Department of Microbiology and Immunology, School of Medicine, Aichi Medical University, Yazako, Nagakute, 480-1195 Aichi, Japan
Correspondence
Tomoaki Yoshida
tomo{at}aichi-med-u.ac.jp
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ABSTRACT |
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INTRODUCTION |
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Previous studies have shown that human umbilical vein endothelial cells (HUVECs) or adult human saphenous vein endothelial cells (HSVECs) are resistant to nanomolar Stx concentrations in vitro (van de Kar et al., 1992; Keusch et al., 1996
). In contrast, a 50 ng Stx1 kg-1 dose, which would lead to picomolar concentrations in tissues, was lethal for primates in vivo (Taylor et al., 1999
). Our previous data indicated that both HUVECs and HSVECs were moderately sensitive to Stxs at picomolar concentrations in the absence of cytokine stimulation at the beginning of the primary culture, whereas they obtained over a 1000-fold resistance to Stxs after only 714 days of in vitro passage (Yoshida et al., 1999
). The HUVEC and HSVEC resistant phenotype after passage was consistent with previous studies and the native response of these endothelial cells in vivo might be different from in vitro results. According to our preliminary trials, the conversion rate toward the toxin-resistant phenotype in vitro was accelerated by IFN addition (T. Yoshida et al., unpublished data), suggesting a possible application to induce such a toxin-resistant phenotype in other cells. The endothelial cells from a microvascular origin stayed highly sensitive to Stxs at femtomolar concentrations during in vitro passage (Ohmi et al., 1998
; Pijpers et al., 2001
). Such sensitivity agreed with the observation of microvascular damage in the in vivo primate model (Siegler et al., 2001
). In this study, IFN-
pre-treatment was shown to induce strong resistance against Stxs in human lung microvascular endothelial cells (HLMECs). This was a peculiar but intriguing phenomenon in that a pro-inflammatory cytokine could reduce, instead of enhance, the lethal response of human cells to Stxs.
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METHODS |
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Cell culture conditions.
Four independent lots of HLMECs (4th passage) were purchased from BioWhittaker and were cultured in MCDB131 medium supplemented with 10 % fetal calf serum, VEGF, IGF-1 and Serum extender (Collaborative Biomedical Products) (complete medium). All plastic culture plates were coated with human fibronectin at 5 µg cm-2 before use. All experiments were done within three generations of passage. The IFN- treatment was performed in sub-confluent conditions in a 24-well plate.
Cell viability analysis.
Cells were harvested from the 24-well plates by using 0·25 % trypsin/1 mM EDTA and plated onto a Terasaki plate (Greiner Labotechnik) at 103 cells per well or in a 96-well plate at 5x103 cells per well and subsequently exposed to Stxs for 2472 h in sub-confluent conditions. Then, the viability of the cells was assessed by the uptake and digestion of calcein/AM (Molecular Probes) as described previously (Yoshida et al., 1999) or by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction. The data obtained on calcein/AM uptake were comparable to the MTT reduction data. The CD50 values were calculated using the ALLFIT program (Guardabasso et al., 1988
) from the means of triplicate data. The cell growth rate was measured by [3H]thymidine uptake for 24 h using 37 kBq per 5x103 cells.
Protein synthesis analysis.
The degree of protein synthesis was assessed by the incorporation of 35S-labelled methionine (Tran35S-label; ICN Biomedicals) into intracellular proteins. HLMECs were plated into a 96-well plate (5x103 cells per well) and treated with 1000 U IFN- ml-1 for 3 days. Cells were exposed to Stx2 for 24 h and pulsed with 37 kBq of 35S-labelled methionine per well for 1 h at 37 °C. The cells were dislodged with 0·25 % trypsin/1 mM EDTA and harvested on a glass fibre filter using 0·5 % EDTA/PBS. After proteins were denatured with 10 % trichloroacetic acid, the filter was rinsed four times and subjected to liquid scintillation counting. Simultaneously, cell viability was determined by calcein/AM uptake after the same pre-treatment with IFN-
and exposure to Stx2.
Toxin binding and uptake analysis.
Stxs (10 pmol) were labelled with 125I by incubation with 18·5 MBq Na125I (ICN Biomedicals) for 2 min at 26 °C in the presence of Iodo-beads (Bio-Rad). The labelled protein was purified on a PD-10 column (Pharmacia-Biotech). The specific radioactivities were 14·0 and 24·6 Bq fmol-1 for Stx1 and Stx2, respectively. To analyse toxin binding, the 125I-labelled Stx1 or Stx2 was added to 105 cells at 1 nM in complete medium, followed by incubation at 4 °C for 3 h. After washing three times, the cells were subjected to -counting. The value obtained in the presence of 50 nM cold Stxs was assumed as the non-specific binding value. For toxin uptake analysis, 3x104 cells were incubated with 109 pM 125I-labelled Stx2 for 20 h at 37 °C in the complete medium, harvested in 0·25 % trypsin/1 mM EDTA, washed four times and then counted.
Subcellular localization of Stx2.
The cell suspension (100 µl) from the toxin uptake analysis was homogenized in the presence of protease inhibitor cocktail (Sigma) and centrifuged at 500 g for 5 min. The supernatant was overlaid on 0·9 ml of 22 % Percol (Pharmacia-Biotech) in 0·25 M sucrose and 15 mM HEPES/NaOH (pH 7·4), and spun at 28 000 g for 30 min in a TLA100.2 rotor in a TL-100 centrifuge (Beckman Instruments). The resultant density gradient was fractionated into 50 µl fractions and counted for radioactivity. The locations of the cytoplasmic and endosomal fractions were determined by the enzymic activities of lactate dehydrogenase and -hexosaminidase, respectively (Wanders et al., 1989
).
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RESULTS |
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Protein synthesis was inhibited by CD50 or a lower dose of Stx2 after IFN- treatment
Since a large amount of Stx2 was incorporated into the cytoplasm even after IFN- treatment, the degree of protein synthesis was measured to confirm the toxin activity inside the cell. When 100 pM of Stx2, an approximate CD50 concentration (lot no. 3), was applied to HLMECs after IFN-
treatment, the rate of protein synthesis was highly suppressed to 11·8 % of the control levels after 24 h exposure (Fig. 3
). Moreover, a 10-fold lower concentration, 10 pM, which allowed for 70 % cell viability, also decreased protein synthesis to 24·8 % (Fig. 3
), indicating that the Stx2 N-glycosidase activity is indeed functioning inside the cells. In contrast, the degree of protein synthesis inhibition in the native HLMECs was consistent with the viability throughout the doses examined (Fig. 3
).
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DISCUSSION |
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Whether the decreases in receptor expression and toxin uptake were responsible for the Stx resistance was examined by comparing the amounts of bound or intracellular Stx before and after IFN- treatment. Stx binding at 4 °C decreased by no greater than twofold after IFN-
treatment, which was measured at the same 1 nM Stx concentration to reflect the receptor content at equilibrium. Next, the amounts of Stx2 uptake were compared at the CD50 toxin concentrations, which damage equivalent proportions of the native and Stx-resistant cells. However, the amount of Stx2 inside the native HLMECs was too small to be detected, even with the radioisotopic labelling. On the other hand, the intracellular toxin in the Stx-resistant phenotype was present at 2·6 fmol per 105 cells and was more than 100-fold greater than in the native ones, even if the native cells incorporated the total toxin input (0·011 fmol). There still remained a possibility that Stxs were transferred to lysozomes and degraded, instead of travelling through the Golgi apparatus to the cytoplasm in an active form (Arab & Lingwood, 1998
). However, a significant proportion of the intracellular Stx2 was recovered from the cytoplasmic fraction without any apparent degradation, where the intracellular toxin inactivated ribosomes by N-glycosidase activity (Endo et al., 1988
). Correspondingly, a remarkable inhibition of protein synthesis was observed, which indicated that the intracellular Stx2 inactivated ribosomes, but did not terminate cell viability. In contrast, the inhibition of protein synthesis coincided well with the cell viability in the case of the native HLMECs, possibly because the viability decrease might have resulted in the overall reduction in protein synthesis. These data indicated that the mode of toxin action might be different in the native and resistant phenotypes and also showed a discrepancy between the ribosome inactivation and the cytotoxicity of Stxs.
To date, many in vitro studies have indicated that pro-inflammatory cytokines are intimately involved in the endothelial damage caused by Stxs, through enhancing receptor expression and sensitizing the target cells (Molostvov et al., 2001; Eisenhauer et al., 2001
). In the particular case of native HLMECs, which exhibit high sensitivity to Stxs in the basal state like other microvascular endothelial cells (Ohmi et al., 1998
; Pijpers et al., 2001
), the CD50 decreased only moderately even after 4 days treatment with a high TNF-
dose (Table 1
) and not at all after 1 day of treatment (data not shown). In contrast, TNF-
addition almost reverted the Stx-resistant phenotype induced by IFN-
to basal sensitivity. The residual resistance of HLMECs would exclude a synergy of these cytokines here. This phenomenon corresponded with other results on the effects of pro-inflammatory cytokines and supports the hypothesis that various endothelial cells in vitro have already obtained a Stx-resistant phenotype, as observed in human umbilical vein endothelial cells, and respond to pro-inflammatory cytokines to a higher degree.
It is reported that IFN-, but not TNF-
, induces apoptosis in human endothelial cells and the degree is enhanced by both IFN-
and TNF-
(Wang et al., 1999
). Correspondingly, the combination of these cytokines reduced HLMEC viability to about 40 % of the control levels (data not shown), although only the growth rate was moderately suppressed by IFN-
alone. The surviving HLMECs from the pre-treatment with these cytokines still showed the weak but significant Stx-resistant phenotype described above. This cytotoxic effect would endanger a simple IFN-
application as a novel therapy. However, the concept of converting the host-cell response rather than inhibiting toxin binding would be feasible, especially given that the factors responsible for the phenotypic difference have been elucidated.
Collectively, a highly resistant phenotype could be induced in human microvascular endothelial cells by IFN- treatment for a few days and neutralized by TNF-
addition. This is the first case of a cytokine reducing the lethal response of human cells to Stxs. It was intriguing that protein synthesis inhibition by Stxs was observed, despite the maintenance of cell viability, suggesting an unknown mechanism of Stx toxicity. In addition, these phenomena raise the possibility of a novel therapeutic approach to focus on the host-cell response to Stxs.
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ACKNOWLEDGEMENTS |
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Received 21 November 2002;
revised 22 April 2003;
accepted 23 May 2003.
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