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 and cath- epsin G, and also an inhibitor of pancreatic enzym es protease inhibitor (SLPI) in human like , 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 . 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 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 G, and a idase, formaldehyde, haematoxylin and Tris-buffered moderate inhibitor of trypsin and .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 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). buffer) for 30 min. This w as follow ed by incubation The PCR-product w as transferred to a Hybond N w ith avidin biotinylated horseradish perox idase com- transfer membrane (Amersham, Buckinghamshire, plex for 30 min. After the sections were rinsed, the UK) according to the method of Maniatis et al.26 After substrate reaction w as performed by incubating the heating the membrane for 30 min at 80°C, it w as sections in 3-amino-9-ethylcarbazole (AEC; Dako® prehybridised for 4 h at 42°C in a solution containing Corporation, Carpinteria, CA, USA) for 25 min. After 53 standard saline citrate (SSC), 50% formamide, final w ashing the specimens w ere rinsed in tap w ater 0.02% (w /v) sodium dodecylsulfate (SDS), 0.1% (w /v) then mounted. All incubations w ere performed at N-lauroylsarcosine and 2% (w /v) blocking reagent room temperature, and specimens w ere w ashed three (Boehringer-Mannheim, Mannheim, Germany). times (5 min) between each step w ith Tris-buffered Hybridisation w as carried out in the same solution, saline. Negative controls w ere alw ays incubated in including 20 ng probe/ml solution overnight at 42°C. parallel w ith adsorbed anti-SLPI antiserum w hich had

148 Mediators of Inflammation ´ Vol 8 ´ 1999 SLPI pro ductio n in hum a n b -cells been produced by affinity chromatography on an ase inhibition, thereby modulating the inflammatory SLPI-conjugated Sepharose 4B column (Pharmacia response. Protection of the ciliated epithelium of the Fine Chemicals, Uppsala, Sw eden). The experiments respiratory tract against the activity of leucocyte w ere repeated three times. proteases released in purulent infection is assumed to be one important function of SLPI.15 New properties (2) Insu lin of this protein have recently been discovered, and The procedure w as identical w ith that described there are now reports indicating antibacterial and above, except that a sheep anti-human insulin anti- antiviral effects.20 –22 In the light of these findings, new body was used in 1:2000 solution (‘The ’, mechanisms for SLPI action may be anticipated. This Birmingham, UK). Incubation w as performed in study show s evidence of SLPI production in the b -cells alkaline phosphatase complex instead of avidin bio- of the islets of Langerhans. Utilising the RT-PCR tinylated horseradish peroxidase. For staining, 22.8 m l technique w e first observed SLPI-mRNA in pancreatic NBT and 17 m l BCIP w ere added to 5 ml buffer. tissue. Using immunohistochemical methods w e w ere then able to confirm that SLPI is produced in the b -cells (3) Dou ble sta ining of the islets of Langerhans. The function of SLPI in the The specimens w ere first stained for insulin, then the islets of Langerlians is far from clear. The question same specimen w as also stained for SLPI, as described arises of w hether SLPI w ith pancreatic origin is meant above. to act extracellularly in the traditional protease inhibitor manner, or w hether it has other functions. The anti HIV-l activity of SLPI has been show n to take Results place early in the viral infection of cells, probably after 22 RT-PCR and Southern blot analysis virus binding and before reverse transcription. Our finding of SLPI in the pancreatic islets of Langerhans Water w as used as a negative control, and trachea indicates an auto-/or paracrine function rather than an tissue as a positive control. A characteristic band w as observed in the trachea tissue lane representing SLPI cDNA fragments. In the lane representing pancreatic tissue there w as specific staining of the correspond- ing amplified cDNA fragments in the same position, but somew hat w eaker than for the positive tracheal control. The bands were distinct and the lanes contained no detectable signs of any other bands. There w ere no detectable bands in the lane contain- ing the negative control (Fig. 1). The pattern was identical in all experiments.

Immunohistochemical staining Immunoreactive SLPI w as found using the human anti-SLPI antibody, resulting in bright red staining clearly concentrated to the islets of Langerhans (Fig. 2A). Using human anti-insulin antibodies, immunor- eactive insulin w as found in the b -ce1ls of the islets of Langerhans, as indicated by distinct blue staining (Fig. 2B). When the specimens were stained first w ith the human anti-insulin antibody, and then w ith the human anti-SLPI antibody, the original staining colours of blue and bright red w ere not observed, but the cells w ere stained purple resulting from insulin and SLPI in the same cell population (Fig. 2C). There w as no sign of SLPI staining w hen using adsorbed anti-SLPI antiserum. The results obtained were verified in FIG. 1. SLPI cDNA fragments were demonstrated in pan- triplicate experiments. creatic tissue using RT-PCR and Southern blot. C, negative control containing water; P, pancreatic tissue; T, tracheal tissue used as a positive control. In the lane representing pancreatic tissue strong specific staining of the correspond- Discussion ing amplified cDNA fragments was observed. The band is at the same position but somewhat weaker than that for the SLPI is found in many different cell types in man. Its positive tracheal control. No bands were detected in the lane generally accepted function has hitherto been prote- containing the negative control.

Mediators of Inflammation ´ Vol 8 ´ 1999 149 M. Ny stro¨ m et al.

A

B

C FIG. 2. Using immunohistochemical methods and staining with different antibody solutions novel colour schemes can be accomplished. (A) The presence of immunoreactive SLPI was demonstrated using a specific human anti-SLPI antibody resulting in a bright red colour in the islets of Langerhans. (B) Human anti-insulin antibodies showed blue staining in the b -cells of pancreas tissue. (C) Staining the specimens first for insulin then for SLPI resulted in a purple colour indicating the presence of insulin and SLPI in the b -ceIls of the islets of Langerhans.

150 Mediators of Inflammation ´ Vol 8 ´ 1999 SLPI pro ductio n in hum a n b -cells

11. Kramps JA, Franken C, Meijer CJ, Dijkman JH. Localization of low exocrine function. In accordance w ith findings of anti molecular w eight protease inhibitor in serous secretory cells of the HIV-1 activity and antibacterial properties, a possible re spiratory tract. J Histio chem Cy to chem 1981; 29 (6): 712–719. 12. De Water R, Willems LN, Van Muijen GN, Franken C, Fransen JA, function might be the protection of the cells against Dijkman JH, e t al. Ultrastructural localization of bronchial antileukopro- viral or bacterial infections. Another possible autocrine tease in central and peripheral human airw ays by a gold-labeling technique using monoclonal antibodies. Am Rev Respir Dis 1986; 133 function could be protease/antiprotease regulation. (5): 882–890. Further studies are, how ever, required to identify the 13. Fryksmark U, Ohlsson K, Polling A Tegner H. Distribution of antileuko- protease in upper respiratory mucosa. Ann Oto l Rhino l La ry ngol 1982: proteases involved. 91 (3 Pt l): 268–271. 14. Bergenfeldt M, Nystrom¨ M, Bohe M, Lindstrom C, Polling A, Ohlsson K. ACKNOWLEDGEMENTS. This study w as supported by grants from the Localization of immunoreactive secretory 1e ukocyte protease inhibitor Sw edish Medical Re search Council (project no. 390), the Medical Faculty, (SLPI) in intestinal mucosa. J Gastro entero l 1996; 31 (1): 18 –23. Lund University, the Albert Pålsson foundation, the University Hospital 15. Ohlsson K, Tegner H. Inhibition of elastase from granulocytes by the low Malmb foundations for medical and cancer research and the Greta and Johan molecular weight bronchial protease inhibitor. Scand J Clin La b Inves t Kock foundations. 1976; 36: 437–445. 16. Hochstrasser K, Re ichert R, Schw arz S, Werle E. Isolierung und charakterisierung eines proteaseninhibitors aus menschlichem bron- References chialsekret. Bio l Chem Ho ppe-Sey le r 1972; 353: 221–226. 17. Hakansson HO, Ohlsson K. Influence of plasma proteinase inhibitors and 1. Thompson RC, Ohlsson K. Isolation properties, and complete amino the secretory leucocyte proteinase inhibitor on pancreatic elastase- acid sequence of human secretory leukocyte protease inhibitor, a potent induced degradation of some plasma proteins. Gas tro entero l Jpn 1992; inhibitor of leukocyte elastase. Pro c Na tl Aca d Sci USA 1986; 83: 27 (5): 652–656. 6692–6696. 18. Fink E, Nettelbeck R, Fritz H. Inhibition of mast cell chymase by Eglin c 2. Haendle H, Ingrisch H, Werle E. A new trypsin-chymotrypsin inhibitor in and antileukoprotease (HUSI-I). Bio l Che m Ho ppe-Sey ler 1986; 367: female cervix secretion. Ho ppe-Sey le r’s Z Phys iol Ch em 1970; 351 (4): 567–571. 545–546. 19. Dietze SC, Sommerhoff CP, Fritz H. Inhibition of histamine release from 3. Wallner O, Fritz H. Characterization of an acid stable proteinase inhibitor human mast cells ex vivo by natural and synthetic chymase inhibitors. in human cervical mucus. Ho ppe-Seyler’s Z Phy sio l Chem 1974; 355: Biol Chem Ho ppe-Se yler 1990: 371 (Suppl): 75–79. 709–715. 20. Masuda K, Kamimura T, Watanabe K, Suga T, Kanesaki M, Takeuchi A, et 4. Cassl´en B, Rosengren M, Ohlsson K. Localization and quantitation of a a l. Pharmacological activity of the C-terminal and N-terminal domains of low molecular w eight proteinase inhibitor, antileukoprotease, in the secretor, leukoprotease inhibitor in vitro. Br J Pharm a co l 1995: 115 (6): human uterus. Ho ppe-Sey ler’s Z Physiol Chem 1981; 362: 953 –961. 883–888. 5. Ohlsson K, Bjartell A, Lilja H. Secretory leucocyte protease inhibitor in 21. Hiemstra PS, Maassen RJ, Stolk J, Heinzel Wieland R, Steffens GJ, Dijkman the male genital tract: PSA-induced proteolytic processing in human JH. Antibacterial activity of antileukoprotease. Infect Im m un 1996; 64 semen and tissue localization. J Andro l 1995; 16 (1): 64–74. (11): 4520 –4524. 6. Haendle H, Fritz H, Trautschold I, Werle E. Ueber einen hormonabh¨angi- 22. McNeely TB, Shugars DC, Rosendahl M, Tucker C, Eisenberg SP, Wahl SM. gen inhibitor fur proteolytische in m¨annlichen accessorischen Inhibition of human immunodeficiency virus type 1 infectivity by geschlechtsdrusen und im sperma. Hoppe-Sey ler’s Z Phy sio l Chem 1965; secretory leukocyte protease inhibitor occurs prior to viral reverse 342: 185–188. transcription. Blood 1997; 90 (3): 1141–1149 7. Schiessler H, Arnhold M, Ohlsson K, Fritz H. Inhibitors of and 23. Nystr¨om M, Bergenfeldt M, Ohlsson K. The elimination of secretory granulocyte proteinases from human genital tract secretions. Ho ppe- leukocyte protease inhibitor (SLPI) from the gastrointestinal tract in man. Seyler’s Z Phy sio l Ch em 1976; 357 (9): 1251–1260. Sca nd J Clin Lab Invest 1997; 57: 119–126. 8. Ohlsson K, Rosengren M, Tegner H. Quantification of granulocyte 24. Maniatis T, Fritsch E, Sambrook J. Transfer of DNA from agarose gels to elastase inhibitors in human mixed saliva and in pure parotid secretion. solid supporters. In: Ford N, Nolan C, Ferguson M, eds. Mo lecula r Ho ppe-Seyler’s Z Phy sio l Ch em 1983; 364: 1323–1328. Cloning: A Labo ra to ry Ma nua l, 2nd edn. New York: Cold Spring Harbor 9. Ohlsson M, Fryksmark U, Polling A, Tegner H, Ohlsson K. Localization of Laboratory Express 1989:34–39. antileukoprotease in the parotid and the submandibular salivary glands. Acta Otola ryngol 1984; 98 (1/2): 147–151. 10. Franken C, Meijer CJLM, Dijkman JH. Tissue distribution of antileukopro- Received 7 March 1999 tease and lysozme in humans. J Histio chem Cy to chem 1989; 37 (4): 493–498. accepted 6 April 1999

Mediators of Inflammation ´ Vol 8 ´ 1999 151 M EDIATORSof INFLAMMATION

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