Purification of Type I Soluble Collagen

Objective-To study collagenase production in labial salivary glands in patients with Sjogren's syndrome (SS). Methods-Collagenases were localised in labial salivary glands by immunohistochemistry. Collagenase activity against triple helical type I collagen monomers in stimulated saliva was measured using sodium dodecyl sulphate polyacrylamide gel electrophoresis and laser densitometry; tissue inhibitor metalloproteinase (TIMP) was measured by enzyme linked immunosorbent assay. Results-Cells containing collagenase of matrix metalloproteinase (MMP)-l type were more frequent and more intensely staining in SS than in healthy glands. Only SS saliva contained functional enzyme (11.7 (6.8) X 106 MU/l). Cells containing MMP-8 type neutrophil collagenase were not found in situ, which was in accordance with sialochemical findings/doxycycline inhibition studies. TIMP was found in both SS and normal saliva. Conclusions-Fibroblast, but not neutrophil type, collagenase is synthesised, secreted, and subsequently activated, but is not inhibited by TIMP in labial salivary glands or saliva in SS. Collagenase may destroy glandular and salivary duct tissue and perturb factors influencing the morphogenetic extracellular matrix. (Ann Rheum Dis 1994; 53: 836-839) Sjogren's syndrome (SS), an autoimmune disease of unknown aetiology, is characterised by keratoconjunctivitis sicca and xerostomia as a result of decreased lachrymal and salivary secretion caused by destruction of the glands by an as yet unknown mechanism.' The main extracellular structural proteins in the lachrymal and salivary glands are type I and III collagen, which are highly susceptible to degradation by specific interstitial collagenases from fibroblasts, matrix metalloproteinase1 (MMP-1), and granulocytes (MMP-8).2 3 Interstitial collagens provide mechanical support and maintain structural integrity, both of which are lost by treatment with high concentrations of collagenases.4 In addition, interstitial collagens at the epithelium-mesenchyme interface provide a substrate for epithelial cell-matrix interactions, which govern embryonic morphogenesis and the continuous renewal of tubulo-alveolar salivary glands.5 6 Loss of structural support, collagenase mediated alterations in the morphogenetic properties of interstitial collagens, or both, might therefore explain the sialectasis, acinar atrophy, and loss of secretory parenchyma which lead to the sicca symptoms characteristic of SS. This study investigated the presence, cellular source and types of collagenases in affected glands and assessed their secretion and state of activation in saliva in patients with SS. Patients and methods PURIFICATION OF TYPE I SOLUBLE COLLAGEN Purification of soluble type I collagen was by the method of Miller and Rhodes,7 from rat tail tendon. Type I collagen contains approximately 13-6% of hydroxyproline and this was used to calculate the collagen content in the sample studied according to the equation: c0= mpflP(1/0 136) AAstd PATIENTS AND SAMPLES We studied eight patients with SS and six healthy controls. Diagnosis of SS was according to the Copenhagen criteria.' Five of the patients had primary SS and three patients had the secondary form of the disease: one patient had underlying rheumatoid arthritis (RA) and two had Reiter's syndrome. All subjects gave their informed consent. Six to eight labial salivary glands (LSG) were obtained from each subject by biopsy (taken under infiltration anaesthesia by blunt dissection), embedded in Tissue-Tek OCT compound, and snap frozen in liquid nitrogen. Stimulated saliva was collected over five minutes from each patient by the same clinician at the same time of the day and under identical conditions. Immediately after the collection of the saliva the samples were centrifuged at 1000 g for five minutes and the supernatants were frozen at -70°C until analysed. IMMUNOHISTOCHEMISTRY Antisera to fibroblast type MMP-1 collagenase and neutrophil type MMP-8 collagenase were produced and characterised as described previously.8 Sections were stained using the avidin-biotin peroxidase complex (ABC).'" To control for method specificity, we compared results from omission of primary antiserum, use of normal rabbit serum (diluted 1:1001:400), use of irrelevant antisera (rabbit antiDepartment of Anatomy, Helsinki University, Helsinki, Finland Y T Konttinen P Kangaspunta M Takagi M Segerberg Department ofMedical Chemistry 0 Lindy Department of Peridontology T Sorsa Department of Biochemistry, University of Bielefeld, Bielefeld, Germany H Tschesche Department of Dermatology, Washington University School ofMedicine, St Louis, MO, USA A Z Eisen Correspondence to: Dr Y T Konttinen, Institute of Biomedicine, Department ofAnatomy, P.O. Box 9, FIN-00014 University of Helsinki, Finland. Accepted for publication 12 August 1994 836 group.bmj.com on May 15, 2017 Published by http://ard.bmj.com/ Downloaded from