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Production of high levels of interleukin 1-like activity by the SPI-802 human leukemia cell line with E receptors
CELLULAR
IMMUNOLOGY
89, 122- 13I ( 1984)
Production of High Levels of lnterleukin l-Like Activity by the SPI-802 Human Leukemia Cell Line with E Receptors ATSUSHIKOMIYAMA, YUKIAKI MIYAGAWA, KOHKI AOYAMA, TARO AKABANE,AND YOSHIOUEHARA Department of Pediatrics, Shinshu University School of Medicine, Asahi 3-I-1, Matsumoto, 390, and Division of Immunochemistry, Suzaka Prefectural Hospital, Suzaka, 382, Japan Received November I, 1983; accepted June 29, 1984 The SPI-802 human leukemia cell line, which possessesE receptors and used to have natural killer activity, has been demonstrated to produce high levels of interleukin 1 (IL-1)-like activity. SPI-802 supematants prepared in 1% serum-containing cultures with lipopolysaccharide stimulation, like similarly prepared adherent-cell-derived IL-l, enhanced phytohemagglutinininduced mouse thymocyte proliferation. When adherent-cell IL-1 gave 50% maximum activity at a reciprocal dilution of 20, SPI-802 supematant gave it at 200, indicating the production of high levels of &l-like activity by the cell line. SPI-802 supematant promoted the production of interleukin 2 (IL-2) by the Jurkat-F1884 T-cell line: Levels of IL-2 activity obtained with 15% SPI-802 supematant were almost equivalent to those obtained with 50% adherent-cell IL-l as estimated by the maximum proliferation of IL-2dependent cytotoxic T cells. SPI-802 supematant by itself exhibited no IL-2 activity. Major IL-l-like activity of SPI-802 supematant was present in fractions from AcA54 columns corresponding to M, 12,000-20,000 and 60,00070,000 and resolved on isoelectrofocusing into two distinct species with pI values of 5.0 and 7.0, being consistent with the results of adherent-cell IL-l. The SPI-802 cell line having E receptors is an ideal source of a soluble factor with the biological and biochemical characteristics of human IL- 1. o 1984 Academic pres* hc.
INTRODUCTION In recent years much attention has been focused on interleukin l(IL-l), which plays a central role in the acquisition and expression of immune responsiveness. Most studies of IL-l have been done using the factor obtained in cultures of adherent mononuclear cells (l-8). In humans, however, the source of IL-l is not satisfactory for its detailed characterization because of the difficulty of obtaining large quantities of purified IL-l from the source. Many investigators have thus been searching for ideal sources of IL-1 (1, 9-16). There is a mouse macrophage cell line (P388Dr) which can releaseimmunostimulatory activity similar to mouse monocytederived IL-l (1, 9, 10). Recently several human cell lines capable of producing IL-l have been reported (12-16). These cell lines, however, produce low levels of IL-l activity (12-16). Only the THP-1, a monocytic leukemia cell line, can produce levels of IL-l activity comparable to those obtainable from peripheral blood monocytes (14). In this article, we report that our SPI-802 human leukemia cell line, which possessesE receptors and used to have natural killer (NK) activity (17), can produce high levels of IL-l-like activity. The SPI-802 cell line is distinct in 122 0008-8749184$3.00 Copyright 8 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
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some property from the monocyte-related human cell lines with IL1 producing ability such as the U937, CM-S, and THP-1 cell lines (12-14) and provides further information concerning cellular characteristics of IL- 1-producing cells. MATERIALS
AND METHODS
Characterization of SPI-802 cells. SPI-802 cells were studied between 22 and 36 months after their culture initiation. During the period, their cytological and functional characteristics were reexamined. Some of the cells were cultured in the presence of 100 rig/ml phorbol myristate acetate (PMA; Sigma, St. Louis, MO.) for 48 hr and processed for the following studies. Morphology, cytochemistry, surface markers, chromosomes, and NK activity of SPI-802 cells were studied as previously described (17). For surface marker studies, monoclonal anti-Leu-5 and anti-Leu-7 (HNK- 1, Becton-Dickenson, Sunnyvale, Calif.), monoclonal OKM 1 (Ortho Pharmaceutical Co., Raritan, N.J.), and monoclonal anti-human monocyte (Bethesda Research Lab., Gaithersburg, Md.) antibodies were commercially obtained. Anti-Ia serum was raised in rabbits (17). To estimate phagocytic ability of SPI-802 cells, the cells were mixed with Staphylococcus aureus and incubated in the presence of fresh human AB serum at 37°C for 30 min. For an adherence test, they were cultured on glass coverslips at 37°C for 30 min. SPI-802 cells were examined for their ability to release plasminogen activators, one of the monocyte functions, by the method of Ragsdale and Arend (18). Preparation of adherent mononuclear cells. Adherent mononuclear cells were obtained by the plastic-adherence procedure from heparinized peripheral blood samples as previously described ( 19, 20). Preparation of SPA802 supernatants and adherent cell-derived IL-l. Crude conditioned media were initially prepared by culturing 2 X 106/ml SPI-802 cells for 2 days in RPM1 1640 medium with 1% fetal calf serum (FCS, Flow Lab., Rockville, Md.) in the presence or absence of 20 pg/ml Escherichia coli lipopolysaccharide (LPS, Difco, Detroit, Mich.). For comparison, crude conditioned media containing IL-l were also prepared from adherent mononuclear cells in similar cultures with 1% FCS and LPS stimulation. The conditioned media were clarified by centrifugation at 4000g for 30 min. Concentration of the media was done by precipitation in a saturated (80%) solution of ammonium sulfate according to the method of Simon and Willoughby (8). In this process the media could be concentrated about lo-fold, with 70-90% recovery of IL-l activity to enhance thymocyte proliferation as described below. The concentrated media were then chromatographed on a 2.6 X 65-cm Sephadex G-100 column equilibrated in 20 mM phosphate-buffered saline (PBS). Columns were calibrated with the following markers: phosphorylase (Mr 92,500), bovine serum albumin (Mr 66,200), ovalbumin (M, 45,000), carbonic anhydrase (Mr 3 l,OOO),soybean trypsin inhibitor (M, 21,500), and lysozyme (Mr 14,400). Each fraction was assayed for its IL-l activity to enhance phytohemagglutinin (PHA)induced mouse thymocyte proliferation. The active fractions were pooled and stored at -40°C until assaysof their biological activities were performed. About 20 ml of the pooled media containing IL-l activity were obtained from 100 ml of the original crude media. The pooled media were used as SPI-802 supernatants and adherent cell IL-l. As a standard IL-l, human IL-l (GUPI-1, IL-l activity > 100 U/ml) was
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commercially obtained from Genzyme Company (Boston, Mass.). The IL-I was diluted 1:10 and used as standard IL-1 (IL 1 activity > 10 U/ml). For biochemical analyses, the crude conditioned media were concentrated by ammonium sulfate precipitation and applied to a 2.6 X 97-cm Ultrogel AcA54 column (LKB, Bromma, Sweden) equilibrated with 20 mM PBS. Columns were calibrated with the molecular markers described above. Aliquots were assayed for their activity toward enhancement of thymocyte proliferation. Following the gel filtration, the fractions containing the low-molecular-weight peak of IL-l activity were pooled, concentrated with an Amicon YM 10 membrane, and processedfor isoelectrofocusing (IEF). The samples were dialyzed against 0.1% polyethylene glycol, M, 6,000. The mixture of each sample, 2% pH 3.5-10 Ampholine (LKB), and 4 g of Ultrodex (LKB) was poured into the tray of an LKB multiphor preparative flat-bed IEF apparatus. Electrofocusing was run with a constant power of 8 W (1200 V) for 15 hr. The fractions were collected, measured for pH, and assayedfor their thymocyte-proliferating activity. Thymocyte-proliferation assay. SPI-802 supematants, adherent cell IL- 1, and standard IL-1 were assayed for their IL-1 activity toward enhancement of PHAinduced mouse thymocyte proliferation according to the method of Meltzer and Oppenheim (1). Briefly, thymocytes were obtained from 4- to 8-week-old C3H/HeJ mice and suspended in RPM1 1640 medium with 10% FCS. The cells were cultured for 3 days at 1 X 106/well in microculture plates in the presence of 1 kg/ml PHA (PHA-P, Difco) and various dilutions of each sample. The cultured cells were pulsed for the final 16 hr of the 72-hr culture with 0.2 &i [3H]thymidine ([3H]TdR, Radiochemical Center, Amersham, England), and they were harvested as previously described (19). [3H]TdR incorporation was measured by the standard procedures. Production of interleukin 2 (IL-2). Since the Jurkat-F1884, a leukemia T-cell line, has the ability to produce IL-2 (21), we used this cell line as an IL-2 producer. In brief, 2 X 106/ml Jurkat cells were cultured with 5 Ilg/ml concanavalin A (Con A, Sigma) plus 10 rig/ml PMA in the presence of various concentrations of LPSstimulated SPI-802 supernatant or adherent cell IL-1 for 2 days, and the culture supernatants were collected. The contaminating PMA in the Jurkat supematants was removed through a charcoal column. The supematants were then processedfor IL-2 activity assay as Jurkat supematants. IL-2 Activity assay. Jurkat supematants and LPS-stimulated SPI-802 supematant were tested for IL2 activity with our IL-2dependent cytotoxic T-cell clone designated as SPY-218. SPY-218 cells were produced from lymphocytes alloactivated in oneway mixed lymphocyte cultures followed by expansion of clonal progeny with IL-2 for 4 weeks in the fashion similar to that described elsewhere (19). For IL-2 activity assay, 2 X lo4 SPY-2 18 cells per well were cultured for 2 days with 10% Jurkat supematant. To examine whether SPI-802 supematant may contain IL-2 activity, SPY-218 cells were similarly cultured with 10% RPM1 1640 medium containing various concentrations of LPS-stimulated SPI-802 supematant. Their [3H]TdR incorporation was measured as described above. RESULTS Cytological and Functional Characterization of SPI-802 Cells Table 1 summarizes cytological and functional features of SPI-802 cells as studied between 22 and 36 months after their culture initiation. SPI-802 cells retained
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TABLE I Cytological and Functional Features of SPI-802 Cells Stimulation of SPI-802 cells with PMA
t-1 Morphology
Blastic cells Granules (-)
(+I Blastic cells Granules (-) Vesicles (+)
Cytochemistry a-Naphthyl butyrate esterase
(-)
t-1
Surface markers (percentage positivity)’ E-rosette Fc-R L4T.W5 Leu-7 OKMl Ia Human monocyte
68 94 72 0 86 96 0
72 92 78 0 84 94 0
Number: 62-68
W
Not studied
NK activity (percentage control)’
0.2
Phagocytosis
t-1
(-)
Adherence
C-J
(-)
Production of plasminogen activators
(-)
t-1
0
“The values for normal peripheral lymphocytes were as follows: E rosette, 72-85%; Fc-R, 24-36%; Leu-5, 74-84%; Leu-7,6-18%; OKMl, 6-192; Ia, 23-50%; human monocyte, 2-4%. bThe chromosome analysis was performed by the G-banding method. c NK activity was determined against K562 by the 5’Cr-releaseassay at a 40: 1 effector:target ratio. The percentage lysis was measured and compared with the control value of normal lymphocytes.
virtually the same cellular characteristics in the morphology, cytochemistry, surface phenotype, and chromosomes as those described previously (17), but lost significant levels of NK activity against K562. The cells had round nuclei and relatively voluminous cytoplasm with many mitochondria and sparseendoplasmic reticulum. They did not contain cytoplasmic granules. They were negative for nonspecific esterase.A large proportion of them formed E rosettes and expressedLeu-5 antigen, certainly demonstrating E receptors on their surfaces. They had IgG-Fc-receptors (Fc-R), OKMl, and Ia antigens as well, but not human monocyte antigen. They were not phagocytic for Staphylococcus aureus and not adherent on glass surfaces. They lacked ability to produce plasminogen activators. The 2day stimulation of SPI-802 cells with PMA did not induce their NK activity, phagocytosis, adherence, and plasminogen-activator production. Biological Activities of SPI-802 Supernatants When the activity of SPI-802 supematants toward enhancement of PHA-induced thymocyte proliferation was analyzed by Sephadex G-100 column chromatography,
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the prominent activity was present in fractions corresponding to M, lO,OOO-20,000, being consistent with the results of adherent cell IL-l. These fractions, therefore, were pooled and used for the present biological assays. Figure 1 shows results of the thymocyte-proliferation assay. The addition of LPSstimulated SPI-802 supematant to PHA-induced mouse thymocyte cultures enhanced the proliferation of the cells, indicating the production of IL-l-like activity by SPI802 cells. Levels of the thymocyte-proliferating activity of the SPI-802 supematant were higher than those of adherent cell IL-l: When standard IL-1 (IL-l activity > 10 U/ml) gave 50% maximum activity at a reciprocal dilution of 60, the SPI-802 supematant and adherent cell IL-l gave it at the dilution of 200 and 20, respectively. Unstimulated SPI-802 supernatant also enhanced the thymocyte proliferation, although to a lesser degree, indicating the spontaneous release of IL-l-like activity from the cell line. In the next experiments SPI-802 supematants were studied for their activity toward promoting the IL-2 production by activated T cells. Con A plus PMAstimulated Jurkat supernatants were prepared in the presence of O-50% LPSstimulated SPI-802 supematant or adherent-cell IL-1 and assayed for their IL-2 activity toward proliferation of IL-2-dependent SPY-2 18 cytotoxic T cells. As shown in Fig. 2, LPS-stimulated SPI-802 supematant as well as adherent cell IL- 1 promoted the IL-2 production. The proliferation of SPY-2 18 cells was increased with the Jurkat supematants obtained with increasing concentrations of the SPI-802 super-
2
8 Reciprocal
32
128 of
sample
512
2,048
dilution
FIG. 1. Thymocyte-proliferation activity of SPI-802 supematants and adherent-cell IL-l. PHA (1 &ml)-induced C3H/HeJ mouse thymocytes (I X 106/well) were cultured for 3 days with serial twofold dilutions of LPS-stimulated (0) or unstimulated (0) SPI-802 supematant or adherent-cell IL-l (X). In parallel experiments, the thymocytes were cultured with standard IL-I containing 710 U/ml of IL-I activity (A). Their [‘H]TdR uptake was measured by the standard procedures.
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> .r c u m
0
0.1
0.2
0.5
1
2
Percent
of
3
4
sample
5
10
15
20
30
50
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FIG. 2. IL-2 activity of Jurkat supematants obtained with SPI-802 supematant or adherent-cell IL-I. IL-2 activity assays were done on the SPY-218 ILZdependent cytotoxic T-cell clone. SPY-218 cells (2 X 104/well) were cultured for 2 days with 10% Con A (5 &ml) plus PMA (10 r&ml)-stimulated Jurkat supematant obtained with 0-50’70of LPS-stimulated SPI-802 supematant (0) or adherent-cell IL1 (X). In parallel experiments, SPY-218 cells were cultured with 10% RPM1 1640 medium containing O50% LPS-stimulated SPI-802 supematant (0). Con A plus PMA-stimulated Jurkat supematants obtained in the absence of the SPI-802 supematant or adherent-cell IL-1 did not have the activity toward the proliferation of SPY-218 cells: cpm < 300.
natant or adherent cell IL- 1, and reached a plateau with those obtained with 15% of the SPI-802 supematant or 50% of adherent cell IL-l. Con A plus PMAstimulated Jurkat supematants obtained in the absence of the SPI-802 supematant or adherent cell IL- 1 did not proliferate SPY-2 18 cells: Counts per minute were less than 300. This result demonstrates the IL-1 dependency of the IL-2 production by our Jurkat cell line and also indicates little or no effect of Con A and of residual PMA, if any, on the SPY-2 18 cell proliferation. Based on these results, it is apparent that SPI-802 cells can produce high levels of IL-l-like activity toward promoting the IL-2 production. LPS-stimulated SPI-802 supematant by itself did not generate the SPY-218 cell proliferation (Fig. 2), excluding the possibility that it may contain IL-2 as well. Biochemical Characterization of IL-l-Like
Activity of SPI-802 Supernatant
For biochemical characterization, conditioned media from LPS-stimulated SPI802 and adherent mononuclear cell cultures in the presence of 1% FCS were chromatographed on an AcA54 column and assayed for their activity toward enhancement of PHA-induced thymocyte proliferation. As shown in Fig. 3, the
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6
I - ia c-2
5
4
3
2
1
7 ; c E
0 60
8 8
5
5
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4
3
3
2
2
1
1
0 50
loo
150
200 Elution
755 volume
360
3kJo
4bo
1
(ml)
Ftc. 3. Ultrogel AcASrlcolumn chromatography of SPI-802 supematant (A) and adherent-cell IL-l (B). Conditioned media were prepared by culturing 2 X 106/ml of SPI-802 cells and adherent mononuclear cells in RPM1 1640 medium with 1% FCS in the presenceof 20 &ml LPS, concentrated by ammonium sulfate precipitation, and applied to a 2.6 X 97-cm Ultrogel AcA54 column equilibrated with 20 mM PBS. Columns were calibrated with the following markers: phosphorylase (M, 92,500), bovine serum albumin (Mr 66,200), ovalbumin (M, U,OOO),carbonic anhydrase (Mr 3 1,OOO),soybean trypsin inhibitor (Mr 2 1,500), and lysozyme (Mr 14,400).
IL- 1-like activity of the SPI-802 supematant was recovered in two fractions, a lowM, form eluting in the range of 12,000-20,000 and a high-M, form of 60,00070,000, being similar to that of adherent cell IL- 1. Following the gel filtration, the IL- 1-like activity containing fractions corresponding to the M, range of 12,000-20,000 were then isoelectrofocused and assayedfor the thymocyte-proliferating activity. The IL- 1-like activity of LPS-stimulated SPI-802 supematant was resolved into two major specieswith pZ values of 5.0 and 7.0 and one minor specieswith a pZ of 4.4 (Fig. 4A). The activity of adherent cell IL-l, on the other hand, was resolved into two major species with pZ values of 5.0 and 7.0 (Fig. 4B). The IL- l-like activity of the SPI-802 supematant was, therefore, consistent with the activity of adherent cell IL-l in the major form of the activity, although somewhat different from that in the minor one. DISCUSSION Several biological activities have been reported for IL-I. For example, IL-I can enhance lectin-induced thymocyte proliferation (1, 2) and promote the production
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number
4. Isoelectrofocusing of Ultrogel AcA54 column-purified thymocyte-proliferating activity of SPI802 supematant (A) and adherent-cell IL-l (B). The fractions containing the low-Mr peak of the activity were pooled, concentrated with an Amicon YM membrane, and subjected to isoelectrofocusing. FIG.
of IL-2 by activated T cells (3, 5, 6). Human IL-1 has M;s between 10,000 and 20,000 and of more than 60,000 (2, 14) and is resolved into two major specieswith p1 values of 5.0 and 7.0 (14). In our present studies, SPI-802 supematants as well as adherent-cell IL-1 had both of these biological activities and biochemical characteristics. Based on these results, it is evident that the SPI-802 cell line can produce a soluble factor with such biological and biochemical characteristics of human IL- 1. Our comparative studies between biological activities of SPI-802 supematants and adherent-cell IL-1 have demonstrated that SPI-802 cells can produce high levels of IL-l-like activity. It is noteworthy that they can spontaneously produce the activity. There are several established human cell lines capable of producing IL-1 ( 12- 16). The U937 and CM-S, macrophage-related cell lines, can produce low levels of IL-l activity (12-14). The THP-1, a monocytic leukemia cell line, can produce levels of IL-l activity comparable to those obtainable from peripheral monocytes ( 14). Fisher et al. ( 15) have recently demonstrated a “dendritic-like” cell line (L428) with IL-l-producing ability. In addition, a keratinocyte cell line capable of producing low levels of IL- 1 activity has been described ( 16). To our knowledge, our SPI-802
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is the only established human cell line that can produce high levels of IL-l-like activity. The production of IL-l is the function associated mainly with monocytes (l-8). SPI-802 cells, however, appear to not be monocytic in nature based on their cytochemical, functional, and immunological characteristics; they were negative for peroxidase (17) and for nonspecific esterase,not phagocytic, not adherent, and did not have human monocyte antigen nor the ability to produce plasminogen activators. It is of interest that SPI-802 cells, unlike the aforementioned human cell lines having IL- 1-producing ability ( 12- 16), certainly possessedE receptorsas demonstrated by their E-rosette formation and Leu-5 expression. The presence of E receptors is one of the surface characteristics of T cells (22). SPI-802 cells, however, did not express any T-cell-associated antigens defined by anti-T-cell antiserum and OKT/Leu monoclonal antibodies (17). Another lineage of cells which have E receptors are NK cells (23, 24). Indeed, SPI-802 cells had Fc-R, OKMl, and Ia in addition to E receptors, being similar to NK cells (23-28). They did not have cytoplasmic granules but resembled NK cells in the abundant cytoplasm with many mitochondria and sparse endoplasmic reticulum (29). In addition, they had spontaneous cytotoxicity against K562 and Molt-3 cells but not against Raji cells as reported elsewhere (17). Such surface phenotype, morphology, and target celldependent spontaneous cytotoxicity of SPI-802 cells favor their NK cell nature, although the exact lineage of human NK cells is still controversial (25). The loss of NK activity of SPI-802 cells demonstrated here may be explained by the gradual decreaseof cytolytic activity during culture as observed in T-killer-cell lines (30, 31). One might question how SPI-802 cells with such cellular characteristics of NK cells have acquired the ability to produce an IL-l-like factor. It is well known that some relevant similarities in the functions as well as the cell markers exist between monocytes and NK cells (25, 26). For example, they share some abilities such as spontaneous cytotoxicity and interferon production (26-28, 32). It thus appears feasible that NK cells, like monocytes, have IL-l-producing ability and that the ability can be augmented under certain conditions, such as the oncogenic event. The recent evidence that large granular lymphocytes (LGL), which are known to be responsible for NK-cell activity (24), are potent IL-1 producers (32, 33) does support the IL-l production by NK cells. Although the exact lineage of our unique SPI-802 cell line remains undetermined, as mentioned above, characterization of the cells certainly provides further information as to cellular characteristics of IL- l-producing cells. With respect to this, the reports by Luger et al. (16, 34) and Fontana et al. (35) are interesting. They have demonstrated that keratinocytes (epidermal cells) can produce IL-l activity (16, 34) and cultured astrocytes and glia cells can secrete IL- l-like activity (35). From these results, it is apparent that besides monocyte-macrophages there are several lineages of cells capable of producing IL-l activity. The SPI-802 cell line can produce high levels of IL-l-like activity, which can promote the IL-2 production by the Jurkat-F1884 cell line. Our SPI-802 leukemia cell line is, therefore, an ideal source of IL-l-like activity and is expected to contribute to further biological and biochemical characterization of human IL-2 as well as IL- 1.
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ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid for Cancer Research (58-26) from the Ministry of Health and Welfare and by a grant (C-59570391) from the Ministry of Education, Culture and Science, Japan.
REFERENCES 1. Meltzer, M. S., and Oppenheim, J. J., J. Immunol. 118, 77, 1977. 2. Togawa, A., Oppenheim, J. J., and Mizel, S. B., J. Immunol. 122, 2112, 1979. 3. Farrar, J. J., Simon, P. L., Farrar, W. L., Koopman, W. J., and Fuller-Bonar, J., Ann. N.Y. Acad. Sci. 332, 303, 1979. 4. Rosenwasser,L. J., Dinarello, C. A., and Rosenthal, A. S., J. Exp. Med. 150, 709, 1979. 5. Larsson, E-L., Iscove, N. N., and Coutinho, A., Nature (London) 283,664, 1980. 6. Smith, K. A., Lachman, L. B., Oppenheim, J. J., and Favata, M. F., J. Exp. Med. 151, 1551, 1980. 7. Maizel, A. J., Mehta, S. R., Ford, R. J., and Lachman, L. B., J. Exp. Med. 153, 470, 1981. 8. Simon, P. L., and Willoughby, W. F., J. Immunol. 126, 1534, 1981. 9. Lachman, L. B., Hacker, M. P., Blyden, G. T., and Handschmacher, R. E., Cell. Immunol. 34, 416, 1977.
10. Mizel, S. B., Oppenheim, J. J., and Rosenstreich, D. L., J. Immunol. 120, 1504, 1978. 11. Lachman, L. B., Moore, J. O., and Metzgar, R. S., Cell. Immunol. 41, 199, 1978. 12. Palacios, R., Ivhed, I., Sideras, P., Nilsson, K., Sugawara, I., and Femandez, C., Eur. J. Immunol. 12, 895, 1982.
13. Butler, R. H., Revoltella, R. P., Musiani, P., and Piantelli, M., Cell. Immunol. 78, 368, 1983. 14. Krakauer, T., and Oppenheim, J. J., CeM.Immunol. 80, 223, 1983. 15. Fisher, R. I., Bostick-Bruton, F., Sauder, D. N., Scala, G., and Diehl, V., J. Immunol. 130, 2666, 1983.
16. Luger, T. A., Stadler, B. M., Luger, B. M., Sztein, M. B., Schmidt, J. A., Hawley-Nelson, P., Grabner, G., and Oppenheim, J. J., J. Invest. Dermatol. 81, 187, 1983. 17. Komiyama, A., Kawai, H., Miyagawa, Y., and Akabane, T., Blood 60, 1429, 1982. 18. Ragsdale, C. G., and Arend, W. P., J. Exp. Med. 149, 954, 1979. 19. Miyagawa, Y., Komiyama, A., Akabane, T., Uehara, Y., and Yano, A., J. Immunol. 129, 1993, 1982.
20. Kawai, H., Hirabayashi, S., Miyagawa, Y., Komiyama, A., and Akabane, T., Cancer 52, 1215, 1983. 21. Gillis, S., and Watson, J., J. Exp. Med. 152, 1709, 1980. 22. Kaplan, M. E., and Clark, C., J. Immunol. Meth. 5, 131, 1974. 23. West, W. H., Cannon, G. B., Kay, H. D., Bonnard, G. D., and Herberman, R. B., J. Immunol. 118, 355, 1977. 24. Timonen, T., Ortaldo, J. R., and Herberman, R. B., J. Exp. Med. 153, 569, 1981. 25. Ortaldo, J. R., Sharrow, S. O., Timonen, T., and Herberman, R. B., J. Immunol. 127, 2401, 1981. 26. Lohmann-Matthes, M-L., Domzig, W., and Roder, J. J., J. Immunol. 123, 1883, 1979. 27. Djeu, J. Y., Heinbaugh, J. A., Holden, H. T., and Herberman, R. B., J. Immunol. 122, 182, 1979. 28. Minato, N., Reid, L., Cantor, H., Lengyel, P., and Bloom, B. R., J. Exp. Med. 152, 124, 1980. 29. Saksela, E., and Timonen, T., In “Natural Cell-Mediated Immunity Against Tumors” (R. B.
Herberman, Ed.), p. 173, Academic Press,New York, 1980. Dennert, G., De Rose.,M., and Allen, R. S., Eur. J. Immunol. 7, 478, 1977. Fathman, C. G., and Hengartner, H., Nature (London) 272, 617, 1978. Kasahara, T., Djeu, J. Y., Dougherty, S. F., and Oppenheim, J. J., J. Immunol. 131, 2379, 1983. Scala, G., Allavena, P., Djeu, J. Y., Kasahara, T., Ortaldo, J. R., Herberman, R. B., and Oppenheim, J. J., Nature (London) 309, 56, 1984. 34. Luger, T. A., Stadler, B. M., Katz, S. I., and Oppenheim, J. J., J. Immunol. 127, 1493, 1981. 35. Fontana, A., Kristensen, F., Dubs, R., Gemsa, D., and Weber, E., J. Immunol. 129, 2413, 1982. 30. 31. 32. 33.
IMMUNOLOGY
89, 122- 13I ( 1984)
Production of High Levels of lnterleukin l-Like Activity by the SPI-802 Human Leukemia Cell Line with E Receptors ATSUSHIKOMIYAMA, YUKIAKI MIYAGAWA, KOHKI AOYAMA, TARO AKABANE,AND YOSHIOUEHARA Department of Pediatrics, Shinshu University School of Medicine, Asahi 3-I-1, Matsumoto, 390, and Division of Immunochemistry, Suzaka Prefectural Hospital, Suzaka, 382, Japan Received November I, 1983; accepted June 29, 1984 The SPI-802 human leukemia cell line, which possessesE receptors and used to have natural killer activity, has been demonstrated to produce high levels of interleukin 1 (IL-1)-like activity. SPI-802 supematants prepared in 1% serum-containing cultures with lipopolysaccharide stimulation, like similarly prepared adherent-cell-derived IL-l, enhanced phytohemagglutinininduced mouse thymocyte proliferation. When adherent-cell IL-1 gave 50% maximum activity at a reciprocal dilution of 20, SPI-802 supematant gave it at 200, indicating the production of high levels of &l-like activity by the cell line. SPI-802 supematant promoted the production of interleukin 2 (IL-2) by the Jurkat-F1884 T-cell line: Levels of IL-2 activity obtained with 15% SPI-802 supematant were almost equivalent to those obtained with 50% adherent-cell IL-l as estimated by the maximum proliferation of IL-2dependent cytotoxic T cells. SPI-802 supematant by itself exhibited no IL-2 activity. Major IL-l-like activity of SPI-802 supematant was present in fractions from AcA54 columns corresponding to M, 12,000-20,000 and 60,00070,000 and resolved on isoelectrofocusing into two distinct species with pI values of 5.0 and 7.0, being consistent with the results of adherent-cell IL-l. The SPI-802 cell line having E receptors is an ideal source of a soluble factor with the biological and biochemical characteristics of human IL- 1. o 1984 Academic pres* hc.
INTRODUCTION In recent years much attention has been focused on interleukin l(IL-l), which plays a central role in the acquisition and expression of immune responsiveness. Most studies of IL-l have been done using the factor obtained in cultures of adherent mononuclear cells (l-8). In humans, however, the source of IL-l is not satisfactory for its detailed characterization because of the difficulty of obtaining large quantities of purified IL-l from the source. Many investigators have thus been searching for ideal sources of IL-1 (1, 9-16). There is a mouse macrophage cell line (P388Dr) which can releaseimmunostimulatory activity similar to mouse monocytederived IL-l (1, 9, 10). Recently several human cell lines capable of producing IL-l have been reported (12-16). These cell lines, however, produce low levels of IL-l activity (12-16). Only the THP-1, a monocytic leukemia cell line, can produce levels of IL-l activity comparable to those obtainable from peripheral blood monocytes (14). In this article, we report that our SPI-802 human leukemia cell line, which possessesE receptors and used to have natural killer (NK) activity (17), can produce high levels of IL-l-like activity. The SPI-802 cell line is distinct in 122 0008-8749184$3.00 Copyright 8 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
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some property from the monocyte-related human cell lines with IL1 producing ability such as the U937, CM-S, and THP-1 cell lines (12-14) and provides further information concerning cellular characteristics of IL- 1-producing cells. MATERIALS
AND METHODS
Characterization of SPI-802 cells. SPI-802 cells were studied between 22 and 36 months after their culture initiation. During the period, their cytological and functional characteristics were reexamined. Some of the cells were cultured in the presence of 100 rig/ml phorbol myristate acetate (PMA; Sigma, St. Louis, MO.) for 48 hr and processed for the following studies. Morphology, cytochemistry, surface markers, chromosomes, and NK activity of SPI-802 cells were studied as previously described (17). For surface marker studies, monoclonal anti-Leu-5 and anti-Leu-7 (HNK- 1, Becton-Dickenson, Sunnyvale, Calif.), monoclonal OKM 1 (Ortho Pharmaceutical Co., Raritan, N.J.), and monoclonal anti-human monocyte (Bethesda Research Lab., Gaithersburg, Md.) antibodies were commercially obtained. Anti-Ia serum was raised in rabbits (17). To estimate phagocytic ability of SPI-802 cells, the cells were mixed with Staphylococcus aureus and incubated in the presence of fresh human AB serum at 37°C for 30 min. For an adherence test, they were cultured on glass coverslips at 37°C for 30 min. SPI-802 cells were examined for their ability to release plasminogen activators, one of the monocyte functions, by the method of Ragsdale and Arend (18). Preparation of adherent mononuclear cells. Adherent mononuclear cells were obtained by the plastic-adherence procedure from heparinized peripheral blood samples as previously described ( 19, 20). Preparation of SPA802 supernatants and adherent cell-derived IL-l. Crude conditioned media were initially prepared by culturing 2 X 106/ml SPI-802 cells for 2 days in RPM1 1640 medium with 1% fetal calf serum (FCS, Flow Lab., Rockville, Md.) in the presence or absence of 20 pg/ml Escherichia coli lipopolysaccharide (LPS, Difco, Detroit, Mich.). For comparison, crude conditioned media containing IL-l were also prepared from adherent mononuclear cells in similar cultures with 1% FCS and LPS stimulation. The conditioned media were clarified by centrifugation at 4000g for 30 min. Concentration of the media was done by precipitation in a saturated (80%) solution of ammonium sulfate according to the method of Simon and Willoughby (8). In this process the media could be concentrated about lo-fold, with 70-90% recovery of IL-l activity to enhance thymocyte proliferation as described below. The concentrated media were then chromatographed on a 2.6 X 65-cm Sephadex G-100 column equilibrated in 20 mM phosphate-buffered saline (PBS). Columns were calibrated with the following markers: phosphorylase (Mr 92,500), bovine serum albumin (Mr 66,200), ovalbumin (M, 45,000), carbonic anhydrase (Mr 3 l,OOO),soybean trypsin inhibitor (M, 21,500), and lysozyme (Mr 14,400). Each fraction was assayed for its IL-l activity to enhance phytohemagglutinin (PHA)induced mouse thymocyte proliferation. The active fractions were pooled and stored at -40°C until assaysof their biological activities were performed. About 20 ml of the pooled media containing IL-l activity were obtained from 100 ml of the original crude media. The pooled media were used as SPI-802 supernatants and adherent cell IL-l. As a standard IL-l, human IL-l (GUPI-1, IL-l activity > 100 U/ml) was
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commercially obtained from Genzyme Company (Boston, Mass.). The IL-I was diluted 1:10 and used as standard IL-1 (IL 1 activity > 10 U/ml). For biochemical analyses, the crude conditioned media were concentrated by ammonium sulfate precipitation and applied to a 2.6 X 97-cm Ultrogel AcA54 column (LKB, Bromma, Sweden) equilibrated with 20 mM PBS. Columns were calibrated with the molecular markers described above. Aliquots were assayed for their activity toward enhancement of thymocyte proliferation. Following the gel filtration, the fractions containing the low-molecular-weight peak of IL-l activity were pooled, concentrated with an Amicon YM 10 membrane, and processedfor isoelectrofocusing (IEF). The samples were dialyzed against 0.1% polyethylene glycol, M, 6,000. The mixture of each sample, 2% pH 3.5-10 Ampholine (LKB), and 4 g of Ultrodex (LKB) was poured into the tray of an LKB multiphor preparative flat-bed IEF apparatus. Electrofocusing was run with a constant power of 8 W (1200 V) for 15 hr. The fractions were collected, measured for pH, and assayedfor their thymocyte-proliferating activity. Thymocyte-proliferation assay. SPI-802 supematants, adherent cell IL- 1, and standard IL-1 were assayed for their IL-1 activity toward enhancement of PHAinduced mouse thymocyte proliferation according to the method of Meltzer and Oppenheim (1). Briefly, thymocytes were obtained from 4- to 8-week-old C3H/HeJ mice and suspended in RPM1 1640 medium with 10% FCS. The cells were cultured for 3 days at 1 X 106/well in microculture plates in the presence of 1 kg/ml PHA (PHA-P, Difco) and various dilutions of each sample. The cultured cells were pulsed for the final 16 hr of the 72-hr culture with 0.2 &i [3H]thymidine ([3H]TdR, Radiochemical Center, Amersham, England), and they were harvested as previously described (19). [3H]TdR incorporation was measured by the standard procedures. Production of interleukin 2 (IL-2). Since the Jurkat-F1884, a leukemia T-cell line, has the ability to produce IL-2 (21), we used this cell line as an IL-2 producer. In brief, 2 X 106/ml Jurkat cells were cultured with 5 Ilg/ml concanavalin A (Con A, Sigma) plus 10 rig/ml PMA in the presence of various concentrations of LPSstimulated SPI-802 supernatant or adherent cell IL-1 for 2 days, and the culture supernatants were collected. The contaminating PMA in the Jurkat supematants was removed through a charcoal column. The supematants were then processedfor IL-2 activity assay as Jurkat supematants. IL-2 Activity assay. Jurkat supematants and LPS-stimulated SPI-802 supematant were tested for IL2 activity with our IL-2dependent cytotoxic T-cell clone designated as SPY-218. SPY-218 cells were produced from lymphocytes alloactivated in oneway mixed lymphocyte cultures followed by expansion of clonal progeny with IL-2 for 4 weeks in the fashion similar to that described elsewhere (19). For IL-2 activity assay, 2 X lo4 SPY-2 18 cells per well were cultured for 2 days with 10% Jurkat supematant. To examine whether SPI-802 supematant may contain IL-2 activity, SPY-218 cells were similarly cultured with 10% RPM1 1640 medium containing various concentrations of LPS-stimulated SPI-802 supematant. Their [3H]TdR incorporation was measured as described above. RESULTS Cytological and Functional Characterization of SPI-802 Cells Table 1 summarizes cytological and functional features of SPI-802 cells as studied between 22 and 36 months after their culture initiation. SPI-802 cells retained
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TABLE I Cytological and Functional Features of SPI-802 Cells Stimulation of SPI-802 cells with PMA
t-1 Morphology
Blastic cells Granules (-)
(+I Blastic cells Granules (-) Vesicles (+)
Cytochemistry a-Naphthyl butyrate esterase
(-)
t-1
Surface markers (percentage positivity)’ E-rosette Fc-R L4T.W5 Leu-7 OKMl Ia Human monocyte
68 94 72 0 86 96 0
72 92 78 0 84 94 0
Number: 62-68
W
Not studied
NK activity (percentage control)’
0.2
Phagocytosis
t-1
(-)
Adherence
C-J
(-)
Production of plasminogen activators
(-)
t-1
0
“The values for normal peripheral lymphocytes were as follows: E rosette, 72-85%; Fc-R, 24-36%; Leu-5, 74-84%; Leu-7,6-18%; OKMl, 6-192; Ia, 23-50%; human monocyte, 2-4%. bThe chromosome analysis was performed by the G-banding method. c NK activity was determined against K562 by the 5’Cr-releaseassay at a 40: 1 effector:target ratio. The percentage lysis was measured and compared with the control value of normal lymphocytes.
virtually the same cellular characteristics in the morphology, cytochemistry, surface phenotype, and chromosomes as those described previously (17), but lost significant levels of NK activity against K562. The cells had round nuclei and relatively voluminous cytoplasm with many mitochondria and sparseendoplasmic reticulum. They did not contain cytoplasmic granules. They were negative for nonspecific esterase.A large proportion of them formed E rosettes and expressedLeu-5 antigen, certainly demonstrating E receptors on their surfaces. They had IgG-Fc-receptors (Fc-R), OKMl, and Ia antigens as well, but not human monocyte antigen. They were not phagocytic for Staphylococcus aureus and not adherent on glass surfaces. They lacked ability to produce plasminogen activators. The 2day stimulation of SPI-802 cells with PMA did not induce their NK activity, phagocytosis, adherence, and plasminogen-activator production. Biological Activities of SPI-802 Supernatants When the activity of SPI-802 supematants toward enhancement of PHA-induced thymocyte proliferation was analyzed by Sephadex G-100 column chromatography,
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the prominent activity was present in fractions corresponding to M, lO,OOO-20,000, being consistent with the results of adherent cell IL-l. These fractions, therefore, were pooled and used for the present biological assays. Figure 1 shows results of the thymocyte-proliferation assay. The addition of LPSstimulated SPI-802 supematant to PHA-induced mouse thymocyte cultures enhanced the proliferation of the cells, indicating the production of IL-l-like activity by SPI802 cells. Levels of the thymocyte-proliferating activity of the SPI-802 supematant were higher than those of adherent cell IL-l: When standard IL-1 (IL-l activity > 10 U/ml) gave 50% maximum activity at a reciprocal dilution of 60, the SPI-802 supematant and adherent cell IL-l gave it at the dilution of 200 and 20, respectively. Unstimulated SPI-802 supernatant also enhanced the thymocyte proliferation, although to a lesser degree, indicating the spontaneous release of IL-l-like activity from the cell line. In the next experiments SPI-802 supematants were studied for their activity toward promoting the IL-2 production by activated T cells. Con A plus PMAstimulated Jurkat supernatants were prepared in the presence of O-50% LPSstimulated SPI-802 supematant or adherent-cell IL-1 and assayed for their IL-2 activity toward proliferation of IL-2-dependent SPY-2 18 cytotoxic T cells. As shown in Fig. 2, LPS-stimulated SPI-802 supematant as well as adherent cell IL- 1 promoted the IL-2 production. The proliferation of SPY-2 18 cells was increased with the Jurkat supematants obtained with increasing concentrations of the SPI-802 super-
2
8 Reciprocal
32
128 of
sample
512
2,048
dilution
FIG. 1. Thymocyte-proliferation activity of SPI-802 supematants and adherent-cell IL-l. PHA (1 &ml)-induced C3H/HeJ mouse thymocytes (I X 106/well) were cultured for 3 days with serial twofold dilutions of LPS-stimulated (0) or unstimulated (0) SPI-802 supematant or adherent-cell IL-l (X). In parallel experiments, the thymocytes were cultured with standard IL-I containing 710 U/ml of IL-I activity (A). Their [‘H]TdR uptake was measured by the standard procedures.
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> .r c u m
0
0.1
0.2
0.5
1
2
Percent
of
3
4
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5
10
15
20
30
50
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FIG. 2. IL-2 activity of Jurkat supematants obtained with SPI-802 supematant or adherent-cell IL-I. IL-2 activity assays were done on the SPY-218 ILZdependent cytotoxic T-cell clone. SPY-218 cells (2 X 104/well) were cultured for 2 days with 10% Con A (5 &ml) plus PMA (10 r&ml)-stimulated Jurkat supematant obtained with 0-50’70of LPS-stimulated SPI-802 supematant (0) or adherent-cell IL1 (X). In parallel experiments, SPY-218 cells were cultured with 10% RPM1 1640 medium containing O50% LPS-stimulated SPI-802 supematant (0). Con A plus PMA-stimulated Jurkat supematants obtained in the absence of the SPI-802 supematant or adherent-cell IL-1 did not have the activity toward the proliferation of SPY-218 cells: cpm < 300.
natant or adherent cell IL- 1, and reached a plateau with those obtained with 15% of the SPI-802 supematant or 50% of adherent cell IL-l. Con A plus PMAstimulated Jurkat supematants obtained in the absence of the SPI-802 supematant or adherent cell IL- 1 did not proliferate SPY-2 18 cells: Counts per minute were less than 300. This result demonstrates the IL-1 dependency of the IL-2 production by our Jurkat cell line and also indicates little or no effect of Con A and of residual PMA, if any, on the SPY-2 18 cell proliferation. Based on these results, it is apparent that SPI-802 cells can produce high levels of IL-l-like activity toward promoting the IL-2 production. LPS-stimulated SPI-802 supematant by itself did not generate the SPY-218 cell proliferation (Fig. 2), excluding the possibility that it may contain IL-2 as well. Biochemical Characterization of IL-l-Like
Activity of SPI-802 Supernatant
For biochemical characterization, conditioned media from LPS-stimulated SPI802 and adherent mononuclear cell cultures in the presence of 1% FCS were chromatographed on an AcA54 column and assayed for their activity toward enhancement of PHA-induced thymocyte proliferation. As shown in Fig. 3, the
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6
I - ia c-2
5
4
3
2
1
7 ; c E
0 60
8 8
5
5
4
4
3
3
2
2
1
1
0 50
loo
150
200 Elution
755 volume
360
3kJo
4bo
1
(ml)
Ftc. 3. Ultrogel AcASrlcolumn chromatography of SPI-802 supematant (A) and adherent-cell IL-l (B). Conditioned media were prepared by culturing 2 X 106/ml of SPI-802 cells and adherent mononuclear cells in RPM1 1640 medium with 1% FCS in the presenceof 20 &ml LPS, concentrated by ammonium sulfate precipitation, and applied to a 2.6 X 97-cm Ultrogel AcA54 column equilibrated with 20 mM PBS. Columns were calibrated with the following markers: phosphorylase (M, 92,500), bovine serum albumin (Mr 66,200), ovalbumin (M, U,OOO),carbonic anhydrase (Mr 3 1,OOO),soybean trypsin inhibitor (Mr 2 1,500), and lysozyme (Mr 14,400).
IL- 1-like activity of the SPI-802 supematant was recovered in two fractions, a lowM, form eluting in the range of 12,000-20,000 and a high-M, form of 60,00070,000, being similar to that of adherent cell IL- 1. Following the gel filtration, the IL- 1-like activity containing fractions corresponding to the M, range of 12,000-20,000 were then isoelectrofocused and assayedfor the thymocyte-proliferating activity. The IL- 1-like activity of LPS-stimulated SPI-802 supematant was resolved into two major specieswith pZ values of 5.0 and 7.0 and one minor specieswith a pZ of 4.4 (Fig. 4A). The activity of adherent cell IL-l, on the other hand, was resolved into two major species with pZ values of 5.0 and 7.0 (Fig. 4B). The IL- l-like activity of the SPI-802 supematant was, therefore, consistent with the activity of adherent cell IL-l in the major form of the activity, although somewhat different from that in the minor one. DISCUSSION Several biological activities have been reported for IL-I. For example, IL-I can enhance lectin-induced thymocyte proliferation (1, 2) and promote the production
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number
4. Isoelectrofocusing of Ultrogel AcA54 column-purified thymocyte-proliferating activity of SPI802 supematant (A) and adherent-cell IL-l (B). The fractions containing the low-Mr peak of the activity were pooled, concentrated with an Amicon YM membrane, and subjected to isoelectrofocusing. FIG.
of IL-2 by activated T cells (3, 5, 6). Human IL-1 has M;s between 10,000 and 20,000 and of more than 60,000 (2, 14) and is resolved into two major specieswith p1 values of 5.0 and 7.0 (14). In our present studies, SPI-802 supematants as well as adherent-cell IL-1 had both of these biological activities and biochemical characteristics. Based on these results, it is evident that the SPI-802 cell line can produce a soluble factor with such biological and biochemical characteristics of human IL- 1. Our comparative studies between biological activities of SPI-802 supematants and adherent-cell IL-1 have demonstrated that SPI-802 cells can produce high levels of IL-l-like activity. It is noteworthy that they can spontaneously produce the activity. There are several established human cell lines capable of producing IL-1 ( 12- 16). The U937 and CM-S, macrophage-related cell lines, can produce low levels of IL-l activity (12-14). The THP-1, a monocytic leukemia cell line, can produce levels of IL-l activity comparable to those obtainable from peripheral monocytes ( 14). Fisher et al. ( 15) have recently demonstrated a “dendritic-like” cell line (L428) with IL-l-producing ability. In addition, a keratinocyte cell line capable of producing low levels of IL- 1 activity has been described ( 16). To our knowledge, our SPI-802
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is the only established human cell line that can produce high levels of IL-l-like activity. The production of IL-l is the function associated mainly with monocytes (l-8). SPI-802 cells, however, appear to not be monocytic in nature based on their cytochemical, functional, and immunological characteristics; they were negative for peroxidase (17) and for nonspecific esterase,not phagocytic, not adherent, and did not have human monocyte antigen nor the ability to produce plasminogen activators. It is of interest that SPI-802 cells, unlike the aforementioned human cell lines having IL- 1-producing ability ( 12- 16), certainly possessedE receptorsas demonstrated by their E-rosette formation and Leu-5 expression. The presence of E receptors is one of the surface characteristics of T cells (22). SPI-802 cells, however, did not express any T-cell-associated antigens defined by anti-T-cell antiserum and OKT/Leu monoclonal antibodies (17). Another lineage of cells which have E receptors are NK cells (23, 24). Indeed, SPI-802 cells had Fc-R, OKMl, and Ia in addition to E receptors, being similar to NK cells (23-28). They did not have cytoplasmic granules but resembled NK cells in the abundant cytoplasm with many mitochondria and sparse endoplasmic reticulum (29). In addition, they had spontaneous cytotoxicity against K562 and Molt-3 cells but not against Raji cells as reported elsewhere (17). Such surface phenotype, morphology, and target celldependent spontaneous cytotoxicity of SPI-802 cells favor their NK cell nature, although the exact lineage of human NK cells is still controversial (25). The loss of NK activity of SPI-802 cells demonstrated here may be explained by the gradual decreaseof cytolytic activity during culture as observed in T-killer-cell lines (30, 31). One might question how SPI-802 cells with such cellular characteristics of NK cells have acquired the ability to produce an IL-l-like factor. It is well known that some relevant similarities in the functions as well as the cell markers exist between monocytes and NK cells (25, 26). For example, they share some abilities such as spontaneous cytotoxicity and interferon production (26-28, 32). It thus appears feasible that NK cells, like monocytes, have IL-l-producing ability and that the ability can be augmented under certain conditions, such as the oncogenic event. The recent evidence that large granular lymphocytes (LGL), which are known to be responsible for NK-cell activity (24), are potent IL-1 producers (32, 33) does support the IL-l production by NK cells. Although the exact lineage of our unique SPI-802 cell line remains undetermined, as mentioned above, characterization of the cells certainly provides further information as to cellular characteristics of IL- l-producing cells. With respect to this, the reports by Luger et al. (16, 34) and Fontana et al. (35) are interesting. They have demonstrated that keratinocytes (epidermal cells) can produce IL-l activity (16, 34) and cultured astrocytes and glia cells can secrete IL- l-like activity (35). From these results, it is apparent that besides monocyte-macrophages there are several lineages of cells capable of producing IL-l activity. The SPI-802 cell line can produce high levels of IL-l-like activity, which can promote the IL-2 production by the Jurkat-F1884 cell line. Our SPI-802 leukemia cell line is, therefore, an ideal source of IL-l-like activity and is expected to contribute to further biological and biochemical characterization of human IL-2 as well as IL- 1.
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ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid for Cancer Research (58-26) from the Ministry of Health and Welfare and by a grant (C-59570391) from the Ministry of Education, Culture and Science, Japan.
REFERENCES 1. Meltzer, M. S., and Oppenheim, J. J., J. Immunol. 118, 77, 1977. 2. Togawa, A., Oppenheim, J. J., and Mizel, S. B., J. Immunol. 122, 2112, 1979. 3. Farrar, J. J., Simon, P. L., Farrar, W. L., Koopman, W. J., and Fuller-Bonar, J., Ann. N.Y. Acad. Sci. 332, 303, 1979. 4. Rosenwasser,L. J., Dinarello, C. A., and Rosenthal, A. S., J. Exp. Med. 150, 709, 1979. 5. Larsson, E-L., Iscove, N. N., and Coutinho, A., Nature (London) 283,664, 1980. 6. Smith, K. A., Lachman, L. B., Oppenheim, J. J., and Favata, M. F., J. Exp. Med. 151, 1551, 1980. 7. Maizel, A. J., Mehta, S. R., Ford, R. J., and Lachman, L. B., J. Exp. Med. 153, 470, 1981. 8. Simon, P. L., and Willoughby, W. F., J. Immunol. 126, 1534, 1981. 9. Lachman, L. B., Hacker, M. P., Blyden, G. T., and Handschmacher, R. E., Cell. Immunol. 34, 416, 1977.
10. Mizel, S. B., Oppenheim, J. J., and Rosenstreich, D. L., J. Immunol. 120, 1504, 1978. 11. Lachman, L. B., Moore, J. O., and Metzgar, R. S., Cell. Immunol. 41, 199, 1978. 12. Palacios, R., Ivhed, I., Sideras, P., Nilsson, K., Sugawara, I., and Femandez, C., Eur. J. Immunol. 12, 895, 1982.
13. Butler, R. H., Revoltella, R. P., Musiani, P., and Piantelli, M., Cell. Immunol. 78, 368, 1983. 14. Krakauer, T., and Oppenheim, J. J., CeM.Immunol. 80, 223, 1983. 15. Fisher, R. I., Bostick-Bruton, F., Sauder, D. N., Scala, G., and Diehl, V., J. Immunol. 130, 2666, 1983.
16. Luger, T. A., Stadler, B. M., Luger, B. M., Sztein, M. B., Schmidt, J. A., Hawley-Nelson, P., Grabner, G., and Oppenheim, J. J., J. Invest. Dermatol. 81, 187, 1983. 17. Komiyama, A., Kawai, H., Miyagawa, Y., and Akabane, T., Blood 60, 1429, 1982. 18. Ragsdale, C. G., and Arend, W. P., J. Exp. Med. 149, 954, 1979. 19. Miyagawa, Y., Komiyama, A., Akabane, T., Uehara, Y., and Yano, A., J. Immunol. 129, 1993, 1982.
20. Kawai, H., Hirabayashi, S., Miyagawa, Y., Komiyama, A., and Akabane, T., Cancer 52, 1215, 1983. 21. Gillis, S., and Watson, J., J. Exp. Med. 152, 1709, 1980. 22. Kaplan, M. E., and Clark, C., J. Immunol. Meth. 5, 131, 1974. 23. West, W. H., Cannon, G. B., Kay, H. D., Bonnard, G. D., and Herberman, R. B., J. Immunol. 118, 355, 1977. 24. Timonen, T., Ortaldo, J. R., and Herberman, R. B., J. Exp. Med. 153, 569, 1981. 25. Ortaldo, J. R., Sharrow, S. O., Timonen, T., and Herberman, R. B., J. Immunol. 127, 2401, 1981. 26. Lohmann-Matthes, M-L., Domzig, W., and Roder, J. J., J. Immunol. 123, 1883, 1979. 27. Djeu, J. Y., Heinbaugh, J. A., Holden, H. T., and Herberman, R. B., J. Immunol. 122, 182, 1979. 28. Minato, N., Reid, L., Cantor, H., Lengyel, P., and Bloom, B. R., J. Exp. Med. 152, 124, 1980. 29. Saksela, E., and Timonen, T., In “Natural Cell-Mediated Immunity Against Tumors” (R. B.
Herberman, Ed.), p. 173, Academic Press,New York, 1980. Dennert, G., De Rose.,M., and Allen, R. S., Eur. J. Immunol. 7, 478, 1977. Fathman, C. G., and Hengartner, H., Nature (London) 272, 617, 1978. Kasahara, T., Djeu, J. Y., Dougherty, S. F., and Oppenheim, J. J., J. Immunol. 131, 2379, 1983. Scala, G., Allavena, P., Djeu, J. Y., Kasahara, T., Ortaldo, J. R., Herberman, R. B., and Oppenheim, J. J., Nature (London) 309, 56, 1984. 34. Luger, T. A., Stadler, B. M., Katz, S. I., and Oppenheim, J. J., J. Immunol. 127, 1493, 1981. 35. Fontana, A., Kristensen, F., Dubs, R., Gemsa, D., and Weber, E., J. Immunol. 129, 2413, 1982. 30. 31. 32. 33.