- Email: [email protected]
Susceptibility of human ocular melanoma cells to spontaneous and interferon-augmented natural cytotoxicity
Cancer Letters, 18 (1983) 205-213 Elsevier Scientific Publishers Ireland Ltd.
205
SUSCEPTIBILITY OF HUMAN OCULAR MELANOMA CELLS TO SPONTANEOUS AND INTERFERON-AUGMENTED NATURAL CYTOTOXICITY
ROBERT C. REESa, IAN G. RENNIEb , ANDREW PLATTSa and LIZA S. CAWOOD’ and ‘Dermatology, The University aDepartmenta of Virology, bOphthalmoIogy Sheffield Medical School, Beech Hill Road, Sheffield SlO 2RX (U.K.)
of
(Received 6 October 1982) (Accepted 18 October 1982)
SUMMARY
Due to the current interest in natural killer (NK) cells as a host defence mechanism against neoplasia, we have investigated the susceptibility of shortterm cultures of human ocular melanoma cells to spontaneous and interferon (IFN)-augmented natural cytotoxicity. Cultures of ocular melanoma cells were readily established and identified as bipolar and multipolar pigmented melanoma cells. In short-term cytotoxicity assays these cell lines demonstrated a weak susceptibility to spontaneous human PBL natural cytotoxicity, and increased killing was observed using IFN-augmented cytotoxic effector cells. These findings may be pertinent in relation to the role of NK cells in vivo.
INTRODUCTION
The supposition that the behaviour of uveal malignant melanomas may be modified by immune mechanisms is supported by the clinical observations of spontaneous regression [ 11,281 and delayed appearance of metastases after the removal of the primary tumour [30]. In attempts to elucidate the nature of this immune response, both humoral and cellular mechanisms have been studied [3,5,6,9,10,13,15,20,24-271. There is evidence to suggest that circulating antibodies to both the surface membranes and cytoplasm of these tumour cells exist [3,8-10,25,27,31] and in vitro and in vivo cellular responses to putative melanoma antigens have been observed [6,16]. It is now apparent that other host cell-mediated defence mechanisms may be important in restricting tumour growth and preventing the establishment of metastases. Of current interest is the role of NK cells in surveillance against neoplasia [ 14,151. In order to postulate a universal role for NK cells in combating malignant disease, it is important to establish whether or not 03043835/83/0000-+30001$03.00 o 1983 Elsevier Scientific Publishers Ireland Ltd. - Published and Printed in Ireland
human tumour cells are sensitive to these effector cells. In the present study, the susceptibility of ocular melanoma targets established in tissue culture, to spontaneous and interferon-activated human NK effector cells was investigated. MATERIALS
AND METHODS
Patients and prepam tion of tumour Tumour samples were obtained from the enucleated eyes of 6 patients. The clinical and histological details are presented in Table 1. In all cases the tumour was located in the choroid. No evidence of metastases was present in any of the patients at the time of enucleation. Immediately after enucleation the globe was opened, the tumour identified and the overlying retina removed. Samples of the tumour were removed, digested with trypsin and suspended in RPM1 1640 medium, supplemented with 20% foetal calf serum, glutamine, penicillin and streptomycin. The cultures were incubated at 37°C and passaged when confluent. All cultures were screened for mycoplasma contamination at the time of assay for susceptibility to natural cytotoxicity. The remainder of the globe and tumour was fixed in 4% formal saline and processed into paraffin wax for histological examination. Preparation of effector cells and nylon wool column fractionation Heparinised peripheral blood samples were obtained from normal healthy individuals. Lymphocyte rich mononuclear cells (effector cells) were isolated by centrifugation of heparinised peripheral blood on lymphocyte separation medium [ 11. Human peripheral blood leucocytes collecting at the interface were harvested and washed 3 times in RPM1 1640 medium supplemented with 10% newborn calf serum (RPMI-CS). Human PBL were fractionated on nylon wool columns as previously described [ 2,161.
TABLE 1 DETAILS OF PATIENTS AND OCULAR MELANOMA CELL CULTURES Patient 1 2 3 4 5 6b
Age 88 73 70 56 .59 67
Sex
Cell typea
F F F M F M
Spindle B cell Mixed cell Mixed cell Mixed cell Spindle B cell Mixed Cell
‘Based on Callender [ 41 classification. bMycoplasma hyorhinis cultivated from cell cultures of patient 6. All other cell cultures were mycoplasma free.
207
Target cells The leukaemic cell line-K562 [ 211, was obtained from Dr. A. Morris (Department of Biological Sciences, The University of Warwick, U.K.). This cell line was cultured in RPMI-CS, and was shown to be free from detectable mycoplasma contamination [ 21 (by direct culture procedures performed at the Mycoplasma Reference Laboratory, Norwich, U.K. by Dr. R. Leach). Interferon and treatment of effector ceils Human freeze-dried lymphoblastoid (Namalva) interferon (HIFN) was kindly provided by Drs. Fantes and Johnson (Wellcome Research Laboratories) and was shown to have a specific activity of approximately 6 X lo5 HIFN units per mg of protein. The HIFN was aliquoted and stored at -80°C at a concentration of 2000 units HIFN per ml in RPMI-CS medium. Nylon wool column non-adherent human PBL effector cells were incubated with or without the addition of HIFN for 18 h at 37°C. One-millilitre cultures containing 5 X 10” effector cells (in RPMI-CS) were incubated in sterile plastic bijoux bottles in a humidified 95% air/Z% COz atmosphere. One hundred units of HIFN (0.1 ml vol.) were added to half the cultures, and an equivalent volume of RPMI-CS added to control cultures. Following overnight incubation at 37”C, HIFN treated and untreated effector cells were washed 3 times in medium and adjusted to the required concentration. Four-hour chromium-51 release assay Target cells (in 0.2 ml RPMI-CS) were labelled with 100 ICi of sodium chromate (Naz5’Cr0,) (Amersham International, Amersham, U.K.). Labelling was performed at 37°C for 1 h, after which the cells were washed 3 times in RPMI-CS and further incubated for 1 h at 37°C. The cells were rewashed 3 times in medium, counted and resuspended at a concentration of 1 X 10’ cells per ml in fresh RPMI-CS medium. All tests were performed in triplicate in round-bottomed Falcon microtest 111 plates (Falcon 3911, Becton Dickinson and Company). One X lo4 target cells (0.1 ml vol.), and effector cells at ratios of 100: 1 to 12 : 1 (in 0.1 ml vol.) were added to microtest plate wells. The cultures were incubated at 37°C (5% CO*) for 4 h, the plates spun at 200 X g for 5 min to sediment the cells, and 0.1 ml of supernatant was removed into separate wells. The plates were dried and sealed with parafilm and the individual wells assessed for ‘lCr content in a gamma spectrophotometer. The percentage chromium-51 release was determined by the following formula: (i SN) X 2 YGslC!rrelease
=
(‘, SN) + (l SN + cells)
where SN = supernatant.
Xl00
The results were expressed
as the percentage
cyto-
208
toxicity
calculated
% cytotoxicity
by the foll =
(test release)
.ila: - (spontaneous
100 - (spontaneous
release)
xlOO
release)
The spontaneous release from target cells in the presence usually within the range 3-10s.
of media alone was
RESULTS
Cell cultures were established from 6 human ocular melanomas as described above and maintained in vitro for up to 205 days before assay for susceptibility to human PBL natural cytotoxicity. In all cases the cultures consisted predominantly of bipolar and multipolar pigmented, melanoma cells. In case 6 (Table 1) a moderate contamination of cultures by fibroblasts was noted, and case 3 demonstrated an extremely low growth pattern, although the cells remained viable. Case 6 was the only cell culture where mycoplasma contamination was detected (Table 1); all other cultures were shown to be free of detectable mycoplasma. Contamination with some strains of mycoplasma has been shown to increase target cell susceptibility to NK cells [ 21, and is an important consideration when assessing target cell sensitivity to natural cytotoxicity. The susceptibility of short term in vitro cultures of ocular melanoma cells to natural cytotoxicity was determined using nylon wool non-adherent human PBL effector cells. In all experiments the NK-sensitive K562 leukaemic cell line was used as an indicator of NK cell activity. In the majority of tests performed, both spontaneous and IFNaugmented cytotoxicity was determined against ocular melanoma and K562 targets. The results of these tests are shown in Table 2. Significant spontaneous NK cell activity (P < 0.001) was demonstrated towards the K562 target cells in all tests performed, and effector cells exposed to human IFN always showed a significant increase in cytotoxicity towards K562 cells (P < 0.001). Using ocular melanoma cells as targets, low levels of spontaneous killing were observed against 2/6 cell cultures tested, although where a cell line proved susceptible to natural killing, not all passage levels showed the same degree of sensitivity. Melanoma cell line 1 when used as a primary culture proved to be insensitive to human PBL effector cells (Table 2, Expt. l), however, in a subsequent test after first passage (PI) (Table 2, Expt. 2) significant natural killing was observed (50 : 1 E/T ratio). This activity was slightly elevated using PBL effecters pre-treated with human IFN. Line 2 target cells were also found to be insensitive to spontaneous natural killing as primary, Pl and P2 targets, but significant cytotoxicity was observed in 3 out of 4 experiments using IFN-treated effector (IFN-E) cells and Pl-2 targets (Table 2, Expts. 3-7). Sensitivity to IFN-E cells appeared to be greater at passage 2, where a significant augmented of killing above the spontaneous value was
1
2
2 2 2 2
3 4
5
6
2
3
4 5 6 7
8 9
10
11
50 : 1 25:l 12:l Pl-14 5O:l 25:l 12 :l Primary-l 0 50 :1 25:l 12:l Pl-14 5O:l Pl-14 5O:l P2-23 5O:l P2-23 50 :l 25:l 12:l Primary-190 30 : 1 Pl-29 5O:l 25:l P2-205’ 100 :l 5O:l 25:l P2-112 1OO:l
Primary-10
Melanoma E/T ratioa Pass no.Days cultured
2.8 9.4d 5.8’ 0 0 0 2.4
5.2’ 3.4 NT 0 0 0 0.8 0.5 0.8 NT
0 0 0
Spontaneous
2.3’ 0.3 4.3d 10.5e 4.3c 3.0 9.3e 17.7e 13.6‘? 0 0 1.9 5.2e
8.0d 6.6d 7.9C NT
NT
IFN-boosted
% Cytotoxicityb melanoma targets
NS 0.05
-
0.01 0.05 0.05
-
0.01 -
0.005 -
NS NS
51.8e 17.6e 6.3’ 35.7e 30.3e 20.1e 35.7e
Spontaneous
% CytoxicityK562 targets
OCULAR MELANOMA
Statistical evaluation of IFN-boosting (P< )
TOWARDS
71.3e 76.8e 73e 79.5e 71e 55.2e 78.1e 68.3e 53Ze 58 .2e 45.6e 28.9= 58.2e
71e 52.2e NT
79.5e
NT
or absence
0.001 0.001 0.001 0.001 0.001 0.001 0.001
-
0.001 0.001 0.001 -
-
0.001 0.001 0.001
-
Statistical evaluation of IFN-boosting (PC)
CELLS
IFN-boosted
TARGET
aE/T = effectorltarget cell ratio. b4-h S’Cr-release assay using nylon wool non-adherent human PBL incubated at 37°C in the presence (IFN-boosted) (spontargus) of 100 units/ml of HIFN. Cl” : o.oJ; PC 0. Oi, eF i 0.001 by Student’s r-test.
1
1
Expt . Patient no.
SPONTANEOUS AND IFN-BOOSTED NK CELL CYTOTOXICITY
TABLE 2
210 TABLE 3 SUMMARY OF SPONTANEOUS AND IFN-BOOSTED NATURAL CYTOTOXICITY TOWARDS CELLS FROM SHORT-TERM OCULAR’MELANOMA CELL LINES Cell passage
No. cell lines demonstrating susceptibility toa PBL-effector
cells
IFN-boosted
Primary Pl-2
O/3 213
I/I 516
Total tests performedb
2/10
712
PBL effector cells
aTest where significant (P = <0.05-
observed (Table 2, Expt. 6, P < 0.01). Similarly, using target cell line 3 and PBL effecters at a 30 : 1, E/T ratio, susceptibility to killing was restricted to IFN-boosted effecters, and represented a signficant increase in killing above the spontaneous value (P < 0.01) (Table 2, Expt. 8). Line 4 target cells at 50 : 1 and 25 : 1 E/T ratios, were insensitive to PBL effector cells (P < 0.05-0.01) (Table 2, Expt. 9), but showed increased sensitivity to cytolysis by IFN-E cells compared with spontaneous PBL killing (P < 0.05-0.01). Melanoma cell lines 5 and 6 were insensitive to spontaneous PBL effector cells, although line 6 proved to be susceptible to killing by PBL effector cells pretreated with human IFN (Table 2, Expt. 10 and 11). These findings are summarised in Table 3, showing that in the majority of tests performed (7/9) IFN-E cells were cytotoxic towards ocular melanoma targets, contrasting with the low level of spontaneous natural cytotoxicity observed (2/10 positive tests). DISCUSSION
The host immune defence mechanisms may play a critical role in preventing tumour metastases by the destruction of cells released from the primary tumour before they develop in distal organs, although the precise immune mechanism responsible for preventing metastases has not been clearly defined. Lymphocytes mediating natural cell-mediated cytotoxicity are thought to play a prominent role, and it is therefore important to determine the susceptibility of human tumour cells to this effector mechanism. There are relatively few reports on natural killing of human solid tumour target cells, due in the main to the failure to obtain suitable fresh, or cultured target cells from biopsy tissue. DeLandazuri et al. [ 71 have shown that NK cell cytotoxicity towards leukaemic and solid tumour target cells is mediated by a discrete sub-population of lymphocytes, now defined morphologically as being large granular lymphocytes (LGL). In another recent study, human
2’11
ovarian carcinoma cells were found to be relatively resistant to spontaneous NK cells although IFN-treated NK effecters were cytotoxic against these targets [ 221. The above findings [2] are similar to those shown here using ocular melanoma cells as targets for natural cytotoxicity. In most tests, ocular melanoma cells were relatively insensitive to spontaneous natural cytotoxicity, but an increase in cytolysis of melanoma targets was observed using effector cells pre-treated with human a-interferon. This agrees with the augmentation of NK cell activity reported against leukaemic/lymphoma target cells [ 8,18,23] suggesting an important role for IFN in the immunoregulation of NK activity. This observation may have significance when considering the potential use of IFN in the treatment of malignant disease. It has been shown that IFN administration in man causes elevated NK cell activity [ 181, and experimentally NK cells appear to be associated with inhibition of tumour metastases [ 11,121. In view of these findings it is relevant to address the question of susceptibility of human tumour targets to spontaneous and HIFN-treated effecters of natural cytotoxicity . Although only a limited number of human ocular melanoma targets were studied for susceptibility to natural killing, the findings may be relevant in the further assessment of the potential significance of natural cell-mediated cytotoxicity in tumour surveillance, and the use of interferon in modulating antitumour immune mechanisms. ACKNOWLEDGEMENTS
The authors thank Messrs. A.J. Dark, H.C. Gupta, J.F. Talbot and A. Zaidi for their assistance in obtaining the pathological material; and Dr. S.S. Bleehan for his help and encouragement. The work was supported by grants from the British National Committee for the Prevention of Blindness and the Yorkshire Cancer Research Campaign. Also thanks to Mrs. Christine Mullan and Miss Tracey Johnson for typing the manuscript. REFERENCES Boyum, A. (1968) Isolation of mononuclear cells and granulocytes from human blood. Stand. J. Clin. Lab. Invest. (Suppl.) 21,77. Brooks, C.G., Rees, R.C. and Leach, R.H. (1979) High non-specific reactivity of normal lymphocytes against mycoplasma-infected target cells in cytotoxicity assays. Eur. J. Immunol., 9,159-165. Brownstein, S., Sheikli, K.M. and Lewis, M.G. (19’77) Immunological studies in patients with malignant melanoma of the uvea. Can. J. Ophthalmol., 12, 16-23. Callender, G.R. (1931) Malignant melanotic tumours of the eye. A study of histologic types in 111 cases. Trans. Am. Acad. Ophthalmol. Otolaryngol., 36, 131-142. Char, D.H., Hollinshead, A., Gogan, D.G., Ballintine, E.J., Hogan, M.J.and Herberman, R .B, (1974) Cutaneous delayed hypersensitivity reactions to soluble melanoma antigen in patients with ocular malignant melanoma. N. Engl. J. Med., 291, 274-277.
212 6 Char, D.M., Jerome,
7
8
9
10
11
12
13 14 15 16 17
18 19 20 21 22
23 24 25 26 27
L., McCoy, J.L. and Herberman, R.B. (1975) Cell-mediated immunity to melanoma-associated antigens in patients with ocular malignant melanoma. Am. J. Ophthalmol., 79,812~816. DeLandazuri, M.O., Lopez-Botet, M., Timonen,T., Ortaldo, J.R. and Herberman, R.B. (1981) Human larger granular lymphocytes: spontaneous and interferon-boosted NK activity against adherent and non-adherent tumour cell lines. J. Immunol., 127, 1380-1383. Einhorn, S., Blomgren, H. and Strander, H. (1980) Interferon and spontaneous cytotoxicity in patients receiving human leucocyte interferon. Int. J. Cancer, 26, 419-428. Federman, J.L., Lewis, M.G. and Clark, W.H. (1974) Tumour-associated antibodies to ocular and cutaneous malignant melanomas: negative interaction with normal choroidal melanocytes. J. Natl. Cancer Inst., 52,587-589. Federman, J.L., Lewis, M.G., Clark, W.H., Egerer, I. and Sorin, L.K. (1974) Tumourassociated antibodies in the serum of ocular melanoma patients. Trans. Am. Acad. Ophthalmol. Otolaryngol., 78, 784-794. Hanna, N. (1980) Expression of metastatic potential of tumour cells in young nude mice is correlated with low levels of natural killer cell-mediated cytotoxicity. Int. J. Cancer, 26, 675-680. Hanna, N. and Fidler, I.J. (1981) Relationship between metastatic potential and resistance to natural killer cell-mediated cytotoxicity in three murine tumour systems J. Natl. Cancer Inst., 66,1183-1190. Hart, D.R.L., Reznitkov, M. and Hughes, L.E. (1968) Cellular resistance to human uveal melanoma. Arch. Ophthalmol., 79,748-754. Herberman, R.B. and Holden, H. (1978) Natural cell-mediated immunity. Adv. Cancer Res., 27,305-377. Howard, G.M. and Spalter, H.F. (1966) Study of autoimmune serologic reactions to ocular melanoma. Arch. Ophthalmol., 76,399-402. Jenson, O.A. and Anderson, S.R. (1974) Spontaneous regression of a malignant melanoma of the choroid. Ophthalmologica, 52,173-182. Julius, M.H., Simpson, E. and Herzenburg, L.A. (1973) A rapid method for the isolation of functional thymus-derived murine lymphocytes. Eur. J. Immunol., 3, 645-649. Karinieni, A.L., Timonen, T. and Kousa, M. (1980) Effect of leucocyte interferon on natural killer cells in healthy volunteers. Stand. J. Immunol., 12, 371-394. Kiessling, R. and Wigzell, H. (1979) An analysis of murine NK cells as to structure function and biological significance. Immunol. Res., 44, 165-208. Lang, J.R., Davidorf, F.H. and Baba, W. (1977) The prognostic significance of lymphocytic infiltration in malignant melanoma of the choroid. Cancer, 40, 2388-2394. Lozzio, C.B. and Lozzio, B.B. (1975) Human chronic myelogenous leukaemia cell line with positive Philadelphia chromsosome. Blood, 45,321-334. Mantovani, A., Allavena, P., Sessa, C., Bolis, G. and Mangioni, C. (1980) Natural killer activity of lymphoid cells isolated from human ascites ovarian tumours. Int. J. Cancer, 25, 573-582. Moore, M. and Potter, M.R. (1980) Enhancement of human natural cell-mediated cytotoxicity by interferon. Br. J. Cancer, 41, 378-387. Paliafito, C.A., Wiikes, M. and Albert, D.M. (1980) Direct identification of T cells in malignant melanoma of the choroid. Int. Ophthalmol. Clin., 20,93-102. Rahi, A.M.S. (1971) Autoimmune reactions in uveal melanoma. Br. J. Ophthalmol., 55,743-807. Rahi, A.H.S. and Agrawal, P.K. (1977) Prognostic parameters in choroidal melanomata. Trans. Ophthslmol. Sot. U.K., 97, 368-372. Rahi, A.H. S., Garner, A. and Mulaty, A.H.A. (1978) Contractible protein antigens in the cells of malignant melanoma of the choroid and their diagnostic significance. Br. J. Ophthalmol., 62,392-401.
213 28 Reese, A.B., Archila, E.A., Jones, IS. and Cooper, W.C. (1965) Necrosis of malignant melanoma of the choroid. Am. J. Ophthalmol., 69,91-104. 29 Rees, R.C., Bray, J., Robins, R.A. and Baldwin, R.W. (1975) Subpopulations of multiparous rat lymph node cells cytotoxic for rat tumour cells and capable of suppressing cytotoxicity in vitro. Int. J. Cancer, 15, 762-772. 30 Wilbur, B.L. and Hartman, M.R. (1931) Malignant melanoma with delayed metastatic growth. Ann. Intern. Med., 5, 201-211. 31 Wong, I.G. and Ostevig, R.M. (1974) Immunofluorescent detection of antibodies to ocular melanomas. Arch. Ophthalmol., 92, 98-102.
205
SUSCEPTIBILITY OF HUMAN OCULAR MELANOMA CELLS TO SPONTANEOUS AND INTERFERON-AUGMENTED NATURAL CYTOTOXICITY
ROBERT C. REESa, IAN G. RENNIEb , ANDREW PLATTSa and LIZA S. CAWOOD’ and ‘Dermatology, The University aDepartmenta of Virology, bOphthalmoIogy Sheffield Medical School, Beech Hill Road, Sheffield SlO 2RX (U.K.)
of
(Received 6 October 1982) (Accepted 18 October 1982)
SUMMARY
Due to the current interest in natural killer (NK) cells as a host defence mechanism against neoplasia, we have investigated the susceptibility of shortterm cultures of human ocular melanoma cells to spontaneous and interferon (IFN)-augmented natural cytotoxicity. Cultures of ocular melanoma cells were readily established and identified as bipolar and multipolar pigmented melanoma cells. In short-term cytotoxicity assays these cell lines demonstrated a weak susceptibility to spontaneous human PBL natural cytotoxicity, and increased killing was observed using IFN-augmented cytotoxic effector cells. These findings may be pertinent in relation to the role of NK cells in vivo.
INTRODUCTION
The supposition that the behaviour of uveal malignant melanomas may be modified by immune mechanisms is supported by the clinical observations of spontaneous regression [ 11,281 and delayed appearance of metastases after the removal of the primary tumour [30]. In attempts to elucidate the nature of this immune response, both humoral and cellular mechanisms have been studied [3,5,6,9,10,13,15,20,24-271. There is evidence to suggest that circulating antibodies to both the surface membranes and cytoplasm of these tumour cells exist [3,8-10,25,27,31] and in vitro and in vivo cellular responses to putative melanoma antigens have been observed [6,16]. It is now apparent that other host cell-mediated defence mechanisms may be important in restricting tumour growth and preventing the establishment of metastases. Of current interest is the role of NK cells in surveillance against neoplasia [ 14,151. In order to postulate a universal role for NK cells in combating malignant disease, it is important to establish whether or not 03043835/83/0000-+30001$03.00 o 1983 Elsevier Scientific Publishers Ireland Ltd. - Published and Printed in Ireland
human tumour cells are sensitive to these effector cells. In the present study, the susceptibility of ocular melanoma targets established in tissue culture, to spontaneous and interferon-activated human NK effector cells was investigated. MATERIALS
AND METHODS
Patients and prepam tion of tumour Tumour samples were obtained from the enucleated eyes of 6 patients. The clinical and histological details are presented in Table 1. In all cases the tumour was located in the choroid. No evidence of metastases was present in any of the patients at the time of enucleation. Immediately after enucleation the globe was opened, the tumour identified and the overlying retina removed. Samples of the tumour were removed, digested with trypsin and suspended in RPM1 1640 medium, supplemented with 20% foetal calf serum, glutamine, penicillin and streptomycin. The cultures were incubated at 37°C and passaged when confluent. All cultures were screened for mycoplasma contamination at the time of assay for susceptibility to natural cytotoxicity. The remainder of the globe and tumour was fixed in 4% formal saline and processed into paraffin wax for histological examination. Preparation of effector cells and nylon wool column fractionation Heparinised peripheral blood samples were obtained from normal healthy individuals. Lymphocyte rich mononuclear cells (effector cells) were isolated by centrifugation of heparinised peripheral blood on lymphocyte separation medium [ 11. Human peripheral blood leucocytes collecting at the interface were harvested and washed 3 times in RPM1 1640 medium supplemented with 10% newborn calf serum (RPMI-CS). Human PBL were fractionated on nylon wool columns as previously described [ 2,161.
TABLE 1 DETAILS OF PATIENTS AND OCULAR MELANOMA CELL CULTURES Patient 1 2 3 4 5 6b
Age 88 73 70 56 .59 67
Sex
Cell typea
F F F M F M
Spindle B cell Mixed cell Mixed cell Mixed cell Spindle B cell Mixed Cell
‘Based on Callender [ 41 classification. bMycoplasma hyorhinis cultivated from cell cultures of patient 6. All other cell cultures were mycoplasma free.
207
Target cells The leukaemic cell line-K562 [ 211, was obtained from Dr. A. Morris (Department of Biological Sciences, The University of Warwick, U.K.). This cell line was cultured in RPMI-CS, and was shown to be free from detectable mycoplasma contamination [ 21 (by direct culture procedures performed at the Mycoplasma Reference Laboratory, Norwich, U.K. by Dr. R. Leach). Interferon and treatment of effector ceils Human freeze-dried lymphoblastoid (Namalva) interferon (HIFN) was kindly provided by Drs. Fantes and Johnson (Wellcome Research Laboratories) and was shown to have a specific activity of approximately 6 X lo5 HIFN units per mg of protein. The HIFN was aliquoted and stored at -80°C at a concentration of 2000 units HIFN per ml in RPMI-CS medium. Nylon wool column non-adherent human PBL effector cells were incubated with or without the addition of HIFN for 18 h at 37°C. One-millilitre cultures containing 5 X 10” effector cells (in RPMI-CS) were incubated in sterile plastic bijoux bottles in a humidified 95% air/Z% COz atmosphere. One hundred units of HIFN (0.1 ml vol.) were added to half the cultures, and an equivalent volume of RPMI-CS added to control cultures. Following overnight incubation at 37”C, HIFN treated and untreated effector cells were washed 3 times in medium and adjusted to the required concentration. Four-hour chromium-51 release assay Target cells (in 0.2 ml RPMI-CS) were labelled with 100 ICi of sodium chromate (Naz5’Cr0,) (Amersham International, Amersham, U.K.). Labelling was performed at 37°C for 1 h, after which the cells were washed 3 times in RPMI-CS and further incubated for 1 h at 37°C. The cells were rewashed 3 times in medium, counted and resuspended at a concentration of 1 X 10’ cells per ml in fresh RPMI-CS medium. All tests were performed in triplicate in round-bottomed Falcon microtest 111 plates (Falcon 3911, Becton Dickinson and Company). One X lo4 target cells (0.1 ml vol.), and effector cells at ratios of 100: 1 to 12 : 1 (in 0.1 ml vol.) were added to microtest plate wells. The cultures were incubated at 37°C (5% CO*) for 4 h, the plates spun at 200 X g for 5 min to sediment the cells, and 0.1 ml of supernatant was removed into separate wells. The plates were dried and sealed with parafilm and the individual wells assessed for ‘lCr content in a gamma spectrophotometer. The percentage chromium-51 release was determined by the following formula: (i SN) X 2 YGslC!rrelease
=
(‘, SN) + (l SN + cells)
where SN = supernatant.
Xl00
The results were expressed
as the percentage
cyto-
208
toxicity
calculated
% cytotoxicity
by the foll =
(test release)
.ila: - (spontaneous
100 - (spontaneous
release)
xlOO
release)
The spontaneous release from target cells in the presence usually within the range 3-10s.
of media alone was
RESULTS
Cell cultures were established from 6 human ocular melanomas as described above and maintained in vitro for up to 205 days before assay for susceptibility to human PBL natural cytotoxicity. In all cases the cultures consisted predominantly of bipolar and multipolar pigmented, melanoma cells. In case 6 (Table 1) a moderate contamination of cultures by fibroblasts was noted, and case 3 demonstrated an extremely low growth pattern, although the cells remained viable. Case 6 was the only cell culture where mycoplasma contamination was detected (Table 1); all other cultures were shown to be free of detectable mycoplasma. Contamination with some strains of mycoplasma has been shown to increase target cell susceptibility to NK cells [ 21, and is an important consideration when assessing target cell sensitivity to natural cytotoxicity. The susceptibility of short term in vitro cultures of ocular melanoma cells to natural cytotoxicity was determined using nylon wool non-adherent human PBL effector cells. In all experiments the NK-sensitive K562 leukaemic cell line was used as an indicator of NK cell activity. In the majority of tests performed, both spontaneous and IFNaugmented cytotoxicity was determined against ocular melanoma and K562 targets. The results of these tests are shown in Table 2. Significant spontaneous NK cell activity (P < 0.001) was demonstrated towards the K562 target cells in all tests performed, and effector cells exposed to human IFN always showed a significant increase in cytotoxicity towards K562 cells (P < 0.001). Using ocular melanoma cells as targets, low levels of spontaneous killing were observed against 2/6 cell cultures tested, although where a cell line proved susceptible to natural killing, not all passage levels showed the same degree of sensitivity. Melanoma cell line 1 when used as a primary culture proved to be insensitive to human PBL effector cells (Table 2, Expt. l), however, in a subsequent test after first passage (PI) (Table 2, Expt. 2) significant natural killing was observed (50 : 1 E/T ratio). This activity was slightly elevated using PBL effecters pre-treated with human IFN. Line 2 target cells were also found to be insensitive to spontaneous natural killing as primary, Pl and P2 targets, but significant cytotoxicity was observed in 3 out of 4 experiments using IFN-treated effector (IFN-E) cells and Pl-2 targets (Table 2, Expts. 3-7). Sensitivity to IFN-E cells appeared to be greater at passage 2, where a significant augmented of killing above the spontaneous value was
1
2
2 2 2 2
3 4
5
6
2
3
4 5 6 7
8 9
10
11
50 : 1 25:l 12:l Pl-14 5O:l 25:l 12 :l Primary-l 0 50 :1 25:l 12:l Pl-14 5O:l Pl-14 5O:l P2-23 5O:l P2-23 50 :l 25:l 12:l Primary-190 30 : 1 Pl-29 5O:l 25:l P2-205’ 100 :l 5O:l 25:l P2-112 1OO:l
Primary-10
Melanoma E/T ratioa Pass no.Days cultured
2.8 9.4d 5.8’ 0 0 0 2.4
5.2’ 3.4 NT 0 0 0 0.8 0.5 0.8 NT
0 0 0
Spontaneous
2.3’ 0.3 4.3d 10.5e 4.3c 3.0 9.3e 17.7e 13.6‘? 0 0 1.9 5.2e
8.0d 6.6d 7.9C NT
NT
IFN-boosted
% Cytotoxicityb melanoma targets
NS 0.05
-
0.01 0.05 0.05
-
0.01 -
0.005 -
NS NS
51.8e 17.6e 6.3’ 35.7e 30.3e 20.1e 35.7e
Spontaneous
% CytoxicityK562 targets
OCULAR MELANOMA
Statistical evaluation of IFN-boosting (P< )
TOWARDS
71.3e 76.8e 73e 79.5e 71e 55.2e 78.1e 68.3e 53Ze 58 .2e 45.6e 28.9= 58.2e
71e 52.2e NT
79.5e
NT
or absence
0.001 0.001 0.001 0.001 0.001 0.001 0.001
-
0.001 0.001 0.001 -
-
0.001 0.001 0.001
-
Statistical evaluation of IFN-boosting (PC)
CELLS
IFN-boosted
TARGET
aE/T = effectorltarget cell ratio. b4-h S’Cr-release assay using nylon wool non-adherent human PBL incubated at 37°C in the presence (IFN-boosted) (spontargus) of 100 units/ml of HIFN. Cl” : o.oJ; PC 0. Oi, eF i 0.001 by Student’s r-test.
1
1
Expt . Patient no.
SPONTANEOUS AND IFN-BOOSTED NK CELL CYTOTOXICITY
TABLE 2
210 TABLE 3 SUMMARY OF SPONTANEOUS AND IFN-BOOSTED NATURAL CYTOTOXICITY TOWARDS CELLS FROM SHORT-TERM OCULAR’MELANOMA CELL LINES Cell passage
No. cell lines demonstrating susceptibility toa PBL-effector
cells
IFN-boosted
Primary Pl-2
O/3 213
I/I 516
Total tests performedb
2/10
712
PBL effector cells
aTest where significant (P = <0.05-
observed (Table 2, Expt. 6, P < 0.01). Similarly, using target cell line 3 and PBL effecters at a 30 : 1, E/T ratio, susceptibility to killing was restricted to IFN-boosted effecters, and represented a signficant increase in killing above the spontaneous value (P < 0.01) (Table 2, Expt. 8). Line 4 target cells at 50 : 1 and 25 : 1 E/T ratios, were insensitive to PBL effector cells (P < 0.05-0.01) (Table 2, Expt. 9), but showed increased sensitivity to cytolysis by IFN-E cells compared with spontaneous PBL killing (P < 0.05-0.01). Melanoma cell lines 5 and 6 were insensitive to spontaneous PBL effector cells, although line 6 proved to be susceptible to killing by PBL effector cells pretreated with human IFN (Table 2, Expt. 10 and 11). These findings are summarised in Table 3, showing that in the majority of tests performed (7/9) IFN-E cells were cytotoxic towards ocular melanoma targets, contrasting with the low level of spontaneous natural cytotoxicity observed (2/10 positive tests). DISCUSSION
The host immune defence mechanisms may play a critical role in preventing tumour metastases by the destruction of cells released from the primary tumour before they develop in distal organs, although the precise immune mechanism responsible for preventing metastases has not been clearly defined. Lymphocytes mediating natural cell-mediated cytotoxicity are thought to play a prominent role, and it is therefore important to determine the susceptibility of human tumour cells to this effector mechanism. There are relatively few reports on natural killing of human solid tumour target cells, due in the main to the failure to obtain suitable fresh, or cultured target cells from biopsy tissue. DeLandazuri et al. [ 71 have shown that NK cell cytotoxicity towards leukaemic and solid tumour target cells is mediated by a discrete sub-population of lymphocytes, now defined morphologically as being large granular lymphocytes (LGL). In another recent study, human
2’11
ovarian carcinoma cells were found to be relatively resistant to spontaneous NK cells although IFN-treated NK effecters were cytotoxic against these targets [ 221. The above findings [2] are similar to those shown here using ocular melanoma cells as targets for natural cytotoxicity. In most tests, ocular melanoma cells were relatively insensitive to spontaneous natural cytotoxicity, but an increase in cytolysis of melanoma targets was observed using effector cells pre-treated with human a-interferon. This agrees with the augmentation of NK cell activity reported against leukaemic/lymphoma target cells [ 8,18,23] suggesting an important role for IFN in the immunoregulation of NK activity. This observation may have significance when considering the potential use of IFN in the treatment of malignant disease. It has been shown that IFN administration in man causes elevated NK cell activity [ 181, and experimentally NK cells appear to be associated with inhibition of tumour metastases [ 11,121. In view of these findings it is relevant to address the question of susceptibility of human tumour targets to spontaneous and HIFN-treated effecters of natural cytotoxicity . Although only a limited number of human ocular melanoma targets were studied for susceptibility to natural killing, the findings may be relevant in the further assessment of the potential significance of natural cell-mediated cytotoxicity in tumour surveillance, and the use of interferon in modulating antitumour immune mechanisms. ACKNOWLEDGEMENTS
The authors thank Messrs. A.J. Dark, H.C. Gupta, J.F. Talbot and A. Zaidi for their assistance in obtaining the pathological material; and Dr. S.S. Bleehan for his help and encouragement. The work was supported by grants from the British National Committee for the Prevention of Blindness and the Yorkshire Cancer Research Campaign. Also thanks to Mrs. Christine Mullan and Miss Tracey Johnson for typing the manuscript. REFERENCES Boyum, A. (1968) Isolation of mononuclear cells and granulocytes from human blood. Stand. J. Clin. Lab. Invest. (Suppl.) 21,77. Brooks, C.G., Rees, R.C. and Leach, R.H. (1979) High non-specific reactivity of normal lymphocytes against mycoplasma-infected target cells in cytotoxicity assays. Eur. J. Immunol., 9,159-165. Brownstein, S., Sheikli, K.M. and Lewis, M.G. (19’77) Immunological studies in patients with malignant melanoma of the uvea. Can. J. Ophthalmol., 12, 16-23. Callender, G.R. (1931) Malignant melanotic tumours of the eye. A study of histologic types in 111 cases. Trans. Am. Acad. Ophthalmol. Otolaryngol., 36, 131-142. Char, D.H., Hollinshead, A., Gogan, D.G., Ballintine, E.J., Hogan, M.J.and Herberman, R .B, (1974) Cutaneous delayed hypersensitivity reactions to soluble melanoma antigen in patients with ocular malignant melanoma. N. Engl. J. Med., 291, 274-277.
212 6 Char, D.M., Jerome,
7
8
9
10
11
12
13 14 15 16 17
18 19 20 21 22
23 24 25 26 27
L., McCoy, J.L. and Herberman, R.B. (1975) Cell-mediated immunity to melanoma-associated antigens in patients with ocular malignant melanoma. Am. J. Ophthalmol., 79,812~816. DeLandazuri, M.O., Lopez-Botet, M., Timonen,T., Ortaldo, J.R. and Herberman, R.B. (1981) Human larger granular lymphocytes: spontaneous and interferon-boosted NK activity against adherent and non-adherent tumour cell lines. J. Immunol., 127, 1380-1383. Einhorn, S., Blomgren, H. and Strander, H. (1980) Interferon and spontaneous cytotoxicity in patients receiving human leucocyte interferon. Int. J. Cancer, 26, 419-428. Federman, J.L., Lewis, M.G. and Clark, W.H. (1974) Tumour-associated antibodies to ocular and cutaneous malignant melanomas: negative interaction with normal choroidal melanocytes. J. Natl. Cancer Inst., 52,587-589. Federman, J.L., Lewis, M.G., Clark, W.H., Egerer, I. and Sorin, L.K. (1974) Tumourassociated antibodies in the serum of ocular melanoma patients. Trans. Am. Acad. Ophthalmol. Otolaryngol., 78, 784-794. Hanna, N. (1980) Expression of metastatic potential of tumour cells in young nude mice is correlated with low levels of natural killer cell-mediated cytotoxicity. Int. J. Cancer, 26, 675-680. Hanna, N. and Fidler, I.J. (1981) Relationship between metastatic potential and resistance to natural killer cell-mediated cytotoxicity in three murine tumour systems J. Natl. Cancer Inst., 66,1183-1190. Hart, D.R.L., Reznitkov, M. and Hughes, L.E. (1968) Cellular resistance to human uveal melanoma. Arch. Ophthalmol., 79,748-754. Herberman, R.B. and Holden, H. (1978) Natural cell-mediated immunity. Adv. Cancer Res., 27,305-377. Howard, G.M. and Spalter, H.F. (1966) Study of autoimmune serologic reactions to ocular melanoma. Arch. Ophthalmol., 76,399-402. Jenson, O.A. and Anderson, S.R. (1974) Spontaneous regression of a malignant melanoma of the choroid. Ophthalmologica, 52,173-182. Julius, M.H., Simpson, E. and Herzenburg, L.A. (1973) A rapid method for the isolation of functional thymus-derived murine lymphocytes. Eur. J. Immunol., 3, 645-649. Karinieni, A.L., Timonen, T. and Kousa, M. (1980) Effect of leucocyte interferon on natural killer cells in healthy volunteers. Stand. J. Immunol., 12, 371-394. Kiessling, R. and Wigzell, H. (1979) An analysis of murine NK cells as to structure function and biological significance. Immunol. Res., 44, 165-208. Lang, J.R., Davidorf, F.H. and Baba, W. (1977) The prognostic significance of lymphocytic infiltration in malignant melanoma of the choroid. Cancer, 40, 2388-2394. Lozzio, C.B. and Lozzio, B.B. (1975) Human chronic myelogenous leukaemia cell line with positive Philadelphia chromsosome. Blood, 45,321-334. Mantovani, A., Allavena, P., Sessa, C., Bolis, G. and Mangioni, C. (1980) Natural killer activity of lymphoid cells isolated from human ascites ovarian tumours. Int. J. Cancer, 25, 573-582. Moore, M. and Potter, M.R. (1980) Enhancement of human natural cell-mediated cytotoxicity by interferon. Br. J. Cancer, 41, 378-387. Paliafito, C.A., Wiikes, M. and Albert, D.M. (1980) Direct identification of T cells in malignant melanoma of the choroid. Int. Ophthalmol. Clin., 20,93-102. Rahi, A.M.S. (1971) Autoimmune reactions in uveal melanoma. Br. J. Ophthalmol., 55,743-807. Rahi, A.H.S. and Agrawal, P.K. (1977) Prognostic parameters in choroidal melanomata. Trans. Ophthslmol. Sot. U.K., 97, 368-372. Rahi, A.H. S., Garner, A. and Mulaty, A.H.A. (1978) Contractible protein antigens in the cells of malignant melanoma of the choroid and their diagnostic significance. Br. J. Ophthalmol., 62,392-401.
213 28 Reese, A.B., Archila, E.A., Jones, IS. and Cooper, W.C. (1965) Necrosis of malignant melanoma of the choroid. Am. J. Ophthalmol., 69,91-104. 29 Rees, R.C., Bray, J., Robins, R.A. and Baldwin, R.W. (1975) Subpopulations of multiparous rat lymph node cells cytotoxic for rat tumour cells and capable of suppressing cytotoxicity in vitro. Int. J. Cancer, 15, 762-772. 30 Wilbur, B.L. and Hartman, M.R. (1931) Malignant melanoma with delayed metastatic growth. Ann. Intern. Med., 5, 201-211. 31 Wong, I.G. and Ostevig, R.M. (1974) Immunofluorescent detection of antibodies to ocular melanomas. Arch. Ophthalmol., 92, 98-102.