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Ca2+independent cytotoxicity of Vibrio parahaemolyticus thermostable direct hemolysin (TDH) on Intestine 407, a cell line derived from human embryonic intestine
ELSEVIER
FEMS Microbiology
Letters 134 (1995) 233-238
Ca’ +-independent cytotoxicity of Vibrio parahaemolyticus thermostable direct hemolysin ( TDHj on Intestine 407, a cell1line derived from human embryonic intestine Guang-Qing Tang, Tetsuya Iida *, Koichiro Yamamoto, Takeshi Honda Received 22 September 1995; accepted I6 October I995
Abstract The effects of Vihrio puruhaemo~~yricusthermostable direct hemolysin on Intestine 407, a cell line derived from the intestine of human embryo, were investigated. The hemolysin was shown to be cytotoxic to Intestine 407. This cy!otoxicity is accompanied by the damage of plasma membrane and lysosomes, as well as cellular degeneration in the form of large transparent blebs. Although an increase in cytosolic free Ca’+ due to the influx of extracellular Ca” was observed in cells treated with thermostable direct hemolysin, it was found to be irrelevant to any of the above effects. These results suggest that the effects of thermostable direct hemolysin observed in this study on Intestine 407 are not mediated by Ca”+-dependent pathways. Keword.\:
Vihrio puruhwmo/~ticus;
Cytotoxicity;
Thermostable direct hemolysin: Intestine 407
1. Introduction Thermostable direct hemolysin (TDH) is considered to be the major virulent factor of Vibrio parukaemolyticus in causing food-borne gastroentetitis [I]. This toxin has shown a diversity of biological activities such as hemolytic activity, enterotoxicity, myocardiotoxicity as well as cytotoxicity on cultured cells [I]. Ca” plays an important role in a variety of cellular processes. In killing rat hepatocytes by various membrane-active toxins, influx of extracellular
‘ Corresponding author. Tel: + 8 I (6) 879 8276; Fax: + 8 I (6) 879 8277.
037%1097/95/$09.50 SSDl
037%1097(95)0041
0
1995 Federation of European Microbiological l-4
Ca’+ (Ca’+o) has been considered to represent or initiate a final common pathway [2]. TDH is a membrane-active toxin [3]. With regard to th!z role of Ca’+ in effects of TDH, Goshima and ccilleagues have shown that TDH induces the influx of Ca” o to mouse myocardial cells and melanoma cells. and have demonstrated that this influx is essentiil for the cell degeneration caused by TDH [4]. Recently we have shown that TDH increases the membtine permeability of human erythrocyte membrane to Ca’+ bivalent cations and propidium iodide [5]. On the other hand, however, even when an influx df Ca” o did not occur, TDH was still able to lyse human erythrocytes [5]. Thus concerning Cal+ dedendency of TDH action, previous studies have come to discrepant conclusions. In this study, we employed a Societies. All rights reserved
cell line derived from the human embryonic intestine, Intestine 407, to investigate whether or not the calcium ion is involved in TDH cytotoxicity.
extracellular sults.
milieu
2.5. Cytotoxicily 2. Materials and methods
2. I. Cell cultures Intestine 407 cells (ATCC CCL 61 were grown in Petri dishes containing Eagle minimal essential medium (EMEM) (Biken, Osaka, Japan) supplemented with 10% calf serum. Cells were cultured at 37°C in a humid atmosphere of 95% air and 5% carbon dioxide. Cells grown to 80-90% confluence were harvested by repeatedly pipetting with EMEM to detach and disperse the cells, followed by centrifugation at 800 X g for IO min. The cell pellet was suspended in phosphate-buffered saline (PBS) supplemented with 5.5 mM glucose (PBS-glucose). The cell suspensions were used for the following experiments. 2.2. Purification
of TDH
TDH was purified
according
to the previous
re-
port [51. 2.3.Measurement
of,free Ca’+ level
Preparations of lo6 cells ml-’ of Intestine 407 cells were loaded with Fluo 3 (5 PM, Flu0 3-AM, Dojindo, Fukuoka, Japan) in the dark at 37°C for 30 min in PBS-glucose. These preparations of Intestine 407 cells loaded with Fluo 3 were allowed to interact with serial dilutions of TDH at 37°C for 30 min in 120 ~1 PBS-glucose containing either 1 mM CaCl, or various concentration of EGTA, and then analysed by flow cytometry as described below. 2.4. Analysis of the role of Ca” Intestine 407
in TDH effects on
The above measurement of Ca’+ i showed that 0.1 mM extracellular EGTA was sufficient to completely prevent the TDH-induced elevation of Ca2+ i. Consequently, in all the following experiments, either 1 mM Ca2+ or 0.1 mM EGTA was added into the
to compare
the subsequent
re-
analysis
Preparations of IO6 cells ml-’ of Intestine 407 cells in a 96-well plate were incubated with serial dilutions of TDH for 30 min at 37°C in 120 ~1 PBS-glucose. Subsequently, 10 ~1 of solution prepared from a cell counting kit (Dojindo, Fukuoka, Japan) were added to each well and incubated for another 2.5 h. Optical density was measured on the Titertek Multiskan MCC/340 MKII (Lab-systems, Finland) at 450 nm. Positive controls were cells treated with 0.2% Triton X-100 and negative controls were those without toxin treatment. Viability was calculated using the optical density of toxintreated COD,), positive control COD,,) and negative control COD,,) samples. The viability formula was (OD, - OD,,)/(OD,, - OD,, 1 X 100%. 2.6. Measurement
of injlax of propidium
iodide (PI)
Preparations of IO6 cells mll ’ of Intestine 407 cells were allowed to interact with serial dilutions of TDH at 37°C for 30 min in 120 ~1 PBS-glucose containing IO pg ml-’ PI and then analysed by flow cytometry as below. 2.7. Neutral red retention assay Preparations of IO6 cells ml-’ of Intestine 407 cells were allowed to interact with serial dilutions of TDH in 120 ~1 PBS-glucose containing 50 pg ml - ’ neutral red at 37°C for 3 h. The cells were washed twice with PBS after which the Neutral red taken up by Intestine 407 was lysed out using 0.5 N HCl/50% ethanol. After centrifugation of the cell debris, the optical density of the supematant was read on the Titertek Multiskan MCC/340 MKII at 540 nm. Positive controls were cells treated with 0.2% Triton X-100 and negative controls were those without toxin treatment. Lysosomal integrity was calculated using the optical density of toxin-treated COD,), positive control (OD,,) and negative control COD,,) samples. The percentage of lysosomal integrity was (OD, - OD,,)/(OD,, - OD,,) X 100%.
G.-Q. Tmg et al. / FEMS Microhiohgy
2.8. Microscopic
25 -
obsenation
Preparations of IO’ cells ml-’ of Intestine 407 cells were loaded with 5 pg ml-’ rhodamine 123 (Calbiochem-Novabiochem Corporation, San Diego, CA) at 37°C for 10 min in PBS-glucose and then allowed to interact with 5 pg ml-’ TDH for 30 min at 37°C in 120 ~1 PBS-glucose. Cell samples were mounted on glass slides, covered with cover slips, and the morphology of the cells was immediately observed and photographed through a fluorescence microscope (Nikon, Tokyo, Japan).
235
Lrttrr.s 134 (IYYS) 233-238
20-
15ii z io-
2.9. Flow cytometry
concentration Fig.
I. Cytosolic
treatment
1
.l
.Oi
,001
Flow cytometry was carried out by using FACScan (Fluorescence-activated cell sorter, BectonDickinson, San Jo&, CA) to acquire 10000 events for each sample. The fluorescence intensity of Fluo 3 was recorded at 5 15-545 nm and that of PI was recorded at 543-627 nm.
free Ca’+
presence of
I
levels in Intestine 407 cells after MFI,
mean fluorescence
units) measured by FACScan.
mM Cal+
; (0)
100
of TDH (pglml)
with serial doses of TDH.
intensity (arbitrary
10
in the presence of 0.
(0)
I
In the
tqM EGTA.
Data are obtained from four independent experiments. For experimental details, bee Materials and methods.
3. Results 3.2. Cytotoxicifi
c$ TDH to Intestine 407
3.1. Change of cytosolic free Ca’ ’ level TDH has previously been shown to induce influx of the extracellular Ca” in the mouse myocardial and melanoma cells [4]. Using Fluo 3, a fluorescent indicator of Ca’+, we monitored the changes in cytosolic Ca’+ levels in Intestine 407 cells treated with TDH in the presence of either Ca2 + or EGTA. As shown in Fig. I, there was an elevation of Ca2+ i level in cytoplasm which was dependent on the dose of TDH in the presence of 1 mM Ca”o. At a dose of 0.3 /*g ml-’ of TDH, 50% of this effect was reached. In contrast, in the absence of Ca’+ o (0. I mM EGTA was added to the reaction solution to chelate possible trace amounts of Calf ), there was no elevation of Ca’+ i levels even after 30 min treatment with TDH. These results indicate that TDH-induced elevations of the Ca’+i level are not due to the release of Ca’+ from intracellular stores but rather due to the influx of Ca’+o. The data also show that increases of Ca2+ i levels could be thoroughly prevented by adding the Ca’+ chelator, EGTA into the extracellular milieu, to eliminate possible trace amounts of Ca’+.
TDH has been shown to be toxic to a variety of cultured cells [I]. Here we examined whether TDH is also cytotoxic to Intestine 407 cells by using a cell counting kit developed by Dojindo. This kitlemploys a tetrazolium reagent which is reduced andlchanged into a soluble Formazan dye under capacity generated by various dehydrogenase activities in viable cells. It can thus be used to count the viable cells 161. As shown in Fig. 2, Intestine 407 underwent a dose-dependent loss of viability due to treatment with TDH. At 0.8 pg ml-‘, TDH accobnplished 50% of this cytotoxic effect. The results in Fig. 2 also show that the cytotoxicity of TDH is not affected in the presence or absence of Ca’+o influx, indicating that TDH cytotoxicity was not due to the increase in Ca’+ i. 3.3. Injlux
of propidium iodide (PI)
Propidium iodide is widely used as an indicator of cell plasma membrane integrity [7]. The plasma membrane of human erythrocyte has been, reported to be damaged by TDH and became permeable to PI
G.-Q.
Tang et al. / FEMS
Microbiology
Letters 134 ( lYYSl233-238 120-
100
100-
60
60-
60
ii
60-
I 40
40-
20
20 1 0 :
0 .Ol
.l
1
concentration
of
10
TDH
100
[5]. Here we analysed the changes in membrane permeability to PI of Intestine 407 under the action of TDH in the presence of either EGTA or Ca2+. As shown in Fig. 3, TDH induced the influx of PI into the cells dose-dependently in the presence of either EGTA or Ca2+. At 0.8 pg ml -I, TDH reached the median level of this effect. These results indicate that Ca*’ o influx is not necessary for TDH to damage the plasma membrane of Intestine 407. 3.4. Loss of lysosomal
..(
.
..m”..,
.Ol
.
.l
concentration
&g/ml)
Fig. 2. Viability of Intestine 407 cells after treatment with TDH. Intestine 407 cells were incubated with serial dilutions of TDH at 37°C with subsequent addition of solution from a cell counting kit. (0) In the presence of 1 mM Ca*+; (0) in the presence of 0.1 mM EGTA. Data are obtained from three independent experiments.
. . . . ..
,001
of
,...
7
.
.
“‘“1
1
10
TDH
@g/ml)
100
Fig. 3. Influx of propidium iodide into Intestine 407 cells after treatment with TDH. MFI, mean fluorescence intensity (arbitrary units) measured by FACScan. (0) In the presence of 1 mM Ca’+; (0) in the presence of 0.1 mM EGTA. Data are obtained from three independent experiments.
TDH lost up to half of their lysosomal integrity compared with the control in the presence of either Cazf or EGTA. The dose of TDH to induce 50% of this effect is 0.8 gg ml - ’ _ The results show that loss
integrity
Eukaryotic cells have a basic set of membranebound organelles. Among them, lysosomes are the major acidic intracellular compartments and play an important role in intracellular digestion [8]. Neutral red is a weak base which is uncharged and membrane-permeant at neutral pH, but membrane-impermeable once protonated. Thus, Neutral red will accumulate within membrane vesicles that have acidic internal pH [9]. Neutral red retention assay has been used to analyse lysosomal integrity [lo]. Employing this assay, we checked whether TDH has any deleterious effect on the lysosomes in Intestine 407 cells. As shown in Fig. 4, Intestine 407 cells treated with
40
! .Ol
. . . ...1
. . . . ...7
.1
concentration
. . . .....(
1
of
. . .mlrq
10
TDH
100
(Fg/ml)
Fig. 4. Lysosome integrity of Intestine 407 cells after treatment with TDH. Intestine 407 cells were incubated with serial dilution of TDH in the presence of 50 pg ml-’ Neutral red for 3 h at 37°C. (0) In the presence of I mM Car+; (0) in the presence of 0. I mM EGTA. Data are obtained from three independent experiments.
237
Fig. 5. Morphological Arrows, mitochondria
changes in Intestine 407 cells following TDH treatment. stained with rhodamine 123; arrow heads. large bleb.
of lysosomal integrity induced by TDH was independent of the influx of Ca*+o. 3.5. Morphological
changes
Loading cells with rhodamine 123, a fluorochrome which selectively stains mitochondria, allows better observation of the morphological changes of Intestine 407 cells induced by TDH through a fluorescence microscope than that through phasecontrast microscope. We stained the Intestine 407 with rhodamine 123 and treated the cells with TDH in the presence of either EGTA or Ca’+. In both cases, TDH caused the plasma membrane to swell out, forming large blebs. These results show that TDH treatment caused the cellular degeneration of Intestine 407 and that these morphological changes were not dependent on the Ca’+o influx (Fig. 5).
4. Discussion In this work, we investigated the effects of TDH on Intestine 407, a cell line derived from human embryonic intestine. Within 30 min of incubation with TDH, levels of cytosolic free Ca2+ (Ca”i) apparently increased in the presence of Ca2+ o. In the absence of extracellular Ca’+ (Ca*+ o), however, no elevation of Ca*+ i was observed (Fig. 1). These data indicate that the plasma membrane became permeable to Ca’+ due to the action of TDH and that increases in Ca’+ i in Intestine 407 were due to an influx of Ca’+ o rather than the release of Ca2+ from intracellular stores.
(A) Cells treated with TDH. (B) Cells not treated with TDH.
The cytotoxicity of TDH to Intestine 407 cells was shown by a drastic loss of the reducing power of the cells which is considered to be essential for cell biosynthesis and for keeping the cellular order [l 11 (Fig. 2). Moreover, other effects such as damage to plasma membranes and lysosomes (Figs. 3 and 4) and morphological degeneration resulting in the formation of large blebs (Fig. 5) were also observed. However, even when the influx of Ca2+ o was inhibited by extracellular Cazc chelator (EGTA), neither the cytotoxicity nor other deleterious effects of TDH was prevented. Thus, these effects were shown to be independent of the elevation of Ca’+i, i.e. irrelevant to any Ca*+-dependent pathway. Ca” o has been reported to be important for the degenerative effects of TDH on mouse myocardial and melanoma cells [4]. The present data seem to be inconsistent with these previous findings. These discrepant results are most likely due to either the different cell types used, or to the differences in the experimental systems. Following the investigation of the effects of various membrane-active toxins on rat hepatocytes, breakdown of the homeostasis of intracellular Ca’+ due to the influx of Ca’+ o has been considered to represent or initiate a final common pathway by which the cells are killed [2]. This Cazf o-influx model is also consistent with the cytotoxic activity of RTX hemolysin family, another group of membrane active agents [ 121. The present study shows, however, that TDH, also a membrane-active toxin, causes cell death without any necessity for Ca’+, an exception to the generally accepted Ca”-dependent cause of cell death referred to as above. This finding might give new insights into the mechanism of cell death
23X
G.-Q. Tang et al. / FEMS Microbiology
by a variety of membrane-active toxins, the mode of action of which is not yet well established.
Acknowledgements
This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.
References [I] Honda,
T. and Iida, T. (19931 The pathogenicity of Vibrio and the role of the thermostable direct haemolysin and related haemolysins. Rev. Med. Microbial. 4, 106-I 13. [2] Schanne, F.A.X., Kane, A.B., Young, E.E. and Farker, J.I. (19791 Calcium dependence of toxic cell death: a final common pathway. Science 206, 700-702. [3] Huntley, J.S. and Hall, A.C. (19941 Aspects of the haemolytic reaction induced by Kanagawa haemolysin of Vibrio pclrahaemo/yticus. Toxicon 32, 1397- 1412. [4] Goshima, K., Owaribe, K., Yamanaka, H. and Yoshino, S. (19781 Requirement of calcium ions for cell degeneration parahaemol~ticus
Letters
134
C19951233-238
with a toxin (vibriolysin) Infect. Immun. 22, x21-832
from
[51 Tang, Cl., Iida, T., Yamamoto, K. and Honda, T. (lYY4) A mutant toxin of Vibrio parahaemolyticus thermostable direct hemolysin which has lost hemolytic activity but retains ability to bind to erythrocytes. Infect. Immun. 62, 3299-3304. [6] Ishiyama, M., Shiga, M., Sasamoto, K., Mizoguchi. M. and He, P. (19931 A new sulfonated tetrazolium salt that produces a highly water-soluble formazan dye. Chem. Pharm. Bull. 46, II 1X-l 122. [7] Jonas, D., Walev, I., Berger, T.. Liebetrau, M., Palmer. M. and Bhakdi, S. (19941 Novel path to apoptosis: small transmembrane pores created by staphylococcal alpha-toxin in T lymphocytes evoke internucleosomal DNA degradation. Infect. Immun. 62, 1304-1312 [Xl Alberts, B.. Bray, D., Lewis, J.. Raff, M., Roberts, K. and Watson, J.D. (19891 Molecular Biology of the Cell, 2nd edn., pp. 459-465. Garland Publishing, New York, NY. 191 Mellman, 1.. Fuchs. R. and Helenius, A. (1986) Acidification of the endocytic and exocytic pathways. Annu. Rev. Biochem. 55, 663-700 [IO] Griffiths, G.D., Lindsay. C.D. and Upshall. D.G. (19941 Examination of the toxicity of several protein toxins of plant origin using bovine pulmonary endothelial cells. Toxicology 90, 1 l-27. [I 11 Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K. and Watson, J.D. (19891 Molecular Biology of the Cell, 2nd edn.. pp. 7 I-78. Garland Publishing, New York, NY. 1121 Welch. R.A. (19941 In: Molecular Genetics of Bacterial Pathogenesis (Miller. V.J., Kaper, J.B., Portnoy, D.A. and Isberg, R.R., Eds.1, pp. 351-364. American Society for Microbiology, Washington, D.C.
FEMS Microbiology
Letters 134 (1995) 233-238
Ca’ +-independent cytotoxicity of Vibrio parahaemolyticus thermostable direct hemolysin ( TDHj on Intestine 407, a cell1line derived from human embryonic intestine Guang-Qing Tang, Tetsuya Iida *, Koichiro Yamamoto, Takeshi Honda Received 22 September 1995; accepted I6 October I995
Abstract The effects of Vihrio puruhaemo~~yricusthermostable direct hemolysin on Intestine 407, a cell line derived from the intestine of human embryo, were investigated. The hemolysin was shown to be cytotoxic to Intestine 407. This cy!otoxicity is accompanied by the damage of plasma membrane and lysosomes, as well as cellular degeneration in the form of large transparent blebs. Although an increase in cytosolic free Ca’+ due to the influx of extracellular Ca” was observed in cells treated with thermostable direct hemolysin, it was found to be irrelevant to any of the above effects. These results suggest that the effects of thermostable direct hemolysin observed in this study on Intestine 407 are not mediated by Ca”+-dependent pathways. Keword.\:
Vihrio puruhwmo/~ticus;
Cytotoxicity;
Thermostable direct hemolysin: Intestine 407
1. Introduction Thermostable direct hemolysin (TDH) is considered to be the major virulent factor of Vibrio parukaemolyticus in causing food-borne gastroentetitis [I]. This toxin has shown a diversity of biological activities such as hemolytic activity, enterotoxicity, myocardiotoxicity as well as cytotoxicity on cultured cells [I]. Ca” plays an important role in a variety of cellular processes. In killing rat hepatocytes by various membrane-active toxins, influx of extracellular
‘ Corresponding author. Tel: + 8 I (6) 879 8276; Fax: + 8 I (6) 879 8277.
037%1097/95/$09.50 SSDl
037%1097(95)0041
0
1995 Federation of European Microbiological l-4
Ca’+ (Ca’+o) has been considered to represent or initiate a final common pathway [2]. TDH is a membrane-active toxin [3]. With regard to th!z role of Ca’+ in effects of TDH, Goshima and ccilleagues have shown that TDH induces the influx of Ca” o to mouse myocardial cells and melanoma cells. and have demonstrated that this influx is essentiil for the cell degeneration caused by TDH [4]. Recently we have shown that TDH increases the membtine permeability of human erythrocyte membrane to Ca’+ bivalent cations and propidium iodide [5]. On the other hand, however, even when an influx df Ca” o did not occur, TDH was still able to lyse human erythrocytes [5]. Thus concerning Cal+ dedendency of TDH action, previous studies have come to discrepant conclusions. In this study, we employed a Societies. All rights reserved
cell line derived from the human embryonic intestine, Intestine 407, to investigate whether or not the calcium ion is involved in TDH cytotoxicity.
extracellular sults.
milieu
2.5. Cytotoxicily 2. Materials and methods
2. I. Cell cultures Intestine 407 cells (ATCC CCL 61 were grown in Petri dishes containing Eagle minimal essential medium (EMEM) (Biken, Osaka, Japan) supplemented with 10% calf serum. Cells were cultured at 37°C in a humid atmosphere of 95% air and 5% carbon dioxide. Cells grown to 80-90% confluence were harvested by repeatedly pipetting with EMEM to detach and disperse the cells, followed by centrifugation at 800 X g for IO min. The cell pellet was suspended in phosphate-buffered saline (PBS) supplemented with 5.5 mM glucose (PBS-glucose). The cell suspensions were used for the following experiments. 2.2. Purification
of TDH
TDH was purified
according
to the previous
re-
port [51. 2.3.Measurement
of,free Ca’+ level
Preparations of lo6 cells ml-’ of Intestine 407 cells were loaded with Fluo 3 (5 PM, Flu0 3-AM, Dojindo, Fukuoka, Japan) in the dark at 37°C for 30 min in PBS-glucose. These preparations of Intestine 407 cells loaded with Fluo 3 were allowed to interact with serial dilutions of TDH at 37°C for 30 min in 120 ~1 PBS-glucose containing either 1 mM CaCl, or various concentration of EGTA, and then analysed by flow cytometry as described below. 2.4. Analysis of the role of Ca” Intestine 407
in TDH effects on
The above measurement of Ca’+ i showed that 0.1 mM extracellular EGTA was sufficient to completely prevent the TDH-induced elevation of Ca2+ i. Consequently, in all the following experiments, either 1 mM Ca2+ or 0.1 mM EGTA was added into the
to compare
the subsequent
re-
analysis
Preparations of IO6 cells ml-’ of Intestine 407 cells in a 96-well plate were incubated with serial dilutions of TDH for 30 min at 37°C in 120 ~1 PBS-glucose. Subsequently, 10 ~1 of solution prepared from a cell counting kit (Dojindo, Fukuoka, Japan) were added to each well and incubated for another 2.5 h. Optical density was measured on the Titertek Multiskan MCC/340 MKII (Lab-systems, Finland) at 450 nm. Positive controls were cells treated with 0.2% Triton X-100 and negative controls were those without toxin treatment. Viability was calculated using the optical density of toxintreated COD,), positive control COD,,) and negative control COD,,) samples. The viability formula was (OD, - OD,,)/(OD,, - OD,, 1 X 100%. 2.6. Measurement
of injlax of propidium
iodide (PI)
Preparations of IO6 cells mll ’ of Intestine 407 cells were allowed to interact with serial dilutions of TDH at 37°C for 30 min in 120 ~1 PBS-glucose containing IO pg ml-’ PI and then analysed by flow cytometry as below. 2.7. Neutral red retention assay Preparations of IO6 cells ml-’ of Intestine 407 cells were allowed to interact with serial dilutions of TDH in 120 ~1 PBS-glucose containing 50 pg ml - ’ neutral red at 37°C for 3 h. The cells were washed twice with PBS after which the Neutral red taken up by Intestine 407 was lysed out using 0.5 N HCl/50% ethanol. After centrifugation of the cell debris, the optical density of the supematant was read on the Titertek Multiskan MCC/340 MKII at 540 nm. Positive controls were cells treated with 0.2% Triton X-100 and negative controls were those without toxin treatment. Lysosomal integrity was calculated using the optical density of toxin-treated COD,), positive control (OD,,) and negative control COD,,) samples. The percentage of lysosomal integrity was (OD, - OD,,)/(OD,, - OD,,) X 100%.
G.-Q. Tmg et al. / FEMS Microhiohgy
2.8. Microscopic
25 -
obsenation
Preparations of IO’ cells ml-’ of Intestine 407 cells were loaded with 5 pg ml-’ rhodamine 123 (Calbiochem-Novabiochem Corporation, San Diego, CA) at 37°C for 10 min in PBS-glucose and then allowed to interact with 5 pg ml-’ TDH for 30 min at 37°C in 120 ~1 PBS-glucose. Cell samples were mounted on glass slides, covered with cover slips, and the morphology of the cells was immediately observed and photographed through a fluorescence microscope (Nikon, Tokyo, Japan).
235
Lrttrr.s 134 (IYYS) 233-238
20-
15ii z io-
2.9. Flow cytometry
concentration Fig.
I. Cytosolic
treatment
1
.l
.Oi
,001
Flow cytometry was carried out by using FACScan (Fluorescence-activated cell sorter, BectonDickinson, San Jo&, CA) to acquire 10000 events for each sample. The fluorescence intensity of Fluo 3 was recorded at 5 15-545 nm and that of PI was recorded at 543-627 nm.
free Ca’+
presence of
I
levels in Intestine 407 cells after MFI,
mean fluorescence
units) measured by FACScan.
mM Cal+
; (0)
100
of TDH (pglml)
with serial doses of TDH.
intensity (arbitrary
10
in the presence of 0.
(0)
I
In the
tqM EGTA.
Data are obtained from four independent experiments. For experimental details, bee Materials and methods.
3. Results 3.2. Cytotoxicifi
c$ TDH to Intestine 407
3.1. Change of cytosolic free Ca’ ’ level TDH has previously been shown to induce influx of the extracellular Ca” in the mouse myocardial and melanoma cells [4]. Using Fluo 3, a fluorescent indicator of Ca’+, we monitored the changes in cytosolic Ca’+ levels in Intestine 407 cells treated with TDH in the presence of either Ca2 + or EGTA. As shown in Fig. I, there was an elevation of Ca2+ i level in cytoplasm which was dependent on the dose of TDH in the presence of 1 mM Ca”o. At a dose of 0.3 /*g ml-’ of TDH, 50% of this effect was reached. In contrast, in the absence of Ca’+ o (0. I mM EGTA was added to the reaction solution to chelate possible trace amounts of Calf ), there was no elevation of Ca’+ i levels even after 30 min treatment with TDH. These results indicate that TDH-induced elevations of the Ca’+i level are not due to the release of Ca’+ from intracellular stores but rather due to the influx of Ca’+o. The data also show that increases of Ca2+ i levels could be thoroughly prevented by adding the Ca’+ chelator, EGTA into the extracellular milieu, to eliminate possible trace amounts of Ca’+.
TDH has been shown to be toxic to a variety of cultured cells [I]. Here we examined whether TDH is also cytotoxic to Intestine 407 cells by using a cell counting kit developed by Dojindo. This kitlemploys a tetrazolium reagent which is reduced andlchanged into a soluble Formazan dye under capacity generated by various dehydrogenase activities in viable cells. It can thus be used to count the viable cells 161. As shown in Fig. 2, Intestine 407 underwent a dose-dependent loss of viability due to treatment with TDH. At 0.8 pg ml-‘, TDH accobnplished 50% of this cytotoxic effect. The results in Fig. 2 also show that the cytotoxicity of TDH is not affected in the presence or absence of Ca’+o influx, indicating that TDH cytotoxicity was not due to the increase in Ca’+ i. 3.3. Injlux
of propidium iodide (PI)
Propidium iodide is widely used as an indicator of cell plasma membrane integrity [7]. The plasma membrane of human erythrocyte has been, reported to be damaged by TDH and became permeable to PI
G.-Q.
Tang et al. / FEMS
Microbiology
Letters 134 ( lYYSl233-238 120-
100
100-
60
60-
60
ii
60-
I 40
40-
20
20 1 0 :
0 .Ol
.l
1
concentration
of
10
TDH
100
[5]. Here we analysed the changes in membrane permeability to PI of Intestine 407 under the action of TDH in the presence of either EGTA or Ca2+. As shown in Fig. 3, TDH induced the influx of PI into the cells dose-dependently in the presence of either EGTA or Ca2+. At 0.8 pg ml -I, TDH reached the median level of this effect. These results indicate that Ca*’ o influx is not necessary for TDH to damage the plasma membrane of Intestine 407. 3.4. Loss of lysosomal
..(
.
..m”..,
.Ol
.
.l
concentration
&g/ml)
Fig. 2. Viability of Intestine 407 cells after treatment with TDH. Intestine 407 cells were incubated with serial dilutions of TDH at 37°C with subsequent addition of solution from a cell counting kit. (0) In the presence of 1 mM Ca*+; (0) in the presence of 0.1 mM EGTA. Data are obtained from three independent experiments.
. . . . ..
,001
of
,...
7
.
.
“‘“1
1
10
TDH
@g/ml)
100
Fig. 3. Influx of propidium iodide into Intestine 407 cells after treatment with TDH. MFI, mean fluorescence intensity (arbitrary units) measured by FACScan. (0) In the presence of 1 mM Ca’+; (0) in the presence of 0.1 mM EGTA. Data are obtained from three independent experiments.
TDH lost up to half of their lysosomal integrity compared with the control in the presence of either Cazf or EGTA. The dose of TDH to induce 50% of this effect is 0.8 gg ml - ’ _ The results show that loss
integrity
Eukaryotic cells have a basic set of membranebound organelles. Among them, lysosomes are the major acidic intracellular compartments and play an important role in intracellular digestion [8]. Neutral red is a weak base which is uncharged and membrane-permeant at neutral pH, but membrane-impermeable once protonated. Thus, Neutral red will accumulate within membrane vesicles that have acidic internal pH [9]. Neutral red retention assay has been used to analyse lysosomal integrity [lo]. Employing this assay, we checked whether TDH has any deleterious effect on the lysosomes in Intestine 407 cells. As shown in Fig. 4, Intestine 407 cells treated with
40
! .Ol
. . . ...1
. . . . ...7
.1
concentration
. . . .....(
1
of
. . .mlrq
10
TDH
100
(Fg/ml)
Fig. 4. Lysosome integrity of Intestine 407 cells after treatment with TDH. Intestine 407 cells were incubated with serial dilution of TDH in the presence of 50 pg ml-’ Neutral red for 3 h at 37°C. (0) In the presence of I mM Car+; (0) in the presence of 0. I mM EGTA. Data are obtained from three independent experiments.
237
Fig. 5. Morphological Arrows, mitochondria
changes in Intestine 407 cells following TDH treatment. stained with rhodamine 123; arrow heads. large bleb.
of lysosomal integrity induced by TDH was independent of the influx of Ca*+o. 3.5. Morphological
changes
Loading cells with rhodamine 123, a fluorochrome which selectively stains mitochondria, allows better observation of the morphological changes of Intestine 407 cells induced by TDH through a fluorescence microscope than that through phasecontrast microscope. We stained the Intestine 407 with rhodamine 123 and treated the cells with TDH in the presence of either EGTA or Ca’+. In both cases, TDH caused the plasma membrane to swell out, forming large blebs. These results show that TDH treatment caused the cellular degeneration of Intestine 407 and that these morphological changes were not dependent on the Ca’+o influx (Fig. 5).
4. Discussion In this work, we investigated the effects of TDH on Intestine 407, a cell line derived from human embryonic intestine. Within 30 min of incubation with TDH, levels of cytosolic free Ca2+ (Ca”i) apparently increased in the presence of Ca2+ o. In the absence of extracellular Ca’+ (Ca*+ o), however, no elevation of Ca*+ i was observed (Fig. 1). These data indicate that the plasma membrane became permeable to Ca’+ due to the action of TDH and that increases in Ca’+ i in Intestine 407 were due to an influx of Ca’+ o rather than the release of Ca2+ from intracellular stores.
(A) Cells treated with TDH. (B) Cells not treated with TDH.
The cytotoxicity of TDH to Intestine 407 cells was shown by a drastic loss of the reducing power of the cells which is considered to be essential for cell biosynthesis and for keeping the cellular order [l 11 (Fig. 2). Moreover, other effects such as damage to plasma membranes and lysosomes (Figs. 3 and 4) and morphological degeneration resulting in the formation of large blebs (Fig. 5) were also observed. However, even when the influx of Ca2+ o was inhibited by extracellular Cazc chelator (EGTA), neither the cytotoxicity nor other deleterious effects of TDH was prevented. Thus, these effects were shown to be independent of the elevation of Ca’+i, i.e. irrelevant to any Ca*+-dependent pathway. Ca” o has been reported to be important for the degenerative effects of TDH on mouse myocardial and melanoma cells [4]. The present data seem to be inconsistent with these previous findings. These discrepant results are most likely due to either the different cell types used, or to the differences in the experimental systems. Following the investigation of the effects of various membrane-active toxins on rat hepatocytes, breakdown of the homeostasis of intracellular Ca’+ due to the influx of Ca’+ o has been considered to represent or initiate a final common pathway by which the cells are killed [2]. This Cazf o-influx model is also consistent with the cytotoxic activity of RTX hemolysin family, another group of membrane active agents [ 121. The present study shows, however, that TDH, also a membrane-active toxin, causes cell death without any necessity for Ca’+, an exception to the generally accepted Ca”-dependent cause of cell death referred to as above. This finding might give new insights into the mechanism of cell death
23X
G.-Q. Tang et al. / FEMS Microbiology
by a variety of membrane-active toxins, the mode of action of which is not yet well established.
Acknowledgements
This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.
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