Function Blocking Autoantibodies Against Matrix Metalloproteinase-1 in Patients with Systemic Sclerosis Shinichi Sato, Ikuko Hayakawa, Minoru Hasegawa,

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Function Blocking Autoantibodies Against Matrix Metalloproteinase-1 in Patients with Systemic Sclerosis Shinichi Sato, Ikuko Hayakawa, Minoru Hasegawa, Kazuhiko Takehara Journal of Investigative Dermatology Volume 120, Issue 4, Pages 542-547 (April 2003) DOI: 10.1046/j.1523-1747.2003.12097.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Anti-MMP-1 antibody levels were elevated in serum samples from patients with SSc, especially dSSc. IgG or IgM anti-MMP-1 antibody levels were determined by ELISA using human recombinant MMP-1 in patients with lSSc, those with dSSc, those with SLE, those with dermatomyositis, and normal controls (CTL). The short bar indicates the mean value in each group. A broken line indicates the cut-off value (mean+2 SD of the control samples). Values in parenthesis represent the dilutions of pooled sera giving half-maximal OD values in ELISA, which were determined by linear regression analysis to generate arbitrary units per milliliter that could be directly compared between each group of patients and normal controls. Journal of Investigative Dermatology 2003 120, 542-547DOI: (10.1046/j.1523-1747.2003.12097.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 IgG anti-MMP-1 antibody levels correlated with the extent of fibrosis in the skin, renal blood vessels, and lung in patients with SSc. We correlated IgG anti-MMP-1 antibody levels (relative OD) with modified Rodnan TSS (A), pulsatility index (B), %DLco (C), or %VC (D). Anti-MMP-1 antibody levels were determined by ELISA using human recombinant MMP-1. The extent of skin sclerosis was measured by modified Rodnan TSS. The 17 anatomic areas were rated as 0 (normal), 1+(mild skin thickening), 2+(moderate), and 3+(severe), and the modified Rodnan TSS was derived by summation of the scores from all 17 areas. The pulsatility index is a parameter for renal vascular resistance determined by color-flow Doppler ultrasonography of the renal interlobar arteries of both kidneys. The extent of lung fibrosis was evaluated by a pulmonary function test, including %DLco and %VC. Journal of Investigative Dermatology 2003 120, 542-547DOI: (10.1046/j.1523-1747.2003.12097.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 The presence of IgG anti-MMP-1 antibody in sera from patients with SSc was confirmed by immunoblotting. Representative immunoblotting of human recombinant MMP-1 with sera from patients or a normal control is shown. Lane 1, colloidal gold-stained MMP-1; lanes 2–4, serum samples from patients with SSc positive for IgG anti-MMP-1 antibody by ELISA; lane 5, a serum sample from the SSc patient positive for anti-topoisomerase I antibody, but not for IgG anti-MMP-1 antibody by ELISA; and lane 6, a normal human serum. Markers for molecular weights (kDa) are shown to the left. The results represent those obtained with 10 SSc positive for IgG anti-MMP-1 antibody by ELISA, nine SSc patients positive for either anti-topoisomerase I antibody, anti-centromere antibody, or anti-U1RNP antibody, but not for IgG anti-MMP-1 antibody by ELISA, and six healthy individuals. Journal of Investigative Dermatology 2003 120, 542-547DOI: (10.1046/j.1523-1747.2003.12097.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 IgG anti-MMP-1 antibody from patients with SSc inhibited MMP-1 collagenase activity. IgG was purified from serum samples of SSc patients positive for IgG anti-MMP-1 antibody by ELISA [anti-MMP-1(+)], those positive for either anti-topoisomerase I antibody, anti-centromere antibody, or anti-U1RNP antibody, but not for IgG anti-MMP-1 antibody by ELISA [anti-MMP-1 (–)], and normal control. Purified IgG was incubated with MMP-1 activated by p-aminophenylmercuric acetate and MMP-1 collagenase activity was determined using biotinylated bovine native collagen as substrate. The amount of cleaved biotinylated fragments of collagen by MMP-1 was measured by ELISA using a biotin-binding microtiter plate. MMP-1 enzymatic activity is shown as a percentage of p-aminophenylmercuric acetate-activated MMP-1 that was defined as 100% (activated MMP-1). Each histogram shows the mean (±SD) results obtained for 10 persons of each group. Journal of Investigative Dermatology 2003 120, 542-547DOI: (10.1046/j.1523-1747.2003.12097.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions