Human microvascular endothelial cells resist Shiga toxins by IFN-{gamma} treatment in vitro

Tomoaki Yoshida, Tsuyoshi Sugiyama, Naoki Koide, Isamu Mori and Takashi Yokochi

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Shiga toxins (Stxs) produced by enterohaemorrhagic Escherichia coli or Shigella dysenteriae damage human endothelial cells predominantly in cooperation with pro-inflammatory cytokines, such as TNF-{alpha}. However, in this study, in vitro IFN-{gamma} pre-treatment resulted in human lung microvascular endothelial cells becoming over 10 000-fold less sensitive to Stxs. In contrast, in their basal condition, they were extremely sensitive to Stxs. Interestingly, TNF-{alpha} addition to IFN-{gamma} reverted the Stx-resistant phenotype, which corresponded with its well-established enhancing effect on Stx toxicity. Toxin binding to the cell was barely affected by IFN-{gamma}. Also, the toxin uptake in the Stx-resistant phenotype was more than 100-fold greater than that of normal cells, when compared at Stx concentrations resulting in equivalent degrees of cell damage. Protein synthesis was inhibited by nearly 90 % in the Stx-resistant phenotype after 24 h toxin exposure. This indicated that the intracellular toxin was active as an N-glycosidase, while cells were still over 60 % viable, suggesting a possible unknown cytotoxic function of Stx. In conclusion, this study shows a unique effect of IFN-{gamma} in the suppression of the toxicity of Stxs in a human microvascular endothelial cell model and the involvement of a novel mechanism in this suppression.


Abbreviations: CD50, 50 % cytotoxic dose; HLMEC, human lung microvascular endothelial cell; IFN-{gamma}, {gamma} interferon; Stx, Shiga toxin; TNF-{alpha}, tumour necrosis factor {alpha}


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Haemolytic uraemic syndrome (HUS) is a serious affliction that involves acute renal failure, coagulation abnormality and blood-cell destruction. Approximately 90 % of HUS cases are preceded by diarrhoea (Decludt et al., 2000; Gianviti et al., 1994), most of which can be attributed to Shiga toxin (Stx)-producing bacteria, such as enterohaemorrhagic Escherichia coli (Karmali et al., 1985; Banatvala et al., 2001) or Shigella dysenteriae (Bhimma et al., 1997). Stxs are believed to damage endothelial cells of the microvascular system especially in the kidney, lung and colon epithelium (Laurence & Mitra, 1997). The endothelial cells express the specific receptor for Stxs, globotriaosyl ceramide (Gb3), and its content is enhanced by various pro-inflammatory cytokines, such as TNF-{alpha} (Molostvov et al., 2001; Eisenhauer et al., 2001). After endocytosis, Stxs travel through the Golgi apparatus (Arab & Lingwood, 1998), are processed by furin (Garred et al., 1995) and then, finally, reach their cellular target, the 60S ribosome. The A-subunit N-glycosidase activity inactivates the ribosome, resulting in protein-synthesis inhibition (Endo et al., 1988).

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 7–14 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-{gamma} 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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Reagents.
Human plasma fibronectin, 0·25 % trypsin/1 mM EDTA solution and recombinant Insulin-like growth factor-1 were purchased from Gibco-BRL. Recombinant human TNF-{alpha} was obtained from PeproTech EC (20 U ng-1). Recombinant vascular endothelial cell growth factor and IFN-{gamma} were from Wako Pure Chemical Industries and Shionogi & Co., respectively. Purified preparations of Stx1 and Stx2, which gave single peaks on HPLC (Kondo et al., 1997) and contained endotoxin at 3x10-6 and 4x10-6 of protein content, respectively, were kindly supplied by the Aichi Prefectural Institute of Public Health, Japan.

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-{gamma} 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 24–72 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-{gamma} 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-{gamma} 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 {gamma}-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 {beta}-hexosaminidase, respectively (Wanders et al., 1989).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
HLMECs became resistant to Stxs by IFN-{gamma}
All four of the employed HLMEC lots were extremely sensitive to Stxs (Table 1, Fig. 1) during three passages without any pre-treatments, in which the CD50 values were comparable with previous reports (Ohmi et al., 1998; Pijpers et al., 2001). The Stx2 toxicity exceeded the toxicity of Stx1, reproducing results from other studies. In our hands, cell death did not apparently involve apoptosis, as suggested by negative TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick end-labelling) staining (data not shown). However, HLMEC responses to Stxs were surprisingly modified by prior IFN-{gamma} treatment. Outstanding resistance to Stx2 was induced by 3–4 days treatment with IFN-{gamma} (1000 U ml-1) by 1000- to 100 000-fold in all tested lots (Table 1, Fig. 1). A lower but significant effect was observed at 100 U IFN-{gamma} ml-1 (Table 1). Interestingly, the degree of CD50 increase was smaller in the case of Stx1 after 3 days treatment, but it surged to a level analogous to that of Stx2 in 6 days. The minimal required pre-treatment period until the appearance of the effect was reproducibly longer than 2 days in multiple experiments (data not shown), as represented by the data in Table 1. Notably, IFN-{gamma} was not required to be present continuously, which was shown by a significant effect of an 8-h pulse and a 3-day incubation without IFN-{gamma} (Table 1). As suggested by the comparable CD50 between 24 h (lot nos 1 and 2) and 48 h (lot no. 3) exposure to Stxs, the apparent toxin resistance was not an artefact due to a slower rate of cell death. The idea was further supported by an increase in viability (30–38 %) from 48 to 72 h exposure to Stx2 at 1 nM, which was presumably because of surviving cell growth. It was unlikely that a novel minor population had been selected during the IFN-{gamma} pre-treatment period, since the relative suppression of cell viability, protein synthesis and [3H]thymidine uptake was only 27·4 % (Fig. 2), 25·6 % and 29 %, respectively, after a 3-day pre-treatment.


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Table 1. CD50 values of Stxs on HLMECs

 


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Fig. 1. Plots of the relative viability of HLMECs versus various doses of Stx2 after pre-treatment with 1000 U TNF-{alpha} ml-1 ({bullet}),1000 U IFN-{gamma} ml-1 ({square}), 1000 U TNF-{alpha} ml-1+1000 U IFN-{gamma} ml-1 ({triangleup}),100 U TNF-{alpha} ml-1+1000 U IFN-{gamma} ml-1 ({blacktriangleup}) or no additions ({circ}). Cells were pre-treated with cytokines for 4 days and exposed to Stx2 for 48 h. The relative viability was determined as the proportion of the value without Stx2 based on calcein/AM uptake and digestion. Representative data from two experiments are depicted as the mean±SD of the triplicate wells.

 


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Fig. 2. Plot of the relative viability of HLMECs during IFN-{gamma} treatment. The relative viability was determined in the same way as in Fig. 1. Representative data from two experiments are depicted as the mean±SD.

 
Stx2 uptake by HLMECs was large enough despite toxin resistance
IFN-{gamma} treatment at 1000 U ml-1 for 3 days decreased the specific binding of 125I-labelled Stx2 less than twofold [no treatment, 14·7±5·0 fmol per 105 cells (at 1 nM); IFN-{gamma} treatment for 3 days, 9·8±3·2 fmol per 105 cells (at 1 nM); data are expressed as the mean±SD from triplicate wells], whereas it induced over a 10 000-fold decrease in the CD50 (Table 1, lot no. 2). A comparable decrease was observed using 125I-labelled Stx1, from 7·2 to 3·7 fmol. It was still possible that the intracellular toxin uptake was highly affected by minute differences in the specific binding. However, the intracellular 125I-labelled Stx2 amounted to 2·64±0·63 fmol per 105 cells by 20 h incubation with IFN-{gamma} at the CD50 concentration of 109 pM (lot no. 3). Furthermore, the intracellular 125I-labelled Stx2 was distributed in the cytoplasmic fraction (37 %), as determined by subcellular fractionation analysis, indicating that the major part of Stx2 was not associating with the plasma membrane. Also, it exhibited an intact molecular size on gel permeation chromatography (data not shown). In contrast, the intracellular Stx2 required to damage 50 % of the native HLMECs should be less than 0·011 fmol per 105 cells, which was the entire Stx2 input at the CD50 concentration, and was far below the detection limit (0·1 fmol). Thus, the amount of intracellular Stx2 required to cause equivalent damage in the IFN-{gamma} pre-treated HLMECs was apparently over 100-fold greater than in the native cells.

Protein synthesis was inhibited by CD50 or a lower dose of Stx2 after IFN-{gamma} treatment
Since a large amount of Stx2 was incorporated into the cytoplasm even after IFN-{gamma} 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-{gamma} 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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Fig. 3. Relative viability (closed symbols) and protein synthesis (open symbols) of HLMECs versus Stx2 before (circles) and after (squares) IFN-{gamma} pre-treatment (1000 U ml-1) were plotted. The cells were pre-treated with IFN-{gamma} for 3 days and exposed to Stx2 for 24 h. The relative viability was determined as in Fig. 1. Protein synthesis was determined by measuring 35S-labelled methionine incorporation as described in Methods. Data from two experiments are depicted as the mean±SD.

 
TNF-{alpha} neutralized the protective effect of IFN-{gamma} against Stxs
A significant but weak sensitization was induced in native HLMECs by a 4-day treatment with a high dose of TNF-{alpha} (1000 U ml-1, 50 ng ml-1) (Table 1, Fig. 1), but not by a 1-day treatment (data not shown). In contrast, TNF-{alpha} addition to IFN-{gamma} treatment reverted the protective effect of IFN-{gamma} by nearly 100- (Stx1) or 1000-fold (Stx2) (Table 1, Fig. 1). It is noteworthy that the resultant CD50 values were still higher than the native ones.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, a representative of adult microvascular endothelial cells, HLMECs, which are quite sensitive to femtomolar doses of Stxs, could be rendered highly resistant to Stxs by IFN-{gamma} treatment. The extent of the sensitivity difference exceeded three to four orders of magnitude in terms of the CD50. The required pre-treatment time of 3 days until the appearance of the effect might suggest that certain sequential signalling steps are required after IFN-{gamma} stimulation. Moreover, a weaker but significant effect induced by an 8-h pulse treatment and incubation without IFN-{gamma} indicated that the initial triggering was the crucial event to induce the phenotype conversion. Although the IFN-{gamma} concentration employed here was relatively high compared with physiological conditions, it is not impractical under pharmacological settings (Wills, 1990).

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-{gamma} treatment. Stx binding at 4 °C decreased by no greater than twofold after IFN-{gamma} 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-{alpha} dose (Table 1) and not at all after 1 day of treatment (data not shown). In contrast, TNF-{alpha} addition almost reverted the Stx-resistant phenotype induced by IFN-{gamma} 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-{gamma}, but not TNF-{alpha}, induces apoptosis in human endothelial cells and the degree is enhanced by both IFN-{gamma} and TNF-{alpha} (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-{gamma} 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-{gamma} 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-{gamma} treatment for a few days and neutralized by TNF-{alpha} 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.


   ACKNOWLEDGEMENTS
 
The authors are indebted to Drs F. Kondo and Y. Suzuki at Aichi Prefectural Institute of Public Health, Japan, for the purified preparations of Stx1 and Stx2. We also thank Ms Akiko Morikawa and Dr Kazuko Takahashi for their technical assistance. This work was partly financed by the Ministry of Education, Culture, Sports Science and Technology, Japan (12670268).


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 21 November 2002; revised 22 April 2003; accepted 23 May 2003.



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