CELLULAR

IMMUNOLOGY

131, 184-190 (1990)

Differential Regulation of Interleukin-1 Gene Expression in Human CD3- Large Granular Lymphocytes MARIA-CHRISTINA GALLI,**' MARKJ. SMYTH,*,~HOWARD A. YOUNG,* CRAIG W. REYNOLDS,~.ANDJOHNR.ORTALDO*,~ *Laboratory

of Experimental Immunology, tBiologica1 Response Modifiers NCI-FCRDC, Frederick, Maryland 21702

Program,

Received May 18, 1990; accepted July 11, 1990

Our laboratory analyzed the expressionof lymphokine and cytokine mRNA in CD3- peripheral blood large granular lymphocytes (LGL). Herein we presentevidencethat this subsetof lymphocytes can synthesize IL- 1fi mRNA constitutively and that the cytoplasmic mRNA levels of IL- l/3 can be increasedrapidly by interleukin (IL)-2. IL-l (YmRNA is expressedconstitutively very infrequently and increasesin IL- 1(YmRNA are seen only after prolonged incubation with IL-2. Furthermore, IL-l activity could not be detected in LGL culture supematants, indicating that other processes may be involved in releasing biologically active IL-l from LGL. In addition, MAb to the p75 IL2 receptor on LGL abrogated IL-2 induction of IL-l@ mRNA, suggestingthat IL-2 signaling via the p75 IL-2 receptor induced IL-lp gene expression in LGL. Since, in contrast to T cells, LGL are capable of mediating effector functions without prior stimulation, they are said to be already “primed” for response.Overall, these data suggestthat constitutive lymphokine gene expression may be involved in the in vivo priming of LGL. o 1990 Academic press, I~C.

INTRODUCTION Large granular lymphocytes (LGL)4 are present at a level of l-3% of total circulating lymphocytes (1). These cells exert natural killer (NK) activity, can be activated to develop lymphokine-activated killer (LAK) activity, and are able to produce a variety of cytokines, including IL2, IL-4, EN-~, and TNF. Therefore, LGL perform cytotoxic and immunoregulatory functions which enable them to play a central role in immune system function. Previous studies on gene regulation in lymphocyte populations have focused on T and B lymphocytes and activated lymphocyte populations such as LAK cells (2, 3) while comparatively few studies have been performed on freshly isolated CD3- LGL. A measurable level of cytokine mRNA is not evident in freshly isolated T lymphocytes, consistent with the in viva-nonactivated state of the great majority of fresh PBMC. LGL, however, are believed to be in vivo “primed” since they readily kill the target ’ Current address:Instituto Sup&ore di Sanita, Lab Biologia Cellulara, Viale Regina Elena, 00 161, Rome, Italy. * Supported by a C. J. Martin travelling fellowship of the National Health and Medical ResearchCouncil of Australia. 3 To whom correspondence should be addressed. 4 Abbreviations used:LGL, large granular lymphocytes; NK, natural killer; IL, interleukin; PBS, phosphatebuffered saline. 184 OOOS-8749190 $3.00 Copyright 0 1990 by Academic Pres$ Inc. All rights of reproduction in any form reserved

IL-I GENE EXPRESSION IN NK CELLS

185

cells without prior stimulation. However, it has previously been demonstrated that LGL IFN-y mRNA is rapidly increased following IL-2 stimulation (4) and that this stimulation can be blocked by agents which inhibit protein kinase C (5). Although previous studies have examined IL- 1-like activity from NK clones and NK cells, in the present study we have further studied LGL gene regulation by examining the expression of IL-I cytokine genes involved in the early phases of cell activation and response. Our data presented herein indicate that human CD3- LGL constitutively expressIL- l/3 mRNA, while IL- 1a!mRNA is not detectable. Moreover, IL-l/3 expression is up-regulated by IL-2 activation of LGL (consistent with the induction observed in LAK populations) (3) and this IL-2 up-regulation is mediated via the ~75 IL-2 receptor constitutively expressedon LGL. Therefore, in human LGL a differential regulation of IL- 1 genesis evident at the cytoplasmic mRNA level consistent with the in vivo primed state of LGL. MATERIALS AND METHODS Cells. Monocytes, T cells, and LGL were purified from peripheral blood of healthy donors according to Timonen et al. (6). The purity of resulting subpopulations was always checked by Giemsa staining and by flow cytometry analysis using a panel of monoclonal antibodies reactive specifically with monocytes, T, B, or NK subsets.In particular, the LGL populations were consistently >93% LGL by morphology and 3000 Ci/ mmol). All lanes were determined to contain equivalent amounts of total cellular RNA as determined by ethidium bromide staining or hybridization to a murine 18s ribosomal RNA cDNA probe (data not shown) unless otherwise indicated. The cDNA probes for human IL-a and -p were gifts from Dr. M. Yamada (Dainippon Pharmaceuticals, Osaka, Japan) and Dr. D. Carter (Upjohn Co., Kalamazoo, MI), respectively, while the cDNA probe for murine 18s ribosomal RNA was provided by Dr. L. Varesio

186

GALL1 ET AL.

2.2 kb

1.6 kb

FIG. I. Expression of IL- 1 mRNA in pooled LGL preparations after IL2 stimulation. Lane 1, unstimulated LGL; lane 2, stimulated with IL-2 for 18 hr; lane 3, stimulated with IL-2 for 2 hr; lane 4, stimulated with IL-2 for 1 hr; lane 5, stimulated with IL-2 for 30 min. The membrane was exposed to X-ray film for 48 hr.

(FCRDC-NCI, Frederick, MD). The anti-IL-2 receptor fi chain antibody was provided by Dr. K. Sugamura, (Tohoku University School of Medicine, Sendai, Japan). RESULTS

IL-l cytoplasmicmRIKAproduction in LGL. LGL were pooled in initial experiments from two separategroups of donors and the RNA was analyzed by hybridization with IL-la and IL- l/3 cDNA probes. As shown in Fig. 1, IL-l/3 mRNA expression was measurable in unstimulated LGL (lane l), reached an increased, steady level after 30 min of IL-2 stimulation (lane 5), and was maintained for at least 18 hr (lane 2). Note that the RNA utilized in lane 1 was partially degraded, which accounts for the smaller size of the hybridizing RNA. However, this hybridization was specific since no binding to IL- 1cxprobes were seen. It should be noted that these culture conditions optimally activated LGL, asdemonstrated by NK activity and IFN-7 production (data not shown) ( 11). In contrast to IL- l,&, IL- 1(YmRNA was detected only after 18 hr of culture (lane 2). Using the same culture conditions, purified T cells from the same pooled donors were negative for both IL-la and -/I gene expression (data not shown). IL-l mRNA expressionin LGL from individual donors.To determine if constitutive IL- 1p mRNA expression was due to a single donor whose LGL were in vivoactivated, we analyzed several individual donors. Highly purified LGL from individual donors showed constitutive expression of IL-lp message(Fig. 2, lane 5; Fig. 3, lane 3; other data not shown). In contrast to untreated and LPS-activated monocytes, CD3- LGL did not express IL- 1LYmRNA. 2 Hour Exposure

2 Day Exposure I

I

I

1234567 IL-la

,“I,

I

1234567 .

2.2 kb

*

1.6 kb

FIG. 2. Comparison of IL-1 mRNA expression in human monocytes and LGL. Northern analysis of total cellular RNA. Lane 1, donor 1 human monocytes after 3 hr adherence;lane 2, donor 1 human monocytesRNA after 18 hr adherence; lane 3, donor 2 monocytes after 18 hr adherence; lane 4, donor 1 monocytes after 18 hr LPS treatment; lane 5, untreated LGL, lane 6, 18-hr IL-2-treated LGL; lane 7, 18-hr LPS-treated LGL. At left, exposure of IL-la and IL-l/3 mRNA for 48 hr. On the right, 2-hr exposure of bottom panel only (IL- 18) hybridization.

IL-I GENE EXPRESSION IN NK CELLS

187

As shown previously with pooled donors, a selective increase in the level of IL-l@ mRNA was observed in LGL from an individual donor after IL-2 stimulation (Fig. 2, lane 6) when compared with unstimulated LGL (Fig. 2, lane 5). The same increase of IL- l/3 mRNA was not detected after LPS stimulation of LGL (Fig. 2, lane 7). As previously seenwith pooled cells, IL- 1(Ygene expression was never observed in donors even after IL-2. As a positive control for IL- 1(Yand IL- l/3 mRNA expression,monocytes from two donors were cultured for various times and/or stimulated with LPS (see Figs. 2 and 3). Lanes 1, 2, and 4 (Fig. 2) depict the level of IL-l@ mRNA in donor 1, while lane 3 (Fig. 2) is donor 2. These data demonstrated that monocytes expressed both IL- 1a! and IL- l/3 mRNA and that these levels were augmented by LPS. (Note: 2-hr exposure for IL-l/3 mRNA only is shown in Fig. 2). Eflect of monocyte contamination on IL-I mRNA analysis. To rule out the possibility that a very small contamination by monocytes could account for the IL-l@ message level, we performed a Northern analysis using RNA from purified T cells (consistently negative) spiked with increasing amounts of RNA from LPS-activated monocytes (Fig. 3). As expected, monocytes alone (Lane 1) or cultured in the presence of LPS (lane 2) expressedIL- 1(Yand IL- l@ mRNA, while untreated LGL expressedonly ILIp mRNA (Lane 3). As shown in Fig. 3, under the same hybridization conditions where IL-l@ mRNA was detected in LGL, IL-l/3 mRNA was only readily detected in T cell preparations that had been intentionally contaminated with RNA equivalent to 10% monocytes (lane 6). Considering the average RNA yield obtained from the two cell populations, a 5% contamination in terms of grams of RNA correspond to a 10% monocyte contamination in terms of cells. At levels of monocyte contamination comparable to that seen in LGL populations (0. l-l .O%,lanes 4 and 5, respectively), IL-18 mRNA was either not detected or present only as a very faint band after 48 hr exposure of the membrane to X-ray film. IL-2 induction of LGL IL-10 mRNA via the ~75 IL-2 receptor. The expression of functional IL-2 receptors on peripheral blood LGL has previously been confirmed by flow cytometric analysis with the TU-27 (anti-p75 IL-2 receptor) MAb (12). In order to determine the involvement of ~75 IL-2 receptors in the induction of IL- l/3 mRNA in human peripheral blood LGL, IL- l@mRNA expressionwas examined after culturing CD3- LGL with IL-2 in the presence or absenceof TU-27 MAb. As shown in Fig. 4, IL-2 induced IL- lp mRNA (lane 3) 4.3-fold above that of the untreated LGL control (lane 1). This induction was almost completely inhibited by TU-27 MAb (lane 4) reducing the density of hybridization from 4.3- to 1.6-fold stimulation compared to lane 1. Analyzing this Northern blot with a murine 18s RNA probe indicated that the apparent small reduction in IL-l@ mRNA in LGL treated with TU-27 MAb alone was due entirely to a reduced amount of total RNA (3-fold less) loaded in lane 2 (data

123456

2.2 kb SW 1.6 kb

FIG. 3. Potential for monocyte contamination in CD3- LGL RNA. Northern analysis of total cellular RNA. Lane 1, monocytes adhered for 3 hr; lane 2, 2-hr LPS-treated adherent monocytes; lane 3, untreated LGL; lane 4, T cells + 0.1% LPS-treated monocytes; lane 5, T cells + 1% LPS-treated monocytes; lane 6, T cells + 5% LPS-treated monocytes.

188

GALL1 ET AL. 1234

1.6 kb

FIG. 4. Effect of anti-p75 IL-2 receptor MAbs on IL-Zinduced IL- 1s mRNA expressionin human peripheral blood CD3- CD56’ LGL. Northern analysis of total cellular RNA. LGL were cultured with: lane 1, 18 hr untreated, lane 2, 18 hr Tu-27 MAb (10 g/ml); lane 3, 18 hr IL-2 (1000 U/ml); and lane 4, 18 hr IL-2 (1000 U/ml) plus Tu-27 MAb (10 g/ml).

not shown). In conclusion, these data indicate that the ~75 IL-2 receptor may be directly involved in IL-2 induction of LGL IL-lp mRNA. LGL production of intracellular IL-I. LGL obtained from four additional donors were also analyzed for intracellular and extracellular IL- 1 activity. A summary of the intracellular data is presented in Table 1. Untreated LGL from all four individuals tested showed a measurable level of intracellular IL-l activity (Table l), this activity was not detectable in the supernatant (not shown). Moreover, IL-2-stimulated LGL had a higher intracellular IL-l activity than untreated LGL. A similar increase in intracellular IL-l was not observed in IL-2-activated T cells (see donor No. 3, Table 1). It should be noted that intracellular IL-l activity was not due to the presence of endogenous IL-2 since the IL-2 was removed and the cells were allowed to incubate TABLE I Production of Intracellular IL- I in IL-2-Stimulated LGL: Correlation with IL- 1(Yor -0 mRNA Expression

Donor

Target cell

Treatment

Incorporation of [‘Hlthymidine (cpm k SD)

No. I

DlO.G4.1

Con A Con A + IL-l” IL-2 IL-2 IL-2 IL-2

999 k 119 47,167 + 1,987 17,183 + 3,440 84,972 k 3,134 31,294 k 4,SS1 55,901 f 7,261 2,280 k 250 1,426 f 171 47,622 + 1,354 59,789 -c 1,038 7,821 f 1,635 77,607 + 12,270

No. 3

No. 4 No. 6

LGL Monocytes LGL LGL T T LGL LGL LGL LGL

Note. Data were obtained after a 140 dilution of the cell lysate. ’ Recombinant IL-I (10 U/ml). b Not analyzed.

Detectable mRNA for IL-l@ N.A.6 N.A. + + + +

N.A. + b

IL-I

GENE

EXPRESSION

IN NK CELLS

189

for 8 hr in the absence of continued stimuli before supernatants were collected. No residual IL-2 activity was detectable in these culture supematants (data not shown). DISCUSSION IL-2-dependent activation of LGL involves multiple mechanisms, some of which have already been described (4, 11). However, the regulation of gene expression in LGL has been only recently addressed (13, 14). We have studied the constitutive expression and IL-2 regulation of IL-l mRNA expression in LGL from eight donors. Although previous results have indicated an IL-l-like activity from NK cells (2), activated lymphocytes (2, 3), or NK clones (l), our results indicate that highly purified human LGL constitutively express mRNA for IL-l@, but not for IL-la. Given that T cells do not expressIL- 1 mRNA, monocytes expressboth IL- 1(Yand IL- l/3 mRNA, and that we have only detected constitutive IL- lp mRNA in LGL, it is clear that major differences exist in the IL-l gene expression of these lymphocyte subsets. In addition, we have demonstrated that IL-2 can increase IL-lp mRNA expression in LGL in sevenof eight donors. Thesedata at the RNA level correlate with the measurable IL-l activity of LGL lysates which were also increased after culturing LGL with IL2. These data confirm and extend a previous study which analyzed the cytokine gene expression pattern of a heterogeneous population of LAK cells (3). In contrast to IL-l@ mRNA, IL- 1(YmRNA was only constitutively expressedin the LGL from one of eight donors. In this donor, neither IL-2 nor LPS could up-regulate this IL-1 a mRNA expression (data not shown). In viva preactivation of LGL may possibly result in constitutive IL- 1cxgene expression; however, this hypothesis requires further investigation. In addition, the observation that IL-la mRNA expression may be increased only after prolonged stimulation by IL-2 suggeststhat other, as yet undefined, pathways ( 15) may also be involved in the control of IL- 1a gene expression. A recent report of Numerof et al. ( 16) indicated that a major source of secretedIL1a//3 from peripheral blood mononuclear cells was the monocytes [LeuM3+, Leu 1l-1. Since most, if not all, of the CD3- LGL IL-l is not secreted after culture, with or without IL-2, the present findings are consistent with the observation that most secreted IL-l is not derived from CD3- LGL. These results are in contrast to the reports of Cuturi ( 17) which did not demonstrate IL- 1 from CD3-, CD56+ cells after stimulation with IL-2. However, the cells employed in this study were long-term cultures of CD3lymphocytes and may represent a selected subset that grows with IL-2, but does not produce IL- 1. This would be consistent with previous data using sorted CD3-, CD 16’ LGL subsets,which were selective in their IL- 1 production to LPS stimulation to cells bearing CD1 1b and HLA-Dr (1). However, the lack of detectable supematant IL-l could be due to inhibitors present after IL-2 stimulation. This possibility is presently under investigation. In addition, the lack of IL- 1p could be due to the lack of specific proteases needed to cleave the IL-l/3 precursor into a mature secreted product ( 18, 19). The present study has not employed RIA or ELISA to measure IL- 1 protein since we were interested in detecting biological active protein and the specificity of the IL1 wasdetermined by the hybridization probes. Previous studies examining LPS-induced LGL (which resulted in secreted IL-l) involved 48 hr stimulation. However, in the present study, our emphasis was on rapidly ( 18 hr) IL-2-induced activation. The mechanism by which IL-2 up-regulates IL- 1 mRNA expression was investigated and it appears from our data (Fig. 4) that IL-2 induction of IL-l/!I mRNA is mediated through the ~75 IL-2 receptor. The ~75 IL-2 receptor has been demonstrated to be

190

GALL1 ET AL.

constitutively expressedon CD3- CD56+ LGL (20,21) and to be functional in IL-2induced activation of NK (18, 19) and LAK (22) activity. These data, implicating that IL-2 activation of NK cells is via the ~75 IL-2 receptor, are further supported by our finding that IL-2 induces IL-lfi mRNA expression in LGL through the ~75 IL-2 receptor. However, these data also suggestthat additional stimuli are needed for IL1p secretion. In summary, the data presented here indicate that in CD3- LGL, IL-lb but not IL- 1CYmRNA was constitutively produced in LGL. IL- l/3 mRNA could be rapidly modulated by IL-2. This finding is in contrast to observations in other lymphocyte populations. Further analysis of the mechanism(s) by which IL- l/3 mRNA levels were increased in LGL will be important in dissecting and understanding the signal transduction events involved in the functional activation of CD3- LGL. ACKNOWLEDGMENTS We thank Drs. Scott Durum and Dan Longo for critical reading of this manuscript and Ms. Susan Charbonneau and Ms. Amy Wenner for secretarial assistance.

REFERENCES 1. Ortaldo, J. R., and Herberman, R. B., In “Annual Review of Immunology” (W. E. Paul, C. G. Fathman, and H. Metzger, Eds.), p. 359. Annual Reviews, Inc., Palo Alto, CA, 1984. 2. Scala, G., Allavena, P., Djeu, J. Y., Kasahara, T., Ortaldo, J. R., Herberman, R. B., and Oppenheim, J. J., Nature (London) 309, 56, 1984. 3. Kovacs, E. J., Beckner, S. K., Longo, D. L., Varesio, L., and Young, H. A., Cancer Res. 49, 940, 1989. 4. Young, H. A., and Ortaldo, J. R., J. Immunol. 139, 124, 1987. 5. Ortaldo, J. R., Young, H. A., and Varesio, L., J. Immunol. 143, 366, 1989. 6. Timonen, T., Ortaldo, J. R., and Herberman, R. B., J. Exp. Med. 153,569, 1981. 7. Kaye, J., Gillis, S., Mizel, S. B., Shevach, E. M., Malek, T. R., Dinarello, C. A., and Lachman, L., J. Immunol. 133, 1339, 1984.

8. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J., Biochemistry 18, 5294, 1979. 9. Glisin, V., Crkvenjakov, R., and Byus, C., Biochemistry 13, 2633, 1974. 10. Thomas, P. S., “Methods in Enzymology” (R. Wu, L. Grossman, and K. Moldave, Eds.), Vol. 100, p. 255. Academic Press,New York, 1983. 1I. Yamada, S., and Ortaldo, J. R., J. Leukocyte Biol. 42, 263, 1987. 12. Yagita, H., Nakata, M., Azuma, A., Nitta, T., Takeshita, T., Sugamura, K., and Okumura, K., J. Exp. Med. 170, 1445, 1989.

13. Anegon, I., Cuturi, M. C., Trinchieri, G., and Perussia,B., J. Exp. Med. 167,452, 1988. 14. Smyth, M. J., Ortaldo, J. R., Shinkai, Y.-I., Yagita, H., Nakata, M., Okumura, K., and Young, H. A., J. Exp. Med. 171, 1269, 1990.

15. Herman, J., and Rabson, A. R., Clin. Immunol. Immunopathol. 38,282, 1986. 16. Numerof, R. P., Aronson, F. R., and Mier, J. W., J. Immunol. 141, 4250, 1988. 17. Cuturi, M. C., et al., J. Exp. Med. 169, 569, 1989. 18. Matsushima, K., Taguchi, M., Kovacs, E. J., Young, H. A., and Oppenheim, J. J., J. Immunol. 136, 2883,1986. 19. Durum, S. K., and Oppenheim, J. J., In “Fundamental Immunology” (W. E. Paul, Ed.), 2nd ed., p.

639. Raven Press,New York, 1989. 20. Phillips, J. H., Takeshita, T., Sugamura, K., and Lanier, L. L., J. Exp. Med. 170, 291, 1989. 21. Takeshita, T., Goto, Y., Tada, K., Nagata, K., Asao, H., and Sugamura, K., J. Exp. Med. 169, 1323, 1989. 22. Ortaldo, J. R., Frey, J., Farrar, W. L., Takeshita, T., and Sugamura, K., Eur. Cytokine Network, 1,27,

1990.