Regulation of hyaluronan-stimulated VCAM-1 expression in murine renal tubular epithelial cells

Aaron Schawalder1, Beat Oertli1, Beatrice Beck-Schimmer1 and Rudolf P. Wüthrich1,2

1 Physiological Institute, University of Zürich-Irchel and 2 Division of Nephrology, Department of Medicine, University Hospital, Zürich, Switzerland

Correspondence and offprint requests to: Rudolf P. Wüthrich, Division of Nephrology, University Hospital, Rämistrasse 100, CH-8091 Zürich, Switzerland.



   Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background. Cytokines stimulate the expression of the adhesion molecule VCAM-1 in renal tubular epithelial cells. We have recently shown that VCAM-1 can also be upregulated by low molecular weight breakdown products of the matrix constituent hyaluronan (HA) (J Immunol 1998; 161: 3431–3437). The mechanisms of VCAM-1 expression in response to HA remain to be defined.

Methods. Using a defined mouse cortical tubular (MCT) cell line we investigated the effect of protein kinase C (PKC) and tyrosine kinase (TK) inhibition on the HA-stimulated VCAM-1 expression by cell ELISA and RT–PCR or Northern blotting. Furthermore, we examined the effect of PKC and TK inhibition on NF-{kappa}B.

Results. We found that the PKC inhibitor GF109203X (acting on conventional, novel and atypical isoforms) inhibited the HA-stimulated VCAM-1 expression in MCT cells dose-dependently up to 90%, whereas chelerythrine (acting on conventional and novel isoforms) had no effect. Downregulation of PKC with PMA did not prevent the HA-stimulated VCAM-1 expression, suggesting that Ca2+- and diacylglycerol-independent (atypical) isoforms of PKC are involved. The TK inhibitor genistein also inhibited the HA-stimulated VCAM-1 expression at the mRNA and protein level up to 70%. Interestingly, the HA-stimulated nuclear translocation of NF-{kappa}B could not be prevented with GF109203X and genistein.

Conclusion. These data demonstrate that the HA-stimulated VCAM-1 expression in MCT cells involves PKC and TK pathways. The absence of an effect of PKC and TK inhibitors on the nuclear translocation of NF-{kappa}B suggests that additional transcription factors are involved for VCAM-1 expression.

Keywords: hyaluronan; ICAM-1; protein kinase C; tyrosine kinase; VCAM-1



   Introduction
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 Abstract
 Introduction
 Methods
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The mechanisms that regulate the expression of adhesion molecules are complex. Numerous studies have documented that pro-inflammatory cytokines and bacterial cell wall products markedly enhance the expression of ICAM-1 and VCAM-1 by endothelial and epithelial cells [1,2]. Additional stimulatory factors have been identified, including products of the coagulation [3] and complement cascade [4], and reactive oxygen species [5]. We recently found that fragments of the extracellular matrix component hyaluronan (HA) could also stimulate the expression of ICAM-1 and VCAM-1 by epithelial and endothelial cells [6].

The composition of the promoter region of adhesion molecule genes is felt to be critical in determining the expression of the various adhesion molecules [7]. A number of transcription factor binding sites have been identified in the ICAM-1 and VCAM-1 promoter, including NF-{kappa}B and AP-1 recognition sites [811]. Furthermore, inhibitory elements have also been recognized whereby shear stress, for example, negatively regulates VCAM-1 expression by endothelial cells [12].

In recent studies we, and others, found that fragmented HA activates the transcription factor NF-{kappa}B [6,13]. We found that inhibition of NF-{kappa}B with the serine protease inhibitor N-tosyL-L-phenylalanine chloromethyl ketone (TPCK) lead to complete inhibition of HA-stimulated ICAM-1 and VCAM-1 expression which suggested that NF-{kappa}B activation plays a key role in this process. However, additional transcription factors are also upregulated in response to HA, including the c-fos/c-jun heterodimer AP-1 [6].

The purpose of the present investigation was to examine the contribution of protein kinase C (PKC) and tyrosine kinase (TK) pathways in the HA-stimulated VCAM-1 expression in renal tubular epithelial cells. Here we show that atypical PKC together with TK activation are required for HA-stimulated VCAM-1 expression. PKC and TK inhibition do not inhibit the nuclear translocation of NF-{kappa}B, suggesting that other nuclear factors are required for the enhanced VCAM-1 expression in response to HA in renal epithelial cells.



   Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Reagents
Tissue culture reagents were obtained from Life Technologies (Gaithersburg, MD) and chemicals from Fluka (Buchs, Switzerland) or Sigma (St Louis, MO). Fragmented HA of intermediate mol.wt derived from human umbilical cord was obtained from Fluka (Buchs, Switzerland) or ICN (Costa Mesa, CA). The PKC inhibitors GF109203X and chelerythrine chloride and the TK inhibitor genistein were obtained from RBI (Natick, MA).

Cell lines and cell culture
The SV40-transformed mouse cortical tubule (MCT) cell line was originally obtained from Dr T. Haverty [14]. MCT cells were grown in DME medium supplemented with 10% FBS, 10 mM HEPES, 100 U/ml penicillin and 100 µg/ml streptomycin. After growing cells to confluence the medium was changed to DME containing 1% FBS for 24 h. Cells were then stimulated with HA (100 µg/ml) for 18 h in DME containing 1% FBS. PKC or TK inhibitors or vehicle were added 60 min prior to and during the 18 h stimulation period. To downregulate PKC, MCT cells were exposed to PMA at 0.75 µM for 48 h. Subsequently, MCT cells were stimulated with HA (100 µg/ml) for 18 h in the presence of PMA, and VCAM-1 expression was then assessed by cell ELISA.

Direct cell ELISA
VCAM-1 expression by MCT cells in response to HA was analysed on adherent cells by direct cell ELISA in 96-well plates as described [6]. After appropriate stimulation the cells were fixed with 3% paraformaldehyde and washed with PBS. Cells were then incubated with the anti-VCAM-1 mAb supernatant MK-2.7 (1:50) in PBS containing 5% FCS for 1 h at 4°C. Negative controls included omission of primary mAb or use of irrelevant mAb. Cells were washed three times and incubated with peroxidase-conjugated sheep anti-rat IgG Fab fragments (Boehringer Mannheim Biochemica, Germany) at 1:1000 in PBS/5% FCS for 1 h at 4°C. Cells were washed again three times and were incubated with the o-phenylenediamine dihydrochloride substrate (Fast OPD tablet sets, Sigma). After 15 min, 20% v/v of concentrated sulfuric acid was added to stop the reaction. The OD in the supernatant was read at 492 nm on a Multiskan RC ELISA reader (Labsystems, Helsinki). Delta ODs were calculated by subtracting the ODs of cells incubated with irrelevant antibody from cells incubated with anti-VCAM-1 mAb. Mean±SEM were then calculated (triplicate determinations). Experiments were performed at least three times.

RNA extraction, RT–PCR and Northern blot analysis
MCT cells were stimulated with HA (100 µg/ml) for 3 h. The PKC and TK inhibitors were added 30 min prior to and during the 3 h stimulation period. Total RNA from cultured MCT cells was then extracted as described [15]. VCAM-1 mRNA expression was analysed either by RT–PCR or by Northern blotting as described [6]. The VCAM-1 primers had the following sequence: forward primer 5'-CCC AAG GAT CCA GAG ATT CA-3', reverse primer 5'-TAA GGT GAG GGT GGC ATT TC-3', resulting in a 489 bp fragment. The following cycling parameters were used: denaturation at 94°C for 40 s, annealing at 58°C for 120 s, extension at 72°C for 150 s, with a terminal extension at 72°C for 7 min. The amplification was performed for 30 cycles. The housekeeping gene GAPDH was also amplified in all RNA samples as described to ensure equal RNA quantities [15]. RT–PCR products were resolved on 1% agarose gels and stained with 0.5 µg/ml ethidium bromide. Gels were then photographed with UV light.

For Northern blotting, total RNA (25 µg) was electrophoresed on 1.5% agarose gels in 20 mM MOPS buffer and blotted onto nylon membranes. Membranes were hybridized overnight at 42°C with an [{alpha}-32P]dCTP labelled VCAM-1 cDNA probe as described [6]. Blots were then washed under stringent conditions (final wash in 0.1x SSC, 1% SDS, 62°C) and were analysed with a PhosphorImager®. Methylene blue staining to detect the 18S and 28S rRNA bands was performed to ensure equal loading.

The presence of mRNA encoding for the atypical PKC {zeta} and {lambda} was assessed by RT–PCR, using the following primers: PKC {zeta} forward 5'-AAG TGG GTG GAC AGT GAA GG-3', reverse 5'-TGC CAT CTA CTG GAG GCT CT-3', yielding a 418 bp fragment [16]; PKC {lambda} forward 5'-AAT GGC CAC ACT TTT CAA GC-3', reverse 5'-CCA CTC TCC CTG GTG TTC AT-3', yielding a 311 bp fragment [17]. Amplification was performed for 40 cycles, using the same cycling parameters as indicated above.

Nuclear extracts
MCT cells were grown to confluence and were then stimulated with HA (100 µg/ml) for 1 h. The PKC and TK inhibitors were added 30 min prior to and during the 1 h stimulation period. Cells were then chilled on ice and were mechanically detached and washed with PBS. Nuclear extracts were then prepared according to Schreiber et al. [18]. Cells were resuspended in 400 µl ice-cold buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF) and incubated for 15 min and were then lysed with 25 µl of 10% NP-40. After centrifugation, the nuclear pellets were resuspended in 50 µl of ice-cold buffer C (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF) and rocked vigorously at 4°C for 1 h. The extracts were then centrifuged for 5 min at 4°C in a microfuge and the supernatants were frozen at -70°C until use. The protein concentration of the extracts was determined using the Bradford method (Bio-Rad, Hercules, CA).

Electrophoretic mobility shift assays (EMSA)
Nuclear extracts were then analysed for the presence of NF-{kappa}B using a 22mer double-stranded oligonucleotide with the sequence 5'-AGT TGA GGG GAC TTT CC

C AGG C-3' (Promega, Madison, WI), the consensus sites being underlined [19]. The oligonucleotide was end-labelled with [{gamma}-32P]ATP using T4 polynucleotide kinase. Extracts were then incubated for 1 h at room temperature with labelled NF-{kappa}B oligonucleotide in binding buffer (5% glycerol, 5 mM DTT, 50 mM NaCl, 50 mM KCl, 1 mM MgCl2, 1 mM EDTA, 10 mM Tris–HCl, pH 7.5) with denatured and sonicated calf thymus DNA (50 µg/ml) in the presence or absence of unlabelled competitor oligonucleotide. DNA–protein complexes were resolved in non-denaturing 5% acrylamide gels in 0.03x TBE buffer at 270 V. Gels were then dried and subjected to PhosphorImager® analysis.



   Results
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 Methods
 Results
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 References
 
PKC and TK inhibitors block VCAM-1 cell surface expression in MCT cells
To investigate the relative contribution of PKC and TK signalling pathways in the expression of VCAM-1 in response to HA, we incubated MCT cells with selective inhibitors of these pathways (Figure 1Go). The broad PKC inhibitor GF109203X (acting on conventional, novel and atypical isoforms [20]) inhibited the HA-stimulated VCAM-1 expression in MCT cells significantly (Figure 1AGo). The effect of GF109203X was dose-dependent between 0.1 and 10 µM, causing a maximal inhibition around 90% (Figure 1BGo). In four separate experiments GF109203X (10 µM) inhibited the HA-stimulated (100 µg/ml) VCAM-1 cell surface expression by 89.5±5.0% (mean±SEM; P<0.0001; two-tailed t-test).



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Fig. 1. Effect of PKC inhibitors GF109203X and chelerythrine, and of TK inhibitor genistein on cell surface VCAM-1 protein expression by MCT cells. VCAM-1 expression was assessed by direct cell ELISA. (A) MCT cells were stimulated with HA (100 µg/ml; ICN) for 18 h. GF109203X (GF) and genistein (G) but not chelerythrine (CL) inhibited HA-stimulated VCAM-1 expression markedly (lane 1, vehicle; lane 2, HA (100 µg/ml); lane 3, HA+CL (2 µM); lane 4, HA+G (25 µM); lane 5, HA+GF (10 µM)). (B–D) MCT cells were stimulated with HA (100 µg/ml; Fluka) for 18 h. GF109203X (B) and genistein (D) but not chelerythrine (C) inhibited VCAM-1 expression dose-dependently. Data represent mean±SEM from typical experiments which were performed at least three times.

 
On the other hand the PKC inhibitor chelerythrine (acting preferentially on conventional and novel isoforms [21]) had no effect on HA-stimulated VCAM-1 expression when used between 0.001 and 2 µM (Figure 1CGo). In four separate experiments chelerythrine (2 µM) did not significantly inhibit VCAM-1 expression (10.0±9.5% inhibition; P>0.05). Together these data suggest that Ca2+- and DAG-independent (atypical) isoforms could be involved in HA-stimulated VCAM-1 expression in these cells.

The TK inhibitor genistein [22] dose-dependently (1–30 µM) inhibited the HA-stimulated VCAM-1 expression (Figure 1DGo). In three separate experiments genistein (25 µM) significantly inhibited the HA-stimulated (100 µg/ml) VCAM-1 cell surface expression by 63.9±11.2% (P<0.01). Similar results were obtained when using two different HA preparations (Figure 1A versus B–DGo).

We detected no toxicity at the above-mentioned inhibitor concentrations. When used for up to 18 h, there was no cell detachment, and trypan blue exclusion was intact. Furthermore, at 3 h there was no sign of mRNA degradation (see below). We also examined the combined effect of GF109203X and genistein on the HA-stimulated VCAM-1 expression by cell ELISA. When used at submaximal concentrations (GF109203X at 5 µM and genistein at 20 µM), the combination of both inhibitors produced a complete inhibition of the HA-stimulated VCAM-1 expression (96.7±3.3% inhibition). Higher concentrations of the two inhibitors produced toxic effects on MCT cells when used together.

In separate experiments we also examined the effects of GF109203X, chelerythrine and genistein on TNF-{alpha}-stimulated (20 ng/ml) VCAM-1 expression by MCT cells. GF109203X (10 µM) but not chelerythrine (2 µM) inhibited the TNF-{alpha}-stimulated VCAM-1 expression significantly. Genistein (25 µM) also significantly inhibited VCAM-1 expression (data not shown). These data suggest that HA and TNF-{alpha} could use similar signalling pathways.

Involvement of atypical PKC in HA-mediated VCAM-1 expression
To confirm that Ca2+- and DAG-independent (atypical) isoforms could be involved in HA-stimulated VCAM-1 expression in these cells, we used downregulation of PKC with the phorbol ester PMA. Figure 2AGo demonstrates that the exposure of MCT cells to 0.75 µM PMA for 48 h did not prevent the stimulation of VCAM-1 with HA. Using RT–PCR we then examined whether the atypical PKC {zeta} and {lambda} were expressed in MCT cells. Figure 2BGo demonstrates that both the {zeta} and the {lambda} isoform are constitutively expressed by MCT cells. HA stimulation did not change steady-state mRNA levels of these two PKC isoforms. These data together with the differential effect of GF109203X and chelerythrine strongly suggest that atypical PKCs are involved in the HA stimulation pathway of VCAM-1.



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Fig. 2. (A) Downregulation with PMA does not prevent HA stimulation of VCAM-1. MCT cells were grown for 48 in PMA (0.75 µM), and were then stimulated overnight for 18 h with HA (100 µg/ml; Fluka) in the presence of 0.75 µM PMA. VCAM-1 expression was assayed by cell ELISA. Data are mean±SEM from a typical experiment. (B) Messenger RNA analysis for the presence of the atypical PKC {zeta} and {lambda} in MCT cells. MCT cells were stimulated with HA (100 µg/ml for 24 h), total RNA was extracted and analysed by RT–PCR.

 
Inhibition of VCAM-1 mRNA expression by PKC and TK inhibitors
We then examined the effect of PKC and TK inhibition on HA-stimulated VCAM-1 expression at the transcriptional level by RT–PCR and Northern blotting. Figure 3AGo demonstrates that the PKC inhibitor GF109203X (10 µM) inhibited the HA-stimulated VCAM-1 mRNA transcript levels markedly, which is concordant with the cell surface expression. Chelerythrine (2 µM) on the other hand did not significantly inhibit steady-state VCAM-1 mRNA expression, also in accord with the lack of inhibition of cell surface VCAM-1 protein. The TK inhibitor genistein also inhibited VCAM-1 mRNA (Figure 3BGo). These data suggest that the inhibition of cell surface VCAM-1 expression by GF109203X and genistein could occur via interference with the transcription of the VCAM-1 gene. Alternatively, the inhibitors could cause a decrease in VCAM-1 mRNA stability, leading indirectly to decreased translation.



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Fig. 3. Effect of PKC inhibitors GF109203X and chelerythrine, and effect of TK inhibitor genistein on steady-state VCAM-1 mRNA levels by MCT cells. (A) RT–PCR analysis for VCAM-1 (top) and GAPDH (bottom) in HA-stimulated (100 µg/ml; Fluka) MCT cells, demonstrating the inhibitory effect of GF109203X (10 µM) but not chelerythrine (2 µM). (B) Northern blot analysis for VCAM-1 (top) and methylene blue (MB) staining for 28S/18S mRNA (bottom) in HA-stimulated (100 µg/ml; Fluka) MCT cells, demonstrating the inhibitory effect of genistein (1–50 µM).

 
Role of NF-{kappa}B in HA-stimulated VCAM-1 expression
It has been shown previously that the mouse and human VCAM-1 promoters contain NF-{kappa}B and AP-1 binding sites [1012,23,24]. In a previous report we have shown that NF-{kappa}B and AP-1 are activated by HA in MCT cells, and that blocking of NF-{kappa}B with the inhibitor TPCK completely inhibited the HA-stimulated VCAM-1 expression in MCT cells, suggesting that NF-{kappa}B activation plays a very important role in the expression of VCAM-1 in response to HA. To elucidate the role of PKC and TK in the HA-stimulated, NF-{kappa}B-dependent VCAM-1 expression, we prepared nuclear extracts from HA-stimulated MCT cells and performed gel-shift assays for NF-{kappa}B. Interestingly, both GF109203X and genistein did not inhibit NF-{kappa}B activation in response to HA (Figure 4Go), which is in striking contrast to the inhibition seen with these two inhibitors at the VCAM-1 mRNA and cell surface protein level (Figures 1 and 3GoGo). These data, together with earlier data with TPCK suggest that NF-{kappa}B is necessary but not sufficient for VCAM-1 expression in response to HA.



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Fig. 4. EMSA for NF-{kappa}B in HA-stimulated MCT cells. The specific bands are marked with an arrow. Several additional non-specific and fainter bands are also found. (A) The PKC inhibitors GF109203X (10 µM) and chelerythrine (2 µM) do not inhibit the nuclear translocation of NF-{kappa}B in response to HA (100 µg/ml; Fluka). (B) The TK inhibitor genistein (30 and 50 µM) also does not inhibit the activation of NF-{kappa}B in response to HA.

 


   Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Our results elucidate several important aspects in the cellular pathway of VCAM-1 activation in response to HA in MCT cells: (i) HA activates an atypical protein kinase C; (ii) HA also uses TK signalling pathways; (iii) HA activates NF-{kappa}B despite PKC and TK inhibition.

Both chelerythrine and GF109203X are potent and specific inhibitors of protein kinase C [25]. The bisindolylmaleimide GF109203X inhibits PKC activity via the ATP-binding site [20]. In contrast, the benzophenanthridine alkaloid chelerythrine acts at the protein substrate binding site, does not block DAG binding to PKC and is a non-competitive inhibitor of ATP binding [21]. GF109203X inhibits all PKC subtypes, including the conventional PKCs {alpha}, ßI, ßII and {gamma} (Ca2+- and DAG-dependent), the novel PKCs {delta} and {varepsilon} (Ca2+-independent, DAG-dependent) and the atypical PKC {zeta} (Ca2+- and DAG-independent) [25,26]. In one study, the IC50 for GF109203X was 8.4 nM for the conventional PKC {alpha} and 5.8 µM for the atypical PKC {zeta} [26]. The concentration of 10 µM which we used should therefore have been effective on all PKC isotypes. Although chelerythrine's inhibitory spectrum on the various PKC isoforms has not been formally determined, it is felt by most that it acts preferentially on the conventional and novel (Ca2+-dependent and -independent, DAG-dependent) PKCs, sparing the atypical forms. The IC50 of chelerythrine on conventional PKC was found to be around 0.7 µM [21]. One report at least documents that a chicken gizzard PKC {zeta} is insensitive to chelerythrine (up to 50 µM) [27]. Therefore, the concentration of 2 µM chelerythrine which we used should have blocked the coventional and novel but not the atypical PKC.

The differential effects of GF109203X and chelerythrine can be used, together with PMA downregulation, to obtain information as to whether atypical PKCs are involved in cellular signalling. Jordan et al. have shown for example that GF109203X prevented the IL-1-induced upregulation of IL-8 in human synovial fibroblasts, whereas chelerythrine had no effect [28]. Likewise, PDGF-induced {alpha}2-integrin expression in human dermal fibroblasts could be inhibited completely with GF109203X, whereas chelerythrine was only partially inhibitory [29]. Using these two PKC inhibitors, it was suggested in both studies that atypical PKCs such as PKC {zeta} were involved. PKC {zeta} activation has been incriminated in many cytokine signalling processes (TNF-{alpha}, IL-1), and our data with PMA downregulation are in agreement with such a pathway and suggest that an atypical PKC is mediating the effects of HA on VCAM-1 expression in MCT cells.

Several studies have shown that TK is important in VCAM-1 expression in endothelial cells [3032]. Few studies have been performed in epithelial cells. IFN-{gamma} (typically acting through the JAK/STAT signalling cascade) does not upregulate VCAM-1 in endothelial cells by itself, but in combination with TNF-{alpha} enhances the effect of IFN-{gamma} [33]. On the other hand IFN-{gamma} is effective alone in tubular epithelial [34] and mesothelial cells [35]. This suggests that the JAK/STAT pathways could be important for epithelial VCAM-1 expression. Further studies will have to examine the involvement of specific JAKs, and will need to test the role of the STAT family of transcription factors in the HA- stimulated VCAM-1 expression.

It is interesting to note that GF109203X did not inhibit NF-{kappa}B activation despite blocking the HA-stimulated VCAM-1 expression. Previously we have shown that blocking of NF-{kappa}B with TPCK inhibited VCAM-1 expression completely [6]. This suggests that NF-{kappa}B is required but not sufficient for VCAM-1 expression. A similar situation has been reported in HUVEC where PKC inhibition did not prevent NF-{kappa}B translocation [36]. This suggests that other transcription factors are required to induce VCAM-1.

In summary, we have shown that the HA-stimulated VCAM-1 expression in a murine renal tubular epithelial cell line depends on an atypical PKC and on a TK pathway. The transcription factor NF-{kappa}B is required but is not sufficient alone to stimulate VCAM-1 expression. Our data highlight the complexity of interactions between PKC, TK and nuclear transcription factors in the HA-stimulated VCAM-1 expression by epithelial cells.



   Acknowledgments
 
We thank C. Gasser for help with the illustrations. This study was supported by the Swiss National Science Foundation (grants No. 32–40390.94 and 32–50721.97 to R.P.W.), the Olga-Mayenfisch Foundation, the Hartmann-Müller Foundation and the Research Foundation of the University of Zürich. B.O. is the recipient of a Postgraduate Fellowship from the University of Zürich and is supported by the Swiss National Science Foundation and the Maurice E. Müller Foundation. B.B.S. is the recipient of a Federal Career Development Award from the Swiss government. R.P.W. is the recipient of a Physician Scientist Award (grant No. 32–38821.93) from the Swiss National Science Foundation.



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 Discussion
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Received for publication: 4. 1.99
Accepted in revised form: 27. 4.99