Research Paper Mediators of Inflammation, 8, 147–151 (1999) SECRETORY leucocyte protease in hibitor (SLPI) is a Production of secretory leucocyte potent in hibitor of granulocyte elastase and cath- epsin G, and also an inhibitor of pancreatic enzym es protease inhibitor (SLPI) in human like trypsin , chym otrypsin and pancreatic elastase. SLPI has also been show n to in hibit HIV-1 in fections pancreatic b -cells by blocking viral DNA synthesis. Since SLPI is an in hibitor of pancreatic proteases we wis hed to in ves- tigate w heth er SLPI was also actually produced in the Max Nystrom,È 1,2 Magnus Bergenfeldt,1,2 pancreas. M-RNA from human pancreatic tissue Irena Ljungcrantz,2 sa Lindeheim2 and show ed evidence of SLPI production usin g the reverse 2,C A transcriptase polym er chain reaction technique (RT- Kjell Ohlsson PCR). Using im m unohistochem ical m ethods SLPI was dem onstrated in the b -ce1ls of the islets of Langer- hans. Th e function could be local protease/anti- 1Department of Surgery, Lund University, Lund, and protease regulation or antiviral/antibacterial defence 2Department of Surgical Pathophysiology, MalmoÈ in the close vicin ity of the cell surface, or even in side University Hospital, SE-205 02 Malmo,È Sweden the b -cell itself. Key words: SLPI, Islets of Langerhans, Pancreas, b -Cell, CA Antiviral, Antibacterial Corresponding Author Tel: (+46) 40 33 31 50/51 Fax: (+46) 40 33 62 40 Introduction blocking DNA synthesis.22 SLPI is probably involved in inflammatory regulation in the intestines as w ell as in SLPI is an acid-resistant 11.8-kDa non-glycosylated the respiratory and urogenital tracts. In patients w ith serine antiprotease. Originally isolated from human ulcerative colitis, active pancreatic proteases have parotid gland secretions,1 it is found in cells on a been found in faeces. Since SLPI is an inhibitor of both variety of mucosal surfaces. For example, the seminal pancreatic and leukocytic proteases, it could be of vesicles, prostate, epididymis and cervix ,2 –7 in serous importance in the pathogenesis of ulcerative colitis. cells of the parotid and submandibular salivary Having recently demonstrated SLPI production in the glands,1,8,9 in the upper respiratory tract, in the intestinal mucosa,14 and know ing that SLPI produced lacrimal glands,10 as w ell as in the Clara cells and in the upper respiratory tract is rapidly degraded in goblet cells of the low er respiratory tract.11–13 SLPI gastric and duodenal juice,23 w e also w anted to has also been found in von Ebner’s glands at the base investigate other possibilities of origin. Earlier studies of the tongue and submucosal cells of the oesopha- failed to show production or presence of SLPI in the gus.10 Recently, SLPI w as found in Paneth cells in the pancreas.10 The aim of this study w as, therefore, to small intestine, and in scattered mucosa cells of reinvestigate the possible presence and production goblet-type in the epithelium of both the large and of SLPI in pancreatic tissue using more sensitive small bow el. In addition, SLPI has frequently been methods. found in colonic adenomas.14 SLPI is thought to be an inflammatory regulator w ith Methods and materials strong inhibitory capacity aimed tow ards proteases present in various body fluids. Thus, SLPI is a potent Xylene, ethanol, hydrochloric acid, hydrogen perox- inhibitor of leucocyte elastase and cathepsin G, and a idase, formaldehyde, haematoxylin and Tris-buffered moderate inhibitor of trypsin and chymotrypsin.1,1 5,1 6 saline (0.05 mol/l Tris in NaCl 0.15 mol/l, pH 7.6) Furthermore, it is a moderate inhibitor of pancreatic w ere standard chemicals for laboratory use. Recombi- elastase,17 and it is also know n to inhibit both mast cell nant human SLPI was a kind gift from Robert chymase activity and histamine release from mast Thompson at Synergen Inc. (Boulder, CO, USA). Goat cells.18 ,19 Its proteinase-inhibitory activity is know n to anti-SLPI antiserum was produced in the laboratory by be located in the second COOH-terminal domain. immunising Swedish landrace goats w ith recombinant Although no inhibitory capacity has been detected in SLPI. Normal goat and rabbit serum w as obtained the NH2-terminal domain, antimicrobial capacity has from Dakopatt AB (Copenhagen, Denmark). Bio- been reported.20,2 1 It has also been reported that SLPI tinylated rabbit anti-goat IgG antibodies and avidin- blocks the human immunodeficiency virus type 1 by biotinylated horseradish peroxidase complex (ABC ISSN 0962-9351 print/ISSN 1466-1861 online/99/030147-05 1999 Taylor & Francis Ltd 147 M. Ny stro¨ m et al. complex) were purchased from Vector Laboratories After hybridisation the membrane w as w ashed in 2 (Burlingame, CA, USA). Diaminobenzidine w as a 3 SSC containing 0.1% SDS, 1 3 15 min, 0.1 3 SSC product of Saveen Biotech AB (Malmo¨ , Sw eden). containing 0.1% SDS, 2 3 15 min, both at room Crystalline3 porcine pepsin w as obtained from Sigma temperature. Detection of the target DNA hybrid w as Chemicals (St. Louis, MO, USA). achieved as follow s. After a brief rinse in 100 mM Tris – HCl, 150 mM NaCl, pH 7.5, the membrane w as incubated in 0.5% (w /v) blocking reagent in the same RT-PCR and Southern blot buffer for 30 min. This w as follow ed by a brief w ash in Human pancreas Poly(A+ ) RNA w as obtained from buffer. Diluted anti-digoxigenin antibody–AP-conju- Clontec (Palo Alto, CA, USA). Specific primers (R&D gate (1:500) w as applied to the membrane for 30 min. Systems Europe Ltd, Abingdon, UK) w ere designed Rinses in buffer, 2 3 15 min follow ed. The staining according to the nucleic acid sequence of SLPI w as carried out by incubating the membrane in a cDNA.24 The upstream and dow nstream primers for solution containing 45 m l Nitroblue Tetrasodium salt amplification of the human SLPI cDNA fragments (NBT), 35 m l 5-bromo-4-chloro-33 indolyl phosphate w ere: 59 -TGT CCT GAC ACT TGT GGC AT-39 and (X-phosphate) in 10 ml buffer (100 mM Tris –HCl, 59 -CGA TCA ACT GGC ACT TCT TG-39 . The oligonu- 100 mM NaCl, 50 mM MgCl2 , pH 9.5). The experiment cleotide used for probing the Southern blot of the RT- w as repeated three times. PCR product had the sequence 59 -GGT TTG GGG TGT CAA CAG GAT CCA GGC AT-39 w ith the modification Immunohistochemical staining 39 +59 digoxigenin.24 The first strand of cDNA w as synthesised using Pancreas specimens from five different patients col- reverse transcriptase, isolated from avian myelo- lected during surgery for pancreatic adenocarcinoma blastosis virus (Appligene, Illkirch, France) in a 50-m l w ere investigated. The specimens were from micro- reaction mixture containing 2.5 m l 400 mM Tris –HCl, and macroscopically normal pancreatic tissue. The pH 8.3, 2.5 m l 400 mM KCl, 1 m l 300 mM MgCl2 , 5 m l patients were two men and three w omen betw een 41 100 mM dithiothretiol (DTT), 5 m l 5 mM 4dNTPmix, and 69 years of age. They had no earlier history of 2 m l 2 mg/ml actinomycin D, 1 m l 4 m M dow nstream pancreatic disease. Informed consent for the proce- primer and approx. 1–2 m g poly(A+ ) RNA (approx. dure w as obtained. The tissue samples w ere cut at 10 m l). The reaction mix ture w as incubated at 42°C for random and fixed in 4% formaldehyde. Paraffin- 60 mm, then diluted w ith 450 m l 10 mM Tris –HCl/ embedded tissue w as routinely stained w ith haema- 10 mM EDTA, pH 7.5. To 5 m l of this diluted mixture toxylin for light microscopic examination. The fixed w ere added 5 m l of each amplification primer, 20 mM specimens were subjected to pepsin digestion (pep- each, 4 m l 5 mM 4dNTPmix, 10 m l 103 amplification sin 4 mg/ml in 0.01 mol/l HCl) for 30 min at 37°C and buffer (500 mM KCl, 100 mM Tris –HCl, pH 8.4, and w ere transferred into Tris-buffered saline. To quench 1 mg/ml gelatine), 1.5 m l 100 mM MgCl2 and 69 m l H2O. endogenous tissue peroxidase activity, in the next The PCR reaction w as performed using the ‘hot start’ step, the sections were incubated w ith 0.3% H2O2 in method w ith paraffin pellets, heating at 94°C for 2 min methanol for 30 min at room temperature. To block follow ed by chilling on ice. Then 2.5 units (0.5 m l) of non-specific staining, the specimens w ere incubated Taq DNA polymerase (Appligene) w ere added to the for 10 min in 5% normal rabbit serum. In order to tubes. Amplification w as carried out in a thermo-cycler show specific staining in the b -cells, the specimens (Hybaid Omnigene, Teddington, UK) w ith the follow - w ere treated as follow s. ing cycling programme: 39 cycles of 2 min at 55°C; 2 min at 72°C and 1 min at 94°C; follow ed by one cycle (1) SLPI of 2 min at 55°C and 7 min at 72°C. The PCR product The slides were incubated w ith goat anti-human SLPI w as then analysed by agarose gel electrophoresis and (1:1000) for 30 min followed by the addition of visualised after ethidium bromide staining (0.8% biotinylated rabbit anti-goat IG antibodies (5 m g/ml agarose gel in 0.53 TBE buffer).