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Human Leukocyte Elastase Induces Proliferation In Vitro and In Vivo

Christina Rogalski, Ulf Meyer-Hoffert, Ehrhardt Proksch, and Oliver Wiedow Department of Dermatology, University of Kiel, Kiel, Germany

Neutrophil in®ltration and epidermal hyperprolifera- Hyperproliferation resulted in a up to 2-fold increase tion are major histopathologic changes observed in of keratinocyte layers. Histologic analysis revealed . Neutrophils contain leukocyte elas- marked vasodilatation but no in¯ammatory in®ltrate. tase, which is thought to be released during neutro- Application of porcine pancreatic elastase (3± phil in®ltration of the . As active human 300 pmol per cm2 ) resulted in similar epidermal leukocyte elastase is known to be present in psoriatic changes as observed for human leukocyte elastase. lesions we were interested whether human leukocyte Hyperproliferative effects of human leukocyte elas- elastase induces hyperproliferation in tase in vitro and in vivo were abolished by the add- in vitro and in vivo. In the cultured murine keratino- ition of elastase inhibitors, such as ela®n, anti- cyte line PAM-212 concentrations of human leu- leukoprotease, and a1- inhibitor. In sum- kocyte elastase from 1 to 30 nM induced signi®cant mary, human leukocyte elastase induces proliferation proliferation as determined by 5-bromo-2¢-deoxy- in murine keratinocytes in concentrations, which can uridine-incorporation. Daily topical application of be found on the skin surface of psoriatic lesions. 0.043±434.8 pmol human leukocyte elastase per cm2 These results may provide an explanation for the epi- skin on hairless mice induced a concentration- dermal hyperproliferation observed in psoriasis. Key dependent epidermal hyperproliferation and an words: elastase/keratinocyte proliferation/psoriasis/serine increase in 5-bromo-2¢-deoxy-uridine incorporation . J Invest Dermatol 118:49±54, 2002 of up to 5-fold in basal keratinocytes within 3 d.

he traditional view that broad-spectrum serine polymorphonuclear leukocytes in very high concentrations (2 pg proteases are mainly degrading extracellular per cell) (Wiedow et al, 1996). Participation of elastase in has changed dramatically with the discovery of signaling processes has been shown in recent investigations. HLE protease receptors that are activated by proteolysis. regulates the expression and secretion of its inhibitor ela®n in the TFollowing proteolysis, a new N-terminus of the alveolar cell line A549 (Reid et al, 1999). Inhibition of HLE- extracellular position of the receptor occurs, which acts as a induced -8 gene expression by urinary trypsin inhibitor tethered ligand. There are four known members of this receptor has been demonstrated in human bronchial epithelial cells family of protease-activated receptors (PAR): PAR-1, PAR-3, and (Nakamura et al, 1997); however, activation of speci®c PAR by PAR-4, which are cleaved and activated by thrombin, and PAR-2, HLE has not been described yet. a receptor for trypsin-like . Recent investigations indicate Psoriasis is morphologically characterized by epidermal hyper- that certain serine proteases are signaling molecules and regulate proliferation and neutrophil in®ltrates in the epidermis. Active multiple cellular functions by activating speci®c PAR. Agonists of HLE is detectable in psoriatic lesions and correlates with skin PAR-1 and PAR-2 stimulate mitogen-activated kinases induration (Wiedow et al, 1992) and disappears with successful and may regulate cellular growth and proliferation (Belham et al, therapy of the disease (Wiedow et al, 1995). Knowing that other 1996; Yu et al, 1997); however, the effects of PAR-2 agonists on serine proteases exhibit mitogen effects we were interested to growth depend on the cell type. Both PAR-2 and PAR-1 agonists determine whether HLE is an important stimulus for keratinocyte stimulate proliferation of endothelial cells and ®broblasts (Mirza et proliferation in vitro and in vivo. al, 1996). In contrast, whereas PAR-1 agonists stimulate growth of keratinocytes, PAR-2 agonists inhibit keratinocyte growth and differentiation (Derian et al, 1997). MATERIALS AND METHODS Human leukocyte elastase (HLE) is a broad-spectrum serine protease (30 kDa) primarily located in the azurophil granules of Materials HLE was obtained from Elastin Products (Paci®c, MO). Active sites were titrated with recombinant eglin C (Ciba Geigy, Basel, Manuscript received May 29, 2001; revised August 7, 2001; accepted for Switzerland). HLE proved to be more than 80% catalytically active. HLE publication October 18, 2001. was dissolved in 0.1 M acetate (pH 5.0) and diluted with Reprint requests to: Dr. Oliver Wiedow, Department of Dermatology, phosphate-buffered saline immediately before application. University of Kiel, Schittenhelmstr. 7, D-24105 Kiel, Germany. Email: Culture of murine keratinocytes The spontaneously transformed [email protected] murine keratinocyte cell line (PAM-212; American Type Culture Abbreviations: BrdU, 5-bromo-2¢-deoxy-uridine; HLE, human leuko- Collection, Rockville, MD) was cultured in RPMI-1640 medium cyte elastase; TEWL, transepidermal loss. (Gibco BRL, Rockville, MD) supplemented with 5% fetal bovine serum

0022-202X/01/$15.00 ´ Copyright # 2002 by The Society for Investigative Dermatology, Inc. 49 50 ROGALSKI ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

and 72 h. The mean values were calculated and the range was determined. The data were recorded as a percentage of the reference (= 100%) immediately after the removal of the skin barrier. Application of enzymes After pretreatment with acetone a marked area (1 cm2) was treated with enzyme and control buffers. HLE concentrations of 0.043±434.8 pmol per 10 ml (4.3 nM±43.48 mM) were 2 applied on 1 cm skin. Porcine pancreatic elastase (Mr 27,000, EC 3.4.21.36, Serva, Heidelberg, Germany) was active site titrated with recombinant ela®n. It was dissolved in 0.1 M sodium acetate (pH 5.0) and diluted with phosphate-buffered saline. Concentrations of 0.3± 300 pmol per 30 ml were applied. The sites of application were covered with adherent aluminum chambers (Finn Chambers on Scanpor, Hermal, Reinbek, Germany). Enzymes and controls were repeatedly applied under occlusion after 0, 24, and 48 h. Furthermore, HLE (434.8 pmol per cm2) was applied in comparison with complexes of HLE and its physiologic inhibitors ela®n (recombinant human ela®n, ICI Pharmaceuticals, Maccles®eld, U.K.), anti-leukoprotease (recombinant human anti-leukoprotease, GruÈnenthal GmbH, Aachen, Germany) and a1-proteinase inhibitor (Sigma, St. Louis, MO). A sample of enzyme was Figure 1. HLE-induced proliferation in murine keratinocytes. titrated with each of the inhibitor until the methoxy-succinyl-alanyl- Proliferation was measured by incorporation of BrdU in the murine alanyl-prolyl-valine-p-nitroanilide hydrolyzing activity was below 0.5% keratinocyte cell line PAM-212 using a cell proliferating enzyme-linked of the enzyme . After 24, 48, and 72 h the erythema was immunosorbent assay. Cells were treated with HLE (0.1±33 nM) for evaluated visually. 24 h. Concentration of 33 nM led to » 50% detachment of cells (#). Histology The tissue was ®xed in formaldehyde and embedded in Increase of proliferation depended on proteolytic activity as shown by paraf®n. Sections were stained with hematoxylin and eosin. The adding HLE (10 nM) preincubated with ela®n (100 nM) or anti- epidermal layers of six slides (®ve visual ®elds, magni®cation 3 40) were leukoprotease (100 nM). Values are mean 6 SD of six separate counted; mean and standard deviation values were calculated. The experiments performed in duplicate. Signi®cant difference compared signi®cance level of given differences was determined using the c2-test. with medium control: *p < 0.001. BrdU incorporation BrdU (3.5 mg; Sigma) was dissolved in 750 ml sodium chloride and injected intraperitoneally, 1 h before the mice were killed by cervical dislocation. Biopsies were taken from the four and antibiotics (100 units penicillin per ml and 100 units streptomycin experimental sites and an untreated control area of each animal. BrdU per ml) and incubated at 37°C in 5% CO2 and humid atmosphere. incorporation into the nuclei of keratinocytes was visualized using the Keratinocyte proliferation assay Proliferation of cultured cells was alkaline phosphatase anti-alkaline phosphatase method (Universal APAAP assessed by 5-bromo-2¢-deoxy-uridine (BrdU) labeling as described Kit mouse, Dakopatts system 40, Dako Diagnostika, Hamburg, previously (Porstmann et al, 1985). Keratinocytes were plated in 96-well Germany). Brie¯y, the slides were covered with 10% normal rabbit ¯at-bottomed microtiter plates at a density of 3000 cells per well and serum (30 min) and then incubated with the primary (aBrdU cultured overnight. Cells were washed twice with phosphate-buffered from mice, Amersham RPN 202, Amersham-Buchler, Braunschweig, saline and the medium was replaced with RPMI-1640 containing 1% Germany) for 1 h, before being cleared with Tris buffer for 15 min. The (wt/vol) bovine serum albumin (Sigma, St. Louis, MO) supplemented slides were then incubated with a second antibody (rabbit anti-mouse with HLE in various concentrations, ela®n (100 nM), anti-leukoprotease IgG) for 1 h, cleared with Tris buffer 15 min, incubated with the (or secretory leukocyte protease inhibitor; 100 nM) or recombinant immune complex (alkaline phosphatase anti-alkaline phosphatase) for 1 h human (100 nM). The cells were then and then cleared with Tris buffer for 15 min. The slides were incubated incubated for 24 h in the presence of 100 mM BrdU. Incorporation of with substrate for 20 min, cleared with Tris for 5 min and then washed BrdU was determined using a cell proliferating enzyme-linked with distilled water for 1 min and lukewarm tap water for 10 min. Counter-staining was performed with hematoxylin Mayer for 15 s. After immunosorbent assay (Proliferation ELISA, Roche, Mannheim, a ®nal wash with distilled water for 1 min the specimens were covered Germany) according to the manufacturer's instructions. The absorbance with a mounting medium (Dako Glycergel Mounting Medium S63, was measured using a spectrophotometer at 450 nm vs 620 nm. Dako). The BrdU-incorporated nuclei appeared brilliant red in this stain. Absorbance was corrected to the measured absorbance of buffer control. The proliferating basal keratinocytes were counted in six slides under a Mean values and standard deviations were calculated, and the signi®cance 2 microscope (3 40), mean value and standard deviation were calculated, level of given differences was determined with the c -test. The index of and the signi®cance level of given differences was determined using the proliferation was calculated by dividing the average of the measured c2-test. The index of proliferation was calculated by dividing the average absorbance after stimulation by the average of the absorbance of of the counted proliferating cells per six high-power ®eld by the average untreated cells. Enzyme activity was con®rmed by substrate assay. of the proliferating cells by the control slides from untreated sites. Animals Hairless male mice [strain SKH I (hr/hr) BR from Charles River, Sulzfeld, Germany] 6±8 wk old and weighing 20±30 g were used. RESULTS All animals were housed in plastic cages and given food and water ad libitum. The treatment was practiced under ether anesthesia. The study HLE stimulates hyperproliferation of keratinocytes was approved by the ethic committee of the Medical Faculty, University in vitro HLE signi®cantly increased BrdU uptake by keratino- of Kiel. cytes in a concentration-dependent manner (Fig 1). Already a concentration of 0.3 nM HLE induced proliferation signi®cantly. Transepidermal water loss (TEWL) The mice were treated with A maximum increase was observed at 3.3 nM reaching a plateau at acetone-soaked cotton balls on both ¯anks to remove the outer of the epidermis and in order to facilitate the penetration of proteins. The 10 nM HLE. Higher concentrations of HLE resulted in a partial to TEWL was measured using an electrolytic water analyzer (MEECO, complete detachment of the cells. The increase in proliferation was Warrington, PE). Dry nitrogen was passed through a Te¯on tube to a comparable with that observed following stimulation with human metal cup placed upon the skin. The nitrogen gas was moistened by epidermal growth factor (100 nM). Applying HLE together with water vapor derived from at the skin surface and conducted elastase inhibitors, ela®n and anti-leukoprotease, abolished HLE- to the implement where the water was split up into oxygen and induced proliferation completely. The inhibitors had no in¯uence hydrogen. An ampere meter registered the electric current resulting from on BrdU incorporation by keratinocytes. this electrolysis. The scale of the electrolytic water analyzer read ppm 2 HLE induces hyperproliferation in vivo Topical application of water by volume (1 ppm = 0.25 g H2O per cm skin surface per h). The skin barrier was considered to be suf®ciently removed when the HLE on hairless mice under occlusion resulted in a pronounced TEWL reached a value of between 400 and 600 ppm. The regeneration induction of proliferation as well as epidermal thickness after 72 h. of the barrier was examined by measuring the TEWL after 0, 24, 48, Hematoxylin and eosin stained biopsies showed marked VOL. 118, NO. 1 JANUARY 2002 ELASTASE INDUCES KERATINOCYTE HYPERPROLIFERATION 51

Figure 2. HLE induced epidermal hyper- proliferation within 3 d. Histologic sections of buffer-treated (A) and HLE-treated (434.8 pmol per cm2) skin (B). BrdU incorporation (C) after application of HLE-treated (34 pmol per cm2) skin. In HLE-treated skin no signi®cant in¯ammatory in®ltrate was visible. Bars: 200 mm.

Table I. TEWL, proliferating keratinocytes, epidermal thickness, and erythema after 3 d of elastase applicationa

TEWL 0 h TEWL 72 h Proliferation Keratinocyte Erythema ppm % Index layers 24 h

Untreated sites 40b ± 1 2.6 6 0.2 0 (30±60) Buffer control 491 13 (11±15) 1 3.2 6 0.2 0 (390±600) Leukocyte elastase 506 22 (17±26) 5.0 6 0.9 5.0 6 0.7 ++ (380±600) Pancreatic elastase 524 18 (17±19) 3.9 6 1.4 5.1 6 0.7 ++ (405±610)

aThe lipids from the skin of hairless mice were removed by acetone pretreatment. Afterwards phosphate-buffered saline (buffer), HLE (434 pmol per cm 2skin), and por- cine pancreatic elastase (300 pmol per cm 2skin) were applied daily under occlusion for 72 h (n = 6). The TEWL was determined after acetone treatment (TEWL 0 h) and after 3 d (TEWL 72 h in % of TEWL 0 h). The number of proliferating keratinocytes (BrdU-positive keratinocytes/6 high-power ®eld) was given as a factor of the un- treated sites (proliferation index). The average number of keratinocyte layers of 6 high-power ®eld was determined for each test-site. bWithout acetone pretreatment. Data are expressed as mean 6 SD (n = 6). Erythema was graded subjectively as absent (0), slight (+/±), moderate (+), and pronounced (++).

vasodilatation but no signi®cant in¯ammatory in®ltrate. HLE- Effect of protease inhibitors Applying HLE together with a treated sites developed a visible and showed a molar excess of the physiologic inhibitors ela®n, anti- curved , suggesting the initial leukoprotease, and a1-proteinase inhibitor abolished the HLE- development of a papilla-like folding of the (Fig 2). induced hyperproliferation completely (Fig 4). In contrast, the Breaking the barrier once with acetone followed by application of addition of a vast molar excess of inhibitors for trypsin-like enzymes the buffers under occlusion did not result in any signi®cant signs of (1 mM n-p-tosyl-l-lysine chloromethyl ketone) as well as for in¯ammation and no induction of epidermal hyperproliferation or chymotrypsin-like enzymes (1 mM n-tosyl-l-phenylalanine increase of epidermal thickness after 72 h (Table I). chloromethyl ketone) did not in¯uence the hyperproliferative The HLE-induced hyperproliferation showed a strong dose± effect of HLE (data not shown). response relationship in regard to the number of proliferating keratinocytes (Fig 3A) and increase of epidermal keratinocyte DISCUSSION layers (Fig 3B). Already a minimal dose of 0.043 pmol HLE per Several cytokines and growth factors are able to modulate cm2 skin increased the proliferation of keratinocytes and thickened 2 keratinocyte proliferation and have been studied in subcon¯uent the epidermis. Application of HLE (434.8 pmol per cm skin) keratinocyte cultures. Interleukin-1 (Ristow, 1987), interleukin-3 increased the number of proliferating basal keratinocytes 5-fold, (Hancock et al, 1988), interleukin-6 (Grossman et al, 1989), whereas the thickness of the epidermis doubled. After a period of 2 interleukin-8 (Tuschil et al, 1992), transforming growth factor-a 24 h following the application of HLE (0.43 pmol per cm skin) an (Coffey et al, 1987), epidermal growth factor (Carpenter and erythema was observed. This became more pronounced with 2 Cohen, 1979), and granulocyte-macrophage colony stimulating increasing concentration of HLE up to 434 pmol per cm factor (Hancock et al, 1988) have been shown to increase (Table I). Comparable effects were observed following the proliferation. -a (Kono et al, 1990), inter- application of porcine pancreas elastase. A signi®cant increase in feron-a (Nickoloff et al, 1984), interferon-b (Nickoloff et al, 1989), proliferation (proliferation index 1.7 6 0.6, n = 6) and a resultant and transforming growth factor-b (Hsuan, 1989) are known to thickening of the epidermis (5.3 6 0.6) was achieved using a inhibit the proliferation of keratinocytes. There is no proof that concentration of 3 pmol pancreas elastase per cm2 skin. The these stimuli are able to induce the proliferation of keratinocytes in maximum concentration of 300 pmol pancreas elastase per cm2 viable skin. Therefore we were interested, whether HLE is capable skin augmented the proliferation index nearly 4-fold and thickened of inducing pronounced keratinocyte proliferation both in vitro and the epidermis from 3.2 6 0.2 to 5.1 6 0.7 epidermal layers. in vivo. Application of the buffers and more pronounced application of In vitro HLE induced proliferation in the murine keratinocyte leukocyte and pancreatic elastase resulted in an increase in TEWL cell line PAM-212. The HLE-mediated proliferative response of 3 d after treatment (Table I). An erythema was visible 24 h after keratinocytes was dependent on the enzymatic activity of HLE at treatment with 30 pmol pancreas elastase per cm2 skin. concentrations from 1 to 30 nM. Application of HLE together with 52 ROGALSKI ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 3. Effects of HLE on the proliferation of keratinocytes and the thickening of the epidermis. (A) Keratinocytes; (B) Figure 4. HLE induced epidermis proliferation in the presence of epidermis. The cells that incorporated BrdU per basal membrane length physiologic inhibitors. HLE-treated (434.8 pmol per cm2) skin was (A) and the epidermal keratinocyte layers were counted. n = 12, compared with HLE + equimolar concentrations of ela®n, anti- controls: n = 30 (A) and n = 42 (B), *p < 0.001. leukoprotease, and a1-proteinase inhibitor. The TEWL (A), the index of proliferation (B), and the number of epidermal layers (C) were determined. n = 6, *p < 0.001. a molar excess of the human keratinocyte-derived protease inhibitors ela®n (Wiedow et al, 1990) and antileukoprotease vasodilatation but no in¯ammatory in®ltrate. This points towards a (Wiedow et al, 1993) inhibited the proliferative effect. The direct proliferative effect of HLE on keratinocytes. Signi®cant concentrations of HLE that induced signi®cant proliferation in cultured keratinocytes were similar to those seen in investigations proliferation of basal keratinocytes with a concomitant signi®cant with other serine-proteases such as thrombin, trypsin, or mast cell increase of epidermal layers could be achieved with 43 fmol HLE per cm2 skin (applied concentration 4.3 nM). The maximum tryptase to activate PAR on keratinocytes. Concentrations of 2 thrombin from 10 pM to 10 nM induced keratinocytes prolifer- increase in epidermal thickness was reached with 4.3 pmol per cm ation by activation of PAR-1 (Derian et al, 1997; Algermissen et al, skin (applied concentration 430 nM). Concentrations of HLE 2000). It has been demonstrated that activation of PAR-2 by necessary for the induction of hyperproliferation in vitro were 20 nM trypsin, 60 nM mast cell tryptase or the PAR-2 activating approximately 10 times less. This might be explained by the fact peptide SLIGRL resulted in decreased keratinocyte proliferation that HLE applied at the skin surface had to diffuse down to basal (Derian et al, 1997; Algermissen et al, 1999); however, it has also keratinocytes, which will be accompanied by a loss of activity. been reported that mast cell tryptase is capable of inducing Application of HLE together with a molar excess of the protease keratinocyte proliferation (Pohlig et al, 1996). In lung ®broblasts inhibitors, antileukoprotease and ela®n, inhibited the proliferative activation of PAR-2 by 5±20 nM trypsin or 2±70 mU tryptase per effect almost completely. The same effect was observed with ml exhibited proliferative effects (Akers et al, 2000) implicating a application of HLE together with human a1-proteinase inhibitor cell type speci®c effect of PAR-2-activation on cell proliferation. (53 kDa). We conceive that the inhibition is caused by inhibiting To the best of our knowledge a receptor-mediated response to the proteolytic activity and not to a reduced penetration due to the HLE in keratinocytes has not been proposed yet. Loew et al (2000) larger molecular weights of enzyme-inhibitor complexes because showed that HLE abolished activation of PAR-1 and decreased the the application of enzyme and inhibitor resulted in unchanged PAR-2 activity in endothelial cells. None of the PAR-1±4 has proliferation both in vitro and in vivo. Moreover, a complex of the been shown to be activated by HLE. reversible inhibitor ela®n (6 kDa) and elastase (30 kDa) with a total Topical application of HLE on viable murine skin induced weight of 36 kDa might have an in¯uence on the penetration. As epidermal hyperproliferation. Histologic analysis revealed marked HLE and pancreatic elastase induced epidermal proliferation in this VOL. 118, NO. 1 JANUARY 2002 ELASTASE INDUCES KERATINOCYTE HYPERPROLIFERATION 53 study the question occurs whether the same ®ndings would have REFERENCES been obtained with other proteolytic enzymes. Controls with Akers I, Parsons M, Hill M, Hollenberg M, Sanjar S, Laurent G, McAnulty R: Mast pancreatic trypsin and chymotrypsin resulted also in pronounced cell tryptase stimulates human lung ®broblast proliferation via protease- epidermal proliferation (data not shown). The biologic meaning activated receptor-2. Am J Physiol Lung Cell Mol Physiol 278:193±201, 2000 remains unclear as these enzymes do not occur to our knowledge in Algermissen B, Hermes B, Feldmann-BoÈddeker I, Bauer F, Henz BM: Mast cell chymase and tryptase during tissue turnoverÐanalysis on in vitro mitogenesis human epidermis. 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