ADONIS
Immunology 1991 72 73-78
001928059100013N
Presence of Ca2 + is obligatory for the cytotoxic activity of dengue virus-induced cytotoxic factor M. KHANNA, U. C. CHATURVEDI, R. DHAWAN, B. L. TEKWANI* & V. C. PANDEY* Postgraduate Department of Microbiology, K.G. Medical College and the *Central Drug Research Institute, Lucknow, India Acceptedfor publication 1I September 1990
SUMMARY The present study was undertaken to investigate the role of calcium ions (Ca2+) in the cytotoxic activity of the cytotoxic factor (CF) produced by T lymphocytes of the dengue type 2 virus (DV)-infected mouse spleen. It was observed that CF prepared in Ca2+-free medium had no cytotoxic activity on normal mouse spleen cells suspended in Ca2 +-free medium but had activity on cells suspended in medium having Ca2+. The cytotoxic activity of CF was restored by substitution with calcium chloride, the optimal dose being 10-' M. CF induced influx of Ca2+ as measured by uptake of radiolabelled calcium chloride (45Ca), in the susceptible target cells, macrophages (MO) and T lymphocytes, but had no effect on CF-resistant B lymphocytes. Calcium channel blocking drugs, like verapamil, nifedipine and diltiazem, inhibited the cytotoxic activity of CF and also the CF-induced influx of 45Ca in MO and T cells. Thus, presence of Ca2+ is obligatory for the cytotoxic activity of CF and the cell death is associated with increased intracellular Ca2+.
lity.3-8 But, the precise mechanism of cell injury during dengue virus infection is not known. Cell cytotoxicity can be mediated by mechanisms which are dependent on calcium ions (Ca2+), while other mechanisms do
INTRODUCTION Dengue virus infection produces immunopathological lesions characterized by lymphoid cell necrosis, thrombocytopenia, alterations in peripheral blood leucocytes and increased capillary permeability. A progressive damage of spleen characterized by hypocellularity, necrosis of cells and disorganization and atrophy of follicles has been demonstrated in dengue type 2 virus (DV)-infected mice. Further, such damage in the spleen cells and peripheral blood leucocytes has been shown to be mediated by a cytokine, the cytotoxic factor (CF), which is produced by T lymphocytes (Ly-1+23-) of the DV-infected mouse spleen (reviewed by Chaturvedi).' 2 CF has been purified by various chromatographic techniques. It separates out in a single band on native polyacrylamide gel electrophoresis (PAGE) at the 43,000 molecular weight (MW) position, and as two bands at the 22-25,000 MW position on SDS-Page. These bands react with similar intensity with the specific anti-CF antibody in the Western blot test. The N' terminal sequence of amino acids of CF has been determined. A comparison of the N' terminal amino acid sequence of CF with that of other cytotoxic protein molecules, such as mouse lymphotoxin, tumour necrosis factor and T-cell serine protease, did not show significant homology (M. Khanna and U. C. Chaturvedi, manuscript submitted for preparation). CF is a highly potent protein molecule which selectively kills T-helper cells (Th), I-Amacrophages and acts on cells capable of liberating histaminelike substances, thus producing increased capillary permeabi-
not require the presence of calcium. A sustained increase in cytosolic Ca2+ has been associated with the onset of cytotoxicity characterized by disruption of cytoskeleton, DNA fragmentation and extensive damage to other subcellular components leading to cell death (reviewed by Orrenius et al.9). The present study was undertaken to investigate the role of Ca2+ in the cytotoxic activity of CF. It demonstrates that the presence of Ca2+ is obligatory for the cytotoxic activity of CF and cell death is associated with influx of Ca2+ in the susceptible target cells, namely T lymphocytes and macrophages (M4).
MATERIALS AND METHODS Animals Inbred Swiss albino mice aged 2-4 months were used in this study and obtained from the colony maintained in this Department. Virus Dengue type 2 virus (DV), strain P23085, was used in the form of infected mouse brain suspension, as described elsewhere.'0
Reagents Verapamil and nifedipine (Sigma Chemicals Co., St Louis, MO) were dissolved in 30% ethyl alcohol as a stock solution; diltiazem hydrochloride (Anglo-French Drug Co. Eastern Ltd,
Correspondence: Professor U. C. Chaturvedi, Dept. of Microbiology, K.G. Medical College, Lucknow-226 003, India.
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Bangalore, India) was dissolved in the medium used. The stock solutions of the drugs were prepared on the day of the experiment and were used in serial dilution. Radiolabelled calcium chloride (45Ca) was obtained from Bhabha Atomic Research Centre, Bombay, India and had a specific activity of 77-4 mCi/gm. It was appropriately diluted and used in doses of 1-3 pCi/ml of cell suspension. Preparation of the cytotoxic factor (CF) CF is secreted by spleen cells obtained from DV-infected mice or can be extracted from such cells."I Briefly, the spleens were removed aseptically from DV-infected moribund mice and the cells were teased out in chilled phosphate-buffered saline, pH 7 (PBS). A single-cell suspension of the cells was prepared in PBS and cell count was adjusted to 2 x 106 cells/ml. The cells were distributed in 5-cm glass Petri dishes in a volume of 4 ml and cultured at 370 in the presence of 5% CO2. After 24 hr the cultures were harvested and the cells were spun down at 2000 g for 10 min. The clear supernatant was tested for cytotoxic activity and stored in small aliquots at -70°. CF was purified by Pharmacia low pressure liquid chromatography (LPLC; Uppsala, Sweden) using Sephacryl S200 gel packed in a 650 x 16 mm column with a bed height of 450 mm. Proteins were eluted with 0-5 M PBS at 40 cm3/hr and the elution fluid was monitored at 280 nm in a UV-Cord. The protein peaks obtained were assayed for cytotoxic activity. The purified CF was vacuum dried or freeze dried in a Speed Vac (Savant Instruments Inc., Farningdale, NY) and stored at -70 .
Preparation of target cells Normal mouse spleen was teased out with forceps in chilled minimum essential medium (MEM) containing 10% bovine serum. A single-cell suspension was treated with Tris ammonium chloride solution to haemolyse the red blood cells. The cells were washed and the viable nucleated cells were counted using the trypan blue dye exclusion technique.'2 Enriched subpopulations of T and B lymphocytes were prepared by filtration of spleen cells through glass-wool and nylon-wool columns. 13.14 The purity of the cell population was screened as described elsewhere.'5 A MO-enriched subpopulation was prepared by collecting glass-adherent cells obtained by layering splenic or peritoneal cells on 5-cm glass Petri dishes and incubating at 37° in the presence of 5% CO2 for 90 min.'6 The non-adherent cells were removed by repeated washing and the glass-adherent cells were scraped off with rubber tipped rod and suspended in MEM after washing three times. About 98% of these cells phagocytosed latex particles and were therefore considered MO enriched subpopulation.'6 In the preliminary experiments identical results were obtained with the splenic and peritoneal M4; therefore, in subsequent experiments peritoneal MO> only were used. Assay of cytotoxic activity of CF Cytotoxic activity of the preparation was assayed using normal mouse spleen cells as target cells. In some experiments MO or T-cell-enriched subpopulations were used. The technique for the assay of cytotoxic activity of CF is described elsewhere.6" Briefly, equal volumes of the test solution and the target cells (2 x 1 06/ml) were mixed in a microtitre U-well perspex plate and
50
.40
8 30 0
at
10 0
2
4 8 16 Dilution of CF
32
Figure 1. Effect of absence of Ca2+ from the milieu on the cytotoxic activity of CF. The activity of CF was assayed using (i) CF prepared in Ca2+-free medium and the target cells suspended in Ca2+-free medium (U); (ii) CF prepared in Ca2 + -free medium and the target cells suspended in the medium containing Ca2+ (A); and (iii) CF and the target cells both, prepared in medium containing Ca2+ (0).
incubated at 4°C for 1 hr. Viable cells were counted using trypan blue dye and the percentage of non-viable cells was calculated after deducting background non-viable cells. Assay of 45Ca uptake Uptake of 45Ca by the target cells was assayed by the technique of Freedman & Khan'7 and Birx et al.'8 Variously treated target cells (I x I07 cells/ml) were incubated with 1-3 pCi of 45Ca/ml at 37° for 30 min. CF kills the target cells equally well both at 4° and 370,11 therefore, the target cells were treated with CF at 4° for 30 min and then transferred to 370 for optimum uptake of 45Ca. The reaction was stopped by putting the tubes in an ice bath and the cells were washed with chilled 0-15 M PBS containing 1-8 mm CaC12 to remove extracellular calcium. The cells were harvested onto a fibre-glass filter using a 10-place filtration assembly (Millipore Co., Bedford, MA) and washed repeatedly with chilled 015 M PBS containing 1-8 mM CaC12. The filters were transferred to glass vials containing 10 ml scintillant fluid and the radioactivity was determined with a LKB (Wallac) Beta counter. The counts per min (c.p.m.) in control and experimental groups were expressed after deducting background c.p.m. The background c.p.m. was obtained after similar procedures from tubes containing medium, CF, drugs and radiolabelled medium without cells. The control group data consisted of c.p.m. in cells and 45Ca without CF or drugs. The experiments were set up in triplicate and were repeated thrice. Mean values + standard error of the mean have been presented. The data were analysed statistically using the Student's t-test for P value. A P value of more than 0-05 was considered insignificant. RESULTS Presence of calcium is essential for cytotoxic activity of CF CF prepared in Ca2+-free medium was assayed for cytotoxic activity on target spleen cells suspended in Ca2+-free medium. In addition, CF was assayed using target cells suspended in MEM (containing Ca2+). In the control, CF prepared in the usual way (in medium containing Ca2+) was assayed on target cells suspended in MEM. The findings summarized in Fig. 1 show that the cytotoxicity of CF was significantly reduced when Ca2+ was absent from both the CF preparation and the test system. The cytotoxic activity of the CF prepared in Ca2+-free medium but tested on target cell suspended in presence of Ca2+ was
Dengue virus-induced cytokine )require Ca2+ for cytotoxicity
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Higher concentration of CaCl2 (10-1 or 10-2 M) alone killed large number of the target cells without the addition of CF.
50
40
30 C 020
OR20
l0
2 3 4 5 6 7 8 910 Molar dilution of CaC12 (log10) Figure 2. Effect of calcium on the cytotoxic activity of CF. The target cells prepared in Ca2+-free medium were treated with various concen-
0
trations of CaCl2 and used to assay the cytotoxic activity ofCF prepared in Ca2+-free medium (0). (-) Cells treated with CaCl2 only.
Table 1. 45Ca influx in CF-treated cells
Cells
c.p.m. Dilution Percent of CF Untreated cells CF treated cells alteration
Macrophages
0 1:32 1:64
3560 +415 3496+650 3356+417
2424+ 306 6363 +809 4015+351
-32 +82 +20
T cells
0 1:32 1:64
3870+ 331 3200+538 3001 +395
2856+225 5762+478 3729+510
-26 +80
0 1:32 1:64
2419+271 3649 +366 3552+784
2318+305 3596+507 3713+409
-4 -2 +4
B cells
+24
Enriched subpopulations of macrophages, T and B cells were treated with various dilutions of CF followed by incubation with I pCi/ml of 45Ca and the c.p.m. were assayed in the cells. Percentage increase (+) or decrease (-) in intracellular 45Ca were calculated as follows: % alteration= 100X c.p.m. of CF treated cells-background c.p.m. _ 100 c.p.m. of untreated cells -background c.p.m.
comparable with the control. Thus the findings showed that calcium is essential for mediation of the cytotoxic activity by CF. Substitution of calcium restores cytotoxic activity of CF To investigate quantitatively the effect of Ca2+, target cells suspended in Ca2+-free medium were treated with graded molar concentrations of CaCI2 for I hr at 37°. To these cells CF (1:2 dilution) prepared in Ca2+-free medium was added and the mixture was further incubated at 40 for 1 hr and the non-viable cells counted. In the presence of I0- or 10-10 M CaCI2, cytotoxic activity of CF was negligible. With 10-8 M the cytotoxic activity increased, reaching levels similar to that of controls with 10-7 M concentrations (Fig. 2). The cytotoxic activity of CF remained at similar levels with concentrations of CaCI2 upto 10-3 M.
Effect of CF on calcium influx into target cells The preceeding experiments indicate Ca2+ is essential for the cytotoxic activity of CF. To investigate whether the target cell killing was due to enhanced Ca2+ ion influx into the target cells, enriched subpopulations of MO, T and B lymphocytes were used and intracellular calcium was measured by estimation of radiolabelled 45Ca. CF was used in three doses, undiluted, 1:32 and 1:64 diluted. For this experiment, 2 x 106 MO were treated with CF and incubated at 4° for 30 min followed by addition of 100 pl of 45Ca. After a further incubation at 37° for 30 min the cells were washed with 1-8 mm cold calcium chloride and the cells were processed for intracellular 45Ca estimation. Table I shows that with a 1: 32 dilution of CF the 45Ca uptake by MO was increased by 82% while that with 1:64 dilution was 20%. Undiluted CF killed all the susceptible target cells quickly, therefore intracellular 45Ca was reduced. The above experiment was repeated using a T-cell enriched subpopulation as target cells. With undiluted CF the decrease in intracellular 45Ca was 26%, while treatment with 1:32 diluted CF increased intracellular 45Ca by 80%. The increase was 24% with 1:64 dilution of CF (Table 1). We have observed that CF does not kill B lymphocytes.3 Therefore, experiments were done to investigate whether alterations in intracellular Ca2+ could be induced by CF in cells which are not adversely affected. Similar experiments with a B-lymphocyte enriched subpopulation showed that no significant alteration occurred in intracellular 45Ca following treatment with various doses of CF (Table 1). Effect of calcium channel blocking drugs on cytotoxicity of CF Drugs are now available which inhibit calcium ion influx through blocking passive 'slow' channels. 19.20 The present experiment was, therefore, carried out to investigate the effect of various calcium channel blocking drugs on the cytotoxic activity of CF. The drugs used were verapamil, nifedipine and diltiazem. Target spleen cells were treated with different concentrations of various calcium channel blocking drugs at 370 for 30 min. The cells were spun down at 2000g for 10 min, resuspended in MEM and used as target cells to assay the cytotoxic activity of CF. The controls included with each test were drug-treated cells alone and untreated spleen cells treated with CF. The data presented in Fig. 3 show that CF killed 45 + 5% of the untreated target spleen cells. Treatment of the target cells with all the three drugs inhibited cytotoxic activity of CF to a similar extent in a dosedependent manner. With I0-I M concentration of verapamil the inhibition was 85%, while that with 10-7 M was 32%. Similar results were obtained by treating target cells with nifedipine or diltiazem. The drugs as such had no adverse effect on the target cells with the concentrations used in the test (data not presented here). The ethanol solution used for dissolving the drugs was similarly diluted and its effect on the target cells was screened. It had no cytotoxic effect on the cells in the dilutions used in the test (Fig. 3). CF kills T cells and MO but has no effect on B cells.3 Further experiments were made using enriched T cells and MO subpopulations at target cells to study the effect of calcium channel
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Cells Drug dilution
40
MO 10-3
20
40
% decrease 60 80
100
verapamil
en
30 c
0o c
MO
10-5
MO
10-7
T
10-3
T
10-5
T
10-7
20
10
4 0
C 10-3 10-5 10-7 Molar conc. of drug
Figure 3. Effect of calcium channel blocking drugs on the cytotoxic activity of CF. Normal mouse spleen cells were treated with various doses of verapamil (A), nifedipine (0) or diltiazem hydrochloride (-) or remained untreated for control (C,O). The cytotoxic activity of CF was assayed using these cells as target. (A) Cells treated with diluent only.
Figure 5. Effect of calcium channel blocking drug verapamil, on CFinduced influx of 45Ca into enriched subpopulations of macrophages (M+) and T lymphocytes (T). The percentage decrease of intracellular 45Ca was calculated as follows: 40r
0/
decrease= c.p.m. of cells treated with - c.p.m. of cells treated
CF
30
drug and with drug
100-
x
c.p.m.
20
100.
of cells treated with CF - c.p.m. of untreated cells
Q)
T5
4 c
20 [
10
10-3 10-5
10-7
C
Cells
Dilution
MO
10-3
MO
10-5
MO
l0o-
T
10-3
T
10-5
T
10-7
20
40
% decreasE 60 80
100
nifedipine
Molar conc. of drug
Figure 4. Effect of calcium channel blocking drugs on the cytotoxic activity of CF on enriched subpopulations of macrophages (MO) and T lymphocytes (T cells). The remaining description is as in Fig. 4.
blocking drugs on the cytotoxic activity of CF. In this experiment verapamil and nifedipine were used. The data presented in Fig. 4a show that CF alone killed 34+4% cells of the MO enriched subpopulation. Treatment of MO with verapamil or nifedipine inhibited the cytotoxicity of CF in a dose-dependent manner; the cytotoxicity being 10 + 2% by treatment with 10-3 M verapamil and 25+3% with 10-7 M of the drug. A similar dose-dependent inhibition of the cytotoxicity of CF on the T-cell enriched subpopulation was observed by treatment of the cells with verapamil or nifedipine (Fig. 4). Effect of calcium channel blocking drugs on CF-induced influx of 45Ca in target cells Further experiments were carried out to investigate the effect of Ca2+ channel blocking drugs on the CF induced calcium ion influx in the target cells. The drugs used were verapamil and nifedipine and the target cells were enriched subpopulations of MO or T lymphocytes. The target cells (2 x 106) were treated with different dilutions of verapamil or nifedipine for 30 min at
Figure 6. Effect of calcium channel blocking drug, nifedipine, on CFinduced influx of 45Ca into enriched subpopulations of the target cells. For details see legend to Fig. 5.
370 followed by addition of CF (1: 32 dilution). After incubation for 30 min at 40 45Ca was added and further incubated at 37° for 30 min. The cells were washed with 18 mm of cold calcium chloride and intracellular 45Ca was measured. The data presented in Fig. 5 show that pretreatment of MO with 10-3 M of verapamil resulted in a 92% inhibition of the influx of CF-induced calcium whereas with 10-5, 10-7 M of verapamil, the inhibition was 62% and 47%, respectively. Similarly, with 10-3 M nifedipine the decrease in calcium uptake was 83%, while with 10-5 and 10-7 M of the drug the decrease was 32% and 41%, respectively (Fig. 6). Similar findings were obtained using a T-cell enriched subpopulation as target cells. It was observed that with 10-3 M
Dengue virus-induced cytokine require Ca2+ for cytotoxicity verapamil the decrease in calcium uptake was 90% while that with I0-I M was 43% (Fig. 5). Therefore, the effect of drug was dose-dependent. Pretreatment of the cells with nifedipine had similar effects (Fig. 6). DISCUSSION The present study indicates that the presence of Ca2+ is obligatory for the cytotoxic activity of CF on the target cells, MO and T cells. These conclusions have been drawn from the experiments which showed that the cytotoxic activity of CF is inhibited by (i) removal of calcium from the milieu; (ii) pretreatment of target cells with calcium channel blocking drugs like, verapamil nifedipine or diltiazem and (iii) substitution of Ca2+ restored the cytotoxic activity of CF. Therefore, killing of susceptible target cells by CF appeared to be a calciumdependent phenomenon. It was observed that removal of Ca2 + from the milieu abrogated the cytotoxic activity of CF. The activity of CF was restored by substitution of Ca2+ in the form of calcium chloride, the optimal dose of which was 10-7 M. Target cell killing by various cytotoxic of factor mechanisms may be Ca2 +-dependent or Ca2 +-independent. Thus, cytotoxic activity of lymphotoxins, tumour necrosis factor, serine esterases and performs is Ca 2+independent. Some clones of cytotoxic T lymphocytes (CTL) are Ca2 +-dependent for their activity, while others are not (reviewed by Clark).2' CF differs from these cytotoxic mechanisms in being Ca2+-dependent for its activity. A progressive damage of spleen occurs in DV-infected mice which is characterized by hypocellularity, necrosis of cells, disorganization and atrophy of follicular architecture. MO and T cells show blebbing of the membrane, hypertrophy of rough endoplasmic reticulum, lobulation of nucleus and cell necrosis and fragmentation.22 Similar subcellular changes leading to cell death are produced in spleen cells by inoculation of CF in mice or by treating these cells in vitro with CF.4 A sustained increase in intracellular Ca2+ is often associated with activation of cytotoxic mechanisms which lead to disruption of cytoskeleton, fragmentation of DNA and damage to other cellular components, resulting into cell death. Recently a number of Ca2+dependent processes have been recognized which lead to cell death; they include activation of proteases, phospholipases and endonucleases, depletion of ATP and thiol modification (reviewed by Orrenius et al.).9 It was observed that CF induced an influx of Ca2+ in the MO and T lymphocytes which was dose dependent, the maximum increase in intracellular 45Ca being about 80% compared to untreated control cells. The role of Ca2+ in cytotoxic activity of CF was further supported by the absence of CF-induced 45Ca influx in B lymphocytes (Table 1), a cell which is not affected by CF.3 CF has been shown to increase capillary permeability through release of histamine.7'8 It is likely that CF-induced increases in Ca2+ in cells (which are sources of vasoactive mediators), as described in the present study, results in their degranulation and release of histamine, etc., increasing the capillary permeability. It has been shown that verapamil, nifedipine and diltiazem inhibit Ca2+ influx into cells through blocking of passive 'slow' channels.'920'23 The data presented here show that calcium channel blocking drugs inhibited the cytotoxic activity of CF in a dose-dependent manner. The effect of all the three drugs used,
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verapamil, nifedipine and diltiazem, was similar. The effect of verapamil and nifedipine was investigated on the CF-induced Ca2+ influx in MO and T cells. It was observed that both the drugs inhibited 45Ca influx to the extent of 90-92% in both types of cells. CF selectively kills I-A negative MO and DV-induced helper T cells (Th) but has no effect on other T cells or B cells.3-5 What determines this selectivity ofCF is not known. It has been shown that a different type of K+ channels are present on CD4+8- cells in comparison with CD4- 8 + cells. It has further been suggested that K+ and Ca2+ influx may be co-ordinately regulated in lymphocytes (reviewed by Klaus).24 It is, therefore, likely that Ca2+ channels which could be activated by CF are present only on Th and I-A- MO, thus determining its selectivity of action. The findings of the present study thus demonstrate that the presence of Ca2+ is obligatory for the mediation of the cytotoxic activity of CF and cell death is associated with increased intracellular Ca2 .
ACKNOWLEDGMENTS The study has been carried out with the financial assistance of the Council of Scientific and Industrial Research, New Delhi.
REFERENCES 1. CHATURVEDI U.C. (1986) Virus-induced cytotoxic factor in AIDS and dengue. Immunol. Today, 7, 159. 2. CHATURVEDI U.C. (1989) Togavirus-induced immunosuppression. In: 'Virus-Induced Immunosuppression.' (eds S. Specter, M. Bendinelli and H. Friedman). p. 253. Plenum Press, New York. 3. CHATURVEDI U.C., MATHUR K.R., GULATI L. & MATHUR A. (1981) Target lymphoid cells for the cytotoxic factor produced in the spleen of dengue virus infected mice. Immunol. Lett. 3, 13. 4. CHATURVEDI U.C., NAGAR R., GULATI L. & MATHUR A. (1987) Variable effects of dengue virus-induced cytotoxic factors on different subpopulations of macrophages. Immunology, 66, 247. 5. KHANNA M., CHATURVEDI U.C. & MATHUR A. (1988) Abrogation of helper T cells by dengue virus-induced cytotoxic factor. Current Sci. 57,411. 6. KHANNA M., CHATURVEDI U.C., SRINIVAsA B.R., SWAMINATHAN K.R. & MATHUR A. (1989) Proteinase-like activity in the cytotoxic factor produced by T-cells during dengue virus infection. Immunology, 67, 32. 7. KHANNA M., CHATURVEDI U.C., SHARMA M., PANDE V.C. & MATHUR A. (1990) Increased capillary permeability mediated by dengue virus induced lymphokine. Immunology, 69, 449. 8. DHAWAN R., KHANNA M., CHATURVEDI U.C. & MATHUR A. (1990) Effect of dengue virus induced cytotoxin on capillary permeability. Br. J. Exp. Path. 71, 83. 9. ORRENIUS S., MCCONKEY J.D., BELLOMO G. & NICOTERA P. (1989) Role of Ca2+ in toxic cell killing. TIPS, 10, 281. 10. CHATURVEDI U.C., TANDON P. & MATHUR A. (1977) Effect of immunosuppression on dengue virus infection in mice. J. Gen. Virol. 36, 449. 11. CHATURVEDI U.C., BHARGAVA A. & MATHUR A. (1980) Production of cytotoxic factor in the spleen of dengue virus infected mice. Immunology, 40, 665. 12. CHATURVEDI U.C., TANDON H.O. & MATHUR A. (1978) Control of in vitro and in vivo spread of Coxsackie B4 virus infection by sensitized spleen cells and antibody. J. Infect. Dis. 138, 181. 13. JULIUS M.H., SIMPSON E. & HERZENBERG L.A. (1973) A rapid method for the isolation of functional thymus derived murine lymphocytes. Eur. J. Immunol. 3, 645.
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14. TRIzIo D. & CUDKOWICZ G. (1974) Separation of T and B lymphocytes by nylon wool columns: Evaluation of efficacy by functional assay in vivo. J. Immunol. 113, 1093. 15. TANDON P., CHATURVEDI U.C. & MATHUR A. (1979) Differential depletion of T lymphocytes in the spleen of dengue virus infected mice. Immunology, 37, 1. 16. CHATURVEDI U.C., SHUKLA M.I. & MATHUR A. (1982) Role of macrophages in the transmission of dengue virus induced-suppressor signal to a subpopulation of T lymphocytes. Ann. Immunol. (Inst. Pasteur), 133, 83. 17. FREEDMAN M.H. & KHAN N.R. (1979) A rapid technique for measuring calcium uptake in mitogen-induced T and B lymphocytes. Can. J. Biochem. 57, 1344. 18. BIRx D., BERGER M. & FLEISHER T.A. (1984) The interference of T cell activation by calcium channel blocking agents. J. Immunol.
133, 2904. 19. BRAUNWALD E. (1982) Mechanism of action at calcium channel blocking agents. N. Engl. J. Med. 307, 1618. 20. NEW W. & TRAUTWEIN W. (1972) The ionic nature at slow inward current and its relation to contraction. Pflugers Arch. 334, 24. 21. CLARK W.R. (1988) Perforin a primary or auxilary lytic mechanism? Immunol. Today, 9, 101. 22. NATH P., TANDON P., GULATI L. & CHATURVEDI U.C. (1983) Histological and ultrastructural study of spleen during dengue virus infection of mice. Indian J. Med. Res. 78, 83. 23. BEELER G.W. & REUTER H. (1970) Membrane calcium current in ventricular myocardium fibres. J. Phyiol. 207, 191. 24. KLAUS G.G.B. (1988) Lymphocyte receptors, signals and cytokines at Newport Beach. Immunol. Today, 9, 157.
Immunology 1991 72 73-78
001928059100013N
Presence of Ca2 + is obligatory for the cytotoxic activity of dengue virus-induced cytotoxic factor M. KHANNA, U. C. CHATURVEDI, R. DHAWAN, B. L. TEKWANI* & V. C. PANDEY* Postgraduate Department of Microbiology, K.G. Medical College and the *Central Drug Research Institute, Lucknow, India Acceptedfor publication 1I September 1990
SUMMARY The present study was undertaken to investigate the role of calcium ions (Ca2+) in the cytotoxic activity of the cytotoxic factor (CF) produced by T lymphocytes of the dengue type 2 virus (DV)-infected mouse spleen. It was observed that CF prepared in Ca2+-free medium had no cytotoxic activity on normal mouse spleen cells suspended in Ca2 +-free medium but had activity on cells suspended in medium having Ca2+. The cytotoxic activity of CF was restored by substitution with calcium chloride, the optimal dose being 10-' M. CF induced influx of Ca2+ as measured by uptake of radiolabelled calcium chloride (45Ca), in the susceptible target cells, macrophages (MO) and T lymphocytes, but had no effect on CF-resistant B lymphocytes. Calcium channel blocking drugs, like verapamil, nifedipine and diltiazem, inhibited the cytotoxic activity of CF and also the CF-induced influx of 45Ca in MO and T cells. Thus, presence of Ca2+ is obligatory for the cytotoxic activity of CF and the cell death is associated with increased intracellular Ca2+.
lity.3-8 But, the precise mechanism of cell injury during dengue virus infection is not known. Cell cytotoxicity can be mediated by mechanisms which are dependent on calcium ions (Ca2+), while other mechanisms do
INTRODUCTION Dengue virus infection produces immunopathological lesions characterized by lymphoid cell necrosis, thrombocytopenia, alterations in peripheral blood leucocytes and increased capillary permeability. A progressive damage of spleen characterized by hypocellularity, necrosis of cells and disorganization and atrophy of follicles has been demonstrated in dengue type 2 virus (DV)-infected mice. Further, such damage in the spleen cells and peripheral blood leucocytes has been shown to be mediated by a cytokine, the cytotoxic factor (CF), which is produced by T lymphocytes (Ly-1+23-) of the DV-infected mouse spleen (reviewed by Chaturvedi).' 2 CF has been purified by various chromatographic techniques. It separates out in a single band on native polyacrylamide gel electrophoresis (PAGE) at the 43,000 molecular weight (MW) position, and as two bands at the 22-25,000 MW position on SDS-Page. These bands react with similar intensity with the specific anti-CF antibody in the Western blot test. The N' terminal sequence of amino acids of CF has been determined. A comparison of the N' terminal amino acid sequence of CF with that of other cytotoxic protein molecules, such as mouse lymphotoxin, tumour necrosis factor and T-cell serine protease, did not show significant homology (M. Khanna and U. C. Chaturvedi, manuscript submitted for preparation). CF is a highly potent protein molecule which selectively kills T-helper cells (Th), I-Amacrophages and acts on cells capable of liberating histaminelike substances, thus producing increased capillary permeabi-
not require the presence of calcium. A sustained increase in cytosolic Ca2+ has been associated with the onset of cytotoxicity characterized by disruption of cytoskeleton, DNA fragmentation and extensive damage to other subcellular components leading to cell death (reviewed by Orrenius et al.9). The present study was undertaken to investigate the role of Ca2+ in the cytotoxic activity of CF. It demonstrates that the presence of Ca2+ is obligatory for the cytotoxic activity of CF and cell death is associated with influx of Ca2+ in the susceptible target cells, namely T lymphocytes and macrophages (M4).
MATERIALS AND METHODS Animals Inbred Swiss albino mice aged 2-4 months were used in this study and obtained from the colony maintained in this Department. Virus Dengue type 2 virus (DV), strain P23085, was used in the form of infected mouse brain suspension, as described elsewhere.'0
Reagents Verapamil and nifedipine (Sigma Chemicals Co., St Louis, MO) were dissolved in 30% ethyl alcohol as a stock solution; diltiazem hydrochloride (Anglo-French Drug Co. Eastern Ltd,
Correspondence: Professor U. C. Chaturvedi, Dept. of Microbiology, K.G. Medical College, Lucknow-226 003, India.
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Bangalore, India) was dissolved in the medium used. The stock solutions of the drugs were prepared on the day of the experiment and were used in serial dilution. Radiolabelled calcium chloride (45Ca) was obtained from Bhabha Atomic Research Centre, Bombay, India and had a specific activity of 77-4 mCi/gm. It was appropriately diluted and used in doses of 1-3 pCi/ml of cell suspension. Preparation of the cytotoxic factor (CF) CF is secreted by spleen cells obtained from DV-infected mice or can be extracted from such cells."I Briefly, the spleens were removed aseptically from DV-infected moribund mice and the cells were teased out in chilled phosphate-buffered saline, pH 7 (PBS). A single-cell suspension of the cells was prepared in PBS and cell count was adjusted to 2 x 106 cells/ml. The cells were distributed in 5-cm glass Petri dishes in a volume of 4 ml and cultured at 370 in the presence of 5% CO2. After 24 hr the cultures were harvested and the cells were spun down at 2000 g for 10 min. The clear supernatant was tested for cytotoxic activity and stored in small aliquots at -70°. CF was purified by Pharmacia low pressure liquid chromatography (LPLC; Uppsala, Sweden) using Sephacryl S200 gel packed in a 650 x 16 mm column with a bed height of 450 mm. Proteins were eluted with 0-5 M PBS at 40 cm3/hr and the elution fluid was monitored at 280 nm in a UV-Cord. The protein peaks obtained were assayed for cytotoxic activity. The purified CF was vacuum dried or freeze dried in a Speed Vac (Savant Instruments Inc., Farningdale, NY) and stored at -70 .
Preparation of target cells Normal mouse spleen was teased out with forceps in chilled minimum essential medium (MEM) containing 10% bovine serum. A single-cell suspension was treated with Tris ammonium chloride solution to haemolyse the red blood cells. The cells were washed and the viable nucleated cells were counted using the trypan blue dye exclusion technique.'2 Enriched subpopulations of T and B lymphocytes were prepared by filtration of spleen cells through glass-wool and nylon-wool columns. 13.14 The purity of the cell population was screened as described elsewhere.'5 A MO-enriched subpopulation was prepared by collecting glass-adherent cells obtained by layering splenic or peritoneal cells on 5-cm glass Petri dishes and incubating at 37° in the presence of 5% CO2 for 90 min.'6 The non-adherent cells were removed by repeated washing and the glass-adherent cells were scraped off with rubber tipped rod and suspended in MEM after washing three times. About 98% of these cells phagocytosed latex particles and were therefore considered MO enriched subpopulation.'6 In the preliminary experiments identical results were obtained with the splenic and peritoneal M4; therefore, in subsequent experiments peritoneal MO> only were used. Assay of cytotoxic activity of CF Cytotoxic activity of the preparation was assayed using normal mouse spleen cells as target cells. In some experiments MO or T-cell-enriched subpopulations were used. The technique for the assay of cytotoxic activity of CF is described elsewhere.6" Briefly, equal volumes of the test solution and the target cells (2 x 1 06/ml) were mixed in a microtitre U-well perspex plate and
50
.40
8 30 0
at
10 0
2
4 8 16 Dilution of CF
32
Figure 1. Effect of absence of Ca2+ from the milieu on the cytotoxic activity of CF. The activity of CF was assayed using (i) CF prepared in Ca2+-free medium and the target cells suspended in Ca2+-free medium (U); (ii) CF prepared in Ca2 + -free medium and the target cells suspended in the medium containing Ca2+ (A); and (iii) CF and the target cells both, prepared in medium containing Ca2+ (0).
incubated at 4°C for 1 hr. Viable cells were counted using trypan blue dye and the percentage of non-viable cells was calculated after deducting background non-viable cells. Assay of 45Ca uptake Uptake of 45Ca by the target cells was assayed by the technique of Freedman & Khan'7 and Birx et al.'8 Variously treated target cells (I x I07 cells/ml) were incubated with 1-3 pCi of 45Ca/ml at 37° for 30 min. CF kills the target cells equally well both at 4° and 370,11 therefore, the target cells were treated with CF at 4° for 30 min and then transferred to 370 for optimum uptake of 45Ca. The reaction was stopped by putting the tubes in an ice bath and the cells were washed with chilled 0-15 M PBS containing 1-8 mm CaC12 to remove extracellular calcium. The cells were harvested onto a fibre-glass filter using a 10-place filtration assembly (Millipore Co., Bedford, MA) and washed repeatedly with chilled 015 M PBS containing 1-8 mM CaC12. The filters were transferred to glass vials containing 10 ml scintillant fluid and the radioactivity was determined with a LKB (Wallac) Beta counter. The counts per min (c.p.m.) in control and experimental groups were expressed after deducting background c.p.m. The background c.p.m. was obtained after similar procedures from tubes containing medium, CF, drugs and radiolabelled medium without cells. The control group data consisted of c.p.m. in cells and 45Ca without CF or drugs. The experiments were set up in triplicate and were repeated thrice. Mean values + standard error of the mean have been presented. The data were analysed statistically using the Student's t-test for P value. A P value of more than 0-05 was considered insignificant. RESULTS Presence of calcium is essential for cytotoxic activity of CF CF prepared in Ca2+-free medium was assayed for cytotoxic activity on target spleen cells suspended in Ca2+-free medium. In addition, CF was assayed using target cells suspended in MEM (containing Ca2+). In the control, CF prepared in the usual way (in medium containing Ca2+) was assayed on target cells suspended in MEM. The findings summarized in Fig. 1 show that the cytotoxicity of CF was significantly reduced when Ca2+ was absent from both the CF preparation and the test system. The cytotoxic activity of the CF prepared in Ca2+-free medium but tested on target cell suspended in presence of Ca2+ was
Dengue virus-induced cytokine )require Ca2+ for cytotoxicity
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Higher concentration of CaCl2 (10-1 or 10-2 M) alone killed large number of the target cells without the addition of CF.
50
40
30 C 020
OR20
l0
2 3 4 5 6 7 8 910 Molar dilution of CaC12 (log10) Figure 2. Effect of calcium on the cytotoxic activity of CF. The target cells prepared in Ca2+-free medium were treated with various concen-
0
trations of CaCl2 and used to assay the cytotoxic activity ofCF prepared in Ca2+-free medium (0). (-) Cells treated with CaCl2 only.
Table 1. 45Ca influx in CF-treated cells
Cells
c.p.m. Dilution Percent of CF Untreated cells CF treated cells alteration
Macrophages
0 1:32 1:64
3560 +415 3496+650 3356+417
2424+ 306 6363 +809 4015+351
-32 +82 +20
T cells
0 1:32 1:64
3870+ 331 3200+538 3001 +395
2856+225 5762+478 3729+510
-26 +80
0 1:32 1:64
2419+271 3649 +366 3552+784
2318+305 3596+507 3713+409
-4 -2 +4
B cells
+24
Enriched subpopulations of macrophages, T and B cells were treated with various dilutions of CF followed by incubation with I pCi/ml of 45Ca and the c.p.m. were assayed in the cells. Percentage increase (+) or decrease (-) in intracellular 45Ca were calculated as follows: % alteration= 100X c.p.m. of CF treated cells-background c.p.m. _ 100 c.p.m. of untreated cells -background c.p.m.
comparable with the control. Thus the findings showed that calcium is essential for mediation of the cytotoxic activity by CF. Substitution of calcium restores cytotoxic activity of CF To investigate quantitatively the effect of Ca2+, target cells suspended in Ca2+-free medium were treated with graded molar concentrations of CaCI2 for I hr at 37°. To these cells CF (1:2 dilution) prepared in Ca2+-free medium was added and the mixture was further incubated at 40 for 1 hr and the non-viable cells counted. In the presence of I0- or 10-10 M CaCI2, cytotoxic activity of CF was negligible. With 10-8 M the cytotoxic activity increased, reaching levels similar to that of controls with 10-7 M concentrations (Fig. 2). The cytotoxic activity of CF remained at similar levels with concentrations of CaCI2 upto 10-3 M.
Effect of CF on calcium influx into target cells The preceeding experiments indicate Ca2+ is essential for the cytotoxic activity of CF. To investigate whether the target cell killing was due to enhanced Ca2+ ion influx into the target cells, enriched subpopulations of MO, T and B lymphocytes were used and intracellular calcium was measured by estimation of radiolabelled 45Ca. CF was used in three doses, undiluted, 1:32 and 1:64 diluted. For this experiment, 2 x 106 MO were treated with CF and incubated at 4° for 30 min followed by addition of 100 pl of 45Ca. After a further incubation at 37° for 30 min the cells were washed with 1-8 mm cold calcium chloride and the cells were processed for intracellular 45Ca estimation. Table I shows that with a 1: 32 dilution of CF the 45Ca uptake by MO was increased by 82% while that with 1:64 dilution was 20%. Undiluted CF killed all the susceptible target cells quickly, therefore intracellular 45Ca was reduced. The above experiment was repeated using a T-cell enriched subpopulation as target cells. With undiluted CF the decrease in intracellular 45Ca was 26%, while treatment with 1:32 diluted CF increased intracellular 45Ca by 80%. The increase was 24% with 1:64 dilution of CF (Table 1). We have observed that CF does not kill B lymphocytes.3 Therefore, experiments were done to investigate whether alterations in intracellular Ca2+ could be induced by CF in cells which are not adversely affected. Similar experiments with a B-lymphocyte enriched subpopulation showed that no significant alteration occurred in intracellular 45Ca following treatment with various doses of CF (Table 1). Effect of calcium channel blocking drugs on cytotoxicity of CF Drugs are now available which inhibit calcium ion influx through blocking passive 'slow' channels. 19.20 The present experiment was, therefore, carried out to investigate the effect of various calcium channel blocking drugs on the cytotoxic activity of CF. The drugs used were verapamil, nifedipine and diltiazem. Target spleen cells were treated with different concentrations of various calcium channel blocking drugs at 370 for 30 min. The cells were spun down at 2000g for 10 min, resuspended in MEM and used as target cells to assay the cytotoxic activity of CF. The controls included with each test were drug-treated cells alone and untreated spleen cells treated with CF. The data presented in Fig. 3 show that CF killed 45 + 5% of the untreated target spleen cells. Treatment of the target cells with all the three drugs inhibited cytotoxic activity of CF to a similar extent in a dosedependent manner. With I0-I M concentration of verapamil the inhibition was 85%, while that with 10-7 M was 32%. Similar results were obtained by treating target cells with nifedipine or diltiazem. The drugs as such had no adverse effect on the target cells with the concentrations used in the test (data not presented here). The ethanol solution used for dissolving the drugs was similarly diluted and its effect on the target cells was screened. It had no cytotoxic effect on the cells in the dilutions used in the test (Fig. 3). CF kills T cells and MO but has no effect on B cells.3 Further experiments were made using enriched T cells and MO subpopulations at target cells to study the effect of calcium channel
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M. Khanna et al. 50
Cells Drug dilution
40
MO 10-3
20
40
% decrease 60 80
100
verapamil
en
30 c
0o c
MO
10-5
MO
10-7
T
10-3
T
10-5
T
10-7
20
10
4 0
C 10-3 10-5 10-7 Molar conc. of drug
Figure 3. Effect of calcium channel blocking drugs on the cytotoxic activity of CF. Normal mouse spleen cells were treated with various doses of verapamil (A), nifedipine (0) or diltiazem hydrochloride (-) or remained untreated for control (C,O). The cytotoxic activity of CF was assayed using these cells as target. (A) Cells treated with diluent only.
Figure 5. Effect of calcium channel blocking drug verapamil, on CFinduced influx of 45Ca into enriched subpopulations of macrophages (M+) and T lymphocytes (T). The percentage decrease of intracellular 45Ca was calculated as follows: 40r
0/
decrease= c.p.m. of cells treated with - c.p.m. of cells treated
CF
30
drug and with drug
100-
x
c.p.m.
20
100.
of cells treated with CF - c.p.m. of untreated cells
Q)
T5
4 c
20 [
10
10-3 10-5
10-7
C
Cells
Dilution
MO
10-3
MO
10-5
MO
l0o-
T
10-3
T
10-5
T
10-7
20
40
% decreasE 60 80
100
nifedipine
Molar conc. of drug
Figure 4. Effect of calcium channel blocking drugs on the cytotoxic activity of CF on enriched subpopulations of macrophages (MO) and T lymphocytes (T cells). The remaining description is as in Fig. 4.
blocking drugs on the cytotoxic activity of CF. In this experiment verapamil and nifedipine were used. The data presented in Fig. 4a show that CF alone killed 34+4% cells of the MO enriched subpopulation. Treatment of MO with verapamil or nifedipine inhibited the cytotoxicity of CF in a dose-dependent manner; the cytotoxicity being 10 + 2% by treatment with 10-3 M verapamil and 25+3% with 10-7 M of the drug. A similar dose-dependent inhibition of the cytotoxicity of CF on the T-cell enriched subpopulation was observed by treatment of the cells with verapamil or nifedipine (Fig. 4). Effect of calcium channel blocking drugs on CF-induced influx of 45Ca in target cells Further experiments were carried out to investigate the effect of Ca2+ channel blocking drugs on the CF induced calcium ion influx in the target cells. The drugs used were verapamil and nifedipine and the target cells were enriched subpopulations of MO or T lymphocytes. The target cells (2 x 106) were treated with different dilutions of verapamil or nifedipine for 30 min at
Figure 6. Effect of calcium channel blocking drug, nifedipine, on CFinduced influx of 45Ca into enriched subpopulations of the target cells. For details see legend to Fig. 5.
370 followed by addition of CF (1: 32 dilution). After incubation for 30 min at 40 45Ca was added and further incubated at 37° for 30 min. The cells were washed with 18 mm of cold calcium chloride and intracellular 45Ca was measured. The data presented in Fig. 5 show that pretreatment of MO with 10-3 M of verapamil resulted in a 92% inhibition of the influx of CF-induced calcium whereas with 10-5, 10-7 M of verapamil, the inhibition was 62% and 47%, respectively. Similarly, with 10-3 M nifedipine the decrease in calcium uptake was 83%, while with 10-5 and 10-7 M of the drug the decrease was 32% and 41%, respectively (Fig. 6). Similar findings were obtained using a T-cell enriched subpopulation as target cells. It was observed that with 10-3 M
Dengue virus-induced cytokine require Ca2+ for cytotoxicity verapamil the decrease in calcium uptake was 90% while that with I0-I M was 43% (Fig. 5). Therefore, the effect of drug was dose-dependent. Pretreatment of the cells with nifedipine had similar effects (Fig. 6). DISCUSSION The present study indicates that the presence of Ca2+ is obligatory for the cytotoxic activity of CF on the target cells, MO and T cells. These conclusions have been drawn from the experiments which showed that the cytotoxic activity of CF is inhibited by (i) removal of calcium from the milieu; (ii) pretreatment of target cells with calcium channel blocking drugs like, verapamil nifedipine or diltiazem and (iii) substitution of Ca2+ restored the cytotoxic activity of CF. Therefore, killing of susceptible target cells by CF appeared to be a calciumdependent phenomenon. It was observed that removal of Ca2 + from the milieu abrogated the cytotoxic activity of CF. The activity of CF was restored by substitution of Ca2+ in the form of calcium chloride, the optimal dose of which was 10-7 M. Target cell killing by various cytotoxic of factor mechanisms may be Ca2 +-dependent or Ca2 +-independent. Thus, cytotoxic activity of lymphotoxins, tumour necrosis factor, serine esterases and performs is Ca 2+independent. Some clones of cytotoxic T lymphocytes (CTL) are Ca2 +-dependent for their activity, while others are not (reviewed by Clark).2' CF differs from these cytotoxic mechanisms in being Ca2+-dependent for its activity. A progressive damage of spleen occurs in DV-infected mice which is characterized by hypocellularity, necrosis of cells, disorganization and atrophy of follicular architecture. MO and T cells show blebbing of the membrane, hypertrophy of rough endoplasmic reticulum, lobulation of nucleus and cell necrosis and fragmentation.22 Similar subcellular changes leading to cell death are produced in spleen cells by inoculation of CF in mice or by treating these cells in vitro with CF.4 A sustained increase in intracellular Ca2+ is often associated with activation of cytotoxic mechanisms which lead to disruption of cytoskeleton, fragmentation of DNA and damage to other cellular components, resulting into cell death. Recently a number of Ca2+dependent processes have been recognized which lead to cell death; they include activation of proteases, phospholipases and endonucleases, depletion of ATP and thiol modification (reviewed by Orrenius et al.).9 It was observed that CF induced an influx of Ca2+ in the MO and T lymphocytes which was dose dependent, the maximum increase in intracellular 45Ca being about 80% compared to untreated control cells. The role of Ca2+ in cytotoxic activity of CF was further supported by the absence of CF-induced 45Ca influx in B lymphocytes (Table 1), a cell which is not affected by CF.3 CF has been shown to increase capillary permeability through release of histamine.7'8 It is likely that CF-induced increases in Ca2+ in cells (which are sources of vasoactive mediators), as described in the present study, results in their degranulation and release of histamine, etc., increasing the capillary permeability. It has been shown that verapamil, nifedipine and diltiazem inhibit Ca2+ influx into cells through blocking of passive 'slow' channels.'920'23 The data presented here show that calcium channel blocking drugs inhibited the cytotoxic activity of CF in a dose-dependent manner. The effect of all the three drugs used,
77
verapamil, nifedipine and diltiazem, was similar. The effect of verapamil and nifedipine was investigated on the CF-induced Ca2+ influx in MO and T cells. It was observed that both the drugs inhibited 45Ca influx to the extent of 90-92% in both types of cells. CF selectively kills I-A negative MO and DV-induced helper T cells (Th) but has no effect on other T cells or B cells.3-5 What determines this selectivity ofCF is not known. It has been shown that a different type of K+ channels are present on CD4+8- cells in comparison with CD4- 8 + cells. It has further been suggested that K+ and Ca2+ influx may be co-ordinately regulated in lymphocytes (reviewed by Klaus).24 It is, therefore, likely that Ca2+ channels which could be activated by CF are present only on Th and I-A- MO, thus determining its selectivity of action. The findings of the present study thus demonstrate that the presence of Ca2+ is obligatory for the mediation of the cytotoxic activity of CF and cell death is associated with increased intracellular Ca2 .
ACKNOWLEDGMENTS The study has been carried out with the financial assistance of the Council of Scientific and Industrial Research, New Delhi.
REFERENCES 1. CHATURVEDI U.C. (1986) Virus-induced cytotoxic factor in AIDS and dengue. Immunol. Today, 7, 159. 2. CHATURVEDI U.C. (1989) Togavirus-induced immunosuppression. In: 'Virus-Induced Immunosuppression.' (eds S. Specter, M. Bendinelli and H. Friedman). p. 253. Plenum Press, New York. 3. CHATURVEDI U.C., MATHUR K.R., GULATI L. & MATHUR A. (1981) Target lymphoid cells for the cytotoxic factor produced in the spleen of dengue virus infected mice. Immunol. Lett. 3, 13. 4. CHATURVEDI U.C., NAGAR R., GULATI L. & MATHUR A. (1987) Variable effects of dengue virus-induced cytotoxic factors on different subpopulations of macrophages. Immunology, 66, 247. 5. KHANNA M., CHATURVEDI U.C. & MATHUR A. (1988) Abrogation of helper T cells by dengue virus-induced cytotoxic factor. Current Sci. 57,411. 6. KHANNA M., CHATURVEDI U.C., SRINIVAsA B.R., SWAMINATHAN K.R. & MATHUR A. (1989) Proteinase-like activity in the cytotoxic factor produced by T-cells during dengue virus infection. Immunology, 67, 32. 7. KHANNA M., CHATURVEDI U.C., SHARMA M., PANDE V.C. & MATHUR A. (1990) Increased capillary permeability mediated by dengue virus induced lymphokine. Immunology, 69, 449. 8. DHAWAN R., KHANNA M., CHATURVEDI U.C. & MATHUR A. (1990) Effect of dengue virus induced cytotoxin on capillary permeability. Br. J. Exp. Path. 71, 83. 9. ORRENIUS S., MCCONKEY J.D., BELLOMO G. & NICOTERA P. (1989) Role of Ca2+ in toxic cell killing. TIPS, 10, 281. 10. CHATURVEDI U.C., TANDON P. & MATHUR A. (1977) Effect of immunosuppression on dengue virus infection in mice. J. Gen. Virol. 36, 449. 11. CHATURVEDI U.C., BHARGAVA A. & MATHUR A. (1980) Production of cytotoxic factor in the spleen of dengue virus infected mice. Immunology, 40, 665. 12. CHATURVEDI U.C., TANDON H.O. & MATHUR A. (1978) Control of in vitro and in vivo spread of Coxsackie B4 virus infection by sensitized spleen cells and antibody. J. Infect. Dis. 138, 181. 13. JULIUS M.H., SIMPSON E. & HERZENBERG L.A. (1973) A rapid method for the isolation of functional thymus derived murine lymphocytes. Eur. J. Immunol. 3, 645.
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14. TRIzIo D. & CUDKOWICZ G. (1974) Separation of T and B lymphocytes by nylon wool columns: Evaluation of efficacy by functional assay in vivo. J. Immunol. 113, 1093. 15. TANDON P., CHATURVEDI U.C. & MATHUR A. (1979) Differential depletion of T lymphocytes in the spleen of dengue virus infected mice. Immunology, 37, 1. 16. CHATURVEDI U.C., SHUKLA M.I. & MATHUR A. (1982) Role of macrophages in the transmission of dengue virus induced-suppressor signal to a subpopulation of T lymphocytes. Ann. Immunol. (Inst. Pasteur), 133, 83. 17. FREEDMAN M.H. & KHAN N.R. (1979) A rapid technique for measuring calcium uptake in mitogen-induced T and B lymphocytes. Can. J. Biochem. 57, 1344. 18. BIRx D., BERGER M. & FLEISHER T.A. (1984) The interference of T cell activation by calcium channel blocking agents. J. Immunol.
133, 2904. 19. BRAUNWALD E. (1982) Mechanism of action at calcium channel blocking agents. N. Engl. J. Med. 307, 1618. 20. NEW W. & TRAUTWEIN W. (1972) The ionic nature at slow inward current and its relation to contraction. Pflugers Arch. 334, 24. 21. CLARK W.R. (1988) Perforin a primary or auxilary lytic mechanism? Immunol. Today, 9, 101. 22. NATH P., TANDON P., GULATI L. & CHATURVEDI U.C. (1983) Histological and ultrastructural study of spleen during dengue virus infection of mice. Indian J. Med. Res. 78, 83. 23. BEELER G.W. & REUTER H. (1970) Membrane calcium current in ventricular myocardium fibres. J. Phyiol. 207, 191. 24. KLAUS G.G.B. (1988) Lymphocyte receptors, signals and cytokines at Newport Beach. Immunol. Today, 9, 157.
