- Email: [email protected]
The role of microtubules in human natural killer cell-mediated cytotoxicity
CELLULAR
IMMUNOLOGY
106,376-386
(1987)
The Role of Microtubules Cell-Mediated
in Human Natural Killer Cytotoxicity
OLLI CARP&N Department ofPathology, University ofHelsinki, Haartmaninkatu
3, 00290 Helsinki, Finland
Received October 26, 1986; accepted January 14, 1987 The effect of four different microtubule (MT) inhibitors on the various stages of human natural killer (NK) cell-mediated cytotoxicity was studied. The MT-disrupting effect of the drugs was monitored by indirect immunofluorescence microscopy and transmission electron microscopy. All the drugs tested, vinblastine sulfate, demecolcine, nocodaxole, and taxol, had only a slight inhibitory effect on NK activity. Cells with nonfunctional MT were capable of normal conjugate formation and polarization of actin-containing microfdaments. Natural killer ccl1cytotoxic fao tor (NKCF) activity produced by cells with nonfunctional MT was slightly diminished. MT disruption caused enlargement of Golgi cistemae, but did not, however, dissociate the overall structural organization of the Golgi complex. The results indicate that fresh human NK cells are capable of lytic activity without functional MT although MT play a small supportive role in production or secretion of NKCF and mediation of the lytic activity. Previous experiments by us and others have strongly suggested that NK cells mediate their cytolytic activity by directed secretion of toxic material. As NK cells with unfunctional microtubules are capable of close to normal secretion the results presented in this report are not inconsistent with the earlier sugQ 1987 Academic PESS, 1,~. gested SthUlUS-SeCretiOn model.
INTRODUCTION Human natural killer (NK) cell activity is mediated by large granular lymphocytes (LGL) that have the ability to recognize their target cells without previous sensitization. Target cell recognition is followed by a set of events in the effector cell that finally result in the lysis of the target cell. The cytoskeletal organization of the NK cell alters upon conjugation with sensitive target cell: actin and vinculin become redistributed into focal contact-like areas (1). Concomitantly to the reorganization of actin microfilaments the cellular shape is changed and the Golgi apparatus becomes polarized toward the target cell (2-5). The experiments with monensin (6), which interrupts the vesicular traffic of Golgi-derived vesicles to cell membrane (7), as well as with other inhibitors of secretory processes (8-12), have strongly suggested that NK cells elicit their lytic activity by a directed secretion. The toxic material apparently binds to the target cell membrane ( 13, 14) and leads to disruption of the target cell. The cytolytic events are suggested to be mediated by natural killer cytotoxic factor (NKCF) (13, 15) or granule cytolysin/perforin (14, 16, 17) that both are produced by cells with NK activity and have the ability to disrupt NK-sensitive target cells. 376 0008-8749187 $3.00 Copyright Q 1987 by Academic Press, Inc. All rights of reproduction in any form -ed.
MICROTUBULES
IN NK CELL-MEDIATED
KILLING
377
Most earlier studies have suggested that intact microtubules (MT) are required in cellular secretory events (18-24), and furthermore, several reports indicate their importance in the maintenance of the organization ofGolgi apparatus (25) see also (26). Some authors have suggested a direct association of microtubules and microtubuleorganizing center (MTOC) in the cytotoxic action mediated by NK cells and T cells (3,9,27-3 1). Our earlier experiments suggested, however, that MT-disrupting agents had only a minor effect on NK cell activity (6). These data appeared to be contradictory to the stimulus-secretion model of NK cell-mediated cytotoxicity which would require an intact secretory apparatus to deliver the lethal hit. Since this important point has remained controversial I designed a series of experiments to further study the role of MT in cytolytic activity of human NK cells. The results suggest, in contrast to some other reports, that MT have only a minor supportive effect on NK activity, and that most of the lytic potential of NK cells is maintained upon dissociation of microtubules. The relevance of these findings to the stimulus-secretion model will be discussed. MATERIALS AND METHODS Efictor and target cells. BufIy coats were obtained from the Finnish Red Cross Blood Transfusion Service. They were purified as described in detail elsewhere (32). Briefly, mononuclear cells were isolated by the Ficoll-Isopaque gradient centrifugation. For removal of adherent cells, the mononuclear cells were incubated in Roux glass bottles, and the nonadherent fraction was run through nylon wool columns. The isolated cells were centrifuged in discontinuous Percoll (Pharmacia Fine Chemicals AB, UppsaIa, Sweden) gradients ranging from 47.5 (v/v) to 37.5% (v/v) with 2.5% diminution steps. The fraction layering at the 37.5% interphase consisted of 75 to 85% of large granular lymphocytes as judged morphologically from Giemsa-stained cytocentrifuge smears, and the NK activity in a 5‘Cr-cytotoxicity assay was also concentrated in this fraction as shown previously (32). K562, the continuous erythroleukemia cell line, was used as a source of NK-sensitive target cells and U937, a histiocytic lymphoma, as target cells in assays for NKCF activity. NK target cell lysis was measured with a standard “Cr-cytotoxicity assay as described previously (1). E:T ratios 40: 1, 20: 1, and 10: 1 were used. The results of E:T ratio 40: 1 are shown but essentially similar results were obtained also with other E:T ratios. Culture conditions. RPM1 1640 (Microbiological Associates, Walkersville, MD), supplemented with 10% heat-inactivated fetal calf serum (Microbiological Associates), 0.29 mg/ml glutamine, and antibiotics, was used as a growth medium if not otherwise stated. Cells were grown and incubated at 37°C in 5% COZ humidified air. Test agents. Vinblastine sulfate (vb) (Eli Lilly, Indianapolis, IN) and demecolcine (dem) (Sigma, St. Louis, MO) were dissolved in PBS in stock concentrations 10 mg/ ml. Nocodazole (not) (Sigma) (stock 10 mg/ml) and tax01 (kindly provided by Dr. Danlos, NCI, Bethesda, MD) (stock 10 mg/ml, 12 mM) were dissolved in DMSO. Subsequent dilutions were made in the growth medium. DMSO did not affect the cytotoxicity within the dilutions used (<1:5000). NK cells were preincubated with the MT inhibitors for 60-90 min and the agents were present in the growth medium during the experiments. Prior to treatment with tax01 the cells were incubated on ice for 10 min to cause dissociation of the MT. The nontoxicity of the agents was confirmed by the trypan blue exclusion test, which showed over 98% viability of the cells
378
OLLI CARP6N
in presence of the drugs. Moreover, the “Cr release of the target cells in presence of MT inhibitors did not exceed 2% when compared to control target cells in a 4-hr assay. NKCFproduction. NKCF was produced by a modification of the method of Wright and Bonavida ( 13). Ficoll-Isopaque centrifuged, glass and nylon wool nonadherent PBL (3 X 106/ml) were incubated in 10% FCS-RPM1 1640 containing MT inhibitors for 90 min. The cells were centrifuged, the supernatants discharged, and the cells were diluted in 1% FCS-RPM1 1640 containing MT inhibitor and K562 target cells (0.3 X 106/ml) or Con A (Pharmacia) ( 1 &ml) as NKCF stimulator. After 6 hr the supcrnatants were collected and filtered through Millex-GS filters (Millipore S. A., Molsheim, France; pore size 0.22 pm). NKCF-containing supernatants were tested immediately for lytic capacity against 5’Cr-labeled U937 target cells in a 18-hr assay by adding 75 ~1 of NKCF-containing supematant to 75 ~1 of U937 cells (5OOO/well) in serum-free RPM1 1640. Spontaneous release of target cells in presence of MT inhibitors was not altered. Controls included also addition of MT inhibitors to control NKCF prior to cytotoxicity assay. Binding assay. The conjugation capability of NK cells was studied as described earlier ( 1). Immunofuorescence staining. LGL or LGL/K562 conjugates were allowed to attach on poly-L-lysine (Sigma)-coated coverslips or alternatively run onto cytocentrifuge slides (800 r-pm, 8 min). The cells were fixed in -20°C methanol (10 min) for visualization of MT or in 3.5% paraformaldehyde (+4’C) in phosphate buffer (pH 7.4) for visualization of microfilaments or the Golgi region. Microtubules were detected by monoclonal antibodies against (Y and fi-tubulin (Radiochemical Centre, Amersham, UK). The cells were first reacted with the antibody for 20 min, washed thoroughly, and reacted with fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG antiserum (Cappel Laboratories, Cochranville, PA). For visualization of actin, NBD-phallacidin (Molecular Probes, Junction City, OR) was used. For detection of Golgi-associated structures the fixed cells were permealized with 0.1% NP-40 for 10 min. Golgi region was subsequently visualized by tetramethylrhodamine (TRITC)-conjugated wheat germ agglutinin (WGA) or Helixpomatia agglutinin (HPA) (33,34). Transmission electron microscopy (EM). Effector cells treated with MT inhibitors for 60 min were conjugated with K562 target cells. A sample of the cell suspension was taken for indirect immunofluorescence (IIF) after 60 min to verify the effect of MT inhibitors and the rest of the cells were fixed in 2.5% glutaraldehyde in phosphate buffer for 60 min at room temperature. The cells were postfixed in 1% osmium tetroxide and embedded in Epon 7 12. Thin sections were poststained with uranyl acetate and lead citrate and were examined in a JEOL JEM- 1200 EX electron microscope. Statistical analysis. The statistical significance of the effect of MT inhibitors on NK cell functions was evaluated by paired t tests. RESULTS The e#ect of MT inhibitors on NK cell-mediated lysis and MT organization. Four MT inhibitors with different modes of action ((cf. (35)) were tested in a 4-hr “Crrelease assay. The microtubule-disrupting effect of the drugs was controlled simulta-
MICROTUBULES
IN NK CELLMEDIATED
KILLING
379
FIG. 1. The effect of MT inhibitors on the MT structural status. The cells were treated with the drugs for 60 min and stained with tubulin antibodies. (A) Fibrillar microtubules in nontreated cells. (B) Tubulinpositive aggregates in vinblastine sulfate (5 &ml)-treated cells. Diffuse tubulin positivity in both (C) demecolcine (5 &ml)- and(D) nocodazole (1 &ml)-treated cells. (E) Strongly tubulin-positive bundles in tax01 (2 &ml)-treated cells. Bar = 2 pm.
neously with the cytotoxicity testing, by immunofluorescence of the effector cells (Fig. 1). The effect of vinblastine sulfate was monitored by the disassembly of MT and formation of paracrystal inclusions visible in the NK cell cytoplasm (Fig. 1B). Demecolcine and nocodazole led to disappearance of MT in IIF (Figs. 1C and 1D). After the treatment MTOC were still visible within part of the cells. In NK cells treated with dem or not a prominent finding was often the appearance of an uropodlike extension within the cytoplasm (Figs. 1C and 1D). As a marker of tax01 effect, aberrant tubulin polymerization was visualized as formations of thick tubulin bundles (Fig. 1E). The MT-disrupting agents, at concentrations shown to be effective in MT disruption, caused only a minor, although significant, inhibition of the NK activity in the cytotoxicity assays as shown in Fig. 2. The effect of different concentrations of vb and not on cytotoxic activity in relation to the MT disassembly was also studied. Figure 3 shows that even concentrations IO-fold over that causing MT disruption brought about only a small inhibition of the cytotoxic activity of NK cells. The e&t of MT inhibitors on conjugateformation. Three agents were tested for their effect on effecter/target cell conjugate formation (Table 1). Not and tax01 did not affect the number of binding effector cells whereas vb treatment resulted in a consistent 25% diminution in the number of conjugating NK cells. NKCF production. NKCF production was induced by two alternative methods, one requiring cell-to-cell interaction with sensitive target cells (K562) and the other
380
OLLI CARPeN %OF
dem
5 /q/ml
(3)
me
2 MI/ml
(6)
tax01
CONTROL
CVTOTOXICITV
2 Pglml(9)
FIG. 2. The effect of MT inhibitors on NK-cell-mediated cytotoxicity. The cells were pretreated with the drugs for 60 min. K562 target cells were added in ET ratio 40: 1, and a 4-hr cytotoxicity assay was performed in presence of the drugs. Mean + SD. The number of experiments is in parenthesis. ** = P < 0.025, NS = not significant.
ligand-receptor binding (Con A). Effector cell stimulation by either method led to three- to fourfold increase in NKCF production in comparison to nonstimulated control cells (Table 2). Treatment of the effector cells with MT inhibitors caused in the average a 14% (range O-23%) decrease of Con A-induced NKCF activity and 24% (range lo-43%) decrease of K562-induced activity (Table 2). The inhibition of the NKCF production was not significant with any of the drugs tested. The eflect of MT inhibitors on microfilament distribution. We have earlier shown that NK cells polarize their actin-containing microfilaments toward the contact area of NK-sensitive target cells ( 1). Such actin redistribution is infrequently seen in conju-
-
+
+
+
“00
vb-
(+I
+
+
+
vb not
0.17 0.1
0.5 0.3
1.7 1
5 3
17 ,.
kwml
FIG. 3. The effect of various concentrations of vb and not on MT structural status and NK-cell-mediated cytotoxicity. The MT organization was studied by staining with tubulin antibodies, + = disruption of MT, (+) = partial disruption of MT, - = no visible effect on MT organ&&on.
MICROTUBULES
IN NK CELLMEDIATED
381
KILLING
TABLE 1 The Effect of MT Inhibitors on NK Cell Conjugate Formation” Percentage of binders among lymphocytesb 32.4k 24.0+ 32.4 + 30.2 +
Control Vb 5 &ml Not 1 &ml Tax01 2 &ml
8.8 6.5 11.3 11.8
Percentage inhibition 26* 0N.Y 7NS
’ NK cells were pretreated with the stated concentrations of the drugs for 60 min, conjugated with K562 target cells (E:T ratio 1: I), and the number of conjugating effector cells was enumerated. b Mean + SD of five experiments. ’ NS = not significant. * p < 0.05.
gates in which the targets are NK cell-resistant, indicating that the microfilament reorganization is associated with the cytotoxic mechanism. On the other hand, also microtubules have been shown to be reoriented during effector:target conjugation (3, 4,3 1). As the microtubule and microfilament systems are closely associated cytoskeletal components I wanted to see how the disruption of MT affected the microhlament organization in E:T conjugates. This was judged by staining of cellular hlamentous actin with NBD phallacidin. The experiments did not, however, show any visible differences in the microfilament distribution in MT-inhibitor-treated effector cells versus nontreated cells conjugating with K562 target cells (data not shown). The effect of MT inhibitors on Go& apparatus. Transmission EM was performed to study the effect of MT inhibitors on the ultrastructure of microtubules and the TABLE 2 The Effect of MT Inhibitors on NKCF Production’ Stimulator
Lysis of u937 cellsb
% Lysis without MT inhibitors
No stimulation Con A Vb5&ml+ConA Dem 5 &ml + Con A Not 1 &ml + Con A Tax01 2 &/ml + Con A
8.4? 26.0+ 21.3 + 27.3 f 19.9 + 21.0&
16.1 6.3 16.8 10.7 6.6 8.2
82 NS 105 NS 77 NS 81NS
K562 Vb 5 &ml + K562 Dem 5 pg/ml + K562 Not 1 &ml + K562 Tax01 2 &ml + K562
32.1 + 18.3+ 23.0+ 28.8 f 28.0+
18.4 9.1 7.0 14.1 5.2
57NS 72 NS 90 NS 87 NS
’ NKCF was produced and the activity was measured as indicated under Materials and Methods. Addition of MT inhibitors to control NKCF did not affect the NKCF-mediated lysis of target cells. b Mean -CSD of eight experiments.
382
OLLI CARPBN
FIG. 4. The effect of MT inhibitors on NK cell Golgi complex and cytoskeletal components. The cells were treated with the inhibitors for 2 hr. (A) Golgi apparatus in nontreated cells. (B) Note the enlarged Golgi cistemae in vinblastine sulfate (5 &m&treated cells. (C) Normal-looking Golgi apparatus in tax01 (2 &nl)-treated cells. (D) Microtubulacontaining paractystals (pc) in vbtreated cells. (E) Coiling of intermediate filaments after treatment with demecolcine (5 &ml). Bar = 0.5 pm. A, B, and C = same magnification.
Golgi complex. The dissociation of MT caused by dem and vb and the formation of tubulin-containing paracrystals in vb-treated cells could be verified by EM (Fig. 4). Also, the coiling of intermediate filaments that occurs parallel to MT disruption (35) was visible (Fig. 4). The inhibitors did not cause dissociation of the NK cell Golgi complex within 2 hr. The only notable effect was a slight enlargement of the Golgi cisternae after vb and dem treatment (Fig. 4). Tax01 did not cause any morphological alterations in the organization of the Golgi apparatus. Fluorescence microscopy did not either indicate any signs of dissociation of the NK cell Golgi apparatus within 4 hr of treatment with MT-disrupting agents (Fig. 5B). On the other hand, MT disruption led to dissociation of the Golgi complex in K562 target cells. This was noticed as diffusely distributed fluorescence of the Golgi elements in the treated cells in comparison to the compact juxtanuclear fluorescence in control cells (Fig. 5). Thus there seemed to be a difference in the requirement of MT in the maintenance of the organization of Golgi apparatus within the two cell types. Fluorescence microscopy further revealed that the Golgi complex of MTdeficient NK cells were normally polarized toward the contact area in NK/K562 conjugates (Fig. 5).
MICROTUBULES
IN NK CELL-MEDIATED
KILLING
383
FIG. 5. Normal polarization of the Golgi apparatus in a vb-treated NK cell conjugating with a K562 target cell. The effector cells were pretreated with MT inhibitors for 1 hr, K562 target cells were added, and the cell conjugates were incubated for additional 3 hr in presence of the drugs. Golgi apparatus were visualized with TRITC-HPA. The compact Golgi-specific fluorescence (arrows) at the cytoplasmic area facing the target cell is visible in both (A) nontrcated cells and (B) vb (5 &ml)-treated NK cells. Bar = 2 pm. The arrowhead shows the staining of the target cell Golgi region in (A). Note the lack of concentrated fluorescence in the target cell following the dissociation of Golgi elements cell after vb treatment.
DISCUSSION These experiments clearly show that MT are not essential for NK cell-mediated lysis. This was confirmed with four different agents that all inhibit MT function by various mechanisms. Vb binds reversibly to tubulin and induces tubulin subunits to aggregate into highly organized paracrystalhne structures discernible in IIF with tubulin antibodies and by transmission EM ((see (35)). Dem and not influence MT organization differently by elevating the critical concentration for the assembly of tubulin subunits leading to dissociation of the MT network (35). In part of the demand not-treated cells, centrioles, which are more resistant to the treatment than MT, were retained after the treatments but all cells had lost their fibrillary microtubule organization as judged by tubulin antibodies in IIF. In parallel to MT disruption the agents also caused coiling of the intermediate filaments visible in transmission EM which gives further evidence for their effect on LGL. Taxol, the fourth agent, is a microcyclic alkaloid that binds specifically and reversibly to polymerized tubulin. It lowers the critical concentration of MT leading to stabilization of existing MT and formation of aberrant MT networks (35). The unfunctional aberrant MT were visible with tubulin antibodies as brightly staining MT bundles. NK cells, when treated with MT inhibiting concentrations of each of these agents, could still continue to lyse the NK target cells at a level close to the nontreated control cells. Evidence from several sources have indicated that NK cells mediate their lytic activity via directed secretion of toxic material (6- 12). Two cellular products, NKCF ( 13, 15) secreted by NK cells (36), and granule cytolysin/perforin (14, 16, 17) obtained from granule preparations of cells with NK activity have been reported to cause dissociation of K562 target cells. The results of this study show that the release of NKCF from NK cells was only insignificantly diminished following MT disrup
384
OLLI CARF’kN
tion. It is possible that the measurement of NKCF activity does not reflect the all cytolytic capacity of NK cells, since additional or interacting molecules may participate in the target cell dusruption. Anyway, the results clearly show that human NK cells retain nearly normal capacity to release secretory products without functional MT and they thus provide evidence that MT would not seem to be universally required for secretion. Analogously, there are reports on T lymphocytes, as well as on other cell types, indicating that cells can continue both intracellular translocation of membrane proteins as well as secretory processes at normal level without functional MT (29,37-39). In a recent study virus-infected normal rat kidney cells were shown to be able to transport virus membrane proteins into the cell surface after disassembly of MT (38). The MT-defective cells, however, were reported to have lost the ability to direct properly the transported material following dispersion of Golgi elements (25). The result is, however, in contrast to a recent study on the biogenesis of epithelial cell surface polarity in which the disruption of neither MT nor microfilaments had any effect on the directed transport of cell surface proteins to distinct domains (40). Ultrastructural studies of the cytolytic action of NK cells have indicated that during conjugation with sensitive target cells the effector cell secretory apparatus is polarized to the cytoplasmic area facing the contact surface (1, 3-5), which allows directed secretion of lytic material (2). The occurrence of such a redistribution, which seems to be important in the cytolytic event, as well as the maintenance of the structural organization of the Golgi complex, has been thought to be regulated by the MT network (see (26)). Therefore, it was important to notice that in contrast to adherent cells the structural organization of NK cell Golgi apparatus was preserved after MT disassembly. The only visible alteration was a slight enlargement of Golgi cisternae in the treated cells. Even the redistribution of Golgi apparatus occurred normally in conjugating cells that lacked functional MT. On the other hand, similar treatment of K562 target cells led to dissociation of the Golgi compartments. Thus, these experiments suggest that there are differences in the regulation of the organization of the Golgi complex in different cell types. The MT inhibitors did not affect other steps of NK lysis than NKCF activity. The only exception was vb that consistently reduced the amount of target-binding cells by 25% in the binding assays. Similarly, NKCF production induced by target cell contact was inhibited 15-33% more by vb than with other MT inhibitors, whereas the inhibition of NKCF production after Con A stimulation was at the same level as with other inhibitors. The nature of the effect of vb on conjugation and contactinduced NKCF production is not known. It is, however, probably not caused directly by MT disassembly but may reflect a different mode of action on MT. In contrast to other used drugs vb leads to formation of large cytoplasmic tubulin-containing paracrystals that may sterically affect cell migration or cell membrane regulation and thus lead to decreased number of conjugating cells. Since our report on the mechanism of NK activity (6) three studies have been published dealing with the requirement of intact MT in NK cell activity. Based on experiments with colchicine (9), or colchicine, vb, and vincristine (30), it was reported that NK cell-mediated lysis is dependent on intact MT complex. In fact, however, in the experiments 10m4M (40 &ml) of colchicine and 10V5 M (9 &ml) of vb caused only a minor (30-35%) inhibition of lytic activity, which was somewhat higher than
MICROTUBULES
IN NK CELL-MEDIATED
KILLING
385
that seen in our experiments at similar concentrations of the drugs. The strong inhibition caused by high concentrations ( low3 M) of colchicine (9,30), which we have also observed (6), may be unrelated to MT disassembly as concentrations several hundred-fold less are sufficient to cause that but may rather reflect the capacity of the agent to interact with various membrane proteins (4 1). Kupfer and others (3) studied localization and requirements of MT in conjugating cloned murine NK cells. In their experiments a combination of not and taxol caused a reversible total inhibition of lytic activity. In similar experiments with fresh human LGL the used concentrations of the drugs caused only 20-30% inhibition of the cytotoxicity (not shown) which may reflect a different requirement for intact MT within the cell types as also discussed by Kupfel et al. (3). In conclusion, the present results show that fresh human NK cells are capable of lysing NK targets without functional MT. Microtubules seem, however, to play a minor supportive role in the cytotoxic action and in the production or secretion of NKCF. As NK cells with disassembled MT maintain their Golgi organization and are able to at least close to normal secretion in response to both ligand-receptor and cell-to-cell contact stimulus the results do not argue against the proposed directed stimulus-secretion model as the mechanism of NK activity. ACKNOWLEDGMENTS I thank Professor Eero Saksela and Dr. Ismo Virtanen for valuable advice and critical comments during the preparation of this manuscript. The excellent technical assistance of Mrs. Maja-Liisa Mantylii and Mrs. Raili Taavela and secretarial help of Ms. MarJa-Leena Rissanen is gratefully acknowledged. The study was supported by grants from the Finnish Cultural Foundation and the Finnish Medical Foundation.
REFERENCES 1. 2. 3. 4. 5. 6. I. 8. 9. 10. 11.
Carp&, O., Virtanen, I., Lehto, V. -P., and Saksela, E.. J. Zmmunol. 131,2695, 1983. Carp&, O., Virtanen, I., and Saksela, E., .I. Immunol. 128,2691, 1982. Kupfer, A., Dennert, G., and Singer, S., Proc. Natl. Acad. Sci. USA 80,7224,1983. Kupfer, A., Dennert, G., and Singer, S., J. Mol. Cell. Immunol. 2,31, 1985. Kupfer, A., Singer, S., and Dennert, G., J. Exp. Med. 163,489, 1986. Carp&, O., Virtanen, I., and Saksela, E., Cell. Immunol. 58,97, I98 1. Ledger, P. W., and Tanzer, M. L., TZBS9,3 15, 1984. Neighbour, P. A., and Huberman, H. S., J. Immunol. 128,1236, 1982. Quan, P. -C., Ishizaka, T., and Bloom, B. R., J. Immunol. 128,1786, 1982. Roder, J. C., and Klein, M., J. Immunol. 123,2785, 1979. Roder, J. C., Argov, S., Klein, M., Petersson, C., Kiessling, R., Andersson, K., and Hansson, M., Immunology40,107, 1980. 12. Verhoef, J., andshanna, S. D., J. Immunol. 131, 125, 1983. 13. Wright, S., and Bonavida, B., J. Immunol. 130,2960, 1983. 14. Blumenthal, R., Millard, P., Henkart, M., Reynolds, C., and He&art, P., Proc. Natl. Acad. Sci. USA 81,5551,1984.
15. 16. 17. 18. 19. 20. 2 I. 22.
Wright, S., and Bonavida, B., J. Immunol. 126, 15 16, 1981. Henkart, P., Millard, P., Reynolds, C., and He&art, M., J. Exp. Med. 160,75, 1984. Young, J. D. -E., Hengartner, H., Podack, E. R., and Cohn, Z. A., Cell 44,849, 1986. Lacy, P., Howell, S., Young, D., and Fink, C., Nature (London) 219, 1177, 1968. Moskalewski, S., Thyberg, J., Lohmander, S., and F&erg, U., Exp. Cell. Res. 95,440, 1975. Patzelt, C., Brown, D., and Jeanrenaud, B., J. Cell Biol. 73,578, 1977. Ehrlich, H., Ross, R., and Bon&in, P., J. Cell Biol. 62,390, 1979. Antoine, J. -C., Maurice, M., Feldmann, G., and Avrameas, S., J. Immunol. 125, 1939, 1980.
386
OLLI CARPEN
23. Bussou-Mabillot, S., Chaubaut-Gu&in, A. -M., Outracht, L., Muller, P., and Rossignol, B., J. Cell Biol. 95, 105, 1982. 24. Bennett, G., Parsons, S., and Carlet, E., Amer. J. Anat. 170,521, 1984. 25. Rogalski, A,, and Singer, S., J. CelI. Biol. 99, 1092, 1984. 26. Thyberg, J., and Moskalewski, S., Exp. Cell Rex 159, 1, 1985. 27. Strom, T., Garovoy, M., Carpenter, C., and Merrill, J., Science 181, 17 1, 1973. 28. Plaut, M., Lichstenstein, L., and Henney, C., J. Zmmunol. 111,77 1, 1973. 29. Henney, C., GaII’ney, J., and Bloom, B., .Z.Exp. Med. 140,837, 1974. 30. Katz, P., Zaytoun, A., and Lee, J., J. Zmmunol. 129,2816, 1982. 3 1. Geiger, B., Rosen, D., and Berke, G., J. Cell Biol. 95, 137, 1982. 32. Timonen, T., and Saksela, E., J. Zmmunol. Methods 36,285,1980. 33. Virtanen, I., Ekblom, P., and Laurila, P., .Z.Cell Biof. 85,429, 1980. 34. Kiiiiriiiinen, L., Virtanen, I., Saraste, J., and Keriinen, S., In “Methods in Enzymology” (S. Fleischer and B. Fleischer, Eds.), Vol. 96, p. 453. Academic Press, Orlando, FL, 1983. 35. DeBrabander, M., Geuens, G., Nuydens, R., Willebrods, R., and DeMeys, Cold Spring Harbor Symp. Quant. Biol. 46,227, 1982. 36. Ortaldo, J., Blanca, I., and Herberman, R., In “Mechanisms of Cytotoxicity by NK Cells” (R. Herberman and D. Callewaert, Eds.), pp. 335-350. Academic Press, Orlando, FL, 1985. 37. K%rklinen, L., Hashimoto, K., Saraste, J., Virtanen, I., and Penttinen, K., .Z.Cell Biol. 87,783, 1980. 38. Rogalski, A., Bergman, J., and Singer, S., J. Cell Biol. 99, 1101, 1984. 39. Virtanen, I., and Vartio, T., Eur. J. Cell Biol. 42,28 1, 1987. 40. Salas, P., Misek, D., VegaSalas, D., Gundersen, D., Cereijido, M., and Rodriquez-Boulan, E., J. Cell Biol. 102, 1853-1867, 1986. 4 1. Stadler, J., and Franke, W., J. Cell Biol. 60,297, 1974.
IMMUNOLOGY
106,376-386
(1987)
The Role of Microtubules Cell-Mediated
in Human Natural Killer Cytotoxicity
OLLI CARP&N Department ofPathology, University ofHelsinki, Haartmaninkatu
3, 00290 Helsinki, Finland
Received October 26, 1986; accepted January 14, 1987 The effect of four different microtubule (MT) inhibitors on the various stages of human natural killer (NK) cell-mediated cytotoxicity was studied. The MT-disrupting effect of the drugs was monitored by indirect immunofluorescence microscopy and transmission electron microscopy. All the drugs tested, vinblastine sulfate, demecolcine, nocodaxole, and taxol, had only a slight inhibitory effect on NK activity. Cells with nonfunctional MT were capable of normal conjugate formation and polarization of actin-containing microfdaments. Natural killer ccl1cytotoxic fao tor (NKCF) activity produced by cells with nonfunctional MT was slightly diminished. MT disruption caused enlargement of Golgi cistemae, but did not, however, dissociate the overall structural organization of the Golgi complex. The results indicate that fresh human NK cells are capable of lytic activity without functional MT although MT play a small supportive role in production or secretion of NKCF and mediation of the lytic activity. Previous experiments by us and others have strongly suggested that NK cells mediate their cytolytic activity by directed secretion of toxic material. As NK cells with unfunctional microtubules are capable of close to normal secretion the results presented in this report are not inconsistent with the earlier sugQ 1987 Academic PESS, 1,~. gested SthUlUS-SeCretiOn model.
INTRODUCTION Human natural killer (NK) cell activity is mediated by large granular lymphocytes (LGL) that have the ability to recognize their target cells without previous sensitization. Target cell recognition is followed by a set of events in the effector cell that finally result in the lysis of the target cell. The cytoskeletal organization of the NK cell alters upon conjugation with sensitive target cell: actin and vinculin become redistributed into focal contact-like areas (1). Concomitantly to the reorganization of actin microfilaments the cellular shape is changed and the Golgi apparatus becomes polarized toward the target cell (2-5). The experiments with monensin (6), which interrupts the vesicular traffic of Golgi-derived vesicles to cell membrane (7), as well as with other inhibitors of secretory processes (8-12), have strongly suggested that NK cells elicit their lytic activity by a directed secretion. The toxic material apparently binds to the target cell membrane ( 13, 14) and leads to disruption of the target cell. The cytolytic events are suggested to be mediated by natural killer cytotoxic factor (NKCF) (13, 15) or granule cytolysin/perforin (14, 16, 17) that both are produced by cells with NK activity and have the ability to disrupt NK-sensitive target cells. 376 0008-8749187 $3.00 Copyright Q 1987 by Academic Press, Inc. All rights of reproduction in any form -ed.
MICROTUBULES
IN NK CELL-MEDIATED
KILLING
377
Most earlier studies have suggested that intact microtubules (MT) are required in cellular secretory events (18-24), and furthermore, several reports indicate their importance in the maintenance of the organization ofGolgi apparatus (25) see also (26). Some authors have suggested a direct association of microtubules and microtubuleorganizing center (MTOC) in the cytotoxic action mediated by NK cells and T cells (3,9,27-3 1). Our earlier experiments suggested, however, that MT-disrupting agents had only a minor effect on NK cell activity (6). These data appeared to be contradictory to the stimulus-secretion model of NK cell-mediated cytotoxicity which would require an intact secretory apparatus to deliver the lethal hit. Since this important point has remained controversial I designed a series of experiments to further study the role of MT in cytolytic activity of human NK cells. The results suggest, in contrast to some other reports, that MT have only a minor supportive effect on NK activity, and that most of the lytic potential of NK cells is maintained upon dissociation of microtubules. The relevance of these findings to the stimulus-secretion model will be discussed. MATERIALS AND METHODS Efictor and target cells. BufIy coats were obtained from the Finnish Red Cross Blood Transfusion Service. They were purified as described in detail elsewhere (32). Briefly, mononuclear cells were isolated by the Ficoll-Isopaque gradient centrifugation. For removal of adherent cells, the mononuclear cells were incubated in Roux glass bottles, and the nonadherent fraction was run through nylon wool columns. The isolated cells were centrifuged in discontinuous Percoll (Pharmacia Fine Chemicals AB, UppsaIa, Sweden) gradients ranging from 47.5 (v/v) to 37.5% (v/v) with 2.5% diminution steps. The fraction layering at the 37.5% interphase consisted of 75 to 85% of large granular lymphocytes as judged morphologically from Giemsa-stained cytocentrifuge smears, and the NK activity in a 5‘Cr-cytotoxicity assay was also concentrated in this fraction as shown previously (32). K562, the continuous erythroleukemia cell line, was used as a source of NK-sensitive target cells and U937, a histiocytic lymphoma, as target cells in assays for NKCF activity. NK target cell lysis was measured with a standard “Cr-cytotoxicity assay as described previously (1). E:T ratios 40: 1, 20: 1, and 10: 1 were used. The results of E:T ratio 40: 1 are shown but essentially similar results were obtained also with other E:T ratios. Culture conditions. RPM1 1640 (Microbiological Associates, Walkersville, MD), supplemented with 10% heat-inactivated fetal calf serum (Microbiological Associates), 0.29 mg/ml glutamine, and antibiotics, was used as a growth medium if not otherwise stated. Cells were grown and incubated at 37°C in 5% COZ humidified air. Test agents. Vinblastine sulfate (vb) (Eli Lilly, Indianapolis, IN) and demecolcine (dem) (Sigma, St. Louis, MO) were dissolved in PBS in stock concentrations 10 mg/ ml. Nocodazole (not) (Sigma) (stock 10 mg/ml) and tax01 (kindly provided by Dr. Danlos, NCI, Bethesda, MD) (stock 10 mg/ml, 12 mM) were dissolved in DMSO. Subsequent dilutions were made in the growth medium. DMSO did not affect the cytotoxicity within the dilutions used (<1:5000). NK cells were preincubated with the MT inhibitors for 60-90 min and the agents were present in the growth medium during the experiments. Prior to treatment with tax01 the cells were incubated on ice for 10 min to cause dissociation of the MT. The nontoxicity of the agents was confirmed by the trypan blue exclusion test, which showed over 98% viability of the cells
378
OLLI CARP6N
in presence of the drugs. Moreover, the “Cr release of the target cells in presence of MT inhibitors did not exceed 2% when compared to control target cells in a 4-hr assay. NKCFproduction. NKCF was produced by a modification of the method of Wright and Bonavida ( 13). Ficoll-Isopaque centrifuged, glass and nylon wool nonadherent PBL (3 X 106/ml) were incubated in 10% FCS-RPM1 1640 containing MT inhibitors for 90 min. The cells were centrifuged, the supernatants discharged, and the cells were diluted in 1% FCS-RPM1 1640 containing MT inhibitor and K562 target cells (0.3 X 106/ml) or Con A (Pharmacia) ( 1 &ml) as NKCF stimulator. After 6 hr the supcrnatants were collected and filtered through Millex-GS filters (Millipore S. A., Molsheim, France; pore size 0.22 pm). NKCF-containing supernatants were tested immediately for lytic capacity against 5’Cr-labeled U937 target cells in a 18-hr assay by adding 75 ~1 of NKCF-containing supematant to 75 ~1 of U937 cells (5OOO/well) in serum-free RPM1 1640. Spontaneous release of target cells in presence of MT inhibitors was not altered. Controls included also addition of MT inhibitors to control NKCF prior to cytotoxicity assay. Binding assay. The conjugation capability of NK cells was studied as described earlier ( 1). Immunofuorescence staining. LGL or LGL/K562 conjugates were allowed to attach on poly-L-lysine (Sigma)-coated coverslips or alternatively run onto cytocentrifuge slides (800 r-pm, 8 min). The cells were fixed in -20°C methanol (10 min) for visualization of MT or in 3.5% paraformaldehyde (+4’C) in phosphate buffer (pH 7.4) for visualization of microfilaments or the Golgi region. Microtubules were detected by monoclonal antibodies against (Y and fi-tubulin (Radiochemical Centre, Amersham, UK). The cells were first reacted with the antibody for 20 min, washed thoroughly, and reacted with fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG antiserum (Cappel Laboratories, Cochranville, PA). For visualization of actin, NBD-phallacidin (Molecular Probes, Junction City, OR) was used. For detection of Golgi-associated structures the fixed cells were permealized with 0.1% NP-40 for 10 min. Golgi region was subsequently visualized by tetramethylrhodamine (TRITC)-conjugated wheat germ agglutinin (WGA) or Helixpomatia agglutinin (HPA) (33,34). Transmission electron microscopy (EM). Effector cells treated with MT inhibitors for 60 min were conjugated with K562 target cells. A sample of the cell suspension was taken for indirect immunofluorescence (IIF) after 60 min to verify the effect of MT inhibitors and the rest of the cells were fixed in 2.5% glutaraldehyde in phosphate buffer for 60 min at room temperature. The cells were postfixed in 1% osmium tetroxide and embedded in Epon 7 12. Thin sections were poststained with uranyl acetate and lead citrate and were examined in a JEOL JEM- 1200 EX electron microscope. Statistical analysis. The statistical significance of the effect of MT inhibitors on NK cell functions was evaluated by paired t tests. RESULTS The e#ect of MT inhibitors on NK cell-mediated lysis and MT organization. Four MT inhibitors with different modes of action ((cf. (35)) were tested in a 4-hr “Crrelease assay. The microtubule-disrupting effect of the drugs was controlled simulta-
MICROTUBULES
IN NK CELLMEDIATED
KILLING
379
FIG. 1. The effect of MT inhibitors on the MT structural status. The cells were treated with the drugs for 60 min and stained with tubulin antibodies. (A) Fibrillar microtubules in nontreated cells. (B) Tubulinpositive aggregates in vinblastine sulfate (5 &ml)-treated cells. Diffuse tubulin positivity in both (C) demecolcine (5 &ml)- and(D) nocodazole (1 &ml)-treated cells. (E) Strongly tubulin-positive bundles in tax01 (2 &ml)-treated cells. Bar = 2 pm.
neously with the cytotoxicity testing, by immunofluorescence of the effector cells (Fig. 1). The effect of vinblastine sulfate was monitored by the disassembly of MT and formation of paracrystal inclusions visible in the NK cell cytoplasm (Fig. 1B). Demecolcine and nocodazole led to disappearance of MT in IIF (Figs. 1C and 1D). After the treatment MTOC were still visible within part of the cells. In NK cells treated with dem or not a prominent finding was often the appearance of an uropodlike extension within the cytoplasm (Figs. 1C and 1D). As a marker of tax01 effect, aberrant tubulin polymerization was visualized as formations of thick tubulin bundles (Fig. 1E). The MT-disrupting agents, at concentrations shown to be effective in MT disruption, caused only a minor, although significant, inhibition of the NK activity in the cytotoxicity assays as shown in Fig. 2. The effect of different concentrations of vb and not on cytotoxic activity in relation to the MT disassembly was also studied. Figure 3 shows that even concentrations IO-fold over that causing MT disruption brought about only a small inhibition of the cytotoxic activity of NK cells. The e&t of MT inhibitors on conjugateformation. Three agents were tested for their effect on effecter/target cell conjugate formation (Table 1). Not and tax01 did not affect the number of binding effector cells whereas vb treatment resulted in a consistent 25% diminution in the number of conjugating NK cells. NKCF production. NKCF production was induced by two alternative methods, one requiring cell-to-cell interaction with sensitive target cells (K562) and the other
380
OLLI CARPeN %OF
dem
5 /q/ml
(3)
me
2 MI/ml
(6)
tax01
CONTROL
CVTOTOXICITV
2 Pglml(9)
FIG. 2. The effect of MT inhibitors on NK-cell-mediated cytotoxicity. The cells were pretreated with the drugs for 60 min. K562 target cells were added in ET ratio 40: 1, and a 4-hr cytotoxicity assay was performed in presence of the drugs. Mean + SD. The number of experiments is in parenthesis. ** = P < 0.025, NS = not significant.
ligand-receptor binding (Con A). Effector cell stimulation by either method led to three- to fourfold increase in NKCF production in comparison to nonstimulated control cells (Table 2). Treatment of the effector cells with MT inhibitors caused in the average a 14% (range O-23%) decrease of Con A-induced NKCF activity and 24% (range lo-43%) decrease of K562-induced activity (Table 2). The inhibition of the NKCF production was not significant with any of the drugs tested. The eflect of MT inhibitors on microfilament distribution. We have earlier shown that NK cells polarize their actin-containing microfilaments toward the contact area of NK-sensitive target cells ( 1). Such actin redistribution is infrequently seen in conju-
-
+
+
+
“00
vb-
(+I
+
+
+
vb not
0.17 0.1
0.5 0.3
1.7 1
5 3
17 ,.
kwml
FIG. 3. The effect of various concentrations of vb and not on MT structural status and NK-cell-mediated cytotoxicity. The MT organization was studied by staining with tubulin antibodies, + = disruption of MT, (+) = partial disruption of MT, - = no visible effect on MT organ&&on.
MICROTUBULES
IN NK CELLMEDIATED
381
KILLING
TABLE 1 The Effect of MT Inhibitors on NK Cell Conjugate Formation” Percentage of binders among lymphocytesb 32.4k 24.0+ 32.4 + 30.2 +
Control Vb 5 &ml Not 1 &ml Tax01 2 &ml
8.8 6.5 11.3 11.8
Percentage inhibition 26* 0N.Y 7NS
’ NK cells were pretreated with the stated concentrations of the drugs for 60 min, conjugated with K562 target cells (E:T ratio 1: I), and the number of conjugating effector cells was enumerated. b Mean + SD of five experiments. ’ NS = not significant. * p < 0.05.
gates in which the targets are NK cell-resistant, indicating that the microfilament reorganization is associated with the cytotoxic mechanism. On the other hand, also microtubules have been shown to be reoriented during effector:target conjugation (3, 4,3 1). As the microtubule and microfilament systems are closely associated cytoskeletal components I wanted to see how the disruption of MT affected the microhlament organization in E:T conjugates. This was judged by staining of cellular hlamentous actin with NBD phallacidin. The experiments did not, however, show any visible differences in the microfilament distribution in MT-inhibitor-treated effector cells versus nontreated cells conjugating with K562 target cells (data not shown). The effect of MT inhibitors on Go& apparatus. Transmission EM was performed to study the effect of MT inhibitors on the ultrastructure of microtubules and the TABLE 2 The Effect of MT Inhibitors on NKCF Production’ Stimulator
Lysis of u937 cellsb
% Lysis without MT inhibitors
No stimulation Con A Vb5&ml+ConA Dem 5 &ml + Con A Not 1 &ml + Con A Tax01 2 &/ml + Con A
8.4? 26.0+ 21.3 + 27.3 f 19.9 + 21.0&
16.1 6.3 16.8 10.7 6.6 8.2
82 NS 105 NS 77 NS 81NS
K562 Vb 5 &ml + K562 Dem 5 pg/ml + K562 Not 1 &ml + K562 Tax01 2 &ml + K562
32.1 + 18.3+ 23.0+ 28.8 f 28.0+
18.4 9.1 7.0 14.1 5.2
57NS 72 NS 90 NS 87 NS
’ NKCF was produced and the activity was measured as indicated under Materials and Methods. Addition of MT inhibitors to control NKCF did not affect the NKCF-mediated lysis of target cells. b Mean -CSD of eight experiments.
382
OLLI CARPBN
FIG. 4. The effect of MT inhibitors on NK cell Golgi complex and cytoskeletal components. The cells were treated with the inhibitors for 2 hr. (A) Golgi apparatus in nontreated cells. (B) Note the enlarged Golgi cistemae in vinblastine sulfate (5 &m&treated cells. (C) Normal-looking Golgi apparatus in tax01 (2 &nl)-treated cells. (D) Microtubulacontaining paractystals (pc) in vbtreated cells. (E) Coiling of intermediate filaments after treatment with demecolcine (5 &ml). Bar = 0.5 pm. A, B, and C = same magnification.
Golgi complex. The dissociation of MT caused by dem and vb and the formation of tubulin-containing paracrystals in vb-treated cells could be verified by EM (Fig. 4). Also, the coiling of intermediate filaments that occurs parallel to MT disruption (35) was visible (Fig. 4). The inhibitors did not cause dissociation of the NK cell Golgi complex within 2 hr. The only notable effect was a slight enlargement of the Golgi cisternae after vb and dem treatment (Fig. 4). Tax01 did not cause any morphological alterations in the organization of the Golgi apparatus. Fluorescence microscopy did not either indicate any signs of dissociation of the NK cell Golgi apparatus within 4 hr of treatment with MT-disrupting agents (Fig. 5B). On the other hand, MT disruption led to dissociation of the Golgi complex in K562 target cells. This was noticed as diffusely distributed fluorescence of the Golgi elements in the treated cells in comparison to the compact juxtanuclear fluorescence in control cells (Fig. 5). Thus there seemed to be a difference in the requirement of MT in the maintenance of the organization of Golgi apparatus within the two cell types. Fluorescence microscopy further revealed that the Golgi complex of MTdeficient NK cells were normally polarized toward the contact area in NK/K562 conjugates (Fig. 5).
MICROTUBULES
IN NK CELL-MEDIATED
KILLING
383
FIG. 5. Normal polarization of the Golgi apparatus in a vb-treated NK cell conjugating with a K562 target cell. The effector cells were pretreated with MT inhibitors for 1 hr, K562 target cells were added, and the cell conjugates were incubated for additional 3 hr in presence of the drugs. Golgi apparatus were visualized with TRITC-HPA. The compact Golgi-specific fluorescence (arrows) at the cytoplasmic area facing the target cell is visible in both (A) nontrcated cells and (B) vb (5 &ml)-treated NK cells. Bar = 2 pm. The arrowhead shows the staining of the target cell Golgi region in (A). Note the lack of concentrated fluorescence in the target cell following the dissociation of Golgi elements cell after vb treatment.
DISCUSSION These experiments clearly show that MT are not essential for NK cell-mediated lysis. This was confirmed with four different agents that all inhibit MT function by various mechanisms. Vb binds reversibly to tubulin and induces tubulin subunits to aggregate into highly organized paracrystalhne structures discernible in IIF with tubulin antibodies and by transmission EM ((see (35)). Dem and not influence MT organization differently by elevating the critical concentration for the assembly of tubulin subunits leading to dissociation of the MT network (35). In part of the demand not-treated cells, centrioles, which are more resistant to the treatment than MT, were retained after the treatments but all cells had lost their fibrillary microtubule organization as judged by tubulin antibodies in IIF. In parallel to MT disruption the agents also caused coiling of the intermediate filaments visible in transmission EM which gives further evidence for their effect on LGL. Taxol, the fourth agent, is a microcyclic alkaloid that binds specifically and reversibly to polymerized tubulin. It lowers the critical concentration of MT leading to stabilization of existing MT and formation of aberrant MT networks (35). The unfunctional aberrant MT were visible with tubulin antibodies as brightly staining MT bundles. NK cells, when treated with MT inhibiting concentrations of each of these agents, could still continue to lyse the NK target cells at a level close to the nontreated control cells. Evidence from several sources have indicated that NK cells mediate their lytic activity via directed secretion of toxic material (6- 12). Two cellular products, NKCF ( 13, 15) secreted by NK cells (36), and granule cytolysin/perforin (14, 16, 17) obtained from granule preparations of cells with NK activity have been reported to cause dissociation of K562 target cells. The results of this study show that the release of NKCF from NK cells was only insignificantly diminished following MT disrup
384
OLLI CARF’kN
tion. It is possible that the measurement of NKCF activity does not reflect the all cytolytic capacity of NK cells, since additional or interacting molecules may participate in the target cell dusruption. Anyway, the results clearly show that human NK cells retain nearly normal capacity to release secretory products without functional MT and they thus provide evidence that MT would not seem to be universally required for secretion. Analogously, there are reports on T lymphocytes, as well as on other cell types, indicating that cells can continue both intracellular translocation of membrane proteins as well as secretory processes at normal level without functional MT (29,37-39). In a recent study virus-infected normal rat kidney cells were shown to be able to transport virus membrane proteins into the cell surface after disassembly of MT (38). The MT-defective cells, however, were reported to have lost the ability to direct properly the transported material following dispersion of Golgi elements (25). The result is, however, in contrast to a recent study on the biogenesis of epithelial cell surface polarity in which the disruption of neither MT nor microfilaments had any effect on the directed transport of cell surface proteins to distinct domains (40). Ultrastructural studies of the cytolytic action of NK cells have indicated that during conjugation with sensitive target cells the effector cell secretory apparatus is polarized to the cytoplasmic area facing the contact surface (1, 3-5), which allows directed secretion of lytic material (2). The occurrence of such a redistribution, which seems to be important in the cytolytic event, as well as the maintenance of the structural organization of the Golgi complex, has been thought to be regulated by the MT network (see (26)). Therefore, it was important to notice that in contrast to adherent cells the structural organization of NK cell Golgi apparatus was preserved after MT disassembly. The only visible alteration was a slight enlargement of Golgi cisternae in the treated cells. Even the redistribution of Golgi apparatus occurred normally in conjugating cells that lacked functional MT. On the other hand, similar treatment of K562 target cells led to dissociation of the Golgi compartments. Thus, these experiments suggest that there are differences in the regulation of the organization of the Golgi complex in different cell types. The MT inhibitors did not affect other steps of NK lysis than NKCF activity. The only exception was vb that consistently reduced the amount of target-binding cells by 25% in the binding assays. Similarly, NKCF production induced by target cell contact was inhibited 15-33% more by vb than with other MT inhibitors, whereas the inhibition of NKCF production after Con A stimulation was at the same level as with other inhibitors. The nature of the effect of vb on conjugation and contactinduced NKCF production is not known. It is, however, probably not caused directly by MT disassembly but may reflect a different mode of action on MT. In contrast to other used drugs vb leads to formation of large cytoplasmic tubulin-containing paracrystals that may sterically affect cell migration or cell membrane regulation and thus lead to decreased number of conjugating cells. Since our report on the mechanism of NK activity (6) three studies have been published dealing with the requirement of intact MT in NK cell activity. Based on experiments with colchicine (9), or colchicine, vb, and vincristine (30), it was reported that NK cell-mediated lysis is dependent on intact MT complex. In fact, however, in the experiments 10m4M (40 &ml) of colchicine and 10V5 M (9 &ml) of vb caused only a minor (30-35%) inhibition of lytic activity, which was somewhat higher than
MICROTUBULES
IN NK CELL-MEDIATED
KILLING
385
that seen in our experiments at similar concentrations of the drugs. The strong inhibition caused by high concentrations ( low3 M) of colchicine (9,30), which we have also observed (6), may be unrelated to MT disassembly as concentrations several hundred-fold less are sufficient to cause that but may rather reflect the capacity of the agent to interact with various membrane proteins (4 1). Kupfer and others (3) studied localization and requirements of MT in conjugating cloned murine NK cells. In their experiments a combination of not and taxol caused a reversible total inhibition of lytic activity. In similar experiments with fresh human LGL the used concentrations of the drugs caused only 20-30% inhibition of the cytotoxicity (not shown) which may reflect a different requirement for intact MT within the cell types as also discussed by Kupfel et al. (3). In conclusion, the present results show that fresh human NK cells are capable of lysing NK targets without functional MT. Microtubules seem, however, to play a minor supportive role in the cytotoxic action and in the production or secretion of NKCF. As NK cells with disassembled MT maintain their Golgi organization and are able to at least close to normal secretion in response to both ligand-receptor and cell-to-cell contact stimulus the results do not argue against the proposed directed stimulus-secretion model as the mechanism of NK activity. ACKNOWLEDGMENTS I thank Professor Eero Saksela and Dr. Ismo Virtanen for valuable advice and critical comments during the preparation of this manuscript. The excellent technical assistance of Mrs. Maja-Liisa Mantylii and Mrs. Raili Taavela and secretarial help of Ms. MarJa-Leena Rissanen is gratefully acknowledged. The study was supported by grants from the Finnish Cultural Foundation and the Finnish Medical Foundation.
REFERENCES 1. 2. 3. 4. 5. 6. I. 8. 9. 10. 11.
Carp&, O., Virtanen, I., Lehto, V. -P., and Saksela, E.. J. Zmmunol. 131,2695, 1983. Carp&, O., Virtanen, I., and Saksela, E., .I. Immunol. 128,2691, 1982. Kupfer, A., Dennert, G., and Singer, S., Proc. Natl. Acad. Sci. USA 80,7224,1983. Kupfer, A., Dennert, G., and Singer, S., J. Mol. Cell. Immunol. 2,31, 1985. Kupfer, A., Singer, S., and Dennert, G., J. Exp. Med. 163,489, 1986. Carp&, O., Virtanen, I., and Saksela, E., Cell. Immunol. 58,97, I98 1. Ledger, P. W., and Tanzer, M. L., TZBS9,3 15, 1984. Neighbour, P. A., and Huberman, H. S., J. Immunol. 128,1236, 1982. Quan, P. -C., Ishizaka, T., and Bloom, B. R., J. Immunol. 128,1786, 1982. Roder, J. C., and Klein, M., J. Immunol. 123,2785, 1979. Roder, J. C., Argov, S., Klein, M., Petersson, C., Kiessling, R., Andersson, K., and Hansson, M., Immunology40,107, 1980. 12. Verhoef, J., andshanna, S. D., J. Immunol. 131, 125, 1983. 13. Wright, S., and Bonavida, B., J. Immunol. 130,2960, 1983. 14. Blumenthal, R., Millard, P., Henkart, M., Reynolds, C., and He&art, P., Proc. Natl. Acad. Sci. USA 81,5551,1984.
15. 16. 17. 18. 19. 20. 2 I. 22.
Wright, S., and Bonavida, B., J. Immunol. 126, 15 16, 1981. Henkart, P., Millard, P., Reynolds, C., and He&art, M., J. Exp. Med. 160,75, 1984. Young, J. D. -E., Hengartner, H., Podack, E. R., and Cohn, Z. A., Cell 44,849, 1986. Lacy, P., Howell, S., Young, D., and Fink, C., Nature (London) 219, 1177, 1968. Moskalewski, S., Thyberg, J., Lohmander, S., and F&erg, U., Exp. Cell. Res. 95,440, 1975. Patzelt, C., Brown, D., and Jeanrenaud, B., J. Cell Biol. 73,578, 1977. Ehrlich, H., Ross, R., and Bon&in, P., J. Cell Biol. 62,390, 1979. Antoine, J. -C., Maurice, M., Feldmann, G., and Avrameas, S., J. Immunol. 125, 1939, 1980.
386
OLLI CARPEN
23. Bussou-Mabillot, S., Chaubaut-Gu&in, A. -M., Outracht, L., Muller, P., and Rossignol, B., J. Cell Biol. 95, 105, 1982. 24. Bennett, G., Parsons, S., and Carlet, E., Amer. J. Anat. 170,521, 1984. 25. Rogalski, A,, and Singer, S., J. CelI. Biol. 99, 1092, 1984. 26. Thyberg, J., and Moskalewski, S., Exp. Cell Rex 159, 1, 1985. 27. Strom, T., Garovoy, M., Carpenter, C., and Merrill, J., Science 181, 17 1, 1973. 28. Plaut, M., Lichstenstein, L., and Henney, C., J. Zmmunol. 111,77 1, 1973. 29. Henney, C., GaII’ney, J., and Bloom, B., .Z.Exp. Med. 140,837, 1974. 30. Katz, P., Zaytoun, A., and Lee, J., J. Zmmunol. 129,2816, 1982. 3 1. Geiger, B., Rosen, D., and Berke, G., J. Cell Biol. 95, 137, 1982. 32. Timonen, T., and Saksela, E., J. Zmmunol. Methods 36,285,1980. 33. Virtanen, I., Ekblom, P., and Laurila, P., .Z.Cell Biof. 85,429, 1980. 34. Kiiiiriiiinen, L., Virtanen, I., Saraste, J., and Keriinen, S., In “Methods in Enzymology” (S. Fleischer and B. Fleischer, Eds.), Vol. 96, p. 453. Academic Press, Orlando, FL, 1983. 35. DeBrabander, M., Geuens, G., Nuydens, R., Willebrods, R., and DeMeys, Cold Spring Harbor Symp. Quant. Biol. 46,227, 1982. 36. Ortaldo, J., Blanca, I., and Herberman, R., In “Mechanisms of Cytotoxicity by NK Cells” (R. Herberman and D. Callewaert, Eds.), pp. 335-350. Academic Press, Orlando, FL, 1985. 37. K%rklinen, L., Hashimoto, K., Saraste, J., Virtanen, I., and Penttinen, K., .Z.Cell Biol. 87,783, 1980. 38. Rogalski, A., Bergman, J., and Singer, S., J. Cell Biol. 99, 1101, 1984. 39. Virtanen, I., and Vartio, T., Eur. J. Cell Biol. 42,28 1, 1987. 40. Salas, P., Misek, D., VegaSalas, D., Gundersen, D., Cereijido, M., and Rodriquez-Boulan, E., J. Cell Biol. 102, 1853-1867, 1986. 4 1. Stadler, J., and Franke, W., J. Cell Biol. 60,297, 1974.