The intracellular 52-kd RoSSA autoantigen in keratinocytes is up-regulated by tumor necrosis factor ╨Ю┬▒ via tumor necrosis factor receptor I.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 52, No. 2, February 2005, pp 531–538 DOI 10.1002/art.20851 © 2005, American College of Rheumatology The Intracellular 52-kd Ro/SSA Autoantigen in Keratinocytes Is Up-Regulated by Tumor Necrosis Factor ␣ Via Tumor Necrosis Factor Receptor I Velia Gerl,1 Björn Hostmann,1 Christa Johnen,2 Aderajew Waka,1 Markus Gerl,1 Frank Schumann,2 Rolf Klein,1 Andreas Radbruch,3 and Falk Hiepe1 Objective. Previous studies have shown that the nuclear Ro/SSA autoantigens involved in photosensitive cutaneous lupus manifestations are regulated by ultraviolet B (UVB) irradiation. UVB exposure triggers the release of tumor necrosis factor ␣ (TNF␣) from keratinocytes in the epidermis and from mast cells in the dermis. The present study aimed to characterize the effect of TNF␣ on messenger RNA (mRNA) and protein expression of the intracellular 52-kd Ro/SSA autoantigen in primary human keratinocytes and to elucidate the TNF␣ receptor (TNFR) signaling pathways mediating this effect. Methods. Expression of 52-kd Ro/SSA mRNA in primary human keratinocytes was investigated by quantitative real-time polymerase chain reaction (LightCycler system) using GAPDH as the housekeeping gene. Expression of 52-kd Ro/SSA protein was studied by flow cytometry after staining intracellular protein with IgG purified from an anti–52-kd Ro/SSA–positive serum. TNFR function was assessed by culturing cells in the presence and absence of neutralizing antibodies directed against the TNFR subunits TNFRI and TNFRII. Results. TNF␣-induced up-regulation of 52-kd Ro/SSA mRNA expression peaked at 4 hours, followed by up-regulation of intracellular 52-kd Ro/SSA protein expression at 24 hours, independently of apoptosis. Between different donors, a high variability of both constitutive expression levels and TNF␣ -induced changes in 52-kd Ro/SSA mRNA and protein expression was observed. The up-regulatory effect of TNF␣ on 52-kd Ro/SSA mRNA and protein expression was inhibited by anti-TNFRI antibodies but enhanced by antiTNFRII antibodies. Conclusion. The finding that TNF␣ up-regulates 52-kd Ro/SSA expression in keratinocytes via TNFRI suggests that it may play a role in the pathogenesis of anti-Ro/SSA–associated cutaneous lupus erythematosus. Autoantibodies directed against the small RNP particle Ro/SSA are mainly associated with connective tissue diseases, especially Sjögren’s syndrome, systemic lupus erythematosus (SLE), subacute cutaneous lupus erythematosus (SCLE), neonatal lupus syndromes, undifferentiated connective tissue disease, and myositis, and are occasionally detected in other autoimmune diseases, such as systemic sclerosis, rheumatoid arthritis, primary biliary cirrhosis, and autoimmune hepatitis (1–4). Anti-Ro/SSA antibodies are often detected together with anti-La/SSB antibodies in sera and are strongly associated with photosensitive cutaneous eruptions in SCLE (5,6), neonatal lupus (7), and other forms of lupus. These autoantibodies are therefore thought to play a key role in the pathogenesis of these skin manifestations, as suggested by the characteristic deposition of immunoglobulins and complement at the epidermal junction. Lee and coworkers experimentally reproduced this pattern of cutaneous IgG deposition by infusing purified anti-Ro/SSA autoantibodies into human skin– grafted mice (8). However, since the targets of these Supported by grants from the Deutsche Forschungsgemeinschaft (SFB 421, C4) and the Competence Network Rheumatology (C3.5). 1 Velia Gerl, PhD, Björn Hostmann, MD, Aderajew Waka, Markus Gerl, MD, Rolf Klein, PhD, Falk Hiepe, MD, PhD: Charité Hospital, Center for University Medicine in Berlin, and German Rheumatism Research Center, Berlin, Germany; 2Christa Johnen, PhD, Frank Schumann: Charité Hospital, Center for University Medicine in Berlin, Berlin, Germany; 3Andreas Radbruch, PhD: German Rheumatism Research Center, Berlin, Germany. Address correspondence and reprint requests to Falk Hiepe, MD, PhD, Charité Hospital, Center for University Medicine in Berlin, Department of Medicine (Rheumatology and Clinical Immunology), Schumannstrasse 20/21, D-10117 Berlin, Germany. E-mail: falk.hiepe@ charite.de. Submitted for publication July 26, 2003; accepted in revised form November 15, 2004. 531 532 GERL ET AL autoantibodies (52- and 60-kd Ro/SSA autoantigens) are located in intracellular compartments, including the nucleus and the cytoplasm (for review, see ref. 9), these intracellular autoantigens must be accessible to the autoantibodies in order to be pathogenic. Several studies revealed that Ro/SSA autoantigens are expressed on the surface of keratinocytes, which are major sites of immunologic damage in photosensitive cutaneous lupus after exposure to stress factors such as ultraviolet (UV) light (10–13), estradiol (14,15), cytotoxic prostaglandins (16), viral infections (17), oxidative stress (18), heat shock (19), and phorbol 12myristate 13-acetate (19). Ro/SSA autoantigens are also present in larger apoptotic blebs (20). Antibodydependent cell-mediated cytotoxicity (ADCC) is an important mechanism by which anti-Ro/SSA autoantibodies can induce keratinocyte cytotoxicity after UVB exposure (21–23). Previous studies have shown that tumor necrosis factor ␣ (TNF␣) can enhance anti-Ro/SSA antibody binding to the surface of keratinocytes (24). Interestingly, UVB irradiation triggers the release of TNF␣ from keratinocytes in the epidermis (25,26) and from mast cells in the dermis (27). Therefore, TNF␣ is thought to be a key mediator in the pathogenesis of both anti-Ro/SSA–associated lupus and photosensitive cutaneous lupus. The objective of the present study was therefore to elucidate the role of TNF␣ in messenger RNA (mRNA) and protein expression of the intracellular 52-kd Ro/SSA autoantigen in primary human keratinocytes. To better understand the regulatory mechanisms underlying 52-kd Ro/SSA autoantigen expression, we also investigated the impact of the TNF␣ receptors I and II (TNFRI and TNFRII) on the up-regulatory effect of TNF␣. MATERIALS AND METHODS Cell culture. Primary human keratinocytes were isolated from the skin of healthy donors and cultured at 1 ⫻ 104 cells/cm2 in 75-cm2 culture flasks containing serum-free keratinocyte growth medium (EpiLife; Tebu-Bio, Offenbach, Germany) supplemented with 60 M CaCl2, EpiLife defined growth supplement (Tebu-Bio), 100 units/ml penicillin, and 100 g/ml streptomycin (Gibco, Paisley, UK) at 37°C in an atmosphere of 5% CO2 in air (28). Cell treatment and analysis of apoptosis. Primary human keratinocytes grown to 80–90% confluence were treated for 1, 2, 4, 8, and 24 hours with 4 ng/ml of human recombinant TNF␣ (Sigma, St. Louis, MO) as described previously (24). Cells were harvested at the specified sampling times, including floating cells detached during the apoptotic process. Intact cells were detached from the flask bottom using Accutase (PAA Laboratories, Linz, Austria), then washed with phosphate buffered saline (Gibco) and stained with annexin V–fluorescein isothiocyanate (FITC) (Becton Dickinson, Heidelberg, Germany) and propidium iodide as recommended by the manufacturer. A FACSCalibur flow cytometer (Becton Dickinson) was used to identify apoptotic cells, which tested positive for annexin V and either negative or positive for propidium iodide. The apoptotic effect of TNF␣ was calculated as the difference between the percentage of apoptotic cells in the TNF␣-treated cell population and the percentage of apoptotic cells in the untreated controls. Since the maximum range of error in repeated independent experiments was ⬃10% (data not shown), all differences ⬎10% were defined as a significant effect. All experiments were carried out in duplicate; mean ⫾ SEM values are indicated. Quantitative real-time polymerase chain reaction (PCR). To keep degraded ribonucleotides from contaminating the samples, annexin V–FITC–positive apoptotic cells were sorted out using a FACSVantage cell sorter (Becton Dickinson). Messenger RNA was isolated from the nonapoptotic cell population using the Oligotex mRNA direct kit (Qiagen, Hilden, Germany). Real-time PCR was performed with a LightCycler (Roche Diagnostics, Mannheim, Germany). Messenger RNA for 52-kd Ro/SSA was amplified and quantitatively analyzed using a 1-step mRNA amplification kit for hybridization probes (Roche Diagnostics). PCR reactions were set up in samples with a final volume of 17 l, including 10 g mRNA from samples or standards, 0.41 M primers, and 2 M hybridization probes. The amplification program for 52-kd Ro/SSA included reverse transcription at 55°C for 15 minutes and initial denaturation at 95°C for 3 minutes, followed by 45 cycles of denaturation at 94°C for 30 seconds, primer annealing at 59°C for 13 seconds, and extension at 72°C for 20 seconds. Fluorescence of the hybridization probe was measured after the annealing step. The specificity of amplification was determined by melting curve analysis. The relative amount of 52-kd Ro/SSA transcripts was normalized to the number of human GAPDH transcripts found in the same reaction tube. Cloned Ro/SSA (29) and GAPDH complementary DNA were used as the quantification standards. Messenger RNA for 52-kd Ro/SSA was amplified using the primer sequences 5⬘-AGCAAAAAAACTTCCTGG TTG-3⬘ (forward) and 5⬘-GAGCTGTGGCACCTTCGATC3⬘ (reverse). GAPDH amplification was carried out using the primer sequences 5⬘-GCAGGGGGGAGCCAAAAG-3⬘ (forward) and 5⬘-AGGCAGGGATGATGTTCTGGA-3⬘ (reverse). The transcripts were detected using fluorescent hybridization probes provided by the manufacturer (TIB-MOLBIOL, Berlin, Germany). Each PCR assay was run in triplicate. The 52-kd Ro/SSA and GAPDH copy numbers were estimated from the threshold amplification cycle numbers using the software supplied with the LightCycler. Intracellular staining of 52-kd Ro/SSA protein. In order to avoid false results due to apoptotic protein degradation, intracellular protein expression was investigated in keratinocytes exhibiting high viability using only the intact cells attached to the flask bottom. Cells were fixed and permeabilized using the Fix & Perm cell permeabilization kit (An-derGrub Bioresearch, Kaumberg, Austria). Specific IgG antibodies purified from the serum of patients with only anti–52-kd UP-REGULATION OF 52-kd Ro/SSA BY TNF␣ VIA TNFRI Ro/SSA antibody reactivity were used to detect intracellular 52-kd Ro/SSA expression. Sole reactivity to 52-kd Ro/SSA was confirmed by immunoblotting using Molt-4 cell extract (30). IgG binding was detected using a phycoerythrin-labeled antihuman IgG (mouse clone G18-145; PharMingen, San Diego, CA) as a secondary antibody. The samples were incubated with the antibodies for 30 minutes at room temperature. Specific binding of the anti-Ro/SSA antibody was confirmed by blocking the samples with the Ro/SSA affinity-purified antigen (Immunovision, Springdale, AR). Stained cells were enumerated using a FACSCalibur flow cytometer. Apoptotic keratinocytes were excluded based on their light-scatter characteristics. Relative changes in expression of 52-kd Ro/SSA antigen in response to TNF␣ treatment were calculated as the ratio of specific antigen staining of treated versus untreated cells, as described elsewhere (31). Studies of TNFRI and TNFRII function. The role of the TNF␣ receptors TNFRI and TNFRII in the up-regulation of 52-kd Ro/SSA autoantigen expression by TNF␣ in human keratinocytes was investigated by pretreating the cells with 0.5 g/ml neutralizing monoclonal antibodies directed against human soluble TNFRI (sTNFRI) (IgG1 clone 16803; R&D Systems, Minneapolis, MN) and/or human sTNFRII (IgG2a clone 22221.311; R&D Systems) for 30 minutes and by further incubating the cells with these antibodies in combination with TNF␣ for up to 24 hours. Intracellular 52-kd Ro/SSA protein and mRNA expression were measured as described above. Statistical analysis. Nonparametric Mann-Whitney tests for comparison of two independent groups were run on GraphPad Prism 3.02 for Windows (GraphPad Software, San Diego, CA). Values were calculated as the mean ⫾ SEM. P values less than 0.05 were defined as statistically significant. RESULTS TNF␣-induced up-regulation of 52-kd Ro/SSA mRNA transcription. Studies of 52-kd Ro/SSA mRNA expression in annexin V–negative (nonapoptotic) human epidermal keratinocytes were carried out by quantitative real-time PCR. Constitutive expression of Ro/SSA was low (2.3 ⫻ 102 copies of 52-kd Ro/SSA mRNA). Cells from 5 donors were treated with TNF␣ to determine the effect of TNF␣ on 52-kd Ro/SSA mRNA expression over time. Ro/SSA expression data were normalized to the number of GAPDH mRNA transcripts. We observed a time-dependent increase in 52-kd Ro/SSA mRNA expression, which peaked at 4 hours. The upregulatory effect of TNF␣ was strong in 2 of the 5 donors, weak in 2 donors, and absent in 1 donor. At the 24-hour sampling time, 52-kd Ro/SSA mRNA expression had decreased sharply and was nearly as low as that in the unstimulated controls (Figure 1). Encouraged by these findings, we investigated 52-kd Ro/SSA mRNA expression in keratinocytes from another 4 donors. Variable degrees of TNF␣-induced up-regulation of 52-kd Ro/SSA mRNA expression were observed at 4 533 Figure 1. Transcription of 52-kd Ro/SSA mRNA up-regulated by tumor necrosis factor ␣ (TNF␣). TNF␣ induced a time-dependent increase in 52-kd Ro/SSA mRNA expression, which peaked at 4 hours. After longer periods of incubation, the expression rate decreased rapidly, falling almost to the level measured in the unstimulated controls. The ratio of 52-kd Ro/SSA mRNA to GAPDH was calculated as the mean of triplicate measurements. The extent of mRNA regulation varied from donor to donor. Strong up-regulation was found in 2 donors (solid circles and open squares), weak up-regulation in another 2 donors (open diamonds and diamonds with an X), and no response in 1 donor (solid triangles). Untreated keratinocytes did not reveal any effects (results not shown). hours. The up-regulatory effect of TNF␣ was strong in 3 of the 9 donors studied (Ro/SSA to GAPDH ratios of 12.8, 24.5, and 38.5), weak in 5 donors (ratios of 3.67, 3.76, 3.05, 4.86, and 2.65), and absent in 1 donor (ratio of 0.95). TNF␣-induced up-regulation of intracellular 52-kd Ro/SSA protein expression. To determine whether up-regulation of 52-kd Ro/SSA mRNA leads to up-regulation of 52-kd Ro/SSA protein, we analyzed samples from 6 additional donors by flow cytometry using anti–52-kd Ro/SSA IgG. As expected, these tests showed that constitutive intracellular expression of 52-kd Ro/SSA protein occurs in primary human keratin- 534 GERL ET AL Figure 2. Slight up-regulation of intracellular 52-kd Ro/SSA autoantigen expression in primary human keratinocytes by tumor necrosis factor ␣ (TNF␣). The cells were cultured with or without TNF␣ for up to 24 hours, fixed, permeabilized, and stained for autoantigen expression. The stained cells were evaluated by fluorescence-activated cell sorting analysis. The living cell fraction was gated according to light-scatter characteristics (results not shown). The staining results for a representative sample (donor 1) are presented as a fluorescence histogram showing the 52-kd Ro/SSA fluorescence of untreated cells (thick black line) and TNF␣-treated cells (thick gray line). 52-kd Ro/SSA stained in the presence of the blocking autoantigen (thin gray line) and fluorescence-conjugated secondary antibodies alone (antihuman IgG–phycoerythrin; thin black line) were used as controls. Mean fluorescence intensities are given. ocytes. Positive staining was neutralized by the affinitypurified Ro/SSA antigen and was therefore judged to be specific. TNF␣ slightly up-regulated 52-kd Ro/SSA protein expression in 4 of 6 donors (Figure 2 and Table 1). Up-regulation of antigen expression was identified as a fluorescence shift in the whole cell population. The baseline level and extent of up-regulation of 52-kd Ro/SSA expression naturally varied from donor to donor. Western blot analyses performed using keratinocyte protein extracts from 2 donors also revealed weak up-regulation of 52-kd Ro/SSA protein expression (data Table 1. Expression of 52-kd Ro/SSA protein in primary human keratinocytes from different donors* Donor Untreated cells TNF␣-treated cells RCE 1 2 3 4 5 6 6.81 ⫾ 0.3 8.25 ⫾ 0.4 14.74 ⫾ 0.1 11.92 ⫾ 4.3 1.05 ⫾ 0.5 0.27 ⫾ 0.0 9.15 ⫾ 0.3 15.25 ⫾ 0.2 9.26 ⫾ 1.1 9.65 ⫾ 0.1 2.36 ⫾ 0.2 1.2 ⫾ 0.0 1.34 1.85 0.63 0.81 2.25 4.44 * Values are the mean ⫾ SEM relative change in mean fluorescence intensity of the positive stained probe minus the control antibody for untreated (baseline) and tumor necrosis factor ␣ (TNF␣)–treated cells. The relative change in expression (RCE) of 52-kd Ro/SSA protein was calculated from protein expression in treated cells divided by protein expression in untreated cells as described in Materials and Methods (values ⬎1 ⫽ up-regulation; values ⬍1 ⫽ down-regulation). Figure 3. Example of the cytometric analysis of the inhibitory effect of neutralizing monoclonal antibodies against tumor necrosis factor ␣ receptor I (anti-TNFRI) on the up-regulation of intracellular 52-kd Ro/SSA protein expression in primary human keratinocytes cultured in the presence of tumor necrosis factor ␣ (TNF␣) for 24 hours. The observed up-regulation was inhibited by pretreating the cells with anti-TNFRI. Results for a representative sample are shown. MFI ⫽ mean fluorescence intensity; PE ⫽ phycoerythrin. not shown). Like flow cytometry, Western blotting revealed variable degrees of constitutive 52-kd Ro/SSA protein expression and up-regulation. TNF␣-induced 52-kd Ro/SSA up-regulation in primary human keratinocytes is mediated by TNFRI signaling and inhibited by TNFRII signaling. To define the roles of TNFRI and TNFRII in TNF␣-induced up-regulation of 52-kd Ro/SSA autoantigen expression, we first reproduced the constitutive surface expression of both TNF␣ receptor subunits. These findings confirmed those of previous studies (32,33). Neutralizing monoclonal antibodies directed against TNFRI inhibited TNF␣-induced up-regulation of intracellular 52-kd Ro/SSA mRNA and protein expression, whereas antiTNFRII antibodies enhanced TNF ␣ -induced upregulation of 52-kd Ro/SSA protein expression (Figures 3 and 4). Treatment of the cells with a combination of anti-sTNFRI and anti-sTNFRII in the presence of TNF␣ resulted in the strongest inhibition of 52-kd Ro/SSA mRNA expression. Significant apoptosis in human keratinocytes not induced by TNF␣. Flow cytometric analysis of primary human keratinocytes stained with annexin V revealed UP-REGULATION OF 52-kd Ro/SSA BY TNF␣ VIA TNFRI Figure 4. Anti-TNFRI antibodies inhibit TNF ␣ -induced upregulation of intracellular 52-kd Ro/SSA mRNA and protein expression in primary human keratinocytes from different donors. The cells were cultured with TNF␣ in the presence or absence of neutralizing monoclonal antibodies directed against TNFRI (␣R⌱) and/or TNFRII (␣R⌱⌱). Neutralization of TNFRII enhanced the up-regulatory effect of TNF␣. Expression of 52-kd Ro/SSA autoantigen was calculated in relation to GAPDH for mRNA and in terms of the MFI for protein. The mRNA values represent the mean and SEM of triplicate measurements, and the protein values are the mean and SEM of duplicate measurements. Messenger RNA and protein expression were determined using cells from different donors. The results were reproduced in 3 serial experiments. See Figure 3 for definitions. the presence of apoptotic cells in all untreated in vitro cultures at 24 hours. The degree of apoptosis varied widely (mean ⫾ SEM 39.8 ⫾ 6.9% [range 13–76%]; n ⫽ 10). After incubating the cells with TNF␣ for up to 24 hours, no significant enhancement of apoptosis was found in 6 of 10 samples, and an antiapoptotic effect was detected in 1 of 10 samples. In 3 samples, expansion of the apoptotic cell fraction was observed after cultivation with TNF␣. The mean ⫾ SEM rate of apoptosis in TNF␣-treated keratinocytes was 40.8 ⫾ 6.8% (range 16–87%; n ⫽ 10). The TNF␣-treated cells and untreated controls did not differ significantly with regard to apoptosis. DISCUSSION Since previous investigations have shown that the proinflammatory cytokine TNF␣ enhances surface expression of 52-kd Ro/SSA (24), the present study aimed to clarify the effect of TNF␣ on the regulation of the 52-kd Ro/SSA autoantigen. The present study is the first to demonstrate that TNF␣ is able to induce the upregulation of 52-kd Ro/SSA mRNA expression in hu- 535 man keratinocytes, and that this effect is followed by an increase in 52-kd Ro/SSA synthesis at the protein level. In contrast to 52-kd Ro/SSA surface expression, which appeared within a maximum of 2 hours, mRNA was up-regulated within 4 hours followed by an intracellular protein increase 24 hours after TNF␣ treatment. This suggests that different mechanisms might underlie antigen expression and translocation and that these processes can occur independently of each other. However, the consequences of the up-regulation of intracellular autoantigen expression and its role in pathogenesis have yet to be investigated. Consistent with reported findings, our data lead us to speculate that TNF␣ might be a mediator of the anti-Ro/SSA antibody–associated lesions occurring in photosensitive cutaneous lupus. UV exposure triggers the release of TNF␣ from keratinocytes (25,26) and mast cells (27). The extent to which TNF␣ secretion can be induced by other factors or other pathways is unknown. The enhancement of 52-kd Ro/SSA expression in keratinocytes may contribute to the development of cutaneous lesions through ADCC (21–23) or complement-dependent cytotoxicity (34). SCLE patients tend to develop such autoimmune pathologies, as reflected by the higher susceptibility of keratinocytes from lupus patients to UV irradiation– induced ADCC compared with that of keratinocytes from normal controls (21). Among other factors, this may be related to the strong association of the ⫺308A polymorphism of the TNF␣ promoter with SLE (35,36) and SCLE (37). The ⫺308A polymorphism was recently shown to be associated with increased TNF␣ accumulation in primary fibroblast cultures and skin from SCLE patients. Other factors may also contribute to TNF␣ production, since SCLE cells secrete more TNF␣ than healthy cells, even when matched for TNF␣ genotype (38). Further, there are indications that keratinocytes derived from lupus patients with photosensitive cutaneous lesions may express more 52-kd Ro/SSA than keratinocytes from healthy donors (34,39). Therefore, it is also conceivable that keratinocytes from lupus patients are more susceptible to TNF␣ in terms of mRNA and protein expression of 52-kd Ro/SSA. In our study, human keratinocytes from healthy donors exhibited a broad variability of baseline and TNF␣-induced Ro/SSA expression. Further investigation of the TNF␣ effect on 52-kd Ro/SSA expression in keratinocytes from SLE patients would be of interest to determine whether this higher susceptibility might be a part of the pathogenetic mechanism in cutaneous lupus lesions. 536 Consequently, TNF␣ blockade might be an effective approach to treating the skin lesions associated with photosensitive cutaneous lupus. If TNF␣ blocking agents are effective in treating photosensitive cutaneous lupus erythematosus lesions, this could confirm that TNF␣ plays a role in the pathogenesis of these lesions. Infliximab, a monoclonal anti-TNF␣ antibody, did in fact lead to an impressive response in one of our patients, a woman with SCLE that was refractory to all conventional drugs (40). Up-regulation of 52-kd and 60-kd Ro/SSA mRNA and protein expression in response to estradiol, which also plays a role in lupus, has already been demonstrated in human keratinocytes and in human cancer cells of the MCF-7 line (41). Since UVB radiation triggers the release of TNF␣, it could lead to up-regulation of Ro/SSA mRNA expression as well. However, Szegedi et al could not find any changes in Ro/SSA mRNA expression in keratinocytes of the HaCaT cell line that were exposed to UV irradiation (42). The most probable explanation for this discrepancy with our results is that Szegedi et al measured mRNA expression after 24–72 hours of UVB radiation, which was too late for detection of changes in Ro/SSA mRNA. Our results clearly demonstrate that the up-regulation of 52-kd Ro/SSA is a fast and transient response, peaking at 4 hours and no longer detectable after 8 hours. The fact that we used the real-time PCR technique that was sensitive enough to detect slight changes in weakly expressed genes (⬍2.5 ⫻ 102 molecules) such as 52-kd Ro/SSA is another important difference. TNF␣ signaling is mediated by both TNFRI, which bears an intracellular death domain, and TNFRII. The present study revealed that the up-regulation of 52-kd Ro/SSA by TNF␣ is mediated by TNFRI signaling and inhibited by TNFRII signaling. Internalization of TNF␣ –TNFR complexes, which has been shown to occur when the ligand binds to keratinocytes, could theoretically be responsible for the transient up-regulation of 52-kd Ro/SSA mRNA (43), because this could lead to termination of TNF␣ signaling and degradation of Ro/SSA mRNA. However, we demonstrated that short-term up-regulation of the autoantigen was sufficient for induction of the protein, which was detectable within 24 hours of TNF␣ treatment. Although the mean up-regulation of mRNA was relatively high, only slight up-regulation of the protein was observed in different donors. One could speculate that expression of the protein is regulated at the translational level or that mRNA is degraded and not all molecules are translated. However, because cell material was lim- GERL ET AL ited, we were unable to analyze 52-kd Ro/SSA expression in keratinocytes from the same donor at both the transcriptional and translational levels. TNF␣ activates several intracellular pathways that can lead to either apoptosis or cell survival. There is evidence suggesting that TNFRII can influence apoptotic signaling, which is mainly promoted by TNFRI. This was demonstrated in T lymphocytes and Jurkat cells in which TNF␣-induced caspase activation mediated by stimulation of TNFRI was potentiated by stimulation with TNFRII (44). TNF␣-specific apoptosis did not occur in most of our samples. Thus, the regulatory intracellular pathway of TNF␣-mediated modulation of 52-kd Ro/SSA expression occurs via TNFRI without the induction of apoptosis. Although both processes are regulated by the same molecules at the receptor level, they may have different intracellular regulatory mechanisms. The unresponsiveness of human keratinocytes to TNF␣ with regard to apoptosis induction has already been shown (33,45). In another study, the investigators postulated that TNF␣mediated apoptosis is counteracted by constitutively expressed antiapoptotic proteins (46). TNFRI-induced apoptosis has been shown to include the activation of FADD/MORT1 (47–49), which in turn recruits FLICE/caspase 8 (47,50). This cysteine protease triggers a proteolytic cascade and the activation of downstream caspases. Another pathway induced by TNF␣ includes the activation of human NF-B (51), a factor that activates the transcription of various genes. In knockout mice, NF-B was shown to be a downstream effector that plays a critical role in protecting cells from TNF␣-induced apoptosis (52). This pathway could be responsible for the unresponsiveness of human keratinocytes to TNF␣-induced apoptosis and may also play a role in the apoptosis-independent regulation of 52-kd Ro/SSA expression seen in our experiments. In conclusion, TNF␣ is able to up-regulate 52-kd Ro/SSA expression in human keratinocytes via TNFRI and to inhibit it via TNFRII. This suggests a possible involvement of TNF␣ in the pathogenesis of anti-Ro/ SSA–associated cutaneous lupus lesions, which will be elucidated in further experiments. TNF␣ targeting should therefore be considered when developing future strategies for the treatment of photosensitive cutaneous lupus. REFERENCES 1. Dorner T, Held C, Trebeljahr G, Lukowsky A, Yamamoto K, Hiepe F. Serologic characteristics in primary biliary cirrhosis UP-REGULATION OF 52-kd Ro/SSA BY TNF␣ VIA TNFRI 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. associated with sicca syndrome. Scand J Gastroenterol 1994;29: 655–60. 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