496 Gut 2001;48:496–502 Parallel expression of macrophage metalloelastase (MMP-12) in duodenal and skin lesions of Gut: first published as 10.1136/gut.48.4.496 on 1 April 2001. Downloaded from patients with dermatitis herpetiformis M T Salmela, SLFPender, T Reunala, T MacDonald, U Saarialho-Kere Abstract intraepithelial lymphocytes.2 The rash and Background—Dermatitis herpetiformis enteropathy improve on a gluten free diet, (DH) is a specific dermatological manifes- implying that DH is a specific skin manifesta- tation of coeliac disease and 80% of DH tion of mainly subclinical coeliac disease patients have gluten sensitive enteropathy (CD).23 manifested by crypt hyperplasia and vil- Matrix metalloproteinases (MMPs) are a lous atrophy. Matrix degradation mediated family of extracellular matrix (ECM) degrad- by collagenase 1 (MMP-1) and stromelysin ing enzymes that are collectively capable of 1 (MMP-3) has previously been implicated degrading essentially all ECM components in the pathobiology of coeliac intestine and and that can further be subdivided into cutaneous DH blisters. collagenases (MMP-1, -8, and -13), gelatinases Aims—To study expression of stromelysin (MMP-2 and -9), stromelysins (MMP-3, -7, 2, metalloelastase, collagenase 3, and mat- -10, and –12), membrane-type MMPs, and rilysin in the intestine and skin of DH other MMPs.45 The extracellular activity of patients. MMPs is regulated by TIMPs 1–4.6 We have Methods—In situ hybridisation using 35S previously reported that expression of MMP-1 labelled cRNA probes was performed on and -3 is enhanced in basal keratinocytes 7 duodenal biopsies of 15 DH patients, three surrounding neutrophil abscesses in DH skin. samples each of control duodenal or Furthermore, when producing experimental jejunal mucosa, fetal ileal explants, le- DH blisters with 50% potassium iodide, uroki- sional DH skin, and 19 serial biopsies of nase plasminogen activator is upregulated at 12 experimental DH blisters. Immunostain- hours, before blisters are seen, while both ing was used to examine type IV collagen, MMP-1 and MMP-3 are abundantly ex- 8 macrophages (CD68), and 92 kDa gelati- pressed in 24 hour biopsies of blisters. In DH, http://gut.bmj.com/ nase (MMP-9) in the specimens. collagenase 1 (MMP-1) and stromelysin 1 Results—Metalloelastase (MMP-12) was (MMP-3) may contribute to formation of blis- abundantly expressed by subepithelial ters by degrading BM components such as type macrophages in both coeliac intestine and VII as well as type IV collagen or laminin 1, spontaneous and induced DH rash. It was respectively. Interestingly, Daum and col- leagues9 have recently demonstrated that these Department of also upregulated in the experimental same MMPs are expressed by subepithelial Dermatology, Helsinki model of coeliac disease (staphylococcal on September 26, 2021 by guest. Protected copyright. University Central endotoxin B stimulated fetal explants). macrophages and fibroblasts in intestinal Hospital, Helsinki, The only other MMP detected was MMP-9 biopsy specimens from patients with CD. Finland which did not colocalise with MMP-12. Tissue transglutaminase (tTG) was recently M T Salmela identified as the endomysial antigen in patients U Saarialho-Kere Conclusions—Upregulation of metallo- 10 elastase is associated with T cell mediated with CD. Patients with DH have elevated immunoglobulin A autoantibodies to tTG Department of immune responses both in the intestine 11 Dermatology, Tampere and skin. In addition to modulating mac- confirming its pathogenic relation with CD. University Hospital, rophage migration, it may contribute to tTG has been shown to be involved in the Tampere, Finland degradation of proteoglycans or basement intermolecular cross linking of type VII colla- T Reunala gen of anchoring fibrils of skin. Interestingly, membrane components in the subepithe- type VII collagen is degraded, at least in Department of lial mucosa. Paediatric (Gut 2001;48:496–502) experimental DH blisters, and expression of Gastroenterology, St both MMP-1 and MMP-3 colocalise in these 8 Bartholomew’s and the Keywords: coeliac disease; metalloproteinase; areas. Royal London School dermatitis herpetiformis The histological changes of CD are charac- of Medicine and terised by deepening of the crypts and flatten- Dentistry, London, UK ing of the villi, and altered turnover of ECM SLFPender Dermatitis herpetiformis (DH) is a chronic 9 T MacDonald has been implicated in these changes. Rapid cutaneous disorder characterised by a blister- collapse of the villus architecture can occur Correspondence to: ing rash on the elbows, knees, and buttocks, Dr U K Saarialho-Kere, and granular IgA deposits in the basement Abbreviations used in this paper: DH, dermatitis Department of Dermatology, membrane (BM) zone.1 Although gastro- herpetiformis; MMPs, matrix metalloproteinases; BM, Helsinki University Central basement membrane; CD, coeliac disease; ECM, Hospital, Meilahdentie 2, intestinal symptoms in DH patients are rare, 00250 Helsinki, Finland. they all have gluten sensitive enteropathy extracellular matrix; SEB, staphylococcal endotoxin B; ulpu.saarialho-kere@helsinki.fi tTG, tissue transglutaminase; IFN-ã, interferon ã; IL, manifested in 80% by crypt hyperplasia or par- interleukin; TNF-á, tumour necrosis factor á; TGF-â, Accepted for publication tial villous atrophy, while the rest have normal transforming growth factor â; GM-CSF, granulocyte 25 September 2000 villous architecture with increased counts of macrophage-colony stimulating factor. www.gutjnl.com MMP-12 in duodenal and skin lesions of patients with dermatitis herpetiformis 497 within hours of administering gliadin to CD Department of Dermatology, Helsinki Univer- patients in vivo.9 Daum and colleagues9 also sity Central Hospital. Informed consent was recently demonstrated in vivo that MMP-1 obtained from individual subjects for all proce- may contribute to degradation of interstitial dures. Gut: first published as 10.1136/gut.48.4.496 on 1 April 2001. Downloaded from collagens and MMP-3 to epithelial cell shed- ding in CD. In an experimental model of IN SITU HYBRIDISATION human fetal small intestine, direct addition of The production and specificity of the antisense activated MMP-3 in explant cultures caused human stromelysin 2, macrophage metallo- collapse of the villi within 24 hours.12 Further- elastase, collagenase 3, and matrilysin probes more, crypt hyperplasia and villous atrophy have been described previously.14 20–22 As a con- rapidly occurred following activation of lamina trol for non-specific hybridisation, sections propria T cells with the bacterial superantigen were hybridised with 35S labelled sense RNA Staphylococcus aureus endotoxin B (SEB).13 We from a bovine tropoelastin cDNA. The validity have shown that various other MMPs such as of this probe as a negative control has been stromelysin 2 (MMP-10), matrilysin (MMP- confirmed by northern23 and in situ hybridisa- 7), collagenase 3 (MMP-13), and macrophage tion24 assays. The cDNAs were transcribed in metalloelastase (MMP-12) are upregulated in vitro using a commercial kit (Promega Corp., another intestinal disorder, inflammatory Madison, Wisconsin, USA) and labelled with bowel disease, characterised by influx of T 35S UTP, as previously described.25 cells, eosinophils, and neutrophils, as well as Following deparaYnisation and rehydration, degradation of the mucosa.14 MMP-10 has a 5 µm sections were pretreated with 1 mg/ml of nearly identical substrate specificity to MMP- proteinase K and washed in 0.1 M trieth- 3.15 MMP-12 is the most elastolytic of the anolamine containing 0.25% acetic anhydride. MMPs but also degrades various other sub- Subsequently, sections were hybridised with strates such as type IV collagen, laminin 1, probes (2.5–5×104 cpm/µl of hybridisation fibronectin, vitronectin, and proteoglycans.16 17 buVer) and washed under stringent conditions, Matrilysin cleaves in vitro collagen type IV, including treatment with RNAse A, as de- proteoglycans, gelatin, aggrecan, fibronectin, scribed previously.25 26 Autoradiography was laminin, tenascin, and elastin.18 Collagenase 3 carried out for 20–40 days. All samples were degrades fibrillar collagens, gelatin, type IV processed in at least two experiments and were collagen, tenascin, and fibronectin.19 The aim independently analysed by two investigators. of this study was to further characterise the Samples previously positive for stromelysin 2 patterns of MMP expression in DH intestine. (chronic wounds), macrophage metalloelastase We compared expression of various MMPs in (sarcoid granulomas), collagenase 3 the intestine and skin of patients with DH, and (squamous cell carcinomas), and matrilysin also used the fetal explant model of enteropa- (sweat gland tumours) were used as positive http://gut.bmj.com/ thy to investigate the link between T cell controls. activation and MMP production. IMMUNOSTAINING Immunostaining was performed using the Methods avidin-biotin-peroxidase complex technique.25 TISSUES Diaminobenzidine or aminoethylcarbazole Fifteen specimens of formalin fixed paraYn (CD20) were used as chromogenic substrates. embedded duodenal biopsies taken during Monoclonal antibodies included MMP-9 (DB- on September 26, 2021 by guest. Protected copyright. upper gastrointestinal endoscopy were ob- 2211; Diabor, Finland)27 and CD68 (KP-1, No tained from the archives of the Department of M814; Dako, Carpinteria, California, USA) to Dermatopathology, University of Helsinki, identify macrophages and type IV collagen Finland. All patients