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Influence of streptolysin S from Streptococcus pyogenes on some functions of lymphocyte membrane
0041-010118110101-0033 f02.00p
Taxicnn, Vol. 19,pp . 33-9 OPerpmw Preattd. 1981 . Printed in Grat Britain.
INFLUENCE OF STREPTOLYSIN S FROM STREPTOCOCCUS PYOGENES ON SOME FUNCTIONS OF LYMPHOCYTE MEMBRANE W. HikYNIEWtcz,* W. ROSZKOWSKI,t S. LIPSKIt and J. JEI .IASZEWICZ* `Department of Bacteriology, National Institute of Hygiene, 24 Chocimska, 00-791 Warsaw tCenter of Radiobiology and Radioprotection, 00-909 Warsaw, Poland
(Acceptedforpublication 1 May 1980) RoSZKOWSKI . S. LIPSKi and J. JELJASZEWICZ. Influence of streptolysin S from on some functions of lymphocyte membrane. Toxicon 19, 33-39, 1981 . -Cytotoxic effect of streptolysin S on murine lymph node lymphocytes was estimated using 6tcr andaaRb release assays . StreptolysinSconcentration of 25 HU/ml produced slight cytotoxic effects in both tests. Influence of streptolysin S on 8 aRb uptake reflecting active potassium transport through lymphocyte membrane was examined . A decrease of active flaRb transport was observed using a streptolysin S concentration not affecting cell viability. No change in cAMP and cGMP levels in lymphocytes exposed to streptolysin S were noted; however, reactivity to isoprenaline, acetylcholine and ConA of streptolysm S-treated lymphocytes was significantly higher. Streptolysin S in subcytotoxic concentrations may alter lymphocyte membrane properties. W . I'IRYNIEWICZ. W .
Saeptoeoccut pyrogenes
INTRODUCTION
S, a hemolytic and cytolytic toxin produced by most strains of Streptococcus pyogenes, was shown to possess a broad spectrum of cytolyticactivity (GINSBURG, 1970 ; BERNHEIMER, 1972; JELIASZEWICZ et al., 1978) . It has been reported recently that streptolysin S exhibits cytotoxic activity toward human and mouse lymphocyte T subpopulations (HRYNIEWICz and PRYJMA, 1977). It has been also shown that when applied in subcytotoxic doses, streptolysin S can influence some T lymphocyte functions in vitro and suppress the immune response to T-dependent antigen in vivo (HRYNIEWICZ and PRYJMA, 1978). The mechanism of this phenomenon is not clear, but impairment of lymphocyte membrane properties has been considered . We decided to test the influence of streptolysin S, applied in subcytotoxic concentrations, on some functions of the murine lymphocyte membrane such as active ion transport, response to mitogenic stimuli and intracellular level of cyclic nucleotides. STREPTOLYSIN
MATERIALS AND METHODS Pritpwadon ofstreptolysin S
This toxin was prepared according to the method of BERNHEIMER (1949) from the supernatant of strain C203S and purified as described elsewhere (HRVNmweez, 1979). The hemolytic activity of the purified toxin was 10,000 hemolytic units/ml as measured by a hemolytictest (HRYNiEwicz et d., 1979). One hemolytic unit (HU) is definedas theamount of streptolysin S in 1 ml sample able to produce 5046 hemolysis of an equal volume of 0896 group0 human erythrocyte suspension afterincubation for30 min at 37"C . of Streptococcus pyogeres,
34
W. HRYNIEWICZ, W. ROSZKOWSKI, S. LIDSKI and J. JELJASZEWICZ
Lymphocytes
Adult, 8-10-weeks old male BALB/c mice were used as a source of lymph node lymphocytes throughout the experiment. The animals were sacrificed by cervical dislocation. Cervical, axillary, inguinal and mesenteric lymph nodes were transferred into sterile cold RPMI-1640 medium (Grand Island Biological Corporation, Grand Island, New York) and thoroughly rinsed . Cell suspensions were prepared by pressing the lymph nodes through a stainless steel screen . The cells were washed, counted in a hemocytometer, andviability estimated by the standard trypan blue exclusion test with a final 0-2% concentration of the dye. For the experiment, lymphocytes were suspended in RPMI-1640 medium supplemented with 1096 fetal calf serum (Grand Island Biological Corporation, Grand Island, New York) and adjusted to 2 x 10' viable cells/ml for measurements of cytotoxicity, ion transportand mitogenic stimulation-or to 107 and 10a cells/ml for measurement of cAMP and cGMP content, respectively . Mitogenic stimulation
To quadruplicate cultures of 1 ml aliquots of lymphocyte suspensions with or without streptolysin S, 5 Ug of concanavalin A (ConA, Calbiochem) was added, and the cells were incubated at 37°C in an atmosphere of 5% CO,. One ILO of 3 H-thymidine (sp. act. = 2 Ci/mmole ; Radiochemical Centre, Amersham) was added per culture 18 hr prior to harvesting . After72 hr culture, the cells were harvested, their viability estimated, and DNA extracted by 10% ice-cold trichloracetic acid, followed by dissolving in TS-1 tissue solubilizer (Koch-Light, Colnbrook, Bucks, England) . The samples were transferred to scintillation vials containing Bray's solution and radioactivity measured. Non-cytotoxicconcentrations of streptolysin S applied in this experiment didnot change lymphocyte viability after 72 hr of culture, as compared to controls. Influx of °°Rb to lymphocytes exposed to streptolysin S
Lymphocyte suspensions were incubated at 37°C for 30 min with various concentrations of streptolysin S diluted in RPMI-1640 medium and containing 5 WCiof NRb at final concentration (wRbCl, Institute of Nuclear Research, Warsaw, Poland ; 135 mCi/1-0 g of Rb). 88 Rb was used instead of yK, since both ions can be taken up by the cell through the active transport system mediated by membranous Na +-K+-ATPase (BONTING . 1970 ; RozENGuRT and HEPPEL, 1975), but the former has a longer half-life. For the ainessment of the Na+-K+ATPase independent transport of rubidium, lymphocytes were incubated as describedabove with iORb and5 x 10 -5 M ouabain (Eli Lilly, Indianapolis, Indiana), a specific inhibitor of rubidium-J)otassium pump (BANERJEE andBosmANN,1976). Afterincubation, the lymphocytes were washed and radioactivityof cell pellet wascounted in a scintillation counterCORUMAT2700 (ICN, Belgium) . Measurement of cytotoxicity by release ofe°Rb and "Cr from prelabeled lymphocytes treated with streptolysin S
ORb as described Prior to streptolysin S treatment, the lymphocytes were incubated at 37°C for 1 hr with either 1 above, or with 20IzCi of s1Cr at final concentration (Na,""CrO),, Institute of NuclearResearch, Warsaw,Poland ; 50-300 mCi/mg Cr). After incubation, the lymphocytes were washed three times, resuspended in medium, and exposed for 30 min to different concentrations of streptolysin S. The cells were collected by centrifugation and radioactivities of the supernatant and the cell pellet were determined . Release of °°Rb and s1Cr from cells was expressed as percentage of total radioactivity (radioactivity of supernatant plus radioactivity of cell pellet) . Cyclic nucleotides assay
Lymphocyte suspensions were incubated at 30°C with different concentrations of streptolysin S for 30 min. Incubation was terminated by addition of TCA (final concentration: 596 w/w). The samples were then centrifuged (3000g,10 min), thesupernatants collected and extracted by ether, anddried in a nitrogen stream. Content of CAMP and cGMP was measured by a radioimmunoassay method (RIA kit, Radiochemical Center, Amersham, England) . RESULTS
Streptolysin S at concentrations higher than 10 HU/ml exhibited cytotoxic effect on murine lymph node lymphocytes, as measured by both mRb and s1Cr release (Table 1). This effect seems to be dose-dependent within the range of applied concentrations . No -triking difference in sensitivity of the two tests was observed . 98Rb uptake was used to determine membrane active transport of ions in the presence of streptolysin S. The toxin diminished the 86Rb uptake (Table 2) at a similar range of concentrations which had a cytotoxic effect . The inhibitory effect of ouabain on total "Rb transport into the lymphocytes wasalso markedly depressed in the presence of streptolysin S. Non-cytotoxic concentrations of streptolysin S did not affect the lymph node lymphocyte stimulation by optimal doses of ConA (Table 3). However, an inhibitory effect of non-cytotoxic concentrations of streptolysin S (10 HU/ml) was observed when the lower
Streptolysin S and Lymphocyte Membrane
35
TABLE 1. CYraroxtcrnrOFSrREP OLYSnv S TOWARDSM[URNE LYMPHOCYTES
Streptolysin S (HU/ml)
NRb release* (%)
5'Cr release' (46)
0 0-5 1.0 5-0 10 .0 25-0 50 .0 100.0
1.0 t 0.3 1.0±0.4 1-0±0.6 1.0 t 0-2 1.8 ± 0.5 12-9 t 1-8t 28-6 ± 1-3t 36.3 ± 2-1t
1.0 ± 0.2 1.0t0 .1 1 .0±0-7 1.0 ± 0-3 1-0 t 0.3 8.3 ± 0-5t 24 .1 ± 1-7t 26 .2 t 1-5t
RU = hemolytic units. 'Means t S.D. based on three determinations . tP < 0-01 as compared to 0value of Streptolysin S. TABLE 2. EFFECT OFSrREProLYsiN S ON NRb urrAKE BY LYMPHOCYTES
Streptolysin S (HU/ml)
N" uptake' (counts/min)
0 1 5 10 50 100
977 ± 41 8711- 70 822 ± 40 708 t 50t 410 t 32t 300 t 34t
e°Rb uptake + Inhibition by 104 ouabain' ouabain (counts/min) (g6) 379 t 38 411 :t 53 451 t 31t 408 t 43t 296 t 38t 211 :t 54t
57 53 45 43 28 30
HU = hemolytic units 'Means ± S.D . based on three determinations. tP < 0-01 as compared to 0 value of streptolysin S.
doses ofConA, resulting in suboptimal lymphocyte stimulation, were used (Table 4). This inhibition was as high as 80 and 100%, whereas stimulation indexes were as low as 4-3 and 1-7, respectively. After addition of streptolysin S (10 HU/ml), a decrease of basal cAMP in lymphocytes was observed (Table 5). However, this concentration of the toxin did not exhibit any cytotoxic effect, as measured by °'Cr and wRb release, but significantly diminished aaRb active transport. On the other hand, incubation of lymphocytes with streptolysin S resulted in a markedly pronounced increase of reactivity of these cells to adrenergic agonists such as isoprenaline (as measured by CAMP content) . This effect was observed with non-cytotoxic doses of streptolysin S. Similarily, cholinergic stimulation of streptolysin S-treated lymphocytes with acetyl-ß-methyl-choline was significantly enhanced (Table 6). Moreover, the same streptolysin S concentrations which inhibitedsuboptimal mitogenic stimulation of lymphocytes with ConA, resulted in enhanced reactivity of cells to this
W. HRYNIEWICZ, W. ROSZKOWSKI, S. LIDSKI and J. JELIASZEWICZ TABLE 3 . EFFECT OF SCREPTOLYSIN SON MURINE LYMPHOCYTE RESPONSES TO CONCANAvALnv A
Streptolysin S (HU/ml)
ConA (10gg/ml) Nonstimulated -stimulated lymphocytes' lymphocytes' (counts/min x 103) (counts/min x 103)
0
4.6±0.7 4.8 ± 0.9 43 t 0.5 4 .9 ± 0 .8 4 .1 ± 0 .4 3 .2 ± 0 .4
1 5 10 50 100
38 .4±13 39 .7 ±I-6 39-6 ± 1 .1 36-2 ± 1-7ß 22 .1 ± 1 04 13 . 4 ± 1-H
sit
Inhibition$ (9b)
83 83 8-5 7"4 5-4 3-9
0 0 7 "3 53-2 69-8
HU = hemolytic units. 'Means ± S.D . are based on three determinations . tSI (Stimulation index) = stimulated lymphocytes (counts/min) nonstimulated lymphocytes (counts/mm) $Inhibition =
stimulated control
-
nonstimulated control
-
stimulated experimental
nonstimulated experimental x 100
(stimulatedcontrol -nonstimulated control) §P < 0.01 as compared to 0 value of streptolysin S. TABLE 4 . EFFECT OFSrREPrOLYSIN S ON CONCANAVALiN A-INDUCED LYMPHOCYTE ACTIVATION
ConA (Lg/ml) 10
5 2-5 1 0
Culture without streptolysinS' (counts/min x 1(') 283 ± 19 .6 ± 133 ± 5 .4 .-t: 3. 1 ±
1 .4 1 .6 0 .8 1-2 0-6
SI'
Culture with_ streptolysin W' (10HU/ml) (counts/min x 103)
Inhibition' (`%)
9 .1 63 43 1-7 1-0
27-4 .-t: 1-9 15 .6 ± 2-1t 5-1 ± 1-2t 3 .2 .t 0-8t 3 .0 ± 0-7t
3-5 24-2 80-4 99 .0 0
HU = hemolytic units. `See footnotes to Table 3. tP < 0"01 as compared to cultures without streptolysin S with the same mitogen concentration . mitogen, as measured by cGMP content. Although the doses of ConA in these experiments were different, there was no significant rise in cGMP level when suboptimal concentrations of the mitogen both in controls and in streptolysin S-treated lymphocytes were applied (unpublished data). DISCUSSION It was found previously that streptolysin S, which is one of the most potent hemolytic toxins, may also affect lymphocyte viability (HRYNiEwICz and PRYJMA,1977) . This toxin
37
Streptolysin S and Lymphocyte Membrane TABLE5 . EFFECrOFSI'REL'IOLYSINSON INTRACELLULAR CAMP CONTENT OF LYI11PHOCYIES cAMP concentration (pmole/10' cells) Streptolysin S (HU/ml) 0 0-5 1 5 10
Lymphocytes' 4-2 t 0-2 4.3 t 0.4 3 .9 * 0.2 3 .6 t 0-3 2-8 ± 0 .2t
Lymphocytes + isoprenalin (10-M)' 6. 2 t 0.2 7 .8 ± 0 .4t 10 .0 ± 0 .5t 9 .0 ± 0.3t 6-6 :t 0.5
* Means :j- S .D. based on three determinations. tP < 0-01 as compared to 0 value of streptolysin S . TABLE 6 . EFFECT OF STREPTOLYSIN S ON INTRACELLULAR CGMP CONTENT OFLYAtPHOCYTFS cGMP concentration (pmole/1(r cells) Streptolysin S (HU/ml) 0 0-5 1 5 10
Lymphocytes + Lymphocytes + ConA acetyl-ß-methylLymphocytes* choline (10',M)' (?S fag/ml)* 3-0 ± 0 .2 3 . 5 ± 0-3 3-0t0-1 3-4 t 0. 3 2.0 t 0.2
3-9 :t 0 .2 5-4 :L 0-2t 5 . 2*0-2t 4 .5 t 0-2t 4 .1 ± 0.4
3-8 :t 0-1 4 . 2 ± 0.3 4 .9t0 .3t 5 .6 ± 0 .5t 5 . 0 t 0 .2t
'Means ± S.D . based on three determinations. tP < 0.01 as compared to 0 value of streptolysin S .
can inhibit T cell rosette formation which suggests changes in the lymphocyte surface structure . Our present investigation demonstrated that the cytotoxic effect of streptolysin S measured by °'Cr and asRb release is preceded by a significant inhibition ofion transport through the cell membrane as determined by NRb uptake . This latter effect was observed in subcytotoxic concentrations of the toxin. The fact that the inhibitory effect of ouabain on the total "Rb transport in streptolysin S-treated lymphocytes was also depressed, indicates that this toxin markedly diminishes activity of Na+-K+ATPase in the lymphocyte membrane . These findings suggest that impairment of ion transport may be involved in the mechanism of the cytotoxic effect. On the other hand, a pronounced inhibitory effect ofthe toxin, applied in subcytotoxicconcentrations, was observed in ConA-induced lymphocyte stimulation, when the effect ofthe mitogen was suboptimal . It has been found that impairment of potassium active transport results in strong inhibition of lymphocyte proliferation (KAY, 1972) . Impairment of ion transport observed in this study may also account for the inhibitory effect ofstreptolysin S on ConA-induced lymphocyte proliferation.
38
W. HRYNIEWICZ, W. ROSZKOWSKI, S. LIDSKI and J . JELJASZEWICZ
Knowing that the streptolysin S effect on lymphocytes is associated with membrane injury, we also searched for the cause of impaired proliferation of cells by observation of changes in cyclic nucleotides content. It is known that cAMP and cGMP are products of enzymes located mostly in cell membranes (PERKINS, 1973 ; GOLDBERG, 1973) and it is also generally accepted that these nucleotides are involved in the cell proliferation control system (PASTAN et al., 1975). HARDEN (1972) and WATSON (1976) reported increased levels of cGMP enhancing mitogenic response of lymphocytes and postulated that a rise in cGMP content is a positive signal for lymphocyte proliferation (COFFEY et al., 1977). We did not find any significant changes in either cAMP and cGMP basal contents caused by streptolysin S when applied in non-cytotoxic concentrations which still influenced lymphocyte proliferation . These results suggest that diminished lymphocyte proliferation is not related to direct changes in basal cyclic nucleotide level. In view of the fact that ConAinduced increase of cGMP level is even significantly enhanced by streptolysin S, it might account for the lack of involvement of cyclic nucleotides in the mechanism of inhibition of lymphocyte proliferation. Otherwise, one would expect a decrease rather than amplification of the response to ConA . Moreover, reactivity of lymphocytes to ß-adrenergic and cholinergic agents, measured by the rise in CAMP and cGMP contents, was also markedly enhanced after streptolysin S treatment. The possibility of an influence of streptolysin S on phosphodiesterase, the enzyme responsible for cyclic nucleotides catabolism, could be excluded in this study as all measurements were performed in the presence of euphiline (an inhibitor of this enzyme). Our data do not explain directly the molecular basis of the observed phenomena but suggest existence of facilitated contact of enzymatic subunits as a result of an increased fluidity of a lipid bilayer of lymphocyte membrane or unmasking of hormonal receptor . It is difficult to evaluate the pathophysiological role of streptolysin S in streptococcal infection. It has been shown that other bacterial toxins may affect lymphocyte membrane as well as immune response (HRYNIEWICZ,1979 ; PALMER and ZAMAN,1979). Our data indicate that streptolysin S may inhibit mitogen-induced lymphocyte proliferation when the stimulation index is low. Lymphocyte stimulation by antigens is rather subtle when compared to non-specific mitogen induced lymphocyte proliferation, since the number of cells participating in the response is much smaller (HIRSCI-MORN and HIRSCHHORN, 1974). It is possible that the observed inhibition of lymphocytes by streptolysin S at low levels of stimulation may also occur in vivo . This cannot, however, be related to a direct effect of streptolysin S on basal cyclic nucleotides content of lymphocytes, as was described for cholera toxin (HOLMGREN, 1978). It has been shown that bacterial infections may be accompanied by stimulation of the adrenergic system with increased catecholamine serum levels (SMrrH,1980) . Streptolysin S-enhanced reactivity of lymphocytes to the adrenergic agonist isoprenaline, has to be considered as an additional possible factor influencing the immune system in vivo, particularly as ß-catecholamines can diminish both T and B cell immune functions (HENNEY et al ., 1972 ; MELmoN et at ., 1974). We postulate that streptolysin S, applied in subcytotoxic concentrations, can directly affect proliferative properties of lymphocytes in vitro. It may also increase lymphocyte susceptibility to adrenergic and cholinergic stimuli which may modulate their functions in vivo . Acbtowledg+anem-This investigation was supported by a research grant 0 .5-339-C fron the U .S . Public Health Service.
39
Streptolysin S and Lymphocyte Membrane REFERENCES
BANEWO S: P. and BosmANN, H. B. (IM) Rubidium transport W ouabain binding in normal and virally transformed mouse fibroblasts. Fxp. Cell Res. 100, 153. BERNHEimmA. W. (1949) Formation a a bacterialtoxin (streptolysin S) by resting celhL L exp. Med. ", 373 Bewvt~wit, A. W. (1972) Hemolysins of streptococci : characterization and effects on biological membranes. In : S&Vtooocci and S&Vtococcal Diseases, p. 19 (WANNAmAxER, L. W. and MATsEN, J. M., Eds.) . New York : Academic Press. %xTnvG . S. 0 (1970) Sodium potassium activated adenosine triphosphatase and cation I Membranes and Ion Douport, Vol. 1, p. 257(BriTAR, E. E., Ed.). NewYork : Wiley. CoFSEY, R. G, HwDDw E M. and HADDEN,J. W. (1977) Evidence forcyclic OMP and calcium mediation of lymphocyte activation by mitogens. J. hnmun. 119, 1387 . CuwTRECwsws, P. (19175) Hormone receptors-their function in cell membranes and some problems; relatedto methodology . In: Advances in Cyclic Nudeodde Research, Vol. 5, p. 5 (GREENGARD . P. and RowwN. G. A., Eds.) . New York : Raven Press. GiNsBuRG, 1. (1970) Streptolysin S. In : Microbial Toxins, Vol . 3, p. 99 (MoNmm, T. C., KwDis, S. andAn., S. J., Eds. New York : Academic Press. GOLDBERG, N. D., 013EA, R. F. and HADDox, M. K. (1973) Cyclic OMP. In : Advances in Cyclic Nudeodde Research, Vol:I p. 05 (GREENGARD, P. andRoBnwN . G. A., Eds.) . New York : RavenPress. HADDEN, J. M., HADDEN, E. M., MADDox, M. K. and Gousses, N. D. (1972) Guanosme 31,5 1-cyclic a possible intraceflularmediator of mitogenicinfluences in lymphocytes, Proc. naft Acad. Sci. U.S.A. 69, 3024 . HE"Y.C S- Bouffi, H. R. andLwwmNPEm,L. M. (1972) Therole ofcyclic3',5 1-adenosiDe motiophosphate in the specific CytOlytiCactivity of lymphocytes . J. human. 1,1526. HrRscmiow K. and FiuescEUt=, R. (1974) Mechanism of lymphocyte activation. In : Medianism of CellMediated Immunity, p.115 (McCLusKEY, R. T. andCotmv, &, New ": Whey . HowGREN, J. (1978) Cholera toxin and the cell In: Bacivrial Toxins and Cell Membranes, p. 333 (JwwszEEwtcz,J. and WwDSt~tBüt, M, W) . "" Academic Press. HRYNIEWIC7- W. (1980) Influence of bacterial toxins on immune mechanisms . Scand. L Infect. Dir., in press. HRYNmvncz. W. and PRyjmA.J. (1977) Effect a "" S on human and mouse T and B lymphocytes. Infect. Immun. 16,730. HRYNmwic7- W. and PRyjmA, J. (1978) Action a streptoWn S on cells concerned in theimmune reaction. In : Patlwgenic "tococci, p . 59 (PARxER, M. T., Ed.) . Chertsey: Reedbooks. HRyNiEwic7- W., GRAY, E. D., Twee . J., WANNAMAKER, L. W., L&uu4 N. and KANcLERsn. K. (1979) Streptolysin S: purification andproperties. L "WW. JE11ASZEWW7. J., Szhuammia, S. and Hxvxn?wtez, W. (1978) Biological effects of staphybcoccal and streptoToxins coccal toxins . In : Bacwfial andCell Alanbraner, p. 10 (JELjAszEwicz.J. and WADrmw T., Eds.) . London : Academic". KAY, J. E. (1972) Lymphocyte stimulation by phytohaemagglutinin : role of the early stimulation of potassium uptake. Erp. Cell Res. 71,245 . MEuAoN. K. L., BouRNE, H. R., WuNnTIN, J.9 SHEARER, G. M., KRAm. J. and BAUMB4004 S. (1974) Hemolytic"e formation leukocytes in vitro. Controlby vasoactive hormones. L W 53,13 . PAwEwD. L. andWAw&N. Depression of cell-mediatedimmunity in chosen. 0, 77. Pwsrwx, 1. H., Jot~sox, G. S. andANDERwoq WB. (1M) Rob 4cyclic nucleotides in "cortroL Ann. Rev. Biodsem. 44,491 . PERKINS. J. P. (1M) "I ". A: Advances in "Nuokodie Rcssmrh, Vol. 3, p. 1 (GR~riGARD, P. and RoBrNsoN, G. A., Eds.). New York: Raven Press. Ro~xc", E. and HEPPeL,L. A. (19175) Scrum rapidly stimulates ouabain-wnsitive =Rb-influx in quiescent 373 cells. Proc. naaL Acad. Sci. U.S.A . 72,4492. SmrrH. 1. M. (1980) Epinephrine and norepiaephrine levels in "challenged with S. awrrrrs and E. ono. In : Proceedings of 4(h International Synrposbon on Spophylowcci and Stalrhylnvoccal Infiacfioras (Jtß.tws~wrcz, J., Ed .) . Stuttgart: Gustav Fischer Verlag, in Press. WAISON .J. (1976) The involvement of cyclic nucleotide metabolism W theinitiation of lymphocyte proliferation induced by mitogens. L Immun. 117, 1656 .
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Taxicnn, Vol. 19,pp . 33-9 OPerpmw Preattd. 1981 . Printed in Grat Britain.
INFLUENCE OF STREPTOLYSIN S FROM STREPTOCOCCUS PYOGENES ON SOME FUNCTIONS OF LYMPHOCYTE MEMBRANE W. HikYNIEWtcz,* W. ROSZKOWSKI,t S. LIPSKIt and J. JEI .IASZEWICZ* `Department of Bacteriology, National Institute of Hygiene, 24 Chocimska, 00-791 Warsaw tCenter of Radiobiology and Radioprotection, 00-909 Warsaw, Poland
(Acceptedforpublication 1 May 1980) RoSZKOWSKI . S. LIPSKi and J. JELJASZEWICZ. Influence of streptolysin S from on some functions of lymphocyte membrane. Toxicon 19, 33-39, 1981 . -Cytotoxic effect of streptolysin S on murine lymph node lymphocytes was estimated using 6tcr andaaRb release assays . StreptolysinSconcentration of 25 HU/ml produced slight cytotoxic effects in both tests. Influence of streptolysin S on 8 aRb uptake reflecting active potassium transport through lymphocyte membrane was examined . A decrease of active flaRb transport was observed using a streptolysin S concentration not affecting cell viability. No change in cAMP and cGMP levels in lymphocytes exposed to streptolysin S were noted; however, reactivity to isoprenaline, acetylcholine and ConA of streptolysm S-treated lymphocytes was significantly higher. Streptolysin S in subcytotoxic concentrations may alter lymphocyte membrane properties. W . I'IRYNIEWICZ. W .
Saeptoeoccut pyrogenes
INTRODUCTION
S, a hemolytic and cytolytic toxin produced by most strains of Streptococcus pyogenes, was shown to possess a broad spectrum of cytolyticactivity (GINSBURG, 1970 ; BERNHEIMER, 1972; JELIASZEWICZ et al., 1978) . It has been reported recently that streptolysin S exhibits cytotoxic activity toward human and mouse lymphocyte T subpopulations (HRYNIEWICz and PRYJMA, 1977). It has been also shown that when applied in subcytotoxic doses, streptolysin S can influence some T lymphocyte functions in vitro and suppress the immune response to T-dependent antigen in vivo (HRYNIEWICZ and PRYJMA, 1978). The mechanism of this phenomenon is not clear, but impairment of lymphocyte membrane properties has been considered . We decided to test the influence of streptolysin S, applied in subcytotoxic concentrations, on some functions of the murine lymphocyte membrane such as active ion transport, response to mitogenic stimuli and intracellular level of cyclic nucleotides. STREPTOLYSIN
MATERIALS AND METHODS Pritpwadon ofstreptolysin S
This toxin was prepared according to the method of BERNHEIMER (1949) from the supernatant of strain C203S and purified as described elsewhere (HRVNmweez, 1979). The hemolytic activity of the purified toxin was 10,000 hemolytic units/ml as measured by a hemolytictest (HRYNiEwicz et d., 1979). One hemolytic unit (HU) is definedas theamount of streptolysin S in 1 ml sample able to produce 5046 hemolysis of an equal volume of 0896 group0 human erythrocyte suspension afterincubation for30 min at 37"C . of Streptococcus pyogeres,
34
W. HRYNIEWICZ, W. ROSZKOWSKI, S. LIDSKI and J. JELJASZEWICZ
Lymphocytes
Adult, 8-10-weeks old male BALB/c mice were used as a source of lymph node lymphocytes throughout the experiment. The animals were sacrificed by cervical dislocation. Cervical, axillary, inguinal and mesenteric lymph nodes were transferred into sterile cold RPMI-1640 medium (Grand Island Biological Corporation, Grand Island, New York) and thoroughly rinsed . Cell suspensions were prepared by pressing the lymph nodes through a stainless steel screen . The cells were washed, counted in a hemocytometer, andviability estimated by the standard trypan blue exclusion test with a final 0-2% concentration of the dye. For the experiment, lymphocytes were suspended in RPMI-1640 medium supplemented with 1096 fetal calf serum (Grand Island Biological Corporation, Grand Island, New York) and adjusted to 2 x 10' viable cells/ml for measurements of cytotoxicity, ion transportand mitogenic stimulation-or to 107 and 10a cells/ml for measurement of cAMP and cGMP content, respectively . Mitogenic stimulation
To quadruplicate cultures of 1 ml aliquots of lymphocyte suspensions with or without streptolysin S, 5 Ug of concanavalin A (ConA, Calbiochem) was added, and the cells were incubated at 37°C in an atmosphere of 5% CO,. One ILO of 3 H-thymidine (sp. act. = 2 Ci/mmole ; Radiochemical Centre, Amersham) was added per culture 18 hr prior to harvesting . After72 hr culture, the cells were harvested, their viability estimated, and DNA extracted by 10% ice-cold trichloracetic acid, followed by dissolving in TS-1 tissue solubilizer (Koch-Light, Colnbrook, Bucks, England) . The samples were transferred to scintillation vials containing Bray's solution and radioactivity measured. Non-cytotoxicconcentrations of streptolysin S applied in this experiment didnot change lymphocyte viability after 72 hr of culture, as compared to controls. Influx of °°Rb to lymphocytes exposed to streptolysin S
Lymphocyte suspensions were incubated at 37°C for 30 min with various concentrations of streptolysin S diluted in RPMI-1640 medium and containing 5 WCiof NRb at final concentration (wRbCl, Institute of Nuclear Research, Warsaw, Poland ; 135 mCi/1-0 g of Rb). 88 Rb was used instead of yK, since both ions can be taken up by the cell through the active transport system mediated by membranous Na +-K+-ATPase (BONTING . 1970 ; RozENGuRT and HEPPEL, 1975), but the former has a longer half-life. For the ainessment of the Na+-K+ATPase independent transport of rubidium, lymphocytes were incubated as describedabove with iORb and5 x 10 -5 M ouabain (Eli Lilly, Indianapolis, Indiana), a specific inhibitor of rubidium-J)otassium pump (BANERJEE andBosmANN,1976). Afterincubation, the lymphocytes were washed and radioactivityof cell pellet wascounted in a scintillation counterCORUMAT2700 (ICN, Belgium) . Measurement of cytotoxicity by release ofe°Rb and "Cr from prelabeled lymphocytes treated with streptolysin S
ORb as described Prior to streptolysin S treatment, the lymphocytes were incubated at 37°C for 1 hr with either 1 above, or with 20IzCi of s1Cr at final concentration (Na,""CrO),, Institute of NuclearResearch, Warsaw,Poland ; 50-300 mCi/mg Cr). After incubation, the lymphocytes were washed three times, resuspended in medium, and exposed for 30 min to different concentrations of streptolysin S. The cells were collected by centrifugation and radioactivities of the supernatant and the cell pellet were determined . Release of °°Rb and s1Cr from cells was expressed as percentage of total radioactivity (radioactivity of supernatant plus radioactivity of cell pellet) . Cyclic nucleotides assay
Lymphocyte suspensions were incubated at 30°C with different concentrations of streptolysin S for 30 min. Incubation was terminated by addition of TCA (final concentration: 596 w/w). The samples were then centrifuged (3000g,10 min), thesupernatants collected and extracted by ether, anddried in a nitrogen stream. Content of CAMP and cGMP was measured by a radioimmunoassay method (RIA kit, Radiochemical Center, Amersham, England) . RESULTS
Streptolysin S at concentrations higher than 10 HU/ml exhibited cytotoxic effect on murine lymph node lymphocytes, as measured by both mRb and s1Cr release (Table 1). This effect seems to be dose-dependent within the range of applied concentrations . No -triking difference in sensitivity of the two tests was observed . 98Rb uptake was used to determine membrane active transport of ions in the presence of streptolysin S. The toxin diminished the 86Rb uptake (Table 2) at a similar range of concentrations which had a cytotoxic effect . The inhibitory effect of ouabain on total "Rb transport into the lymphocytes wasalso markedly depressed in the presence of streptolysin S. Non-cytotoxic concentrations of streptolysin S did not affect the lymph node lymphocyte stimulation by optimal doses of ConA (Table 3). However, an inhibitory effect of non-cytotoxic concentrations of streptolysin S (10 HU/ml) was observed when the lower
Streptolysin S and Lymphocyte Membrane
35
TABLE 1. CYraroxtcrnrOFSrREP OLYSnv S TOWARDSM[URNE LYMPHOCYTES
Streptolysin S (HU/ml)
NRb release* (%)
5'Cr release' (46)
0 0-5 1.0 5-0 10 .0 25-0 50 .0 100.0
1.0 t 0.3 1.0±0.4 1-0±0.6 1.0 t 0-2 1.8 ± 0.5 12-9 t 1-8t 28-6 ± 1-3t 36.3 ± 2-1t
1.0 ± 0.2 1.0t0 .1 1 .0±0-7 1.0 ± 0-3 1-0 t 0.3 8.3 ± 0-5t 24 .1 ± 1-7t 26 .2 t 1-5t
RU = hemolytic units. 'Means t S.D. based on three determinations . tP < 0-01 as compared to 0value of Streptolysin S. TABLE 2. EFFECT OFSrREProLYsiN S ON NRb urrAKE BY LYMPHOCYTES
Streptolysin S (HU/ml)
N" uptake' (counts/min)
0 1 5 10 50 100
977 ± 41 8711- 70 822 ± 40 708 t 50t 410 t 32t 300 t 34t
e°Rb uptake + Inhibition by 104 ouabain' ouabain (counts/min) (g6) 379 t 38 411 :t 53 451 t 31t 408 t 43t 296 t 38t 211 :t 54t
57 53 45 43 28 30
HU = hemolytic units 'Means ± S.D . based on three determinations. tP < 0-01 as compared to 0 value of streptolysin S.
doses ofConA, resulting in suboptimal lymphocyte stimulation, were used (Table 4). This inhibition was as high as 80 and 100%, whereas stimulation indexes were as low as 4-3 and 1-7, respectively. After addition of streptolysin S (10 HU/ml), a decrease of basal cAMP in lymphocytes was observed (Table 5). However, this concentration of the toxin did not exhibit any cytotoxic effect, as measured by °'Cr and wRb release, but significantly diminished aaRb active transport. On the other hand, incubation of lymphocytes with streptolysin S resulted in a markedly pronounced increase of reactivity of these cells to adrenergic agonists such as isoprenaline (as measured by CAMP content) . This effect was observed with non-cytotoxic doses of streptolysin S. Similarily, cholinergic stimulation of streptolysin S-treated lymphocytes with acetyl-ß-methyl-choline was significantly enhanced (Table 6). Moreover, the same streptolysin S concentrations which inhibitedsuboptimal mitogenic stimulation of lymphocytes with ConA, resulted in enhanced reactivity of cells to this
W. HRYNIEWICZ, W. ROSZKOWSKI, S. LIDSKI and J. JELIASZEWICZ TABLE 3 . EFFECT OF SCREPTOLYSIN SON MURINE LYMPHOCYTE RESPONSES TO CONCANAvALnv A
Streptolysin S (HU/ml)
ConA (10gg/ml) Nonstimulated -stimulated lymphocytes' lymphocytes' (counts/min x 103) (counts/min x 103)
0
4.6±0.7 4.8 ± 0.9 43 t 0.5 4 .9 ± 0 .8 4 .1 ± 0 .4 3 .2 ± 0 .4
1 5 10 50 100
38 .4±13 39 .7 ±I-6 39-6 ± 1 .1 36-2 ± 1-7ß 22 .1 ± 1 04 13 . 4 ± 1-H
sit
Inhibition$ (9b)
83 83 8-5 7"4 5-4 3-9
0 0 7 "3 53-2 69-8
HU = hemolytic units. 'Means ± S.D . are based on three determinations . tSI (Stimulation index) = stimulated lymphocytes (counts/min) nonstimulated lymphocytes (counts/mm) $Inhibition =
stimulated control
-
nonstimulated control
-
stimulated experimental
nonstimulated experimental x 100
(stimulatedcontrol -nonstimulated control) §P < 0.01 as compared to 0 value of streptolysin S. TABLE 4 . EFFECT OFSrREPrOLYSIN S ON CONCANAVALiN A-INDUCED LYMPHOCYTE ACTIVATION
ConA (Lg/ml) 10
5 2-5 1 0
Culture without streptolysinS' (counts/min x 1(') 283 ± 19 .6 ± 133 ± 5 .4 .-t: 3. 1 ±
1 .4 1 .6 0 .8 1-2 0-6
SI'
Culture with_ streptolysin W' (10HU/ml) (counts/min x 103)
Inhibition' (`%)
9 .1 63 43 1-7 1-0
27-4 .-t: 1-9 15 .6 ± 2-1t 5-1 ± 1-2t 3 .2 .t 0-8t 3 .0 ± 0-7t
3-5 24-2 80-4 99 .0 0
HU = hemolytic units. `See footnotes to Table 3. tP < 0"01 as compared to cultures without streptolysin S with the same mitogen concentration . mitogen, as measured by cGMP content. Although the doses of ConA in these experiments were different, there was no significant rise in cGMP level when suboptimal concentrations of the mitogen both in controls and in streptolysin S-treated lymphocytes were applied (unpublished data). DISCUSSION It was found previously that streptolysin S, which is one of the most potent hemolytic toxins, may also affect lymphocyte viability (HRYNiEwICz and PRYJMA,1977) . This toxin
37
Streptolysin S and Lymphocyte Membrane TABLE5 . EFFECrOFSI'REL'IOLYSINSON INTRACELLULAR CAMP CONTENT OF LYI11PHOCYIES cAMP concentration (pmole/10' cells) Streptolysin S (HU/ml) 0 0-5 1 5 10
Lymphocytes' 4-2 t 0-2 4.3 t 0.4 3 .9 * 0.2 3 .6 t 0-3 2-8 ± 0 .2t
Lymphocytes + isoprenalin (10-M)' 6. 2 t 0.2 7 .8 ± 0 .4t 10 .0 ± 0 .5t 9 .0 ± 0.3t 6-6 :t 0.5
* Means :j- S .D. based on three determinations. tP < 0-01 as compared to 0 value of streptolysin S . TABLE 6 . EFFECT OF STREPTOLYSIN S ON INTRACELLULAR CGMP CONTENT OFLYAtPHOCYTFS cGMP concentration (pmole/1(r cells) Streptolysin S (HU/ml) 0 0-5 1 5 10
Lymphocytes + Lymphocytes + ConA acetyl-ß-methylLymphocytes* choline (10',M)' (?S fag/ml)* 3-0 ± 0 .2 3 . 5 ± 0-3 3-0t0-1 3-4 t 0. 3 2.0 t 0.2
3-9 :t 0 .2 5-4 :L 0-2t 5 . 2*0-2t 4 .5 t 0-2t 4 .1 ± 0.4
3-8 :t 0-1 4 . 2 ± 0.3 4 .9t0 .3t 5 .6 ± 0 .5t 5 . 0 t 0 .2t
'Means ± S.D . based on three determinations. tP < 0.01 as compared to 0 value of streptolysin S .
can inhibit T cell rosette formation which suggests changes in the lymphocyte surface structure . Our present investigation demonstrated that the cytotoxic effect of streptolysin S measured by °'Cr and asRb release is preceded by a significant inhibition ofion transport through the cell membrane as determined by NRb uptake . This latter effect was observed in subcytotoxic concentrations of the toxin. The fact that the inhibitory effect of ouabain on the total "Rb transport in streptolysin S-treated lymphocytes was also depressed, indicates that this toxin markedly diminishes activity of Na+-K+ATPase in the lymphocyte membrane . These findings suggest that impairment of ion transport may be involved in the mechanism of the cytotoxic effect. On the other hand, a pronounced inhibitory effect ofthe toxin, applied in subcytotoxicconcentrations, was observed in ConA-induced lymphocyte stimulation, when the effect ofthe mitogen was suboptimal . It has been found that impairment of potassium active transport results in strong inhibition of lymphocyte proliferation (KAY, 1972) . Impairment of ion transport observed in this study may also account for the inhibitory effect ofstreptolysin S on ConA-induced lymphocyte proliferation.
38
W. HRYNIEWICZ, W. ROSZKOWSKI, S. LIDSKI and J . JELJASZEWICZ
Knowing that the streptolysin S effect on lymphocytes is associated with membrane injury, we also searched for the cause of impaired proliferation of cells by observation of changes in cyclic nucleotides content. It is known that cAMP and cGMP are products of enzymes located mostly in cell membranes (PERKINS, 1973 ; GOLDBERG, 1973) and it is also generally accepted that these nucleotides are involved in the cell proliferation control system (PASTAN et al., 1975). HARDEN (1972) and WATSON (1976) reported increased levels of cGMP enhancing mitogenic response of lymphocytes and postulated that a rise in cGMP content is a positive signal for lymphocyte proliferation (COFFEY et al., 1977). We did not find any significant changes in either cAMP and cGMP basal contents caused by streptolysin S when applied in non-cytotoxic concentrations which still influenced lymphocyte proliferation . These results suggest that diminished lymphocyte proliferation is not related to direct changes in basal cyclic nucleotide level. In view of the fact that ConAinduced increase of cGMP level is even significantly enhanced by streptolysin S, it might account for the lack of involvement of cyclic nucleotides in the mechanism of inhibition of lymphocyte proliferation. Otherwise, one would expect a decrease rather than amplification of the response to ConA . Moreover, reactivity of lymphocytes to ß-adrenergic and cholinergic agents, measured by the rise in CAMP and cGMP contents, was also markedly enhanced after streptolysin S treatment. The possibility of an influence of streptolysin S on phosphodiesterase, the enzyme responsible for cyclic nucleotides catabolism, could be excluded in this study as all measurements were performed in the presence of euphiline (an inhibitor of this enzyme). Our data do not explain directly the molecular basis of the observed phenomena but suggest existence of facilitated contact of enzymatic subunits as a result of an increased fluidity of a lipid bilayer of lymphocyte membrane or unmasking of hormonal receptor . It is difficult to evaluate the pathophysiological role of streptolysin S in streptococcal infection. It has been shown that other bacterial toxins may affect lymphocyte membrane as well as immune response (HRYNIEWICZ,1979 ; PALMER and ZAMAN,1979). Our data indicate that streptolysin S may inhibit mitogen-induced lymphocyte proliferation when the stimulation index is low. Lymphocyte stimulation by antigens is rather subtle when compared to non-specific mitogen induced lymphocyte proliferation, since the number of cells participating in the response is much smaller (HIRSCI-MORN and HIRSCHHORN, 1974). It is possible that the observed inhibition of lymphocytes by streptolysin S at low levels of stimulation may also occur in vivo . This cannot, however, be related to a direct effect of streptolysin S on basal cyclic nucleotides content of lymphocytes, as was described for cholera toxin (HOLMGREN, 1978). It has been shown that bacterial infections may be accompanied by stimulation of the adrenergic system with increased catecholamine serum levels (SMrrH,1980) . Streptolysin S-enhanced reactivity of lymphocytes to the adrenergic agonist isoprenaline, has to be considered as an additional possible factor influencing the immune system in vivo, particularly as ß-catecholamines can diminish both T and B cell immune functions (HENNEY et al ., 1972 ; MELmoN et at ., 1974). We postulate that streptolysin S, applied in subcytotoxic concentrations, can directly affect proliferative properties of lymphocytes in vitro. It may also increase lymphocyte susceptibility to adrenergic and cholinergic stimuli which may modulate their functions in vivo . Acbtowledg+anem-This investigation was supported by a research grant 0 .5-339-C fron the U .S . Public Health Service.
39
Streptolysin S and Lymphocyte Membrane REFERENCES
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