pi MCA. cd was variably expressed. Some cases showed weak positivity of most cells whereas in others many of the chondrocytes were negative. Only occasional cells expressed a3. There was no staining with antibodies to a2 or a4.
expression of the alternative fibronectin receptor has not been excluded by this study. Chondrocytes in vivo do not appear to express the |32 subfamily (LeuCAMs LFA-1, Mac-1) or members of the |33 subfamily which includes the classical vitronectin receptor p3aV [18]. VLA-5 is widely distributed, being expressed by epithelial cells, leucocytes and fibroblasts [19, 20] and is not known to bind any ligand other than fibronectin. The presence of a5 and |31 subunits on the cell surface of human articular chondrocytes would suggest that interactions between chondrocytes and fibronectin may be important in vivo. Fibronectin has a number of effects on cultured chondrocytes including stimulating attachment and spreading, altering morphology and inhibiting synthesis of type II collagen and type 1 proteoglycan [21,22]. Bovine articular cartilage chondrocytes adhere to fibronectin coated plastic with a higher avidity than to albumin-coated plates. This attachment can be inhibited by competition with an RGDcontaining peptide [23], suggesting the requirement for an RGD sequence binding interaction which characterizes the binding of VLA-5 to fibronectin. Biochemical and immunohistochemical studies have demonstrated the presence offibronectinin cartilage in vivo [24-26]. The precise distribution of fibronectin in articular cartilage, however, is uncertain. Predominantly pericellular [24], or predominantly interterritorial matrix distribution [25] have both been reported. Increased amounts of fibronectin are present in the superficial layers of osteoarthrotic cartilage [25] which may in part be related to increased de novo synthesis by
SALTER ETAL.:
INTEGRIN EXPRESSION BY HUMAN ARTICULAR CHONDROCYTES
ACKNOWLEDGEMENTS
We would like to thank Miss Julie McLachlan for typing the manuscripts and local orthopaedic and vascular surgeons and their theatre staff for the provision of fresh material. The work of the laboratory is generously supported by the Arthritis and Rheumatism Council for Research. REFERENCES
1. Hamerman D. The biology of osteoarthritis. N EnglJ Med 1989;320:1322-30. 2. Pfaffle M, Ruggerio F, Hofmann H, et al. Biosynthesis, secretion and extracellular localization of anchorin CII, a collagen-binding protein of the calpactin family. EMBO J 1988;7:2335-42. 3. Hynes RO. Integrins, a family of cell surface receptors. Cell 1987;48:549-55. 4. Hemler ME. VLA proteins in the integrin family: structures, functions and their role on leukocytes. A Rev Immunol 1990;8:365-40,0. 5. Albelda SM, Buck CA.' Integrins and other cell adhesion molecules. FASEB J 1990;4:2868-80.
6. Hemler ME, Sanchez-Madrid F, Flotle TJ, et al. Glycoproteins of 210 000 and 130 000 MW on activated T cells: cell distribution and antigenic relations to components on resting cells and T cell lines. J Immunol 1984;132:3011-18. 7. Hildreth JEK, Gotch FM, Hildreth PD, McMichael AJ. A human lymphocyte-associated antigen involved in cell-mediated lympholysis. Eur J Immunol 1983;13:202-8. 8. Gatter KC, Cordell JL, Turley H, et al. The immunohistochemical detection of platelets, megakaryocytes and thrombi in routinely processed specimens. Histopathology 1988;13:257-67. 9. Kiefel V, Santoso S, Katzmann B, Mueller-Eckhardt C. The Br7Brh alloantigen system on human platelets. Blood 1989;73:2219-23. 10. Wayner EA, Carter WG, Piotrowicz RS, Kunick TJ. The function of multiple extracellular matrix receptors in mediating cell adhesion to extracellular matrix: preparation of monoclonal antibodies to the fibronectin receptor that specifically inhibit cell adhesion to fibronectin and react with platelet glycoprotein Ic-IIa. J Cell Biol 1988;107:1881-91. 11. Werb Z, Tremble PM, Behrendtsen O, Crowley G, Damsky CW. Signal transduction through the fibronectin receptor induces collagenase and stromelysin gene expression. J Cell Biol 1989;109:877-89. 12. Sanchez-Madrid F, De Landazuri MO, Morago G, Lebrian M, Acevedo A, Bernabeu C. VLA-3: a novel polypeptide association within the VLA molecular complex: cell distribution and biochemical characterization . Eur J Immunol 1986;16:1343-9. 13. Salter DM, Krajewski AS, Dewar AE. Immunohistochemical staining of non-Hodgkin's lymphoma with monoclonal antibodies specific for the leucocyte common antigen. / Pathol 1985;146:345-53. 14. Norton AJ, Isaacson PG. Detailed phenotype analysis of B-cell lymphoma using a panel of antibodies reactive in routinely fixed wax embedded tissue. Am J Pathol 1987;129:434-40. 15. Pytela R, Pierschbacher MD, Rouslahti E. Identification and isolation of a 140kD cell surface glycoprotein with properties expected of a fibronectin receptor. Cell 1985;40:191-8. 16. Sonnenberg A, Modderman PW, Hogervorst F. Laminin receptor on platelets is the integrin VLA-6. Nature 1988,360:487-9. 17. Vogel B, Tarone G, Giancotti FG, Gailit J, Ruoslahti E. A novel fibronectin receptor with an unexpected subunit composition (aV(31). J Biol Chem 1990;26S:5934-7. 18. Phillips DR, Charo IF, Parise LV, Fitzgerald LA. The platelet membrane glycoprotein Ilb-IIIa complex. Blood 1988;71:831-43. 19. Hemler ME, Huang C, Schwartz L. The VLA protein family. Characterization of five distinct cell surface heterodimers each with a common 130,000 weight beta subunit. J Biol Chem 1987;262:3300-9. 20. Adams JC, Watt FM. Changes in keratinocyte adhesion during terminal differentiation: reduction in fibronectin binding precedes a5|31 integrin loss from the cell surface. Cell 1990;63:425-35. 21. Pennypacker JP, Wassell JR, Yamada KM, Pratt RM. The influence of an adhesive cell surface protein on chondrogenic expression in vitro. Exp Cell Res 1979,121:411-15. 22. West CM, De Weard H, Dowdy K, de la Paz A. Aspecificity for cellular fibronectin in its affects on
Downloaded from http://rheumatology.oxfordjournals.org/ at New York University on July 7, 2015
chondrocytes [26]. It is unclear whether these changes in fibronectin concentration are associated with alterations of the chondrocyte phenotype in vivo. Immunoreactivity for a l and a3 suggests that chondrocytes also express VLA-1 and VLA-3. VLA-1 is a collagen receptor [27] and VLA-3 binds to collagen, fibronectin and laminin. Both receptors appear to function independently of RGD peptides. Further studies will be required to elucidate the role of these molecules in articular cartilage. However, the sporadic distribution of their expression appears to represent a further example of phenotypic heterogeneity in what has frequently been regarded as a homogeneous tissue. Integrin-matrix interactions are believed to be important in the control of diverse cell functions over and above simple cell anchorage. Modification of these interactions following either altered extracellular matrix composition as recognized in osteoarthrosis or altered expression of membrane receptors can result in changes in cell behaviour. Preliminary studies on osteoarthrotic cartilage show increased immunoreactivity for a l , a3 and a5 subunits especially in superficial zone cells and cell clusters in areas of fibrillation (Salter etal. unpublished observations), raising the possibility that altered expression of these molecules may be associated with progression of cartilage damage and altered chondrocyte function. In conclusion, this study demonstrates the expression of proteins binding well characterized monoclonal antibodies against a l , a3, a5 and pi (CD29) integrin subunits by normal human adult articular cartilage. (31 and a5 were strongly expressed, suggesting that human articular chondrocytes express the (31 a5 integrin complex (VLA-5) which constitutes the classical fibronectin receptor. Immunoreactivity for a l and a3 was less consistently seen, suggesting heterogeneity of expression of VLA-1 and VLA-3. Further work is underway to define more accurately the role of these molecules in chondrocyte-matrix interaction and to assess their expression in osteoarthrotic cartilage.
233
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BRITISH JOURNAL OF RHEUMATOLOGY VOL. XXXI NO. 4
cultured chondroblasts. Differentiation 1984;27: 67-73. 23. Sommerin Y, Larsson TL, Heinegard D. Chondrocyte-matrix interactions. Attachment to proteins isolated from cartilage. Exp Cell Res 1989; 184: 181-92. 24. Burton-Wurster N, Horn VJ, Lust G. Immunohistochemical localization of fibronectin and chondronectin in canine articular cartilage. J Histochem Cytochem 1988;36:581-8.
25. Jones KL, Brown M, Ali SY, Brown RA. An immunohistochemical study of fibronectin in human osteoarthritic and disease free articular cartilage. Ann Rheum Dis 1987;46:809-15. 26. Brown RA, Jones KC. The synthesis and accumulation of fibronectin by human articular cartilage. J Rheumatol 1990;17:65-72. 27. Kramer RH, Marks N. Identification of integrin collagen receptors on human melanoma cells. J Biol Chem 1989;264:4684-8.
ROYAL NATIONAL HOSPITAL FOR RHEUMATIC DISEASES A 2-DAY INTENSIVE COURSE IN METROLOGY Dates: Thursday, 7 May and Friday, 8 May 1992 The course is aimed at nurses, physiotherapists and clinically based personnel, working in the field of rheumatology, who are interested in the use of the measurement and assessment of patients as a tool for better treatment or research. Further information: Dr Joan Davies, MB, BCh, DPH (tel.: 0225 465941, ext. 208).
Downloaded from http://rheumatology.oxfordjournals.org/ at New York University on July 7, 2015
NOTICE
INTEGRIN EXPRESSION BY HUMAN ARTICULAR CHONDROCYTES BY D. M. SALTER, D. E. HUGHES, R. SIMPSON AND D. L. GARDNER Department of Pathology, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG SUMMARY Expression of integrins, a family of cell adhesion proteins, by human articular cartilage chondrocytes in vivo has been studied by immunohistological techniques using cryostat sections and a panel of anti-integrin monoclonal antibodies. Chondrocytes strongly expressed (31 and a5 integrin subunits, known to form the classical fibronectin receptor, VLA-5, strongly, a l was expressed more weakly and variably; a3 was expressed by occasional cells only. Chondrocytes did not express P2, p3 or other integrin a subunits tested. KEY WORDS:
Cartilage, Immunohistology, Monoclonal antibodies, Cell ahesion molecules.
theatre or autopsy room, the joints opened and disarticulated. A coronal block was obtained by sawing through the articular surface and underlying bone. A 5 mm wide strip of full thickness articular cartilage was removed using a scalpel, placed in OCT and snapfrozen in liquid nitrogen. Cartilage was not taken from joints with macroscopic evidence of osteoarthrosis. Immunohistology Four pirn frozen sections were cut with a Bright cryostat and mounted on poly-L-lysine coated glass slides, allowed to come to room temperature and fixed in acetone. Cases were stained with a panel of well characterized monoclonal antibodies (MCA) (Table I) against pi (CD29) [6], |J2 (CD18) [7], P3 (CD61) [8] and cd-5 integrin subunits (CD49a-e) [6, 9-12] by an avidin-biotin-complex (ABC) technique [13,14] using a Shandon sequenza. Positive controls were sections of human tonsil. Negative controls were provided by omitting the primary antibody. TS2/16, TS2/7, HP2/1 were gifts from F Sanchez-Madrid, University of Madrid, Gi9 and Gil4 were gifts from S Santoso, Institute of Clinical Immunology and Transfusion Medicine, Giessen. AO43 and BIIG2 were gifts from S Dedhar, University of British Columbia, MHM23 and Y2/51 were obtained from Dako Ltd. RESULTS The results are summarized in Table I. Cartilage from all cases and joint surfaces gave similar results.
MATERIALS AND METHODS
P Chains Positive staining for pi integrin (CD29) was seen in TABLE I
Tissues Fresh human articular cartilage was obtained from medial femoral condyles (11 cases), medial tibial plateaux (13 cases), distal tibia (four cases) or taluses (eight cases) of below (seven cases) or above (four cases) knee amputations for peripheral vascular disease; at autopsy from sudden deaths (nine cases), or from the femoral head (one case) removed at hemiarthroplasty for osteoporotic neck fracture. Fourteen cases were male (age range 16-80, mean 48 years), seven were female (age range 35-89, mean 70.9 years). Cartilage was sampled in the following way. Resections and intact joints were received directly from
INTEGRIN EXPRESSION BY ARTICULAR CARTILAGE CHONDROCYTES
Submitted 17 January; revised version accepted 16 May 1991. Correspondence to Dr D. Salter.
MCA
Specificity Dilution
TS2/16 MHM23 Y2/51 TS2/7 Gi 14 Gi9 AO43 HP2/1 BIIG2
P1/CD29 P2/CD18 P3/CD61 cd/CD49a a2/CD49b «2/CD49b «3/CD49c a4/CD49d «5/CD49e
1/40 1/100 1/400 1/10 1/100 1/100 1/400 1/10 1/400
Superficial Mid Deep Calcified zone zone zone zone
±1-
±/-
±/-
±/-
+ , All cells positive; ±, positive and negative cells, ±_, occasional positive cells/most cells negative, - , all cells negative. 231
Downloaded from http://rheumatology.oxfordjournals.org/ at New York University on July 7, 2015
ARTICULAR cartilage consists of cells, chondrocytes, embedded in a proteoglycan- and collagen-rich matrix. Normal adult cartilage shows zonal variation with differences in chondrocyte morphology, biochemical composition and metabolism [1]. Control of chondrocyte function is central to the maintenance and integrity of structure and organization of this tissue. In a relatively paucicellular tissue such as cartilage, cellmatrix interactions are potentially important in the regulation of chondrocyte metabolism. Anchorin CII has been shown to be expressed by chondrocytes and to bind type II collagen in vitro [2], but other molecules are likely to be involved in chondrocyte-matrix adhesion. A number of cell adhesion receptors have now been recognized which may regulate cell-matrix and cell-cell adhesion, but it is unclear which, if any, of these participate in chondrocytematrix binding. One potentially important group is the integrins [3,4], a family of heterodimeric transmembrane glycoproteins. Integrins are distributed widely in many tissues; through interactions with matrix components including collagen, fibronectin and vitronectin, they may modulate cell function [3-5]. This study was undertaken to determine the expression of integrins by human articular chondrocytes in vivo and so increase our understanding of the molecular basis of chondrocyte-matrix interactions.
232
BRITISH JOURNAL OF RHEUMATOLOGY VOL. XXXI NO. 4
all cases, chondrocytes in all anatomical zones showing uniform cytoplasmic membrane positivity using the ABC immunostaining technique (Fig. la). Initial studies with the less sensitive indirect immunoperoxidase technique demonstrated positive and negative cells at a lower titre (1/40) of primary antibody and strong and weak staining of the majority of cells when the primary antibody was used at a higher titre (1/10) suggesting variable expression of this molecule (results not shown). There was no reactivity with anti-p*2 and anti-p3 MCA.
DISCUSSION This study shows that chondrocytes of adult human articular cartilage express pi integrin (CD29) and a5 (CD49e) in a similar distribution. This suggests that the majority of articular chondrocytes express the pia5 complex, VLA-5, which constitutes the classical fibronectin receptor [15]. Expression of al and a3 is less consistent and weaker. We were not able to test other integrin subunits such as a6 (VLA-6) and aV which are known to associate with (31 [16,17] and therefore
\
a
*
b
FIG. 1.—Human articular cartilage reacted with (a) TS2/16, anti-pi integrin, (b) BII G2, anti-a5, (c) negative control. There is a positive reaction product at the cytoplasmic membrane of chondrocytes in cartilage stained with TS2/16 and BII G2 (arrows). ABC-immunoperoxidase. haematoxylin counterstain: (a) x500. (b) X500, (c) x500.
Downloaded from http://rheumatology.oxfordjournals.org/ at New York University on July 7, 2015
a Chains Positive cytoplasmic membrane staining of chondrocytes for a5 was seen in all cases (Fig. lb) in a similar distribution to that observed with the anti-pi MCA. cd was variably expressed. Some cases showed weak positivity of most cells whereas in others many of the chondrocytes were negative. Only occasional cells expressed a3. There was no staining with antibodies to a2 or a4.
expression of the alternative fibronectin receptor has not been excluded by this study. Chondrocytes in vivo do not appear to express the |32 subfamily (LeuCAMs LFA-1, Mac-1) or members of the |33 subfamily which includes the classical vitronectin receptor p3aV [18]. VLA-5 is widely distributed, being expressed by epithelial cells, leucocytes and fibroblasts [19, 20] and is not known to bind any ligand other than fibronectin. The presence of a5 and |31 subunits on the cell surface of human articular chondrocytes would suggest that interactions between chondrocytes and fibronectin may be important in vivo. Fibronectin has a number of effects on cultured chondrocytes including stimulating attachment and spreading, altering morphology and inhibiting synthesis of type II collagen and type 1 proteoglycan [21,22]. Bovine articular cartilage chondrocytes adhere to fibronectin coated plastic with a higher avidity than to albumin-coated plates. This attachment can be inhibited by competition with an RGDcontaining peptide [23], suggesting the requirement for an RGD sequence binding interaction which characterizes the binding of VLA-5 to fibronectin. Biochemical and immunohistochemical studies have demonstrated the presence offibronectinin cartilage in vivo [24-26]. The precise distribution of fibronectin in articular cartilage, however, is uncertain. Predominantly pericellular [24], or predominantly interterritorial matrix distribution [25] have both been reported. Increased amounts of fibronectin are present in the superficial layers of osteoarthrotic cartilage [25] which may in part be related to increased de novo synthesis by
SALTER ETAL.:
INTEGRIN EXPRESSION BY HUMAN ARTICULAR CHONDROCYTES
ACKNOWLEDGEMENTS
We would like to thank Miss Julie McLachlan for typing the manuscripts and local orthopaedic and vascular surgeons and their theatre staff for the provision of fresh material. The work of the laboratory is generously supported by the Arthritis and Rheumatism Council for Research. REFERENCES
1. Hamerman D. The biology of osteoarthritis. N EnglJ Med 1989;320:1322-30. 2. Pfaffle M, Ruggerio F, Hofmann H, et al. Biosynthesis, secretion and extracellular localization of anchorin CII, a collagen-binding protein of the calpactin family. EMBO J 1988;7:2335-42. 3. Hynes RO. Integrins, a family of cell surface receptors. Cell 1987;48:549-55. 4. Hemler ME. VLA proteins in the integrin family: structures, functions and their role on leukocytes. A Rev Immunol 1990;8:365-40,0. 5. Albelda SM, Buck CA.' Integrins and other cell adhesion molecules. FASEB J 1990;4:2868-80.
6. Hemler ME, Sanchez-Madrid F, Flotle TJ, et al. Glycoproteins of 210 000 and 130 000 MW on activated T cells: cell distribution and antigenic relations to components on resting cells and T cell lines. J Immunol 1984;132:3011-18. 7. Hildreth JEK, Gotch FM, Hildreth PD, McMichael AJ. A human lymphocyte-associated antigen involved in cell-mediated lympholysis. Eur J Immunol 1983;13:202-8. 8. Gatter KC, Cordell JL, Turley H, et al. The immunohistochemical detection of platelets, megakaryocytes and thrombi in routinely processed specimens. Histopathology 1988;13:257-67. 9. Kiefel V, Santoso S, Katzmann B, Mueller-Eckhardt C. The Br7Brh alloantigen system on human platelets. Blood 1989;73:2219-23. 10. Wayner EA, Carter WG, Piotrowicz RS, Kunick TJ. The function of multiple extracellular matrix receptors in mediating cell adhesion to extracellular matrix: preparation of monoclonal antibodies to the fibronectin receptor that specifically inhibit cell adhesion to fibronectin and react with platelet glycoprotein Ic-IIa. J Cell Biol 1988;107:1881-91. 11. Werb Z, Tremble PM, Behrendtsen O, Crowley G, Damsky CW. Signal transduction through the fibronectin receptor induces collagenase and stromelysin gene expression. J Cell Biol 1989;109:877-89. 12. Sanchez-Madrid F, De Landazuri MO, Morago G, Lebrian M, Acevedo A, Bernabeu C. VLA-3: a novel polypeptide association within the VLA molecular complex: cell distribution and biochemical characterization . Eur J Immunol 1986;16:1343-9. 13. Salter DM, Krajewski AS, Dewar AE. Immunohistochemical staining of non-Hodgkin's lymphoma with monoclonal antibodies specific for the leucocyte common antigen. / Pathol 1985;146:345-53. 14. Norton AJ, Isaacson PG. Detailed phenotype analysis of B-cell lymphoma using a panel of antibodies reactive in routinely fixed wax embedded tissue. Am J Pathol 1987;129:434-40. 15. Pytela R, Pierschbacher MD, Rouslahti E. Identification and isolation of a 140kD cell surface glycoprotein with properties expected of a fibronectin receptor. Cell 1985;40:191-8. 16. Sonnenberg A, Modderman PW, Hogervorst F. Laminin receptor on platelets is the integrin VLA-6. Nature 1988,360:487-9. 17. Vogel B, Tarone G, Giancotti FG, Gailit J, Ruoslahti E. A novel fibronectin receptor with an unexpected subunit composition (aV(31). J Biol Chem 1990;26S:5934-7. 18. Phillips DR, Charo IF, Parise LV, Fitzgerald LA. The platelet membrane glycoprotein Ilb-IIIa complex. Blood 1988;71:831-43. 19. Hemler ME, Huang C, Schwartz L. The VLA protein family. Characterization of five distinct cell surface heterodimers each with a common 130,000 weight beta subunit. J Biol Chem 1987;262:3300-9. 20. Adams JC, Watt FM. Changes in keratinocyte adhesion during terminal differentiation: reduction in fibronectin binding precedes a5|31 integrin loss from the cell surface. Cell 1990;63:425-35. 21. Pennypacker JP, Wassell JR, Yamada KM, Pratt RM. The influence of an adhesive cell surface protein on chondrogenic expression in vitro. Exp Cell Res 1979,121:411-15. 22. West CM, De Weard H, Dowdy K, de la Paz A. Aspecificity for cellular fibronectin in its affects on
Downloaded from http://rheumatology.oxfordjournals.org/ at New York University on July 7, 2015
chondrocytes [26]. It is unclear whether these changes in fibronectin concentration are associated with alterations of the chondrocyte phenotype in vivo. Immunoreactivity for a l and a3 suggests that chondrocytes also express VLA-1 and VLA-3. VLA-1 is a collagen receptor [27] and VLA-3 binds to collagen, fibronectin and laminin. Both receptors appear to function independently of RGD peptides. Further studies will be required to elucidate the role of these molecules in articular cartilage. However, the sporadic distribution of their expression appears to represent a further example of phenotypic heterogeneity in what has frequently been regarded as a homogeneous tissue. Integrin-matrix interactions are believed to be important in the control of diverse cell functions over and above simple cell anchorage. Modification of these interactions following either altered extracellular matrix composition as recognized in osteoarthrosis or altered expression of membrane receptors can result in changes in cell behaviour. Preliminary studies on osteoarthrotic cartilage show increased immunoreactivity for a l , a3 and a5 subunits especially in superficial zone cells and cell clusters in areas of fibrillation (Salter etal. unpublished observations), raising the possibility that altered expression of these molecules may be associated with progression of cartilage damage and altered chondrocyte function. In conclusion, this study demonstrates the expression of proteins binding well characterized monoclonal antibodies against a l , a3, a5 and pi (CD29) integrin subunits by normal human adult articular cartilage. (31 and a5 were strongly expressed, suggesting that human articular chondrocytes express the (31 a5 integrin complex (VLA-5) which constitutes the classical fibronectin receptor. Immunoreactivity for a l and a3 was less consistently seen, suggesting heterogeneity of expression of VLA-1 and VLA-3. Further work is underway to define more accurately the role of these molecules in chondrocyte-matrix interaction and to assess their expression in osteoarthrotic cartilage.
233
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BRITISH JOURNAL OF RHEUMATOLOGY VOL. XXXI NO. 4
cultured chondroblasts. Differentiation 1984;27: 67-73. 23. Sommerin Y, Larsson TL, Heinegard D. Chondrocyte-matrix interactions. Attachment to proteins isolated from cartilage. Exp Cell Res 1989; 184: 181-92. 24. Burton-Wurster N, Horn VJ, Lust G. Immunohistochemical localization of fibronectin and chondronectin in canine articular cartilage. J Histochem Cytochem 1988;36:581-8.
25. Jones KL, Brown M, Ali SY, Brown RA. An immunohistochemical study of fibronectin in human osteoarthritic and disease free articular cartilage. Ann Rheum Dis 1987;46:809-15. 26. Brown RA, Jones KC. The synthesis and accumulation of fibronectin by human articular cartilage. J Rheumatol 1990;17:65-72. 27. Kramer RH, Marks N. Identification of integrin collagen receptors on human melanoma cells. J Biol Chem 1989;264:4684-8.
ROYAL NATIONAL HOSPITAL FOR RHEUMATIC DISEASES A 2-DAY INTENSIVE COURSE IN METROLOGY Dates: Thursday, 7 May and Friday, 8 May 1992 The course is aimed at nurses, physiotherapists and clinically based personnel, working in the field of rheumatology, who are interested in the use of the measurement and assessment of patients as a tool for better treatment or research. Further information: Dr Joan Davies, MB, BCh, DPH (tel.: 0225 465941, ext. 208).
Downloaded from http://rheumatology.oxfordjournals.org/ at New York University on July 7, 2015
NOTICE
