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(The FASEB Journal. 2000;14:565-571.)
© 2000 FASEB

Response of keratinocytes from normal and psoriatic epidermis to interferon-{gamma} differs in the expression of zinc-{alpha}2-glycoprotein and cathepsin D

SAN-HWAN CHEN*, ISTVAN ARANY{dagger},1, NARIN APISARNTHANARAX{dagger}, SRINIVASAN RAJARAMAN{ddagger}, STEPHEN K. TYRING*,{dagger}, TOSHIO HORIKOSHI**, HENRY BRYSK* and MIRIAM M. BRYSK*,{dagger},§,12

* Departments of Dermatology,
{dagger} Microbiology and Immunology,
{ddagger} Pathology, and
§ Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555, USA; and
** Basic Research Laboratory, Kanebo Ltd., Odawara, Kanagawa, 250-0002 Japan

2Correspondence: Department of Dermatology, University of Texas Medical Branch, Galveston, Texas 77555-0783, USA. E-mail: mibrysk{at}utmb.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Psoriasis is a T cell-mediated inflammatory disease characterized by hyperproliferation and by aberrant differentiation. We found cathepsin D and zinc-{alpha}2-glycoprotein, two catalytic enzymes associated with apoptosis and desquamation, to be present in the stratum corneum of the normal epidermis but absent from the psoriatic plaque. Psoriasis is characterized by an altered response to interferon-{gamma} (IFN-{gamma}), including the induction of apoptosis in normal but not in psoriatic keratinocytes, often with opposite effects on gene expression of suprabasal proteins. We found that IFN-{gamma} binding and signaling were attenuated in psoriasis: The IFN-{gamma} receptor, the signal transducer and activator of transcription STAT-1, and the interferon regulatory factor IRF-1 were strongly up-regulated by IFN-{gamma} in normal keratinocytes, but not in psoriatic ones. IFN-{gamma} strongly up-regulated the expression of the catalytic enzymes cathepsin D and zinc-{alpha}2-glycoprotein in normal keratinocytes but down-regulated them in psoriatic ones; the reverse was true of the apoptotic suppressor bcl-2. We believe that the aberrant response to IFN-{gamma} plays a central role in the pathophysiology of psoriasis, particularly the disruption of apoptosis and desquamation.—Chen, S.-H., Arany, I., Apisarnthanarax, N., Rajaraman, S., Tyring, S. K., Horikoshi, T., Brysk, H., Brysk, M. M. Response of keratinocytes from normal and psoriatic epidermis to interferon-{gamma} differs in the expression of zinc-{alpha}2-glycoprotein and cathepsin D.


Key Words: interferon-{gamma} receptors • interferon regulatory factor • interferon signal transducer and activator • epidermal differentiation • apoptosis • bcl-2.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE EPIDERMIS, THE most differentiated epithelium, consists of up to 20 cell layers with distinct morphologies. Its terminal differentiation includes keratinization into a well-formed granular layer and a stratum corneum in which the nuclei disappear and the cells cornify. The outermost squames are then shed in an orderly process of desquamation. Psoriasis is an inflammatory disease characterized by hyperplasia of normally quiescent basal keratinocytes (1) . The psoriatic plaque is characterized by hyperproliferation and by a precocious pattern of terminal differentiation (2) in which a granular layer is not formed and nuclei persist in the stratum corneum. Differences in apoptosis have been reported between normal and psoriatic epidermis. A block in bcl-2 and bcl-x expression is required to initiate apoptosis (3 , 4) . The expression of bcl-2 and bcl-x is aberrant in psoriasis (5 6 7) . Consistent with the abnormalities of the stratum corneum, desquamation is also premature in psoriasis, as the cohesive forces holding together adjacent squames are altered. Many structural and regulatory proteins associated with terminal differentiation are abnormally expressed in psoriasis. In particular, there is a precocious expression of involucrin and transglutaminase (8 , 9) and a strongly reduced expression of filaggrin (2 , 10) .

The level of interferon-{gamma} (IFN-{gamma}) is over 10-fold greater in the psoriatic epidermis than in normal epidermis, as measured by immunocytochemistry (11) and by polymerase chain reaction (PCR) (12) . In evolving psoriatic lesions, activated T-lymphocytes accumulate early and colocalize with lesional keratinocytes expressing ICAM-1 (13) . IFN-{gamma} down-regulates the expression of MHC class II and ICAM-I molecules in psoriatic keratinocytes, whereas it up-regulates them in normal keratinocytes (13) . Whereas IFN-{gamma} promotes growth arrest of normal keratinocytes (14 , 15) , it induces proliferation of psoriatic keratinocytes (16 , 17) . This abnormal response is believed to contribute to the hyperproliferation observed in psoriatic epidermis. Because IFN-{gamma} induces often opposite effects in normal and psoriatic keratinocytes, we believe that IFN-{gamma} plays a central role in the pathophysiology of psoriasis.

For IFN-{gamma} to exert its pleiotropic functions, it must first bind to its receptor (18) . This receptor (IFNGR-1)has been cloned and localized to chromosome 6 (19) . It needs a species-specific accessory factor (IFNGR-2), which has been cloned and localized to chromosome 21 (20) . Both receptor subunits interact to transduce the IFN-{gamma} signal (21) by phosphorylating specific proteins, such as STAT-1, which translocate to the nucleus and bind to cognate cis-acting IFN-{gamma} activation sequences (GAS sites) (22) . The IFN regulatory transcription factor (IRF-1), regulated by STAT-1, binds to an interferon stimulated response element (ISRE) in the promoter region of IFN-{gamma}-inducible genes. Stimulation by IFN-{gamma} has been shown to induce binding of both IRF-1 and STAT-1 proteins to cognate DNA elements in normal but not in psoriatic keratinocytes (23) . The transcription of IRF-1 and STAT-1 and of the IFN-{gamma} receptors in psoriasis has not been investigated.

Cathepsin D (CatD) is an aspartic protease, ubiquitously expressed in mammalian cells, which is thought to play a role in protein catabolism via the degradation of intracellular and extracellular endocytosed proteins (24) . The human enzyme is synthesized as a 52 kDa proenzyme that undergoes subsequent proteolytic processing to produce the intermediate activated 48 kDa form, which is then cleaved into a double chain, the mature 33 kDa form and a 14 kDa fragment (25 , 26) . Antibodies to CatD differ in their recognition of the various isoforms, and conflicts among clinical studies have been ascribed to consequent discrepancies in immunochemical response (27) . Its activation is associated with the lysosomes (28) . In the epidermis, the greatest lysosomal activity is in the granular layer, where most of the intracellular macromolecules are degraded prior to cornification. In the stratum corneum, where the epidermis cornifies, we detected CatD biochemically entirely in its active isoforms and confirmed its presence by immunolocalization (29) . Earlier papers using different antibodies had failed to recognize CatD in the stratum corneum, with one exception (30) . CatD activity was found to be stimulated by IFN-{gamma} in macrophages (31) , but there have been no reports on the effect of IFN-{gamma} on CatD expression in epithelial cells.

Zinc-{alpha}2-glycoprotein (ZAG) is found as a soluble protein in body fluids and in glandular epithelia (32) . A significant association has been observed between ZAG levels and the histological grade of tumors, with higher levels found in well-differentiated tumors than in poorly differentiated ones (33 34 35) . We have found ZAG to be present also in stratified epithelia and cloned it from a library of human epidermal keratinocytes that had been treated with IFN-{gamma} (34) . Its gene expression was higher in the epidermis than in any other stratified epithelium. The epidermal nucleotide sequence was identical to that previously reported for ZAG cloned from prostate (36) and breast (37) . The sequence was also found to be identical to that of desquamin, a glycoprotein that we had earlier isolated from the terminally differentiated epidermis (38) . Consequently, we now refer to the epidermal molecule as ZAG. The expression of ZAG in psoriasis has not been previously examined.

We believe that a defect in IFN-{gamma} binding and signaling is a crucial characteristic of psoriasis, and that the altered response to IFN-{gamma} affects the expression of functional molecules playing a role in terminal differentiation and apoptosis, including the enzymes CatD and ZAG that are found in the stratum corneum. We study the transcription of the IFN-{gamma} receptor (IFNGR-1), its accessory factor (IFNGR-2), and the signaling regulatory factors STAT-1 and IRF-1 in keratinocytes derived from normal and psoriatic epidermis that have been treated with IFN-{gamma}. We compare the effect of IFN-{gamma} on the expression of CatD, ZAG, bcl-2, and involucrin. We also examine by immunohistochemistry the distribution of CatD and ZAG in normal and psoriatic epidermis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Immunohistochemical staining
Human normal and psoriatic lesional skin was obtained by biopsy and fixed in formalin for 24 h and embedded in paraffin. Deparaffinized sections were sequentially incubated with 10 µg/ml of rabbit anti-CatD antibody (IM16; Calbiochem, San Diego, Calif.) and 10 µg/ml of rabbit anti-ZAG antibody (kindly provided by Dr. Okhubo), both for 1 h, then with 1:50 biotinylated goat anti-rabbit IgG (Dako, Santa Barbara, Calif.) for 1 h, and 1:100 avidin-peroxidase conjugate (Dako) for 1 h, with frequent washes in PBS between incubations. The reaction production was visualized by incubation with 0.05% diaminobenzidine and 0.5% H2O2. Controls included omission of primary and secondary antibodies, and substitution of primary antibody with non-immune rabbit serum.

Cell culture
The epidermis from fresh human skin biopsies was separated from the dermis by incubation with trypsin (0.25% of 1:250, in Hanks’ salt solution) overnight at 4°C. A series of punch biopsies (0.2 mm), from five lesional psoriatic patients were similarly incubated in trypsin to dissociate the epidermal cells. All keratinocytes were cultured in keratinocyte growth medium (KGM; Clonetics, San Diego, Calif.) nearly to confluence, subcultured and grown in 25 cm2 flasks till confluence, then switched to KGM supplemented to 2 mM CaCl2 and 10% fetal bovine serum. A day later, IFN-{gamma} (1000 U/ml) was added to some of the cells, whereas untreated cells served as controls. Cells were grown in IFN-{gamma} for 3 days in a 5% CO2 incubator at 37°C.

Quantitative analysis of mRNA levels
Cultured cells were washed with PBS, then scraped from the culture vessels. After low-speed centrifugation, RNA was extracted from the cell pellets using Tri-Reagent (Molecular Research Center, Cincinnati, Ohio). To determine gene transcripts, one µg of RNA was subjected to cDNA synthesis at 42°C for 1 h, using Superscript RNase H- reverse transcriptase (Gibco BRL, Grand Island, N.Y.) and random hexamer priming (Promega, Madison, Wis.) in the presence of deoxynucleotide triphosphates (dNTP; Perkin-Elmer Cetus, Norwalk, Conn.) for the first-strand synthesis. This cDNA mixture was aliquoted, and PCR amplifications were performed under the same conditions using different gene-specific primer pairs. To verify the integrity of the RNA and cDNA for each experimental sample, a separate control amplification using {alpha}-tubulin was included in the PCR runs; {alpha}-tubulin also served as a constitutively expressed internal control, and target gene mRNA levels were normalized to the levels of {alpha}-tubulin. The specificity of PCR was confirmed by gene-specific oligonucleotide hybridization after agarose gel electrophoresis and Southern transfer. We have previously published fuller details of the RNA processing, reverse transcription, design of specific primer pairs, and PCR steps, including appropriate controls (39) .


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunohistochemistry
The immunolocalization of CatD in normal and psoriatic epidermis is illustrated in Fig. 1 . In the normal epidermis, it is expressed in the upper granular layer and throughout the stratum corneum (Fig. 1a ). In contrast, in psoriatic skin, CatD staining is seen in all the viable cell layers but not in the stratum corneum (Fig. 1b ).



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  		Figure 1. The immunolocalization of CatD in normal and psoriatic epidermis. In the normal epidermis, it is expressed in the upper granular layer and throughout the stratum corneum (a). In contrast, in psoriatic skin, CatD staining is seen in all the viable cell layers but not in the stratum corneum (b).

The immunolocalization of ZAG in normal and psoriatic epidermis is illustrated in Fig. 2 . ZAG is present only in the upper granular layer and throughout the stratum corneum in the normal epidermis (Fig. 2a ). In contrast, there is no ZAG staining in any epidermal cell layers in the psoriatic plaque (Fig. 2b ).



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  		Figure 2. The immunolocalization of ZAG in normal and psoriatic epidermis. ZAG is present only in the upper granular layer and throughout the stratum corneum in the normal epidermis (a). In contrast, there is no ZAG staining in any epidermal cell layers in the psoriatic plaque (b).

Gene expression
We cultured keratinocytes from normal and lesional psoriatic epidermis and exposed them for 3 days to IFN-{gamma}, along with untreated controls. Gene expression levels were obtained by reverse transcriptase-PCR for ZAG, CatD, involucrin, bcl-2, and the IFN-{gamma} binding and regulatory factors IFNGR-1, IFNGR-2, STAT-1, and IRF-1, together with {alpha}-tubulin serving as a constitutively expressed internal control. Results, accumulated over three runs, are displayed from densitometer scans of mRNA levels. Gene levels for untreated normal and psoriatic cells are shown in Fig. 3 . The up-regulation by IFN-{gamma} is displayed quantitatively in Fig. 4 as ratios of mRNA levels for treated cells as against untreated ones.



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  		Figure 3. Comparison of mRNA levels (as determined by RT-PCR, normalized to the expression level of {alpha}-tubulin, which served as an internal control) in primary cultures of human keratinocytes derived from normal (striped bar) and psoriatic (black bar) epidermis. Each histogram represents the mean of three runs, with the standard deviation indicated atop each bar.



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  		Figure 4. Effect of IFN-{gamma} treatment on normal and psoriatic epidermis. Ratio of mRNA levels for IFN-{gamma} treated/untreated keratinocytes, normal (striped bar) and psoriatic (black bar). Statistics as in Fig. 3 .

Without IFN-{gamma} treatment, the mRNA levels of CatD and ZAG are much higher in psoriatic than in normal cells, whereas differences in the other genes are less prominent. IFN-{gamma} treatment of normal keratinocytes up-regulates the expression of all but one of the genes, ranging from 11-fold for ZAG to a marginal change for IFNGR2, and down-regulates bcl-2 very substantially. On the other hand, CatD and ZAG are drastically down-regulated by IFN-{gamma} in psoriatic keratinocytes (to levels well below those in the normal cells), whereas bcl-2 is fivefold up-regulated. The other genes undergo changes of some 50% or less, ranging from involucrin on the upside to STAT-1 on the downside.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The psoriatic plaque is abnormal in that the granular and transitional cell layers are attenuated. Organelles, including the nuclei, are normally degraded in these layers; nuclei are retained in the psoriatic stratum corneum. Involucrin is cross-linked by transglutaminase in the granular cells into the cornified envelope (40) and has been localized to the upper spinous and granular layers of the normal epidermis (8) . It is expressed precociously in the psoriatic epidermis (8) . This premature expression has been associated with hyperproliferation (41) . Starting in the granular layer, aspartic proteinases are activated in the lysosomes. As CatD has been implicated in the activation of transglutaminase (42) , it may contribute to catalyzing enzymatic processes during cornification. The cohesive forces holding together adjacent squames of the stratum corneum are altered in psoriasis; this leads to premature desquamation. We have shown biochemically that CatD is present in the stratum corneum of normal skin and participates in its desquamation (29 , 43) . The active forms of CatD immunolocalize to the transition layer and the stratum corneum in the normal epidermis; they do not label the stratum corneum of the psoriatic plaque. CatD, like involucrin, is precociously expressed in psoriatic cells.

Another enzyme that we have shown to participate in desquamation of normal skin is ZAG (38) . We had previously shown that ZAG expresses enzymatic activity (44) and that it plays a role in stratum corneum cohesion (45) . ZAG also has ribonuclease activity (46) and it participates in the destruction of nuclei in terminally differentiating keratinocytes (47) . ZAG immunolocalizes to the transition layer and the stratum corneum in the normal epidermis. Its expression is suppressed in psoriasis, like that of filaggrin (2) . In the normal epidermis, filaggrin immunolocalizes to the granular layer and the lower stratum corneum. Because of its ability to aggregate intermediate filaments, filaggrin is thought to stabilize keratin filaments in the stratum corneum (48) .

Whereas the concentration of IFN-{gamma} is very low in normal skin, it is 10-fold higher in the psoriatic plaque (11) . When IFN-{gamma} is injected into normal skin, it induces a psoriatic-like lesion at the injection site (49) . The epidermis of transgenic mice overexpressing IFN-{gamma} exhibits increased proliferation, epidermal thickening, retention of nuclei, and flaky skin lesions, conditions similar to those observed in psoriasis (50) . Normal differentiating epidermal keratinocytes in culture undergo apoptosis when they are stimulated by IFN-{gamma} (51) . On the other hand, psoriatic keratinocytes fail to undergo apoptosis even when activated by IFN-{gamma} (6) . In the normal epidermis, it is primarily the Langerhans cells that produce and secrete IFN-{gamma}; the increased level of IFN-{gamma} in the psoriatic plaque is derived from activated T cells. By using cultured keratinocytes, free of contaminating cells that secrete IFN-{gamma}, we can directly examine the effect of IFN-{gamma} on epidermal differentiation and apoptosis in normal and psoriatic keratinocytes.

Involucrin is a suprabasal differentiation marker that is prematurely expressed in psoriasis (8) . Its gene expression is quadrupled in normal keratinocytes by IFN-{gamma}, doubled in psoriatic ones. The reduced but still positive up-regulation represents a trade-off between hyperproliferation and the disruption of differentiation. It contrasts with the suppression of CatD and ZAG, which are associated with later stages of differentiation.

Most discussions of cell death have tended to focus on endonucleases, but specific exogenous RNases have also been linked to these processes. Onconase, a cytotoxic ribonuclease, kills tumor cells in vivo and in vitro (52) . Similarly, bovine seminal ribonuclease destroys thyroid epithelial tumors (53) . The association of RNase activity with senescence has been reported for the RNase RNS2 (54) . RNases can inhibit the cell’s translational machinery, hence the biosynthesis of new proteins. This impairment can lead to organelle degradation and ultimate cell death. In the epidermis in vivo, the destruction of nuclei and other organelles occurs in the transition zone between the granular layer and the stratum corneum (55) . As ZAG is first expressed in this region and has RNase activity (46) , we suspect that it has some connection to these processes. Nuclei are degraded when epidermal keratinocytes are grown in the presence of ZAG (47) or of IFN-{gamma} (51) . IFN-{gamma} stimulates ZAG expression in differentiating keratinocytes (56 , 57) . We have found here that ZAG expression is up-regulated 11-fold by IFN-{gamma} in normal keratinocytes, but is drastically down-regulated by it in psoriatic keratinocytes.

Apoptosis can be initiated by IFN-{gamma} in T cells (58) . CatD activity is stimulated by IFN-{gamma} in macrophages (31) . PC-12 cells overexpressing CatD after transfection exhibit an increase in apoptosis (59) . Antisense RNA to CatD, as well the CatD inhibitor pepstatin A, protects HeLa cells from apoptosis by IFN-{gamma} (60) . These results associate IFN-{gamma} and CatD with promotion of apoptosis. IFN-{gamma} triggers apoptosis in terminally differentiated normal epidermal keratinocytes (51) . Psoriatic keratinocytes, however, fail to undergo apoptosis even when activated by IFN-{gamma} (6) . Consistently, we found that CatD expression was up-regulated sixfold by IFN-{gamma} in normal keratinocytes, but comparably down-regulated in psoriatic keratinocytes. Contrariwise, we found that the expression of bcl-2, which blocks apoptosis, was up-regulated fivefold by IFN-{gamma} in psoriatic keratinocytes, but similarly down-regulated in normal cells.

As psoriasis is characterized by an altered response of epidermal keratinocytes to IFN-{gamma}, it is logical to expect the signaling pathways transduced by IFN-{gamma} to also be aberrant. The receptor subunits IFNGR-1 and IFNGR-2 interact to transduce the IFN-{gamma} signal (61) . Immunochemical studies of normal and psoriatic skin have led to conflicting conclusions as to the distribution of IFNGR-1: one (62) found staining to be even throughout the viable layers in normal epidermis, but largely confined to the lower layers in psoriatic epidermis; another (63) reported staining limited to the basal cells in normal epidermis, but variably extending to the suprabasal layers as well in psoriatic skin. The expression of IFNGR-2 has not been previously studied in the epidermis. We found that exogenous IFN-{gamma} up-regulated IFNGR-1 gene expression in normal keratinocytes fourfold, but only weakly in psoriatic keratinocytes. It had no effect on the expression levels of IFNGR-2.

After binding to its receptor, IFN-{gamma} phosphorylates signal transducers and activators of transcription (STATs), which then migrate to the nucleus (64) where they induce the expression of many genes that mediate cellular responses (22) . The IFN-inducible proteins include a family of interferon regulatory factors (65) , of which the anti-oncogene IRF-1 is one of the best-characterized (66) . IFN-inducible genes contain special DNA sequences in their promoter region, designated as ISRE and GAS elements (67) , which bind STATs and/or IRF-1. We have noticed that ZAG and CatD contain such putative binding sequences. Binding of IRF-1 and STAT-1 to cognate DNA elements after stimulation by IFN-{gamma} is induced in normal but not in psoriatic keratinocytes (23) . We found that treatment with IFN-{gamma} doubled gene expression of STAT-1 in normal keratinocytes, but halved it in psoriatic ones. There was an eightfold up-regulation of IRF-1 in normal keratinocytes, but little response to IFN-{gamma} in psoriatic ones.

The focus of this study has been on our conviction of the central role of the aberrant response of the psoriatic plaque to the IFN-{gamma} stimulus. We found that IFN-{gamma} binding and signaling were attenuated in psoriasis: The IFN-{gamma} receptor and regulatory factors were strongly up-regulated by IFN-{gamma} in normal keratinocytes, but were insensitive to IFN-{gamma} in psoriatic keratinocytes. We demonstrated by immunolocalization that the catalytic enzymes CatD and ZAG, associated with apoptosis and desquamation, were present in the stratum corneum of normal epidermis but absent from the stratum corneum of the psoriatic plaque. We found that CatD and ZAG were strongly up-regulated by IFN-{gamma} in normal keratinocytes, but suppressed by it in psoriatic keratinocytes. Consistently, the apoptotic suppressor bcl-2 had just the opposite response in both cases. The decreased apoptosis in response to IFN-{gamma} in psoriatic keratinocytes and the failure of IFN-{gamma} to induce the growth suppressors IRF-1 and STAT-1 concur with the pathophysiology of this disease.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the supply of ZAG polyclonal antibody by Prof. Iwao Ohkubo (Shiga University of Medical Science, Seta, Japan).


   FOOTNOTES
 
1 These two authors contributed equally to this work.

Received for publication April 23, 1999. Revised for publication October 6, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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