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Human Leukocyte Elastase Induces Keratinocyte Proliferation In Vitro and In Vivo ARTICLE in JOURNAL OF INVESTIGATIVE DERMATOLOGY · FEBRUARY 2002 Impact Factor: 7.22 · DOI: 10.1046/j.0022-202x.2001.01650.x · Source: PubMed
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Human Leukocyte Elastase Induces Keratinocyte Proliferation In Vitro and In Vivo Christina Rogalski, Ulf Meyer-Hoffert, Ehrhardt Proksch, and Oliver Wiedow Department of Dermatology, University of Kiel, Kiel, Germany
Hyperproliferation resulted in a up to 2-fold increase of keratinocyte layers. Histologic analysis revealed marked vasodilatation but no in¯ammatory in®ltrate. Application of porcine pancreatic elastase (3± 300 pmol per cm2 skin) resulted in similar epidermal changes as observed for human leukocyte elastase. Hyperproliferative effects of human leukocyte elastase in vitro and in vivo were abolished by the addition of elastase inhibitors, such as ela®n, antileukoprotease, and a1-protease inhibitor. In summary, human leukocyte elastase induces proliferation in murine keratinocytes in concentrations, which can be found on the skin surface of psoriatic lesions. These results may provide an explanation for the epidermal hyperproliferation observed in psoriasis. Key words: elastase/keratinocyte proliferation/psoriasis/serine proteases. J Invest Dermatol 118:49±54, 2002
Neutrophil in®ltration and epidermal hyperproliferation are major histopathologic changes observed in psoriasis. Neutrophils contain human leukocyte elastase, which is thought to be released during neutrophil in®ltration of the epidermis. As active human leukocyte elastase is known to be present in psoriatic lesions we were interested whether human leukocyte elastase induces hyperproliferation in keratinocytes in vitro and in vivo. In the cultured murine keratinocyte cell line PAM-212 concentrations of human leukocyte elastase from 1 to 30 nM induced signi®cant proliferation as determined by 5-bromo-2¢-deoxyuridine-incorporation. Daily topical application of 0.043±434.8 pmol human leukocyte elastase per cm2 skin on hairless mice induced a concentrationdependent epidermal hyperproliferation and an increase in 5-bromo-2¢-deoxy-uridine incorporation of up to 5-fold in basal keratinocytes within 3 d.
T
polymorphonuclear leukocytes in very high concentrations (2 pg enzyme per cell) (Wiedow et al, 1996). Participation of elastase in signaling processes has been shown in recent investigations. HLE regulates the expression and secretion of its inhibitor ela®n in the alveolar cell line A549 (Reid et al, 1999). Inhibition of HLEinduced interleukin-8 gene expression by urinary trypsin inhibitor has been demonstrated in human bronchial epithelial cells (Nakamura et al, 1997); however, activation of speci®c PAR by HLE has not been described yet. Psoriasis is morphologically characterized by epidermal hyperproliferation and neutrophil in®ltrates in the epidermis. Active HLE is detectable in psoriatic lesions and correlates with skin induration (Wiedow et al, 1992) and disappears with successful therapy of the disease (Wiedow et al, 1995). Knowing that other serine proteases exhibit mitogen effects we were interested to determine whether HLE is an important stimulus for keratinocyte proliferation in vitro and in vivo.
he traditional view that broad-spectrum serine proteases are mainly degrading extracellular proteins has changed dramatically with the discovery of protease receptors that are activated by proteolysis. Following proteolysis, a new N-terminus of the extracellular position of the receptor occurs, which acts as a tethered ligand. There are four known members of this receptor family of protease-activated receptors (PAR): PAR-1, PAR-3, and PAR-4, which are cleaved and activated by thrombin, and PAR-2, a receptor for trypsin-like enzymes. Recent investigations indicate that certain serine proteases are signaling molecules and regulate multiple cellular functions by activating speci®c PAR. Agonists of PAR-1 and PAR-2 stimulate mitogen-activated protein kinases and may regulate cellular growth and proliferation (Belham et al, 1996; Yu et al, 1997); however, the effects of PAR-2 agonists on growth depend on the cell type. Both PAR-2 and PAR-1 agonists stimulate proliferation of endothelial cells and ®broblasts (Mirza et al, 1996). In contrast, whereas PAR-1 agonists stimulate growth of keratinocytes, PAR-2 agonists inhibit keratinocyte growth and differentiation (Derian et al, 1997). Human leukocyte elastase (HLE) is a broad-spectrum serine protease (30 kDa) primarily located in the azurophil granules of
MATERIALS AND METHODS Materials HLE was obtained from Elastin Products (Paci®c, MO). Active sites were titrated with recombinant eglin C (Ciba Geigy, Basel, Switzerland). HLE proved to be more than 80% catalytically active. HLE was dissolved in 0.1 M sodium acetate (pH 5.0) and diluted with phosphate-buffered saline immediately before application.
Manuscript received May 29, 2001; revised August 7, 2001; accepted for publication October 18, 2001. Reprint requests to: Dr. Oliver Wiedow, Department of Dermatology, University of Kiel, Schittenhelmstr. 7, D-24105 Kiel, Germany. Email:
[email protected] Abbreviations: BrdU, 5-bromo-2¢-deoxy-uridine; HLE, human leukocyte elastase; TEWL, transepidermal water loss. 0022-202X/01/$15.00
Culture of murine keratinocytes The spontaneously transformed murine keratinocyte cell line (PAM-212; American Type Culture Collection, Rockville, MD) was cultured in RPMI-1640 medium (Gibco BRL, Rockville, MD) supplemented with 5% fetal bovine serum
´ Copyright # 2002 by The Society for Investigative Dermatology, Inc. 49
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ROGALSKI ET AL
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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.
Figure 1. HLE-induced proliferation in murine keratinocytes. Proliferation was measured by incorporation of BrdU in the murine keratinocyte cell line PAM-212 using a cell proliferating enzyme-linked immunosorbent assay. Cells were treated with HLE (0.1±33 nM) for 24 h. Concentration of 33 nM led to » 50% detachment of cells (#). Increase of proliferation depended on proteolytic activity as shown by adding HLE (10 nM) preincubated with ela®n (100 nM) or antileukoprotease (100 nM). Values are mean 6 SD of six separate experiments performed in duplicate. Signi®cant difference compared with medium control: *p < 0.001.
and antibiotics (100 units penicillin per ml and 100 units streptomycin per ml) and incubated at 37°C in 5% CO2 and humid atmosphere. Keratinocyte proliferation assay Proliferation of cultured cells was assessed by 5-bromo-2¢-deoxy-uridine (BrdU) labeling as described previously (Porstmann et al, 1985). Keratinocytes were plated in 96-well ¯at-bottomed microtiter plates at a density of 3000 cells per well and cultured overnight. Cells were washed twice with phosphate-buffered saline and the medium was replaced with RPMI-1640 containing 1% (wt/vol) bovine serum albumin (Sigma, St. Louis, MO) supplemented with HLE in various concentrations, ela®n (100 nM), anti-leukoprotease (or secretory leukocyte protease inhibitor; 100 nM) or recombinant human epidermal growth factor (100 nM). The cells were then incubated for 24 h in the presence of 100 mM BrdU. Incorporation of BrdU was determined using a cell proliferating enzyme-linked immunosorbent assay (Proliferation ELISA, Roche, Mannheim, Germany) according to the manufacturer's instructions. The absorbance was measured using a spectrophotometer at 450 nm vs 620 nm. Absorbance was corrected to the measured absorbance of buffer control. Mean values and standard deviations were calculated, and the signi®cance level of given differences was determined with the c2-test. The index of proliferation was calculated by dividing the average of the measured absorbance after stimulation by the average of the absorbance of untreated cells. Enzyme activity was con®rmed by substrate assay. 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. All animals were housed in plastic cages and given food and water ad libitum. The treatment was practiced under ether anesthesia. The study was approved by the ethic committee of the Medical Faculty, University of Kiel. Transepidermal water loss (TEWL) The mice were treated with acetone-soaked cotton balls on both ¯anks to remove the outer lipids of the epidermis and in order to facilitate the penetration of proteins. The TEWL was measured using an electrolytic water analyzer (MEECO, Warrington, PE). Dry nitrogen was passed through a Te¯on tube to a metal cup placed upon the skin. The nitrogen gas was moistened by water vapor derived from perspiration at the skin surface and conducted to the implement where the water was split up into oxygen and hydrogen. An ampere meter registered the electric current resulting from this electrolysis. The scale of the electrolytic water analyzer read ppm water by volume (1 ppm = 0.25 g H2O per cm2 skin surface per h). The skin barrier was considered to be suf®ciently removed when the TEWL reached a value of between 400 and 600 ppm. The regeneration of the barrier was examined by measuring the TEWL after 0, 24, 48,
Application of enzymes After pretreatment with acetone a marked area (1 cm2) was treated with enzyme solutions and control buffers. HLE concentrations of 0.043±434.8 pmol per 10 ml (4.3 nM±43.48 mM) were applied on 1 cm2 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 titrated with each of the inhibitor until the methoxy-succinyl-alanylalanyl-prolyl-valine-p-nitroanilide hydrolyzing activity was below 0.5% of the enzyme solution. After 24, 48, and 72 h the erythema was evaluated visually. Histology The tissue was ®xed in formaldehyde and embedded in paraf®n. Sections were stained with hematoxylin and eosin. The epidermal layers of six slides (®ve visual ®elds, magni®cation 3 40) were counted; mean and standard deviation values were calculated. The signi®cance level of given differences was determined using the c2-test. 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 experimental sites and an untreated control area of each animal. BrdU incorporation into the nuclei of keratinocytes was visualized using the alkaline phosphatase anti-alkaline phosphatase method (Universal APAAP Kit mouse, Dakopatts system 40, Dako Diagnostika, Hamburg, Germany). Brie¯y, the slides were covered with 10% normal rabbit serum (30 min) and then incubated with the primary antibody (aBrdU from mice, Amersham RPN 202, Amersham-Buchler, Braunschweig, Germany) for 1 h, before being cleared with Tris buffer for 15 min. The slides were then incubated with a second antibody (rabbit anti-mouse IgG) for 1 h, cleared with Tris buffer 15 min, incubated with the immune complex (alkaline phosphatase anti-alkaline phosphatase) for 1 h and then cleared with Tris buffer for 15 min. The slides were incubated with substrate for 20 min, cleared with Tris for 5 min and then washed with distilled water for 1 min and lukewarm tap water for 10 min. Counter-staining was performed with hematoxylin Mayer for 15 s. After a ®nal wash with distilled water for 1 min the specimens were covered with a mounting medium (Dako Glycergel Mounting Medium S63, Dako). The BrdU-incorporated nuclei appeared brilliant red in this stain. The proliferating basal keratinocytes were counted in six slides under a microscope (3 40), mean value and standard deviation were calculated, and the signi®cance level of given differences was determined using the c2-test. The index of proliferation was calculated by dividing the average of the counted proliferating cells per six high-power ®eld by the average of the proliferating cells by the control slides from untreated sites.
RESULTS HLE stimulates hyperproliferation of keratinocytes in vitro HLE signi®cantly increased BrdU uptake by keratinocytes in a concentration-dependent manner (Fig 1). Already a concentration of 0.3 nM HLE induced proliferation signi®cantly. A maximum increase was observed at 3.3 nM reaching a plateau at 10 nM HLE. Higher concentrations of HLE resulted in a partial to complete detachment of the cells. The increase in proliferation was comparable with that observed following stimulation with human epidermal growth factor (100 nM). Applying HLE together with elastase inhibitors, ela®n and anti-leukoprotease, abolished HLEinduced proliferation completely. The inhibitors had no in¯uence on BrdU incorporation by keratinocytes. HLE induces hyperproliferation in vivo Topical application of HLE on hairless mice under occlusion resulted in a pronounced induction of proliferation as well as epidermal thickness after 72 h. Hematoxylin and eosin stained biopsies showed marked
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Figure 2. HLE induced epidermal hyperproliferation 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
Untreated sites Buffer control Leukocyte elastase Pancreatic elastase
TEWL 0 h ppm
TEWL 72 h %
Proliferation Index
Keratinocyte layers
Erythema 24 h
40b (30±60) 491 (390±600) 506 (380±600) 524 (405±610)
±
1
2.6 6 0.2
0
13 (11±15)
1
3.2 6 0.2
0
22 (17±26)
5.0 6 0.9
5.0 6 0.7
++
18 (17±19)
3.9 6 1.4
5.1 6 0.7
++
a The lipids from the skin of hairless mice were removed by acetone pretreatment. Afterwards phosphate-buffered saline (buffer), HLE (434 pmol per cm 2skin), and porcine 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 untreated sites (proliferation index). The average number of keratinocyte layers of 6 high-power ®eld was determined for each test-site. b Without 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. HLEtreated sites developed a visible stratum granulosum and showed a curved dermoepidermal junction, suggesting the initial development of a papilla-like folding of the dermis (Fig 2). Breaking the barrier once with acetone followed by application of the buffers under occlusion did not result in any signi®cant signs of in¯ammation and no induction of epidermal hyperproliferation or increase of epidermal thickness after 72 h (Table I). The HLE-induced hyperproliferation showed a strong dose± response relationship in regard to the number of proliferating keratinocytes (Fig 3A) and increase of epidermal keratinocyte layers (Fig 3B). Already a minimal dose of 0.043 pmol HLE per cm2 skin increased the proliferation of keratinocytes and thickened the epidermis. Application of HLE (434.8 pmol per cm2 skin) increased the number of proliferating basal keratinocytes 5-fold, whereas the thickness of the epidermis doubled. After a period of 24 h following the application of HLE (0.43 pmol per cm2 skin) an erythema was observed. This became more pronounced with increasing concentration of HLE up to 434 pmol per cm2 (Table I). Comparable effects were observed following the application of porcine pancreas elastase. A signi®cant increase in proliferation (proliferation index 1.7 6 0.6, n = 6) and a resultant thickening of the epidermis (5.3 6 0.6) was achieved using a concentration of 3 pmol pancreas elastase per cm2 skin. The maximum concentration of 300 pmol pancreas elastase per cm2 skin augmented the proliferation index nearly 4-fold and thickened the epidermis from 3.2 6 0.2 to 5.1 6 0.7 epidermal layers. Application of the buffers and more pronounced application of leukocyte and pancreatic elastase resulted in an increase in TEWL 3 d after treatment (Table I). An erythema was visible 24 h after treatment with 30 pmol pancreas elastase per cm2 skin.
Effect of protease inhibitors Applying HLE together with a molar excess of the physiologic inhibitors ela®n, antileukoprotease, and a1-proteinase inhibitor abolished the HLEinduced hyperproliferation completely (Fig 4). In contrast, the addition of a vast molar excess of inhibitors for trypsin-like enzymes (1 mM n-p-tosyl-l-lysine chloromethyl ketone) as well as for chymotrypsin-like enzymes (1 mM n-tosyl-l-phenylalanine chloromethyl ketone) did not in¯uence the hyperproliferative effect of HLE (data not shown). DISCUSSION Several cytokines and growth factors are able to modulate keratinocyte proliferation and have been studied in subcon¯uent keratinocyte cultures. Interleukin-1 (Ristow, 1987), interleukin-3 (Hancock et al, 1988), interleukin-6 (Grossman et al, 1989), interleukin-8 (Tuschil et al, 1992), transforming growth factor-a (Coffey et al, 1987), epidermal growth factor (Carpenter and Cohen, 1979), and granulocyte-macrophage colony stimulating factor (Hancock et al, 1988) have been shown to increase proliferation. Tumor necrosis factor-a (Kono et al, 1990), interferon-a (Nickoloff et al, 1984), interferon-b (Nickoloff et al, 1989), and transforming growth factor-b (Hsuan, 1989) are known to inhibit the proliferation of keratinocytes. There is no proof that these stimuli are able to induce the proliferation of keratinocytes in viable skin. Therefore we were interested, whether HLE is capable of inducing pronounced keratinocyte proliferation both in vitro and in vivo. In vitro HLE induced proliferation in the murine keratinocyte cell line PAM-212. The HLE-mediated proliferative response of keratinocytes was dependent on the enzymatic activity of HLE at concentrations from 1 to 30 nM. Application of HLE together with
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ROGALSKI ET AL
Figure 3. Effects of HLE on the proliferation of keratinocytes and the thickening of the epidermis. (A) Keratinocytes; (B) epidermis. The cells that incorporated BrdU per basal membrane length (A) and the epidermal keratinocyte layers were counted. n = 12, controls: n = 30 (A) and n = 42 (B), *p < 0.001.
a molar excess of the human keratinocyte-derived protease inhibitors ela®n (Wiedow et al, 1990) and antileukoprotease (Wiedow et al, 1993) inhibited the proliferative effect. The concentrations of HLE that induced signi®cant proliferation in cultured keratinocytes were similar to those seen in investigations with other serine-proteases such as thrombin, trypsin, or mast cell tryptase to activate PAR on keratinocytes. Concentrations of thrombin from 10 pM to 10 nM induced keratinocytes proliferation by activation of PAR-1 (Derian et al, 1997; Algermissen et al, 2000). It has been demonstrated that activation of PAR-2 by 20 nM trypsin, 60 nM mast cell tryptase or the PAR-2 activating peptide SLIGRL resulted in decreased keratinocyte proliferation (Derian et al, 1997; Algermissen et al, 1999); however, it has also been reported that mast cell tryptase is capable of inducing keratinocyte proliferation (Pohlig et al, 1996). In lung ®broblasts activation of PAR-2 by 5±20 nM trypsin or 2±70 mU tryptase per ml exhibited proliferative effects (Akers et al, 2000) implicating a cell type speci®c effect of PAR-2-activation on cell proliferation. To the best of our knowledge a receptor-mediated response to HLE in keratinocytes has not been proposed yet. Loew et al (2000) showed that HLE abolished activation of PAR-1 and decreased the PAR-2 activity in endothelial cells. None of the PAR-1±4 has been shown to be activated by HLE. Topical application of HLE on viable murine skin induced epidermal hyperproliferation. Histologic analysis revealed marked
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Figure 4. HLE induced epidermis proliferation in the presence of physiologic inhibitors. HLE-treated (434.8 pmol per cm2) skin was compared with HLE + equimolar concentrations of ela®n, antileukoprotease, 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.
vasodilatation but no in¯ammatory in®ltrate. This points towards a direct proliferative effect of HLE on keratinocytes. Signi®cant proliferation of basal keratinocytes with a concomitant signi®cant increase of epidermal layers could be achieved with 43 fmol HLE per cm2 skin (applied concentration 4.3 nM). The maximum increase in epidermal thickness was reached with 4.3 pmol per cm2 skin (applied concentration 430 nM). Concentrations of HLE necessary for the induction of hyperproliferation in vitro were approximately 10 times less. This might be explained by the fact that HLE applied at the skin surface had to diffuse down to basal keratinocytes, which will be accompanied by a loss of activity. Application of HLE together with a molar excess of the protease inhibitors, antileukoprotease and ela®n, inhibited the proliferative effect almost completely. The same effect was observed with application of HLE together with human a1-proteinase inhibitor (53 kDa). We conceive that the inhibition is caused by inhibiting the proteolytic activity and not to a reduced penetration due to the larger molecular weights of enzyme-inhibitor complexes because the application of enzyme and inhibitor resulted in unchanged proliferation both in vitro and in vivo. Moreover, a complex of the reversible inhibitor ela®n (6 kDa) and elastase (30 kDa) with a total weight of 36 kDa might have an in¯uence on the penetration. As HLE and pancreatic elastase induced epidermal proliferation in this
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study the question occurs whether the same ®ndings would have been obtained with other proteolytic enzymes. Controls with pancreatic trypsin and chymotrypsin resulted also in pronounced epidermal proliferation (data not shown). The biologic meaning remains unclear as these enzymes do not occur to our knowledge in human epidermis. Whether epidermis derived proteases such as stratum corneum chymotryptic enzyme and stratum corneum tryptic enzyme, which exhibit trypsin-like and chymotrypsin-like activity, are able to cause epidermal hyperproliferation has not been investigated so far. The concentrations of HLE, which were tested on hairless mice and cultured keratinocytes in this study, are comparable with those found on the skin surface of psoriatic lesions (Wiedow et al, 1995). Measuring the skin surface proteolytic activity of HLE on psoriatic lesions the 95% con®dence interval was between 5 and 50 fmol HLE per cm2 skin the range between 0.5 fmol and 40 pmol HLE per cm2 skin. The high variation re¯ects the variability of the neutrophil in®ltrate in psoriatic lesions. Elastase might be an important stimulus for epidermal proliferation in early stage psoriasis as HLE was detected predominantly in acute lesions (Glinski et al, 1984). It is believed that psoriatic lesions enlarge by centrifugal expansion (Ragaz and Ackerman, 1979; Gerritsen et al, 1997). The margin of spreading plaques represents the most active site, as indicated by a higher density of spongiform pustules of Kogoj and microabscesses of Munro (Beurskens et al, 1989; Van de Kerkhof and Chang, 1989). Studies that demonstrated that ela®n is increased within the psoriatic plaque (Schalkwijk et al, 1993) and signi®cantly more in the region of the center than in the margin (Van de Kerkhof et al, 1991) provide evidence that elastase may also be a crucial factor for the enlargement of psoriatic plaques. Quanti®cation of epidermal cell populations in the center and margin of stable psoriatic plaques, which had not spread the last 2 wk, exhibited no signi®cant changes for in¯ammation and proliferation with a great heterogeneity (Mommers et al, 1999). Further quantitative studies are needed to substantiate the differences between the margin and the center of spreading psoriatic plaques and if the release of HLE is involved in this process. It is conceivable that proteolytic enzymes such as serine proteases are important stimuli for the induction of keratinocyte hyperproliferation. It has been shown that the hyperproliferation of human skin induced by minimal trauma such as removal of horny layers by tape stripping is suppressed by trypsin inhibitors (Denda et al, 1997). In morphologic investigations on human epidermal sheets by electron microscopy, structural changes of keratinocytes in response to HLE such as mitochondrial swelling, intracytoplasmatic lipid inclusions, and building of microtubuli were detected (Ludolph-Hauser et al, 1999). HLE has been shown to detach cultured keratinocytes and to degrade hemidesmosomes and components of the basal membrane in biopsies of human skin (Briggaman et al, 1984; Heck et al, 1990; Glinski et al, 1991; Katayama et al, 1994). These changes indicate that keratinocytes respond actively to HLE. In summary, we were able to demonstrate that HLE can induce hyperproliferation in keratinocytes both in vitro and in vivo. A comparison between murine and human epidermis remains speculative. The concentrations necessary for the hyperproliferative response were similar to those observed in psoriatic lesions, whereas the concentrations of cytokines and growth factors capable of inducing keratinocyte proliferation are largely unknown in the psoriatic epidermis. Experiments with human keratinocytes and human epidermis are in progress to clarify the role of leukocyte elastase as a driving force of keratinocyte proliferation in psoriatic patients. We thank Mrs B. Bargmann and Mrs C. Neumann for their technical assistance. This research was supported by a grant of the Deutsche Forschungsgemeinschaft Ch 37-7/1 and SFB-415.
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