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The role of protein synthesis in antibody-dependent cell-mediated cytotoxicity
CELLULARIMMUNOLOGY31, 332-339 (1977)
The Role of Protein Synthesis in Antibody-Dependent Cell-Mediated Cytotoxicityl MARTIN A. ISTURIZ AND RITA L. CARDONI 2* 3 Imtituto
de Iwucstigaciom+s
Mkdicas, Universidad de Bl4enos Aires, BUENOS Aires 1427, Argentina Received
January
Domto
Alvarez
3000,
4, 1977
Antibody-depedent cell-mediated cytotoxicity (ADCC) could be initiated without protein synthesis [human peripheral blood lymphocytes as effector cells incubated with IO-” M cycloheximide, (Cy) 1, although the reaction did not achieve its full lytic ability. This partial inhibition of ADCC was dependent on the dose of Cy. Both ADCC and protein synthesis returned to normal values after removal of the inhibitor. The kinetics of the reaction carried out by Cy-treated effector cells for short periods was similar to that of controls. After this time, the percentage of lysed target cells increased continuously in controls, while the cytotoxiciy of Cy-treated effector cells reached a plateau. When effector cells carried out ADCC in the presence of Cy, their lytic mechanism was “wasted,” and it could be recovered only by removal of the inhibitor. Our results indicate that effector cells have a preformed lytic mechanism operative in ADCC. This lytic mechanism is consumed during the reaction and its recovery requires protein synthesis.
INTRODUCTION Previous studies have shown that antibody-dependent cell-mediated cytotoxicity (ADCC) may be an important process of the host’s immune surveillance system, since this activity has been demonstrated against virus-infected cells (l-3), neoplastic cells (4, 5), allogeneic graft rejection (6), and protozoa (7, 8). It is a very efficient system, attacking target cells sensitized with very low concentrations of antibody (9). Furthermore, effector cells and antibody can be reutilized to carry out cytolysis with a new batch of target cells (10). It is well established that the reaction requires close contact between sensitized target and effector cells, and it results in immediate damage of the former (11). However, it is not clear what the relationship is between protein synthesis and ADCC. While some investigators report dose-dependent inhibition of both ADCC and protein synthesis by cycloheximide (Cy) and puromycin (12), others show that nonsensitized lymphocytes are equipped for ADCC and that they expressed their activity even at concentrations of the same inhibitors which block [‘“Clleucine incorporation into proteins (13). The present paper was done in order to explore the role of protein synthesis in 1 This work was supported by CONICET Grant No. 5840. * To whom reprint requests should be directed. 3 Research Fellow from National Council for Scientific and Technical Research (CONICET)
.
332 Copyright All rights
@ 1977 by Academic Press, of reproduction in any form
Inc. reserved.
ISSN 000888749
PROTEIN
SYNTHESIS
IN
333
ADCC
the effector phase of the ADCC reaction. The problem was approached by combining the use of Cy, a reversible inhibitor of the translation step of protein synthesis, and an experimental tlesign that allowetl us to tlivitle the reaction into “wasting” and effector stages. MATEKIALS
AND
METHODS
Human ly~phoid cells. Peripheral blood lymphocytes (PBL) were isolated from human volunteers as has been described (14). The resultant cell suspension, composed of 95-98s lymphocytes, was washed three times with medium 199 (DIFCO Laboratories, Detroit, Michigan) containing 100 units/ml of penicillin, 100 pg/ml of streptomycin, and 2.5% heat-inactivated fetal calf serum (TC-199). The same tissue culture medium (TC-199) was used in all cytotoxicity experiments. Cell viability was assayed by the trypan blue exclusion test. The concentration of lymphocytes was determined by counting the cell suspensions stained with Turk’s solution. Antibody-dependent cell-mcdintcd cytotoxicit~l (ADCC). ADCC was measured utilizing j’Cr-labeled chicken red blood cells (“‘Cr-CRBC) sensitized with rabbit antibody to CRBC at a subagglutinating dose (Ab-“ICr-CRBC) Rabbit antibodies to CRBC and Yr-CRBC were prepared as described elsewhere (15). ADCC was assayed by culturing mixtures containing 2.5 x 10” PBL and 5 X 10’ Ab-“‘CrCRBC in a 5 : 1 effector to target cell ratio in a final volume of 1 ml, unless otherwise stated (16). The mixtures were centrifuged at 200g for 10 min in an International PR-2 centrifuge in order to establish contact between effector and target cells. The pellets were then resuspended and incubatcl for 1 hr at 37°C. The initiation of this incubation period was considered as time zero. At the end of the reaction, the mixtures were centrifuged at 400g for 15 niin and 0.5 ml of supernatant was transferred to another test tube. The radioactivity of both the supernatant and the pellet was measured in a Packard 3320 gama counter. The percentage of s’Cr released, representing ADCC, was calculated as follows : %Vr
released =
supernatant activity total activity
X 2
x 100.
This value was corrected by subtracting the percentage of “‘Cr released in control cultures in the absence of antibody (l-2% alCr released). Percentage of “‘Cr released ranged between 40 and 60%. ADCC results are expressed as: %ADCC
y051Cr released in reaction tubes = %51c r re 1eased in the absence of inhibitors
x 100.
Protein synthesis by lzuman lymplzoid cells. The protein synthesis in PBL was evaluated by measuring the incorporation of [l”C]lysine into proteins. PBL were incubated with 0.5 PC1 of [‘“Cl lysine (specific activity 301 mCi/mmol, Amersham/ Searle Corp.) for 1 hr at 37°C under the same conditions as for the ADCC assays. After incubation the cells were washed three times in saline solution at 4°C. The pellet was suspended in distilled water for lysis, and trichloroacetic acid (TCA, Baker Analyzed Reagent) was added to a final concentrations of 7.570 ; the reaction was heated at 80°C for 15 min to hydrolyze aminoacyl-tRNA. TCA-precipitable material was washed three times with 5% TCA, solubilized in formic acid, dissolved in scintillation fluid (PPO, 4 g ; POPOP, 0.2 g; toluene, 200 ml ; ab-
334
ISTURIZ
AND
CARDONI
solute ethyl alcohol, 300 ml), and counted in a Packard 3320 beta counter. The percentage of [‘Xllysine incorporated in samples from a 1-hr pulse, representing protein synthesis, was calculated as follows: O/o[14C]lysine incorporated
=
sample activity control activity without
Cy
x 100
= y0 protein synthesis. [14C]-Lysine was not incorporated when cells were incubated at 4°C up to 2 hr. Cycloheximide (Cy) (Sigma Chemical Co., St. Louis, Missouri) was dissolved in 0.15 M sodium chloride at different concentrations. Cy at a concentration of 10m3M completely inhibited protein synthesis 10 min after addition. Experiments for determination of both protein synthesis and ADCC were performed at least four times, and a representative one is shown under Results. RESULTS Relationship
between ADCC and Protein Synthesis
These experiments were designed to establish the role of protein synthesis in the initiation step of the ADCC reaction. As shown in Fig. 1, when ADCC was performed with Cy throughout the reaction, the cytolytic ability of the cells was not fully expressed, but protein synthesis was completely inhibited. On the other hand, when Cy was removed from the reaction, both ADCC and protein synthesis reached control values, The relation between protein synthesis and ADCC was investigated. ADCC was assayed with Cy at concentrations shown in Fig. 2. The results obtained demonstrate that ADCC and protein synthesis are closely related. Similar results were obtained if the ADCC was done in the same way except that the reaction mixtures were incubated for 18 hr. This indicated that Cy did not delay the Yr released from damaged Ab-Yr-CRBC. The results shown in Figs. 1 and 2 demonstrate that in the presence of Cy, the effector cells displayed a significant level of ADCC in spite of the sharp decrease in protein synthesis.
0
AOCC
I%! Protein
0
(a) with
Cy
synthesis
1 b) Washed
FIG. 1. The effects of cycloheximide (Cy) on antibody-dependent cell-mediated cytotoxicity (ADCC) and protein synthesis and its reversibility upon the ,removal of Cy. (a) 2.5 X lo0 were preincubated with lo-’ M Cy for 15 min human peripheral blood lymphocytes (PBL) at 37°C. Cy remained during the ADCC reaction. (b) 2.5 X 10” PBL were lpreincubated with lo-’ M Cy for 15 min at 37°C and then washed three times at 37°C with medium 199. Then PBL of either group were assayed for both ADCC and protein synthesis as detailed under Materials and Methods.
PROTEIX
S’I’NTIIESTS
IN
335
ADCC
100
0"
0
AOCC
@I
Protein
synthesis
," 0' * 50 2 k? s 0
CYCLOHEXIMIDE, molar
FIG. 2. Dose-dependent effect of cycloheximide (Cy) on antibody-dependent cell-mediated cy:otoxicity (ADCC) and protein synthesis. 2.5 X 10’ human peripheral blood lymphocytes (PBL) were preincubated for 15 min with lo-“, lo-“, lo-“, and 10e3 M Cy. ADCC and protein synthesis were tested as described under Materials and Methods. Cy rmemained in the medium throughout the reaction.
In order to know why ADCC underwent partial inhibition, kinetics experiments were carried out with and without Cy. The results shown in Fig. 3 demonstrate that the ADCC reaction with Cy-treated cells had the same slope as that of control Instead, Cy-treated cells cells at short times (up to 30 min in this experiment). displayed a decreased cytotoxic effect when later stages were considered (after 30 min in this case). It is clearly shown that Cy did not delay the onset of the reaction. Consumption
and Recovery
of ADCC
As ADCC of effector cells with impaired protein synthesis appears to be consumed after a short time of reaction, the following experiments were designed to provide evidence that lytic mechanisms can be “wasted” during a previous phase of exposure to target cells. It is also well known that effector cells that have performed ADCC can be reutilized after removal of target cells by different procedures ( 10, 16).
UNTREATED
Cy TREATEO
OLIfI
’
’
0 5 IO 20 30 INCUBATION,
1
60
I 120
minutes
FIG. 3. The effect of cycloheximide (Cy) on antibody-dependent cell-mediated cytotoxicity (ADCC) kinetics. 2.5 X IO” human peripheral blood lymphocytes (PBL) were pretreated with lo-’ M Cy for 15 min at 37°C. Cy remained in the medium during the reaction. ADCC was determined at different incubation times. Untreated effector cells (PBL) were assayed at the same time. ADCC was determined as detailed under Mat’erials and Methods.
336
ISTURIZ
AND
CARDONI STEP
STEP
2
Ab-CRBC*
100 I
/Ab-CRBG
E 50 7 (0) ," 0 _ (b) 2
**--
.I-’
_e--
-eAb-CRBC+Cy
,N ,,t-.-.-.-.,Ab-CRBC+Cy
Ab-CRBC*
Ab-CR&y
.' Cc).'
O-
120 INCUBATION,minutes
FIG. 4. Consumption and recovery of antibody-dependent cell-mediated cytotoxicity (ADCC) in the Ipresence of cycloheximide (Cy). (a) In the first step, 5 X 10” human peripheral blood lymphocytes (PBL) and unlabeled antibody-coated chicken erythrocytes (Ab-CRBC) in a 3: 1 effector to target cells ratio were centrifuged at 2OOg for 10 min, resuspended, and incubated for 1 hr at 37°C. Then the residual CRBC were haemolyzed by exposure to distilled water for 80 set at 4°C and the immediate addition of concentrated saline solution to restore isotonicity. The PBL were washed with medium 199 three times at 4°C (-). (b) In the first step, 5 X 10’ PBL and unlabeled Ab-CRBC in a 3: 1 effector to target cells ratio in the presence of 10m3iW Cy were centrifuged at 2OOg for 10 min, resuspendmed,and incubated for 1 hr at 37°C in the presence of lo-” M Cy. After osmotic shock and restoration of isotonicity as in the former experiment, the cells were washed with medium 199 three times at 4°C (- - -). (c) In the first step, 5 X 10” PBL were treated in the same way as in (a) and (b), but after restoring isotonicity the cells were washed three times at 4°C with medium 199 containing 10m3M Cy (.-.-*-.-). In the second step, “Cr-labeled-Ab-CRBC (Ab-Yr-CRBC) in a 10: 1 effector to target cells ratio were added to either group and ADCC as assayed at different times as detailed under Materials and Methods. ADCC was calculated arbibrarily considering that lysis obtained with cells exposed in the ficst step to Ab-CRBC during 120 min was 100%. Reactions (a) and (b) were (performed in 1 ml of medium 199 and reaction (c) was carried out in 1 ml of medium 199 containing lo-” M Cy. Ab-CRBC* represents Ab-“CrCRBC.
The experiment deta.iled in Fig. 4 depicts the behavior of effector cells when a second set of target cells is added. In the first step of this experiment, ADCC was performed with unlabeled Ab-CRBC, and then the remaining target cells were hemolyzed (step 1). In a second step, Ab-51Cr-CRBC were added and ADCC was assayed at different times (Fig. 4a). Control tests made simultaneously with labeled target cells revealed that cytolysis took place on step 1 and that protein synthesis was functioning (Table 1). Removal of unlabeled Ab-CRBC was done by osmotic shock in order to avoid blockade by target cell-antibody complexes when Ab-51Cr-CRBC were added in the second step. Cells were washed at 4°C to avoid protein synthesis. Previous experiments showed that effector cells incubated in the first step with unsensitized CRBC or CRBC plus cycloheximide had the same ADCC potential in the second step as untreated cells. On the other hand, pretreatment with Ab-CRBC always gave some degree of inhibition (lO-20%). Therefore, to evaluate waste of the cytolytic mechanism (Fig. 4), ADCC values were compared to the ADCC ability of effector cells pretreated with Ab-CRBC in the absence of protein synthesis inhibitors. In Fig. 4b, the experiments were outlined in the same way but Cy was added in the first step and removed by washing. Cytotoxicity was lower than in the absence
PROTEIN
CONSUMPTION
(C)
@=f-)+cV
SYNTHESIS
IN
337
ADCC RECOVERY
WASH
STEP
-04)
TC-199
t
AND LYSIS
Cy
Ab-CRBC*
PBL-Ab-CRBC
FIG. 5. Schematic diagram of consumption and recovery of cytolytic ability of effector cells (peripheral blood lymphocytes, PBL). (a) During the consumption step, PBL have performed antibody-dependent cell-mediated cytotoxicity (ADCC) and, after removal of the target, they are able to react again with new target cells [represents curve (a) in Fig. 41. (b) ADCC is performed by PBL with Cy in the culture medium. When Cy was removed, protein synthesis was restored and ADCC activity was gradually recovered. When a 15-hr assay was performed, ADCC reached the same values as untreated effector cells [represents curve (b) in Fig. 41. (c) Lytic capacity has been consumed in the first stage, when PBL have carried out ADCC with Cy, and only a residual activity was developed [represents curve (c) in Fig. 41. Filled circles represent cytolytic ability. The consumption step represents step 1 in Fig. 4, and recovery and lysis represent step 2 in Fig. 4. Ab-CRBC* represents Ab-“Cr-CRBC.
of Cy. However, when Cy was added in the first step and allowed to remain throughout the reaction, ADCC in the second step was lowest and a plateau was promptly reached (Fig. 4~). These results indicate that the lytic mechanism of ADCC can be consumed by previous exposure to sensitized target cells and that full recovery of ADCC depends on protein synthesis. TABLE Antibody-Dependent
1
Cell-Mediated Cytotoxicity and Protein of Peripheral Blood Lymphocytes
Incubation of 2.5 X 108PBL witha Vr-CRBC Ab-6rCr-CRBC
Protein synthesis* 100 98 99
Synthesis
ADCCc (70) 1 100
a Peripheral blood lymphocytes (PBL) were incubated for 1 hr at 37’C with 5 X lo6 61Crlabeled chicken red blood cells (61Cr-CRBC) or antibody-coated SICr-CRBC (AbNJr-CRBC) or without target cells. b Protein synthesis was assayed as described under Materials and Methods. The results are expressed as percentages of [Xllysine incorporated in the absence of target cells. c Antibody-dependent cell-mediated cytotoxicity (ADCC) was tested as described under Materials and Methods.
338
ISTURIZ
AND
CARDONI
DISCUSSION The aim of the present work was to study the relationship between ADCC and protein synthesis. In order to investigate the role of protein synthesis in the cytolytic process, cycloheximide (Cy), a specific inhibitor of the translation step (17), was used. Cy was selected because it affects protein synthesis in a reversible way. Our results indicate that protein synthesis is not necessary for the ADCC reaction to start, but the lytic ability of the effector cells is not fully expressed in the presence of Cy (Figs. l-3). The inhibition of ADCC and protein synthesis is dose dependent and reversible (Figs. 1 and 2) ; hence, it cannot be explained by a lethal effect of the drug on the effector cells. Furthermore, Cy does not delay 51Cr release from damaged target cells since, after prolonged incubation of ADCC reactions in the presence of Cy, the results are similar to those obtained in the 1-hr assay. These results are in accordance with previous reports (13, 18, 19) suggesting that ADCC may function in the presence of inhibitors of protein synthesis. However, a dose-dependent inhibition was observed using both cycloheximide and puromycin (12). In our experiments, the inhibition was evident only when an appropriate ratio (5 : 1) of effector to target cells was used. If the ratio of effector to target cells was increased, the inhibitory effect of Cy on ADCC was masked (data not shown). This may be the cause of the controversial results reported by others (12, 13, 18, 19). Our kinetic experiments provide further evidence for the independence of the initial phase of ADCC from protein synthesis. Thus, there was no lag in the cytotoxic reaction with or without Cy (Fig. 3). On the contrary, in this experiment, after 30 min the reaction of the Cy-treated effector cells reached a plateau. This can be explained if a preformed lytic mechanism has been “wasted” in the initial phase of ADCC and cannot be replaced in the absence of protein synthesis. This possibility was explored by performing the ADCC in two sequential steps. In the first one, effector cells were exposed to unlabeled target cells and allowed to accomplish one cytotlytic cycle with or without an inhibitor of protein synthesis (Fig. 4). In the second step, after removal of the remaining target cells by osmotic lysis, the same effector cells were exposed to labeled sensitized target cells, and the amount of %r released in the presence or absence of Cy was measured. The results shown in Fig. 4 indicate that the presence of Cy throughout the reaction yields maximum inhibition (Fig. 4~). If the inhibitor is removed before the second step, the cells gradually recover their lytic ability (Fig. 4b). Indeed, in long-term experiments, effector cells exposed to Cy in the first step achieved the same cytotoxic levels as the controls. The results depicted in Fig. 4 may be interpreted as shown in Fig. 5. In the first step, the effector cells established a close contact with target cells (Ab-CRBC) in the presence or absence of Cy. When Cy was in the culture medium the effector cells wasted their cytotoxic mechanism (represented by filled circles), and since they required protein synthesis to replace it, they remained inhibited (Figs. 5b and c). However, when the first cycle of ADCC was performed in the absence of Cy, the effector cells wasted their cytotoxic mechanism, but this was restored by a de nova protein synthesis (Fig. Sa). In the second step, uninhibited effector cells produced a maximum lytic activity (Fig. 5a). PBL that had been exposed to a first cycle of ADCC in the presence
PROTEIN
SYNTIIESIS
IN
AIXC
339
of Cy can gradually recover their lytic activity if the second step is performed in the absence of the protein synthesis inhibitor (Fig. Sb). In this case, the lytic ability expressed in step 2 was the result of a recovery of the lytic mechanism allowed by an active protein synthesis. On the other hand, effector cells that were permanently inhibited (Fig. 5~) displayed, in the second cycle, the residual ADCC activity that was not consumed in step 1, and as Cy was also present in step 2, the cytotoxic mechanism could not be restored by protein synthesis. Since our results and those of others (13, 18, 19) indicate that cytolysis can be initiated in the absence of protein synthesis, ADCC appears to be an extremely efficient mechanism that, according to previous reports (II), may act immediately after triggering. In conclusion, we propose that ADCC can be started by a preformed lytic mechanism that can be wasted during the reaction and then restored by active protein synthesis. These results, however, do not provide evidence to establish the nature of the lytic component(s) involved in the ADCC process. ACKNOWLEDGMENTS We wish to thank Dr. M. M. E. de Bracco and Dr. M. Michel for Ms. A. M. Pizzi for technical assistance, and Ms. P. P&a for secretarial
helpful discussions, assistance.
REFERENCES 1. Shore, S. L., Black, C. M., Melewicz, F. M., Wood, P. A., and Nahmias, A. J., J. Z?rznzz~zol. 116, 194, 1976. 2. Rager-Zisman, B., and Bloom, B. R., Nature (Loxdon) 251, 542, 1974. 3. Rouse, B. T., Wardley, R. C., and Babiuk, L. A., Cell. Imnzunol. 22, 182, 1976. 4. Pollak, S., Heppner, G., Brown, R. J., and Nelson, K., Zltt. J. Cancer, 9, 316, 1972. 5. Skurzak, H. M., Klein, E., Yoshida, T. O., and Lamon, E. W., J. Exp. Med. 135, 997, 1972. 6. Hersey, P., Cullen, P., and MacLennan, I. C. M., Trmzspluxtation 16, 9, 1973. 7. Mkwanazi, J. B., Frank, D., and Baker, J. R., Nature (Zmzdm) 259, 403, 1976. 8. Butterworth, A. E., Sturrock, R. F., Houba, V., and Rees, P. H., Nufzve (Loudox) 252, 503, 1974. 9. Perlmann, P., and Helm, G., Advau. Z~mmr~ol. 11, 117, 1969. 10. Perlmann, P., Perlmann, H., and Biberfeld, P., J. Zlrzmz~zol. 108, 558, 1972. 11. Scornik, J. C., J. Z~U~ZZCIIO~. 113, 1519, 1974. 12. Strom, T. B., Garovoy, M. R., Bear, R., Gribik, M., Carpenter, C. B., and Merrill, J. P., Fed. Pvoc. 33, 780, 1974 (abst.). 13. Wunderlich, J. R., Rosenberg, E. B., and Connolly, S. M., In “Progress in Immunology I” (B. Amos, Ed.), p. 473. Academic Press, New York, 1971. 14. Isturiz, M. A., de Bracco, M. M. E., and Manni, J. A., Cell. Inzvzztrtol. 16, 82, 1975. 15. Manni, J. A., de Bracco, M. M. E., and Patrucco, A., Mcdicim (Buenos Aires) 34, 185, 1974. 16. de Bracco, M. M. E., Isturiz, M. A., and Manni, J. A., Cell. Zmmuno1. 30, 325, 1976. 17. Grollman, A. P., Pvoc. Nat. Acad. Sci. USA 56, 1867, 1966. 18. MacLennan, I. C. M., and Harding, B., Zmnzu~ology 18, 405, 1970. 19. Dickmeiss, E., Scmd. J. Zwwmnol. 3, 817, 1974.
The Role of Protein Synthesis in Antibody-Dependent Cell-Mediated Cytotoxicityl MARTIN A. ISTURIZ AND RITA L. CARDONI 2* 3 Imtituto
de Iwucstigaciom+s
Mkdicas, Universidad de Bl4enos Aires, BUENOS Aires 1427, Argentina Received
January
Domto
Alvarez
3000,
4, 1977
Antibody-depedent cell-mediated cytotoxicity (ADCC) could be initiated without protein synthesis [human peripheral blood lymphocytes as effector cells incubated with IO-” M cycloheximide, (Cy) 1, although the reaction did not achieve its full lytic ability. This partial inhibition of ADCC was dependent on the dose of Cy. Both ADCC and protein synthesis returned to normal values after removal of the inhibitor. The kinetics of the reaction carried out by Cy-treated effector cells for short periods was similar to that of controls. After this time, the percentage of lysed target cells increased continuously in controls, while the cytotoxiciy of Cy-treated effector cells reached a plateau. When effector cells carried out ADCC in the presence of Cy, their lytic mechanism was “wasted,” and it could be recovered only by removal of the inhibitor. Our results indicate that effector cells have a preformed lytic mechanism operative in ADCC. This lytic mechanism is consumed during the reaction and its recovery requires protein synthesis.
INTRODUCTION Previous studies have shown that antibody-dependent cell-mediated cytotoxicity (ADCC) may be an important process of the host’s immune surveillance system, since this activity has been demonstrated against virus-infected cells (l-3), neoplastic cells (4, 5), allogeneic graft rejection (6), and protozoa (7, 8). It is a very efficient system, attacking target cells sensitized with very low concentrations of antibody (9). Furthermore, effector cells and antibody can be reutilized to carry out cytolysis with a new batch of target cells (10). It is well established that the reaction requires close contact between sensitized target and effector cells, and it results in immediate damage of the former (11). However, it is not clear what the relationship is between protein synthesis and ADCC. While some investigators report dose-dependent inhibition of both ADCC and protein synthesis by cycloheximide (Cy) and puromycin (12), others show that nonsensitized lymphocytes are equipped for ADCC and that they expressed their activity even at concentrations of the same inhibitors which block [‘“Clleucine incorporation into proteins (13). The present paper was done in order to explore the role of protein synthesis in 1 This work was supported by CONICET Grant No. 5840. * To whom reprint requests should be directed. 3 Research Fellow from National Council for Scientific and Technical Research (CONICET)
.
332 Copyright All rights
@ 1977 by Academic Press, of reproduction in any form
Inc. reserved.
ISSN 000888749
PROTEIN
SYNTHESIS
IN
333
ADCC
the effector phase of the ADCC reaction. The problem was approached by combining the use of Cy, a reversible inhibitor of the translation step of protein synthesis, and an experimental tlesign that allowetl us to tlivitle the reaction into “wasting” and effector stages. MATEKIALS
AND
METHODS
Human ly~phoid cells. Peripheral blood lymphocytes (PBL) were isolated from human volunteers as has been described (14). The resultant cell suspension, composed of 95-98s lymphocytes, was washed three times with medium 199 (DIFCO Laboratories, Detroit, Michigan) containing 100 units/ml of penicillin, 100 pg/ml of streptomycin, and 2.5% heat-inactivated fetal calf serum (TC-199). The same tissue culture medium (TC-199) was used in all cytotoxicity experiments. Cell viability was assayed by the trypan blue exclusion test. The concentration of lymphocytes was determined by counting the cell suspensions stained with Turk’s solution. Antibody-dependent cell-mcdintcd cytotoxicit~l (ADCC). ADCC was measured utilizing j’Cr-labeled chicken red blood cells (“‘Cr-CRBC) sensitized with rabbit antibody to CRBC at a subagglutinating dose (Ab-“ICr-CRBC) Rabbit antibodies to CRBC and Yr-CRBC were prepared as described elsewhere (15). ADCC was assayed by culturing mixtures containing 2.5 x 10” PBL and 5 X 10’ Ab-“‘CrCRBC in a 5 : 1 effector to target cell ratio in a final volume of 1 ml, unless otherwise stated (16). The mixtures were centrifuged at 200g for 10 min in an International PR-2 centrifuge in order to establish contact between effector and target cells. The pellets were then resuspended and incubatcl for 1 hr at 37°C. The initiation of this incubation period was considered as time zero. At the end of the reaction, the mixtures were centrifuged at 400g for 15 niin and 0.5 ml of supernatant was transferred to another test tube. The radioactivity of both the supernatant and the pellet was measured in a Packard 3320 gama counter. The percentage of s’Cr released, representing ADCC, was calculated as follows : %Vr
released =
supernatant activity total activity
X 2
x 100.
This value was corrected by subtracting the percentage of “‘Cr released in control cultures in the absence of antibody (l-2% alCr released). Percentage of “‘Cr released ranged between 40 and 60%. ADCC results are expressed as: %ADCC
y051Cr released in reaction tubes = %51c r re 1eased in the absence of inhibitors
x 100.
Protein synthesis by lzuman lymplzoid cells. The protein synthesis in PBL was evaluated by measuring the incorporation of [l”C]lysine into proteins. PBL were incubated with 0.5 PC1 of [‘“Cl lysine (specific activity 301 mCi/mmol, Amersham/ Searle Corp.) for 1 hr at 37°C under the same conditions as for the ADCC assays. After incubation the cells were washed three times in saline solution at 4°C. The pellet was suspended in distilled water for lysis, and trichloroacetic acid (TCA, Baker Analyzed Reagent) was added to a final concentrations of 7.570 ; the reaction was heated at 80°C for 15 min to hydrolyze aminoacyl-tRNA. TCA-precipitable material was washed three times with 5% TCA, solubilized in formic acid, dissolved in scintillation fluid (PPO, 4 g ; POPOP, 0.2 g; toluene, 200 ml ; ab-
334
ISTURIZ
AND
CARDONI
solute ethyl alcohol, 300 ml), and counted in a Packard 3320 beta counter. The percentage of [‘Xllysine incorporated in samples from a 1-hr pulse, representing protein synthesis, was calculated as follows: O/o[14C]lysine incorporated
=
sample activity control activity without
Cy
x 100
= y0 protein synthesis. [14C]-Lysine was not incorporated when cells were incubated at 4°C up to 2 hr. Cycloheximide (Cy) (Sigma Chemical Co., St. Louis, Missouri) was dissolved in 0.15 M sodium chloride at different concentrations. Cy at a concentration of 10m3M completely inhibited protein synthesis 10 min after addition. Experiments for determination of both protein synthesis and ADCC were performed at least four times, and a representative one is shown under Results. RESULTS Relationship
between ADCC and Protein Synthesis
These experiments were designed to establish the role of protein synthesis in the initiation step of the ADCC reaction. As shown in Fig. 1, when ADCC was performed with Cy throughout the reaction, the cytolytic ability of the cells was not fully expressed, but protein synthesis was completely inhibited. On the other hand, when Cy was removed from the reaction, both ADCC and protein synthesis reached control values, The relation between protein synthesis and ADCC was investigated. ADCC was assayed with Cy at concentrations shown in Fig. 2. The results obtained demonstrate that ADCC and protein synthesis are closely related. Similar results were obtained if the ADCC was done in the same way except that the reaction mixtures were incubated for 18 hr. This indicated that Cy did not delay the Yr released from damaged Ab-Yr-CRBC. The results shown in Figs. 1 and 2 demonstrate that in the presence of Cy, the effector cells displayed a significant level of ADCC in spite of the sharp decrease in protein synthesis.
0
AOCC
I%! Protein
0
(a) with
Cy
synthesis
1 b) Washed
FIG. 1. The effects of cycloheximide (Cy) on antibody-dependent cell-mediated cytotoxicity (ADCC) and protein synthesis and its reversibility upon the ,removal of Cy. (a) 2.5 X lo0 were preincubated with lo-’ M Cy for 15 min human peripheral blood lymphocytes (PBL) at 37°C. Cy remained during the ADCC reaction. (b) 2.5 X 10” PBL were lpreincubated with lo-’ M Cy for 15 min at 37°C and then washed three times at 37°C with medium 199. Then PBL of either group were assayed for both ADCC and protein synthesis as detailed under Materials and Methods.
PROTEIX
S’I’NTIIESTS
IN
335
ADCC
100
0"
0
AOCC
@I
Protein
synthesis
," 0' * 50 2 k? s 0
CYCLOHEXIMIDE, molar
FIG. 2. Dose-dependent effect of cycloheximide (Cy) on antibody-dependent cell-mediated cy:otoxicity (ADCC) and protein synthesis. 2.5 X 10’ human peripheral blood lymphocytes (PBL) were preincubated for 15 min with lo-“, lo-“, lo-“, and 10e3 M Cy. ADCC and protein synthesis were tested as described under Materials and Methods. Cy rmemained in the medium throughout the reaction.
In order to know why ADCC underwent partial inhibition, kinetics experiments were carried out with and without Cy. The results shown in Fig. 3 demonstrate that the ADCC reaction with Cy-treated cells had the same slope as that of control Instead, Cy-treated cells cells at short times (up to 30 min in this experiment). displayed a decreased cytotoxic effect when later stages were considered (after 30 min in this case). It is clearly shown that Cy did not delay the onset of the reaction. Consumption
and Recovery
of ADCC
As ADCC of effector cells with impaired protein synthesis appears to be consumed after a short time of reaction, the following experiments were designed to provide evidence that lytic mechanisms can be “wasted” during a previous phase of exposure to target cells. It is also well known that effector cells that have performed ADCC can be reutilized after removal of target cells by different procedures ( 10, 16).
UNTREATED
Cy TREATEO
OLIfI
’
’
0 5 IO 20 30 INCUBATION,
1
60
I 120
minutes
FIG. 3. The effect of cycloheximide (Cy) on antibody-dependent cell-mediated cytotoxicity (ADCC) kinetics. 2.5 X IO” human peripheral blood lymphocytes (PBL) were pretreated with lo-’ M Cy for 15 min at 37°C. Cy remained in the medium during the reaction. ADCC was determined at different incubation times. Untreated effector cells (PBL) were assayed at the same time. ADCC was determined as detailed under Mat’erials and Methods.
336
ISTURIZ
AND
CARDONI STEP
STEP
2
Ab-CRBC*
100 I
/Ab-CRBG
E 50 7 (0) ," 0 _ (b) 2
**--
.I-’
_e--
-eAb-CRBC+Cy
,N ,,t-.-.-.-.,Ab-CRBC+Cy
Ab-CRBC*
Ab-CR&y
.' Cc).'
O-
120 INCUBATION,minutes
FIG. 4. Consumption and recovery of antibody-dependent cell-mediated cytotoxicity (ADCC) in the Ipresence of cycloheximide (Cy). (a) In the first step, 5 X 10” human peripheral blood lymphocytes (PBL) and unlabeled antibody-coated chicken erythrocytes (Ab-CRBC) in a 3: 1 effector to target cells ratio were centrifuged at 2OOg for 10 min, resuspended, and incubated for 1 hr at 37°C. Then the residual CRBC were haemolyzed by exposure to distilled water for 80 set at 4°C and the immediate addition of concentrated saline solution to restore isotonicity. The PBL were washed with medium 199 three times at 4°C (-). (b) In the first step, 5 X 10’ PBL and unlabeled Ab-CRBC in a 3: 1 effector to target cells ratio in the presence of 10m3iW Cy were centrifuged at 2OOg for 10 min, resuspendmed,and incubated for 1 hr at 37°C in the presence of lo-” M Cy. After osmotic shock and restoration of isotonicity as in the former experiment, the cells were washed with medium 199 three times at 4°C (- - -). (c) In the first step, 5 X 10” PBL were treated in the same way as in (a) and (b), but after restoring isotonicity the cells were washed three times at 4°C with medium 199 containing 10m3M Cy (.-.-*-.-). In the second step, “Cr-labeled-Ab-CRBC (Ab-Yr-CRBC) in a 10: 1 effector to target cells ratio were added to either group and ADCC as assayed at different times as detailed under Materials and Methods. ADCC was calculated arbibrarily considering that lysis obtained with cells exposed in the ficst step to Ab-CRBC during 120 min was 100%. Reactions (a) and (b) were (performed in 1 ml of medium 199 and reaction (c) was carried out in 1 ml of medium 199 containing lo-” M Cy. Ab-CRBC* represents Ab-“CrCRBC.
The experiment deta.iled in Fig. 4 depicts the behavior of effector cells when a second set of target cells is added. In the first step of this experiment, ADCC was performed with unlabeled Ab-CRBC, and then the remaining target cells were hemolyzed (step 1). In a second step, Ab-51Cr-CRBC were added and ADCC was assayed at different times (Fig. 4a). Control tests made simultaneously with labeled target cells revealed that cytolysis took place on step 1 and that protein synthesis was functioning (Table 1). Removal of unlabeled Ab-CRBC was done by osmotic shock in order to avoid blockade by target cell-antibody complexes when Ab-51Cr-CRBC were added in the second step. Cells were washed at 4°C to avoid protein synthesis. Previous experiments showed that effector cells incubated in the first step with unsensitized CRBC or CRBC plus cycloheximide had the same ADCC potential in the second step as untreated cells. On the other hand, pretreatment with Ab-CRBC always gave some degree of inhibition (lO-20%). Therefore, to evaluate waste of the cytolytic mechanism (Fig. 4), ADCC values were compared to the ADCC ability of effector cells pretreated with Ab-CRBC in the absence of protein synthesis inhibitors. In Fig. 4b, the experiments were outlined in the same way but Cy was added in the first step and removed by washing. Cytotoxicity was lower than in the absence
PROTEIN
CONSUMPTION
(C)
@=f-)+cV
SYNTHESIS
IN
337
ADCC RECOVERY
WASH
STEP
-04)
TC-199
t
AND LYSIS
Cy
Ab-CRBC*
PBL-Ab-CRBC
FIG. 5. Schematic diagram of consumption and recovery of cytolytic ability of effector cells (peripheral blood lymphocytes, PBL). (a) During the consumption step, PBL have performed antibody-dependent cell-mediated cytotoxicity (ADCC) and, after removal of the target, they are able to react again with new target cells [represents curve (a) in Fig. 41. (b) ADCC is performed by PBL with Cy in the culture medium. When Cy was removed, protein synthesis was restored and ADCC activity was gradually recovered. When a 15-hr assay was performed, ADCC reached the same values as untreated effector cells [represents curve (b) in Fig. 41. (c) Lytic capacity has been consumed in the first stage, when PBL have carried out ADCC with Cy, and only a residual activity was developed [represents curve (c) in Fig. 41. Filled circles represent cytolytic ability. The consumption step represents step 1 in Fig. 4, and recovery and lysis represent step 2 in Fig. 4. Ab-CRBC* represents Ab-“Cr-CRBC.
of Cy. However, when Cy was added in the first step and allowed to remain throughout the reaction, ADCC in the second step was lowest and a plateau was promptly reached (Fig. 4~). These results indicate that the lytic mechanism of ADCC can be consumed by previous exposure to sensitized target cells and that full recovery of ADCC depends on protein synthesis. TABLE Antibody-Dependent
1
Cell-Mediated Cytotoxicity and Protein of Peripheral Blood Lymphocytes
Incubation of 2.5 X 108PBL witha Vr-CRBC Ab-6rCr-CRBC
Protein synthesis* 100 98 99
Synthesis
ADCCc (70) 1 100
a Peripheral blood lymphocytes (PBL) were incubated for 1 hr at 37’C with 5 X lo6 61Crlabeled chicken red blood cells (61Cr-CRBC) or antibody-coated SICr-CRBC (AbNJr-CRBC) or without target cells. b Protein synthesis was assayed as described under Materials and Methods. The results are expressed as percentages of [Xllysine incorporated in the absence of target cells. c Antibody-dependent cell-mediated cytotoxicity (ADCC) was tested as described under Materials and Methods.
338
ISTURIZ
AND
CARDONI
DISCUSSION The aim of the present work was to study the relationship between ADCC and protein synthesis. In order to investigate the role of protein synthesis in the cytolytic process, cycloheximide (Cy), a specific inhibitor of the translation step (17), was used. Cy was selected because it affects protein synthesis in a reversible way. Our results indicate that protein synthesis is not necessary for the ADCC reaction to start, but the lytic ability of the effector cells is not fully expressed in the presence of Cy (Figs. l-3). The inhibition of ADCC and protein synthesis is dose dependent and reversible (Figs. 1 and 2) ; hence, it cannot be explained by a lethal effect of the drug on the effector cells. Furthermore, Cy does not delay 51Cr release from damaged target cells since, after prolonged incubation of ADCC reactions in the presence of Cy, the results are similar to those obtained in the 1-hr assay. These results are in accordance with previous reports (13, 18, 19) suggesting that ADCC may function in the presence of inhibitors of protein synthesis. However, a dose-dependent inhibition was observed using both cycloheximide and puromycin (12). In our experiments, the inhibition was evident only when an appropriate ratio (5 : 1) of effector to target cells was used. If the ratio of effector to target cells was increased, the inhibitory effect of Cy on ADCC was masked (data not shown). This may be the cause of the controversial results reported by others (12, 13, 18, 19). Our kinetic experiments provide further evidence for the independence of the initial phase of ADCC from protein synthesis. Thus, there was no lag in the cytotoxic reaction with or without Cy (Fig. 3). On the contrary, in this experiment, after 30 min the reaction of the Cy-treated effector cells reached a plateau. This can be explained if a preformed lytic mechanism has been “wasted” in the initial phase of ADCC and cannot be replaced in the absence of protein synthesis. This possibility was explored by performing the ADCC in two sequential steps. In the first one, effector cells were exposed to unlabeled target cells and allowed to accomplish one cytotlytic cycle with or without an inhibitor of protein synthesis (Fig. 4). In the second step, after removal of the remaining target cells by osmotic lysis, the same effector cells were exposed to labeled sensitized target cells, and the amount of %r released in the presence or absence of Cy was measured. The results shown in Fig. 4 indicate that the presence of Cy throughout the reaction yields maximum inhibition (Fig. 4~). If the inhibitor is removed before the second step, the cells gradually recover their lytic ability (Fig. 4b). Indeed, in long-term experiments, effector cells exposed to Cy in the first step achieved the same cytotoxic levels as the controls. The results depicted in Fig. 4 may be interpreted as shown in Fig. 5. In the first step, the effector cells established a close contact with target cells (Ab-CRBC) in the presence or absence of Cy. When Cy was in the culture medium the effector cells wasted their cytotoxic mechanism (represented by filled circles), and since they required protein synthesis to replace it, they remained inhibited (Figs. 5b and c). However, when the first cycle of ADCC was performed in the absence of Cy, the effector cells wasted their cytotoxic mechanism, but this was restored by a de nova protein synthesis (Fig. Sa). In the second step, uninhibited effector cells produced a maximum lytic activity (Fig. 5a). PBL that had been exposed to a first cycle of ADCC in the presence
PROTEIN
SYNTIIESIS
IN
AIXC
339
of Cy can gradually recover their lytic activity if the second step is performed in the absence of the protein synthesis inhibitor (Fig. Sb). In this case, the lytic ability expressed in step 2 was the result of a recovery of the lytic mechanism allowed by an active protein synthesis. On the other hand, effector cells that were permanently inhibited (Fig. 5~) displayed, in the second cycle, the residual ADCC activity that was not consumed in step 1, and as Cy was also present in step 2, the cytotoxic mechanism could not be restored by protein synthesis. Since our results and those of others (13, 18, 19) indicate that cytolysis can be initiated in the absence of protein synthesis, ADCC appears to be an extremely efficient mechanism that, according to previous reports (II), may act immediately after triggering. In conclusion, we propose that ADCC can be started by a preformed lytic mechanism that can be wasted during the reaction and then restored by active protein synthesis. These results, however, do not provide evidence to establish the nature of the lytic component(s) involved in the ADCC process. ACKNOWLEDGMENTS We wish to thank Dr. M. M. E. de Bracco and Dr. M. Michel for Ms. A. M. Pizzi for technical assistance, and Ms. P. P&a for secretarial
helpful discussions, assistance.
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