- Email: [email protected]
Increased serum levels of soluble vascular cell adhesion molecule 1 and E-selectin in patients with localized scleroderma
Increased serum levels of soluble vascular cell adhesion molecule 1 and E-selectin in patients with localized scleroderma Kenichi Yamane, MD, Hironobu Ihn, MD, PhD, Masahide Kubo, MD, Norihito Yazawa, MD, Kanako Kikuchi, MD, PhD, Yoshinao Soma, MD, PhD, and Kunihiko Tamaki, MD, PhD Tokyo, Japan Background: The serum levels of soluble forms of vascular cell adhesion molecule 1 (sVCAM-1) and Eselectin (sE-selectin) can be used as a marker of endothelial activation. Objective: We investigated whether the serum level of sVCAM-1 and sE-selectin in patients with localized scleroderma (LSc) was correlated with the clinical or serologic features of this disease. Methods: Serum samples from 59 patients with LSc, 20 patients with systemic sclerosis (SSc) and 29 healthy controls were examined using specific enzyme-linked immunosorbent assays. Results: The serum levels of sVCAM-1 and sE-selectin in patients with LSc were significantly higher than those of the healthy controls. The serum levels of sVCAM-1 and sE-selectin were correlated with both the number of sclerotic lesions and the number of involved areas. Conclusion: These results indicate that the serum levels of sVCAM-1 and sE-selectin may reflect the extent of the skin involvement (ie, the disease severity) and may be useful for monitoring the in vivo states of endothelial activation in LSc. (J Am Acad Dermatol 2000;42:64-9.)
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ocalized scleroderma (LSc) is a connective tissue disorder usually limited to the skin and subcutaneous tissue, but it sometimes involves the muscle beneath the cutaneous lesion. The absence of Raynaud’s phenomenon, acrosclerosis, and internal organ involvement differentiates LSc from systemic sclerosis (SSc).1 Generalized morphea, which is the most severe form of LSc, has been reported to be accompanied by a variety of abnormal immune reactions, such as the presence of antinuclear antibody (ANA), the lupus erythematosus (LE) cell phenomenon, rheumatoid factor (RF), anti-single-stranded DNA antibody (anti-ssDNA) and antihistone antibody (AHA).2-8 We have recently demonstrated additional immunologic abnormalities in patients with LSc: elevated serum levels of interleukin 2 (IL-2), IL-4, IL-6, soluble IL-2 receptor, and soluble intercellular adhesion molecule 1 (sICAM-1).9-11 From the Department of Dermatology, Faculty of Medicine, University of Tokyo. Accepted for publication Aug 3, 1999. Reprint requests: H. Ihn, MD, Department of Dermatology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: [email protected]. Copyright © 2000 by the American Academy of Dermatology, Inc. 0190-9622/2000/$12.00 + 0 16/1/102363
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Vascular cell adhesion molecule 1 (VCAM-1) is a member of the immunoglobulin gene superfamily and is induced on the surface of endothelial cells within hours of stimulation by the inflammatory cytokines, such as IL-1 or tumor necrosis factor (TNF) α.12 VCAM-1 is expressed by activated endothelial cells and several extravascular cell types, including lymphoid dendritic cells, murine bone marrow stromal cells, proximal tubule cells, and synovial lining cells.13-16 VCAM-1 is a ligand for the integrinα4β1 (VLA-4) and binds weakly to α4β7.17,18 E-selectin is a member of the selectin family and is induced on vascular endothelial cells by cytokines such as IL-1, TNF-α, interferon gamma (IFN-γ), and IL4.19,20 Unlike other endothelial adhesion molecules such as VCAM-1, E-selectin has been reported to be expressed only on endothelial cells. E-selectin acts as a ligand for the sialyl-Lewisx and sialyl-Lewisa.21,22 Recently, soluble forms of various adhesion molecules have been reported.23,24 Increased expression levels of soluble VCAM-1 (sVCAM-1) and soluble Eselectin (sE-selectin) have been described in various inflammatory and neoplastic diseases.25-30 In this study, we measured the serum levels of sVCAM-1 and of sE-selectin in patients with LSc and
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investigated whether these levels would serve as useful markers of the disease activity.
MATERIAL AND METHODS Patients The 3 study groups consisted of 59 patients with LSc, 20 patients with SSc, and 29 healthy controls. The patients with LSc were classified into 3 subgroups: (1) those with generalized morphea (GM, n = 21), (2) those with linear scleroderma (LS, n = 26), (3) those with morphea (n = 12). If patients had both morphea and linear lesions, they were classified as having GM. The number of sclerotic lesions larger than 3 cm in diameter was counted in each patient when the serum samples were collected. The number of involved areas of the body was also counted; we divided the body into 7 areas: (1) head and neck, (2) anterior part of the trunk, (3) posterior part of the trunk, (4) right upper extremity, (5) left upper extremity, (6) right lower extremity, and (7) left lower extremity. All patients with SSc met the American College of Rheumatology criteria for the diagnosis of SSc (scleroderma).31 Enzyme-linked immunosorbent assays for soluble adhesion molecules Aliquots of serum were frozen at –80°C until assayed. According to the manufacturer’s instructions, specific enzyme-linked immunosorbent assay (ELISA) kits were used for the determination of sVCAM-1 (sensitivity, 0.5 ng/mL; British Bio-technology Products, Oxford, UK) and sE-selectin levels (sensitivity, 0.5 ng/mL; Bender MedSystems, Vienna, Austria). Serum sVCAM-1 or sE-selectin levels more than 2 standard deviations (SDs) greater than the levels of the normal control subjects were regarded as elevated. ANA and RF ANAs were detected by an indirect immunofluorescence method with HEp-2 cells.4 RF was measured by the tube latex agglutination test, as described previously.9 Immunodiffusion Anti-U1 ribonucleoprotein antibody, anti-Sm antibody, and anti-topoisomerase I antibody were detected by double immunodiffusion as described previously.4 ELISAs for AHAs and anti-ssDNA antibodies The serum levels of AHAs were measured with an ELISA, as described previously.8 Absorbance values exceeding the mean plus 3 SDs for the normal control subjects were considered positive. For antissDNA antibodies, the wells were pretreated for 1
hour with 0.1% protamine sulfate (Sigma Chemical Co, St Louis, Mo). After the wells were rinsed with phosphate-buffered saline (PBS), calf thymus ssDNA (Sigma) was added at 1 µg/mL in PBS. An ELISA was then performed, as described previously.7 Statistical analysis The statistical analysis was carried out with Student t test for the comparison of means and Fisher’s exact probability test for the analysis of frequency. Correlations with clinical data were assessed by Spearman’s rank correlation coefficient. Two-tailed P values less than .05 were considered significant.
RESULTS Serum levels of sVCAM-1 The serum levels of sVCAM-1 were significantly higher in the LSc patients compared with the healthy controls (1077.4 ± 574.6 ng/mL vs 753.3 ± 380.9 ng/mL; P < .01). The sVCAM-1 levels in sera from the patients with GM (1365.1 ± 633.4 ng/mL) were significantly higher than those of the patients with morphea (748.9 ± 439.0 ng/mL; P < .01). However, the serum levels of sVCAM-1 in the patients with LS were not significantly higher than those of the patients with morphea. The SSc patients also had significantly higher serum sVCAM-1 levels compared with the healthy controls (1203.3 ± 473.9 ng/mL vs 753.3 ± 380.9 ng/mL; P < .001). The cut-off value (mean + 2 SD) was set at 1515.1 ng/mL, based on the data of the 29 healthy control sera. Elevated serum levels of sVCAM-1 were found in 6 of the 21 patients (29%) with GM, 4 of the 26 patients (15%) with LS, and 1 of the 12 patients (8%) with morphea (Fig 1). Seven of the 20 patients (35%) with SSc had serum sVCAM-1 levels greater than the cut-off value. Elevated serum levels of sVCAM-1 were found in 4 of the 10 patients (40%) with diffuse cutaneous SSc and 3 of the 10 patients (30%) with limited cutaneous SSc. Serum levels of soluble E-selectin Compared with the levels in the control subjects (66.6 ± 31.6 ng/mL), the serum levels of sE-selectin in the patients with LSc were significantly elevated (90.8 ± 48.1 ng/mL; P < .02). The sE-selectin levels in sera from the patients with GM (110.4 ± 57.1 ng/mL) were significantly higher than those of the patients with morphea (66.4 ± 30.6 ng/mL; P < .02). The serum levels of sE-selectin in the patients with LS, however, were not significantly higher than those in the patients with morphea. The patients with SSc had significantly higher serum sE-selectin levels than those of the healthy controls (101.4 ± 40.4 ng/mL vs 66.6 ± 31.6 ng/mL; P < .01). The cut-off value (mean + 2 SD) was set at 129.8 ng/mL, based on the data of
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Fig 1. Serum levels of soluble VCAM-1 (sVCAM-1). Dashed line represents the cut-off value (mean + 2 SD), which was set as 1515.1 ng/mL, based on data of the 29 healthy control sera. Elevated serum levels of sVCAM-1 were found in 6 of the 21 patients (29%) with GM, 4 of the 26 patients (15%) with LS, and 1 of the 12 patients (8%) with morphea. Seven of the 20 patients (35%) with SSc had serum sVCAM-1 levels greater than the cut-off value.
Fig 2. Serum levels of soluble E-selectin (sE-selectin). Dashed line represents the cut-off value (mean + 2 SD), which was set as 129.8 ng/mL, based on data of the 29 healthy control sera. Elevated serum levels of sE-selectin were found in 7 of the 21 patients (33%) with GM, 4 of the 26 patients (15%) with LS, and 1 of the 12 patients (8%) with morphea. Eight of the 20 patients (40%) with SSc had serum sE-selectin levels greater than the cut-off value.
the 29 healthy controls. Elevated serum levels of sEselectin were found in 7 of the 21 patients (33%) with GM, 4 of the 26 patients (15%) with LS, and 1 of the 12 patients (8%) with morphea (Fig 2). Eight of the 20 patients (40%) with SSc had serum sE-selectin levels greater than the cut-off value. Elevated serum levels of sE-selectin were found in 4 of the 10 patients (40%) with diffuse cutaneous SSc and 4 of the 10 patients (40%) with limited cutaneous SSc.
nificant difference between these groups in sex, age, or duration of the disease. The number of sclerotic lesions was significantly greater in the patients with elevated levels of sVCAM-1 than in those with normal levels (4.3 ± 1.8 vs 2.6 ± 1.9, P < .02). The number of involved areas was also significantly greater in the patients with elevated levels of sVCAM-1 than in those with normal levels (2.7 ± 1.4 vs 1.8 ± 1.1, P < .02). In addition, the number of sclerotic lesions and the number of involved areas were significantly greater in the patients with elevated levels of sEselectin than in those with normal levels (4.8 ± 3.3 vs 2.9 ± 2.7, P < .05; 2.8 ± 1.6 vs 1.7 ± 1.0, P < .01, respectively). The presence of RF was found in a significantly greater proportion of patients with elevated levels of sVCAM-1 than in patients with normal
Correlation of serum soluble adhesion molecules with clinical and serologic features of patients with LSc The clinical and serologic features of patients with elevated or normal levels of soluble adhesion molecules are shown in Tables I and II. There was no sig-
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Table I. Correlation of serum sVCAM-1 levels with clinical and serologic features of patients with localized scleroderma Patients with elevated sVCAM-1 levels (n = 11)
Patients with normal sVCAM-1 levels (n = 48)
2:9 28.7 ± 21.4 4.2 ± 4.5 4.3 ± 1.8* 2.7 ± 1.4*
12:36 22.5 ± 15.7 4.5 ± 5.6 2.6 ± 1.9* 1.8 ± 1.1*
Sex (M:F) Age (y) Duration of disease (y) No. of sclerotic lesions No. of involved areas Proportion of patients positive for ANA Anti-ssDNA RF AHA IgG AHA IgM
91% 36% 64%* 50% 33%
55% 17% 24%* 34% 34%
*P < .02.
Table II. Correlation of serum sE-selectin levels with clinical and serologic features of patients with localized scleroderma Patients with elevated sE-selectin levels (n = 12)
Patients with normal sE-selectin levels (n = 47)
3:9 21.3 ± 21.4 2.9 ± 2.2 4.8 ± 3.3* 2.8 ± 1.6†
11:36 22.5 ± 15.7 4.9 ± 5.9 2.9 ± 2.7* 1.7 ± 1.0†
83% 33% 50% 50% 50%
62% 28% 27% 13% 31%
Sex (M:F) Age (y) Duration of disease (y) No. of sclerotic lesions No. of involved areas Proportion of patients positive for ANA Anti-ssDNA RF AHA IgG AHA IgM *P < .05. †P < .01.
levels (64% vs 24%; P < .02). Moreover, the serum sVCAM-1 levels were significantly correlated with the serum sE-selectin levels (r = 0.26; P < .02). The presence of ANA was found in a greater proportion in patients with elevated sVCAM-1 levels (91%) than in those with normal sVCAM-1 levels (55%), but there is no significant difference between these two groups (P = .54). In addition, there were no significant correlations between the serum sVCAM-1 levels and the presence of anti-ssDNA, AHA IgG, or AHA IgM. There were also no significant correlations between the sE-selectin levels and the presence of ANA, RF, anti-ssDNA, AHA IgG, or AHA IgM. There were no specific autoantibodies, such as anti-topoisomerase I antibody or anti-centromere antibody, in
sera from patients with LSc by indirect immunofluorescence or immunodiffusion.
DISCUSSION In this study, we found that the serum levels of sVCAM-1 and sE-selectin were significantly higher in patients with LSc than in healthy controls. This suggests VCAM-1 and E-selectin upregulation in patients with LSc because the amounts of soluble adhesion molecules are correlated with the level of their surface expression. sVCAM-1 and sE-selectin are shed mainly by activated endothelial cells.23,24 E-selectin has been reported to be expressed only by activated endothelial cells. The results of the present study indicate that high levels of sVCAM-1 and sE-selectin
68 Yamane et al
may be correlated with endothelial activation in LSc, and they suggest the clinical importance of these soluble adhesion molecules in patients with LSc. sVCAM-1 and sE-selectin contain most of the structure and exhibit most of the function of the extracellular portion of cell-bound adhesion molecules. For example, it has been reported that the membrane-anchored form of sVCAM-1 is converted to the soluble form by a regulated proteolytic cleavage process involving metalloproteinase.24 It appears that the sources of sVCAM-1 and sE-selectin in vivo may be mainly cell-bound adhesion molecules expressed on activated endothelial cells. VCAM-1 and E-selectin are expressed on activated endothelial cells after stimulation with IL-1, TNF-α, and IFN-γ.32,33 These findings suggest that the elevated serum levels of sVCAM-1 and sE-selectin may be a consequence of long-term exposure to these cytokines in patients with LSc. Elevated serum levels of sVCAM-1 and sE-selectin have been described in various autoimmune disorders, such as rheumatoid arthritis, systemic lupus erythematosus, and SSc.25-30 In our study, as in others, the serum levels of sVCAM-1 and sE-selectin in patients with SSc were higher than those of healthy controls. Previous studies demonstrated that serum levels of soluble adhesion molecules are useful markers of the disease activity. For example, the serum levels of sVCAM-1 and sE-selectin correlated with clinical disease activity in SSc.27-30 In rheumatoid arthritis, high levels of sVCAM-1 were detected in rheumatoid synovial fluid and plasma samples and were correlated with disease severity.25,26 In the present study, patients with GM, the most severe form of LSc, had the highest levels of sVCAM-1 and sEselectin. In addition, the serum levels of these soluble adhesion molecules were significantly correlated with both the number of sclerotic lesions and the number of involved areas. These results suggest that the serum levels of sVCAM-1 and sE-selectin may reflect the extent of the skin involvement, that is, the severity of LSc. In conclusion, we have demonstrated the elevation of sVCAM-1 and sE-selectin in patients with LSc. Serum levels of sVCAM-1 and sE-selectin may reflect the severity of the disease and may be useful for monitoring in vivo states of endothelial activation. Further studies are needed to document the potential role of sVCAM-1 and sE-selectin in LSc. REFERENCES 1. Rodnan GP. When is scleroderma not scleroderma? Bull Rheum Dis 1981;31:7-10. 2. Scarola JA, Shulman LE. Serologic abnormalities and their significance in localized scleroderma [abstract]. Arthritis Rheum 1975;18:526.
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3. Rodnan GP, Rabin BS, Reichlin M. Eosinophilia and serological abnormalities in linear localized scleroderma [abstract]. Arthritis Rheum 1977;20:133. 4. Takehara K, Moroi Y, Nakabayashi Y, Ishibashi Y. Antinuclear antibodies in localized scleroderma. Arthritis Rheum 1983;26: 612-6. 5. Woo TY, Rasmussen JE. Juvenile linear scleroderma associated with serological abnormalities. Arch Dermatol 1985;121:14035. 6. Falanga V, Medsger TA Jr, Reichlin M, Rodnan GP. Linear scleroderma: clinical spectrum, prognosis, and laboratory abnormalities. Ann Intern Med 1986;104:849-57. 7. Takehara K, Kikuchi K, Soma Y, Igarashi A, Ishibashi Y. Anti-singlestrand DNA antibody and muscle involvement in localized scleroderma. Arch Dermatol 1990;126:1368-9. 8. Sato S, Ihn H, Soma Y, Igarashi A, Tamaki T, Kikuchi K et al. Antihistone antibodies in localized scleroderma. Arthritis Rheum 1993;36:1137-41. 9. Ihn H, Sato S, Fujimoto M, Kikuchi K, Takehara K. Demonstration of interleukin-2, interleukin-4 and interleukin-6 in sera from patients with localized scleroderma. Arch Dermatol Res 1995;287:193-7. 10. Ihn H, Sato S, Fujimoto M, Kikuchi K, Takehara K. Clinical significance of serum levels of soluble interleukin-2 receptor in patients with localized scleroderma. Br J Dermatol 1996;134: 843-7. 11. Ihn H, Fujimoto M, Sato S , Kikuchi K, Igarashi A, Soma Y, et al. Increased levels of circulating intercellular adhesion molecule1 in patients with localized scleroderma. J Am Acad Dermatol 1994;31:591-5. 12. Osborn L, Hession C,Tizard R,Vassallo C, Luhowskyj S, Chi-Rosso G, et al. Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes. Cell 1989;59:1203-11. 13. Rice GE, Munro JM, Corless C, Bevilacqua MP. Vascular and nonvascular expression of INCAM-110: a target for mononuclear leukocyte adhesion in normal and inflamed human tissues. Am J Pathol 1991;138:385-93. 14. Miyake K, Medina K, Ishihara K, Kimoto M, Auerbach R, Kincade PW. A VCAM-like adhesion molecule on murine bone marrow stromal cells mediates binding of lymphocyte precursors in culture. J Cell Biol 1991;114:557-65. 15. Seron D, Cameron JS, Haskard DO. Expression of VCAM-1 in the normal and diseased kidney. Nephrol Dial Transplant 1991;6:917-22. 16. Morales-Ducret J, Wayner E, Elices MJ, Alvaro-Gracia JM, Zvaifler NJ, Firestein GS. α4/β1 Integrin (VLA-4) ligands in arthritis: vascular cell adhesion molecule-1 expression in synovium and on fibroblast-like synoviocytes. J Immunol 1992;149:1424-31. 17. Elices MJ, Osborn L, Takada Y , Crouse C, Luhowskyj S, Hemler ME, et al. VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/ fibronectin binding site. Cell 1990;60:577-84. 18. Ruegg C, Postigo AA, Sikorski EE, Butcher EC, Pytela R, Erle DJ. Role of integrin α4β7/α4βP in lymphocyte adherence to fibronectin and VCAM-1 and in homotypic cell clustering. J Cell Biol 1992;117:179-89. 19. Bevilacqua MP, Pober JS, Mendrick DL, Cotran RS, Gimbrone MA Jr. Identification of an inducible endothelial-leukocyte adhesion molecule. Proc Natl Acad Sci 1989;84:9238-42. 20. Bevilacqua MP, Stengelin S, Gimbrone MA Jr, Seed B. Endothelial leukocyte adhesion molecule 1: an inducible receptor of neutrophils related to complement regulatory proteins and lectins. Science 1989;243:1160-5. 21. Phillips ML, Nudelman E, Gaeta FC, Perez M, Singhal AK,
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Hakomori S, et al. ELAM-1 mediates cell adhesion by recognition of a carbonhydrate ligand, sialyl-Lex. Science 1990;250: 1130-2. Berg EL, Robinson MK, Mansson O, Butcher EC, Magnani JL. A carbohydrate domain common to both sialyl-Lea and sialyl-Lex is recognized by the endothelial cell leucocyte adhesion molecule ELAM-1. J Biol Chem 1991;266:14869-72. Newman W, Beall LD, Carson CW , Hunder GG, Graben N, Randhawa ZI, et al. Soluble E-selectin is found in supernatants of activated endothelial cells and is elevated in the serum of patients with septic shock. J Immunol 1993;150:644-54. Leca G, Mansur SE, Bensussan A. Expression of VCAM-1 (CD106) by a subset of TCRδ-bearing lymphocyte clones: involvement of a metalloprotease in the specific hydrolytic release of the soluble isoform. J Immunol 1995;154:1069-77. Masson JC, Kapahi P, Haskard DO. Detection of increased levels of circulating intercellular adhesion molecules in some patients with rheumatoid arthritis but not in patients with systemic lupus erythematosus: lack of correlation with levels of circulating vascular cell adhesion molecule 1. Arthritis Rheum 1993; 36:519-27. Carson CW, Beall LD, Hunder GG, Johnson CM, Newman W. Soluble E-selectin is increased in inflammatory synovial fluid. J Rheumatol 1994;21:605-11. Gruschwitz MS, Hornstein OP, von den Driesch P. Correlation of soluble adhesion molecules in the peripheral blood of sclero-
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derma patients with their in situ expression and with disease activity. Arthritis Rheum 1995;38:184-9. Carson CW, Beall LD, Hunder GG, Johnson CM, Newman W. Serum ELAM-1 is increased in vasculitis, scleroderma, and systemic lupus erythematosus. J Rheumatol 1993;20:809-14. Hebbar M, Lassalle P, Jannin A, Vanhee D, Bisiau S, Hatron PY, et al. E-selectin expression in salivary endothelial cells and sera from patients with systemic sclerosis: role of resident mast cellderived tumor necrosis factor. Arthritis Rheum 1995;38:406-12. Ihn H, Sato S, Fujimoto M,Takehara K,Tamaki K. Increased serum levels of soluble vascular cell adhesion molecule-1 and Eselectin in patients with systemic sclerosis. Br J Rheumatol 1998;37:1188-92. Subcommittee for Scleroderma Criteria of the ARA Diagnostic and Therapeutic Criteria Committee. Preliminary criteria for the classification of systemic sclerosis (scleroderma). Arthritis Rheum 1980;23:581-9. Thornhill MH, Haskard DO. IL-4 regulates endothelial cell activation by IL-1, tumor necrosis factor, or IFN-γ. J Immunol 1990; 145:865-72. Wellicome SM, Thornhill MH, Pitzalis C, Thomas DS, Lanchbury JS, Panayi GS, et al. A monoclonal antibody that detects a novel antigen on endothelial cells that is induced by tumor necrosis factor, IL-1, or lipopolysaccharide. J Immunol 1990;144:2558-65.
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ocalized scleroderma (LSc) is a connective tissue disorder usually limited to the skin and subcutaneous tissue, but it sometimes involves the muscle beneath the cutaneous lesion. The absence of Raynaud’s phenomenon, acrosclerosis, and internal organ involvement differentiates LSc from systemic sclerosis (SSc).1 Generalized morphea, which is the most severe form of LSc, has been reported to be accompanied by a variety of abnormal immune reactions, such as the presence of antinuclear antibody (ANA), the lupus erythematosus (LE) cell phenomenon, rheumatoid factor (RF), anti-single-stranded DNA antibody (anti-ssDNA) and antihistone antibody (AHA).2-8 We have recently demonstrated additional immunologic abnormalities in patients with LSc: elevated serum levels of interleukin 2 (IL-2), IL-4, IL-6, soluble IL-2 receptor, and soluble intercellular adhesion molecule 1 (sICAM-1).9-11 From the Department of Dermatology, Faculty of Medicine, University of Tokyo. Accepted for publication Aug 3, 1999. Reprint requests: H. Ihn, MD, Department of Dermatology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: [email protected]. Copyright © 2000 by the American Academy of Dermatology, Inc. 0190-9622/2000/$12.00 + 0 16/1/102363
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Vascular cell adhesion molecule 1 (VCAM-1) is a member of the immunoglobulin gene superfamily and is induced on the surface of endothelial cells within hours of stimulation by the inflammatory cytokines, such as IL-1 or tumor necrosis factor (TNF) α.12 VCAM-1 is expressed by activated endothelial cells and several extravascular cell types, including lymphoid dendritic cells, murine bone marrow stromal cells, proximal tubule cells, and synovial lining cells.13-16 VCAM-1 is a ligand for the integrinα4β1 (VLA-4) and binds weakly to α4β7.17,18 E-selectin is a member of the selectin family and is induced on vascular endothelial cells by cytokines such as IL-1, TNF-α, interferon gamma (IFN-γ), and IL4.19,20 Unlike other endothelial adhesion molecules such as VCAM-1, E-selectin has been reported to be expressed only on endothelial cells. E-selectin acts as a ligand for the sialyl-Lewisx and sialyl-Lewisa.21,22 Recently, soluble forms of various adhesion molecules have been reported.23,24 Increased expression levels of soluble VCAM-1 (sVCAM-1) and soluble Eselectin (sE-selectin) have been described in various inflammatory and neoplastic diseases.25-30 In this study, we measured the serum levels of sVCAM-1 and of sE-selectin in patients with LSc and
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investigated whether these levels would serve as useful markers of the disease activity.
MATERIAL AND METHODS Patients The 3 study groups consisted of 59 patients with LSc, 20 patients with SSc, and 29 healthy controls. The patients with LSc were classified into 3 subgroups: (1) those with generalized morphea (GM, n = 21), (2) those with linear scleroderma (LS, n = 26), (3) those with morphea (n = 12). If patients had both morphea and linear lesions, they were classified as having GM. The number of sclerotic lesions larger than 3 cm in diameter was counted in each patient when the serum samples were collected. The number of involved areas of the body was also counted; we divided the body into 7 areas: (1) head and neck, (2) anterior part of the trunk, (3) posterior part of the trunk, (4) right upper extremity, (5) left upper extremity, (6) right lower extremity, and (7) left lower extremity. All patients with SSc met the American College of Rheumatology criteria for the diagnosis of SSc (scleroderma).31 Enzyme-linked immunosorbent assays for soluble adhesion molecules Aliquots of serum were frozen at –80°C until assayed. According to the manufacturer’s instructions, specific enzyme-linked immunosorbent assay (ELISA) kits were used for the determination of sVCAM-1 (sensitivity, 0.5 ng/mL; British Bio-technology Products, Oxford, UK) and sE-selectin levels (sensitivity, 0.5 ng/mL; Bender MedSystems, Vienna, Austria). Serum sVCAM-1 or sE-selectin levels more than 2 standard deviations (SDs) greater than the levels of the normal control subjects were regarded as elevated. ANA and RF ANAs were detected by an indirect immunofluorescence method with HEp-2 cells.4 RF was measured by the tube latex agglutination test, as described previously.9 Immunodiffusion Anti-U1 ribonucleoprotein antibody, anti-Sm antibody, and anti-topoisomerase I antibody were detected by double immunodiffusion as described previously.4 ELISAs for AHAs and anti-ssDNA antibodies The serum levels of AHAs were measured with an ELISA, as described previously.8 Absorbance values exceeding the mean plus 3 SDs for the normal control subjects were considered positive. For antissDNA antibodies, the wells were pretreated for 1
hour with 0.1% protamine sulfate (Sigma Chemical Co, St Louis, Mo). After the wells were rinsed with phosphate-buffered saline (PBS), calf thymus ssDNA (Sigma) was added at 1 µg/mL in PBS. An ELISA was then performed, as described previously.7 Statistical analysis The statistical analysis was carried out with Student t test for the comparison of means and Fisher’s exact probability test for the analysis of frequency. Correlations with clinical data were assessed by Spearman’s rank correlation coefficient. Two-tailed P values less than .05 were considered significant.
RESULTS Serum levels of sVCAM-1 The serum levels of sVCAM-1 were significantly higher in the LSc patients compared with the healthy controls (1077.4 ± 574.6 ng/mL vs 753.3 ± 380.9 ng/mL; P < .01). The sVCAM-1 levels in sera from the patients with GM (1365.1 ± 633.4 ng/mL) were significantly higher than those of the patients with morphea (748.9 ± 439.0 ng/mL; P < .01). However, the serum levels of sVCAM-1 in the patients with LS were not significantly higher than those of the patients with morphea. The SSc patients also had significantly higher serum sVCAM-1 levels compared with the healthy controls (1203.3 ± 473.9 ng/mL vs 753.3 ± 380.9 ng/mL; P < .001). The cut-off value (mean + 2 SD) was set at 1515.1 ng/mL, based on the data of the 29 healthy control sera. Elevated serum levels of sVCAM-1 were found in 6 of the 21 patients (29%) with GM, 4 of the 26 patients (15%) with LS, and 1 of the 12 patients (8%) with morphea (Fig 1). Seven of the 20 patients (35%) with SSc had serum sVCAM-1 levels greater than the cut-off value. Elevated serum levels of sVCAM-1 were found in 4 of the 10 patients (40%) with diffuse cutaneous SSc and 3 of the 10 patients (30%) with limited cutaneous SSc. Serum levels of soluble E-selectin Compared with the levels in the control subjects (66.6 ± 31.6 ng/mL), the serum levels of sE-selectin in the patients with LSc were significantly elevated (90.8 ± 48.1 ng/mL; P < .02). The sE-selectin levels in sera from the patients with GM (110.4 ± 57.1 ng/mL) were significantly higher than those of the patients with morphea (66.4 ± 30.6 ng/mL; P < .02). The serum levels of sE-selectin in the patients with LS, however, were not significantly higher than those in the patients with morphea. The patients with SSc had significantly higher serum sE-selectin levels than those of the healthy controls (101.4 ± 40.4 ng/mL vs 66.6 ± 31.6 ng/mL; P < .01). The cut-off value (mean + 2 SD) was set at 129.8 ng/mL, based on the data of
66 Yamane et al
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Fig 1. Serum levels of soluble VCAM-1 (sVCAM-1). Dashed line represents the cut-off value (mean + 2 SD), which was set as 1515.1 ng/mL, based on data of the 29 healthy control sera. Elevated serum levels of sVCAM-1 were found in 6 of the 21 patients (29%) with GM, 4 of the 26 patients (15%) with LS, and 1 of the 12 patients (8%) with morphea. Seven of the 20 patients (35%) with SSc had serum sVCAM-1 levels greater than the cut-off value.
Fig 2. Serum levels of soluble E-selectin (sE-selectin). Dashed line represents the cut-off value (mean + 2 SD), which was set as 129.8 ng/mL, based on data of the 29 healthy control sera. Elevated serum levels of sE-selectin were found in 7 of the 21 patients (33%) with GM, 4 of the 26 patients (15%) with LS, and 1 of the 12 patients (8%) with morphea. Eight of the 20 patients (40%) with SSc had serum sE-selectin levels greater than the cut-off value.
the 29 healthy controls. Elevated serum levels of sEselectin were found in 7 of the 21 patients (33%) with GM, 4 of the 26 patients (15%) with LS, and 1 of the 12 patients (8%) with morphea (Fig 2). Eight of the 20 patients (40%) with SSc had serum sE-selectin levels greater than the cut-off value. Elevated serum levels of sE-selectin were found in 4 of the 10 patients (40%) with diffuse cutaneous SSc and 4 of the 10 patients (40%) with limited cutaneous SSc.
nificant difference between these groups in sex, age, or duration of the disease. The number of sclerotic lesions was significantly greater in the patients with elevated levels of sVCAM-1 than in those with normal levels (4.3 ± 1.8 vs 2.6 ± 1.9, P < .02). The number of involved areas was also significantly greater in the patients with elevated levels of sVCAM-1 than in those with normal levels (2.7 ± 1.4 vs 1.8 ± 1.1, P < .02). In addition, the number of sclerotic lesions and the number of involved areas were significantly greater in the patients with elevated levels of sEselectin than in those with normal levels (4.8 ± 3.3 vs 2.9 ± 2.7, P < .05; 2.8 ± 1.6 vs 1.7 ± 1.0, P < .01, respectively). The presence of RF was found in a significantly greater proportion of patients with elevated levels of sVCAM-1 than in patients with normal
Correlation of serum soluble adhesion molecules with clinical and serologic features of patients with LSc The clinical and serologic features of patients with elevated or normal levels of soluble adhesion molecules are shown in Tables I and II. There was no sig-
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Table I. Correlation of serum sVCAM-1 levels with clinical and serologic features of patients with localized scleroderma Patients with elevated sVCAM-1 levels (n = 11)
Patients with normal sVCAM-1 levels (n = 48)
2:9 28.7 ± 21.4 4.2 ± 4.5 4.3 ± 1.8* 2.7 ± 1.4*
12:36 22.5 ± 15.7 4.5 ± 5.6 2.6 ± 1.9* 1.8 ± 1.1*
Sex (M:F) Age (y) Duration of disease (y) No. of sclerotic lesions No. of involved areas Proportion of patients positive for ANA Anti-ssDNA RF AHA IgG AHA IgM
91% 36% 64%* 50% 33%
55% 17% 24%* 34% 34%
*P < .02.
Table II. Correlation of serum sE-selectin levels with clinical and serologic features of patients with localized scleroderma Patients with elevated sE-selectin levels (n = 12)
Patients with normal sE-selectin levels (n = 47)
3:9 21.3 ± 21.4 2.9 ± 2.2 4.8 ± 3.3* 2.8 ± 1.6†
11:36 22.5 ± 15.7 4.9 ± 5.9 2.9 ± 2.7* 1.7 ± 1.0†
83% 33% 50% 50% 50%
62% 28% 27% 13% 31%
Sex (M:F) Age (y) Duration of disease (y) No. of sclerotic lesions No. of involved areas Proportion of patients positive for ANA Anti-ssDNA RF AHA IgG AHA IgM *P < .05. †P < .01.
levels (64% vs 24%; P < .02). Moreover, the serum sVCAM-1 levels were significantly correlated with the serum sE-selectin levels (r = 0.26; P < .02). The presence of ANA was found in a greater proportion in patients with elevated sVCAM-1 levels (91%) than in those with normal sVCAM-1 levels (55%), but there is no significant difference between these two groups (P = .54). In addition, there were no significant correlations between the serum sVCAM-1 levels and the presence of anti-ssDNA, AHA IgG, or AHA IgM. There were also no significant correlations between the sE-selectin levels and the presence of ANA, RF, anti-ssDNA, AHA IgG, or AHA IgM. There were no specific autoantibodies, such as anti-topoisomerase I antibody or anti-centromere antibody, in
sera from patients with LSc by indirect immunofluorescence or immunodiffusion.
DISCUSSION In this study, we found that the serum levels of sVCAM-1 and sE-selectin were significantly higher in patients with LSc than in healthy controls. This suggests VCAM-1 and E-selectin upregulation in patients with LSc because the amounts of soluble adhesion molecules are correlated with the level of their surface expression. sVCAM-1 and sE-selectin are shed mainly by activated endothelial cells.23,24 E-selectin has been reported to be expressed only by activated endothelial cells. The results of the present study indicate that high levels of sVCAM-1 and sE-selectin
68 Yamane et al
may be correlated with endothelial activation in LSc, and they suggest the clinical importance of these soluble adhesion molecules in patients with LSc. sVCAM-1 and sE-selectin contain most of the structure and exhibit most of the function of the extracellular portion of cell-bound adhesion molecules. For example, it has been reported that the membrane-anchored form of sVCAM-1 is converted to the soluble form by a regulated proteolytic cleavage process involving metalloproteinase.24 It appears that the sources of sVCAM-1 and sE-selectin in vivo may be mainly cell-bound adhesion molecules expressed on activated endothelial cells. VCAM-1 and E-selectin are expressed on activated endothelial cells after stimulation with IL-1, TNF-α, and IFN-γ.32,33 These findings suggest that the elevated serum levels of sVCAM-1 and sE-selectin may be a consequence of long-term exposure to these cytokines in patients with LSc. Elevated serum levels of sVCAM-1 and sE-selectin have been described in various autoimmune disorders, such as rheumatoid arthritis, systemic lupus erythematosus, and SSc.25-30 In our study, as in others, the serum levels of sVCAM-1 and sE-selectin in patients with SSc were higher than those of healthy controls. Previous studies demonstrated that serum levels of soluble adhesion molecules are useful markers of the disease activity. For example, the serum levels of sVCAM-1 and sE-selectin correlated with clinical disease activity in SSc.27-30 In rheumatoid arthritis, high levels of sVCAM-1 were detected in rheumatoid synovial fluid and plasma samples and were correlated with disease severity.25,26 In the present study, patients with GM, the most severe form of LSc, had the highest levels of sVCAM-1 and sEselectin. In addition, the serum levels of these soluble adhesion molecules were significantly correlated with both the number of sclerotic lesions and the number of involved areas. These results suggest that the serum levels of sVCAM-1 and sE-selectin may reflect the extent of the skin involvement, that is, the severity of LSc. In conclusion, we have demonstrated the elevation of sVCAM-1 and sE-selectin in patients with LSc. Serum levels of sVCAM-1 and sE-selectin may reflect the severity of the disease and may be useful for monitoring in vivo states of endothelial activation. Further studies are needed to document the potential role of sVCAM-1 and sE-selectin in LSc. REFERENCES 1. Rodnan GP. When is scleroderma not scleroderma? Bull Rheum Dis 1981;31:7-10. 2. Scarola JA, Shulman LE. Serologic abnormalities and their significance in localized scleroderma [abstract]. Arthritis Rheum 1975;18:526.
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