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SYNERGISTIC PROLIFERATION AND ACTIVATION OF NATURAL KILLER CELLS BY INTERLEUKIN 12 AND INTERLEUKIN 18
Article No. cyto.1999.0501, available online at http://www.idealibrary.com on
SYNERGISTIC PROLIFERATION AND ACTIVATION OF NATURAL KILLER CELLS BY INTERLEUKIN 12 AND INTERLEUKIN 18 Bernard R. Lauwerys,1 Jean-Christophe Renauld,2,3 Fre´de´ric A. Houssiau1 We investigated the effects of IL-12 and IL-18 on unstimulated murine splenocytes and observed that the two cytokines strongly synergized for their proliferation, whereas IL-12 and IL-18 alone were essentially inactive in this respect. Phenotypical and functional analyses of cells proliferating in response to IL-12 and IL-18 revealed that large granular Ly-49C + DX5 + CD3 NK blasts were expanded in these cultures and that they displayed cytotoxic activity against Yac-1 cells, a murine NK cell target. Further analyses indicated three major differences between NK cells appearing in response to IL-12 and IL-18 and those derived in the presence of other NK cell growth factors, such as IL-2 or IL-15. First, a population of T-NK cells, i.e. expressing T cell (TCR, CD3) and NK cell (Ly-49) markers, was detected amongst cells growing in IL-2 or IL-15 but not in cultures supplemented with IL-12 and IL-18. Second, most NK cells derived with IL-2 or IL-15 expressed the NK1.1 antigen, while those derived with IL-12 and IL-18 did not. Finally, striking differences were observed regarding cytokine production. Cells stimulated with IL-12 and IL-18 in combination, but not with IL-2 or IL-15, produced IFN-, IL-3, IL-6 and TNF. IFN- was not involved in the response of NK cells to IL-12 and IL-18, as indicated by experiments demonstrating that the combination of the two cytokines displayed similar effects on spleen cells from IFN-R-knock-out mice. Receptor (IL-12R1, IL-12R2 and IL-18R) gene expression studies did not indicate that the mechanism underlying the synergy between IL-12 and IL-18 involved reciprocal induction of their receptors. Taken together, our results demonstrate that IL-12 and IL-18 exert striking synergistic activities for NK cell proliferation and activation, distinct from those induced by IL-2 or IL-15. 1999 Academic Press
Interleukin (IL)12 is a heterodimeric cytokine produced by B cells, macrophages and dendritic cells that plays a well-known immunoregulatory role by promoting cell-mediated immunity, in particular through upregulation of IFN- production.1 Another phagocyte-derived cytokine displaying a potent IFN-inducing activity, provisionally called IGIF (IFN-inducing factor), was recently cloned2 and re-named IL-18.3. Interestingly, IL-12 and IL-18 were found to synergize for IFN- production by human and murine T cells.4–6 We and others unmasked a similar synergy between the two cytokines for IFN- production by B From the 1Rheumatology and 2Experimental Medicine Units, Christian de Duve Institute of Cellular Pathology, Universite´ catholique de Louvain, Bruxelles, Belgium; and 3Ludwig Institute for Cancer Research, Brussels Branch, Belgium Correspondence to: Fre´de´ric A. Houssiau, Rheumatology Unit, UCL 5390, Avenue Hippocrate 10, B-1200 Bruxelles, Belgium; E-mail: [email protected] Received 24 September 1998; accepted for publication 11 January 1999 1999 Academic Press 1043–4666/99/110822+09 $30.00/0 KEY WORDS: Interleukin 12/Interleukin 18/NK cells 822
cells. Thus, murine naive B cells stimulated with antiCD40 mAb and IL-4 produce huge amounts of IFN- in response to the combination of IL-12 and IL-18.7 Similarly, activated B cells isolated from mice with chronic graft-vs-host disease produce IFN- after stimulation with both cytokines.8 In these two experimental settings, the synergy between IL-12 and IL-18 for IFN- induction accounted for the strong inhibition of immunoglobulin (Ig) production exerted by the two cytokines.7,8 These results, by indicating that the synergy between IL-12 and IL-18 was operating on at least two distinct lymphocyte subsets, prompted us to investigate further the effects of IL-12 and IL-18 on normal murine splenocytes. This approach led to the intriguing observation that the two cytokines strongly synergized for the proliferation of unstimulated murine spleen cells, whereas IL-12 and IL-18 alone were inactive in this respect. Characterization of splenocytes proliferating in response to IL-12 and IL-18 indicated that they belonged to the natural killer (NK) cell lineage and CYTOKINE, Vol. 11, No. 11 (November), 1999: pp 822–830
Synergistic activation of NK cells by IL-12 and IL-18 / 823
Surface markers analysis of cells proliferating in response to IL-12 and IL-18, IL-2 or IL-15 The phenotype of cells proliferating in response to the combination of IL-12 and IL-18 was further studied and compared to that of cells cultured in the presence of IL-2 or IL-15, two other well-known NK cell growth factors. Double-labelling experiments revealed that Ly-49C + cells proliferating in response to IL-12 and IL-18 consisted of a homogeneous population of Ly-49C + NK1.1 CD3 NK cells (Figs 2A and 2C). While a population of Ly-49C + blasts was also detected in cultures supplemented with IL-2, these cells differed from those appearing in cultures supplemented with IL-12 and IL-18 regarding CD3 and
IL-2 IL-12 IL-18
IL-12 + IL-18 0
5
10
15
20
25
30
35
40
45
3 [ H]Thymidine incorporation (kcpm)
B
No stimulation
IL-12 + IL-18
SSC
Nylon wool-filtered splenocytes were cultured in the presence or absence of IL-12 and/or IL-18. As indicated in Figure 1A, addition of IL-12 and IL-18 alone had no effect on cell proliferation. By contrast, the combination of the two cytokines induced as strong a proliferation as that observed with IL-2 (50 U/ml), with half-maximal stimulation being obtained with 10 pg/ml of IL-12 and 50 ng/ml of IL-18. To characterize the cells responding to IL-12 and IL-18, we performed FACS analyses with T cell (CD3), B cell (CD19), monocyte (Mac-1) and NK cell (Ly-49C, DX-5) markers. These experiments revealed that a population of large (high FSC), granular (high SSC), Ly-49C + DX5 + CD3 NK blasts was present in cultures stimulated with IL-12 and IL-18 (Fig. 1B and 1C). Similar results were obtained with nylon woolfiltered splenocytes prepared from C57BL/6 and 129 mice. In order to confirm that cells proliferating in response to the combination of IL-12 and IL-18 belonged to the NK cell lineage, we performed cytotoxity assays using NK-sensitive Yac-1 cells as targets and nylon wool-filtered splenocytes as effectors. Spleen cells cultured with IL-12 and IL-18 in combination, exerted a strong cytotoxic activity, comparable to that observed with cells supplemented with IL-2. Again, a strong synergy between IL-12 and IL-18 was unmasked (Table 1). Cells proliferating in response to IL-12 and IL-18 were sorted by flow cytometry on the basis of their FSC/SSC pattern (upper-right region in Figure 1B) and they also displayed a potent cytotoxic activity on Yac-1 targets (Table 1).
Cytokines added
Synergistic proliferation and activation of NK cells induced by IL-12 and IL-18
–
SSC
RESULTS
A
FSC
FSC
C
DX5
Cell counts
that these NK cells were phenotypically and functionally different from those derived in the presence of IL-2 or IL-15.
Ly-49C
Log fluorescence Figure 1.
Proliferation of NK cells.
Nylon wool-filtered Balb/c splenocytes were cultured in the presence of the indicated cytokine(s), as described in Materials and Methods. Proliferations (mean kcpmSEM) were measured by [3H]thymidine incorporation (A). The plots shown in (B) indicate the distribution of cells (cultured without or with IL-12 and IL-18) along the forward scatter channel (FSC) and the side scatter channel (SSC) on flow cytometric analysis. FACS analyses (DX5, Ly-49C) shown in (C) were gated on large granular cells appearing in response to IL-12 and IL-18, as defined by the area depicted in (B). Shaded areas correspond to labelling obtained with an isotype-matched control mAb. The data presented in A, B and C are representative of 12, 10 and 8 experiments, respectively.
NK1.1 expression. Thus, most (80%) Ly-49C + cells present in IL-2 cultures were NK1.1 + (Fig. 2B) and could be categorized according to CD3 expression into a Ly-49C + CD3 + subset of T-NK cells and a Ly-49C + CD3 subset of NK cells (Fig. 2D). These experiments
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TABLE 1.
CYTOKINE, Vol 11, No. 11 (November, 1999: 822–830)
Lytic activity of NK cell populations Cytokines added
Cells Total N + cells
Sorted cells*
E/T ratio
None
IL-12
IL-18
IL-12 IL-18
IL-2
100 30 10 3 1 30 10 3 1
0 0 0 0 0 ND ND ND ND
19 4 0 0 0 ND ND ND ND
0 0 0 0 0 ND ND ND ND
58 41 18 3 2 69 27 17 6
62 36 33 16 6 61 52 27 13
Figures represent percentage of specific lysis (SD<10%) of 51Cr-labelled Yac-1 cells (1103 cells) measured in quadruplicate (for total N + cells) or triplicate (for sorted cells) experiments; effector cells used in the cytotoxity assays have been previously stimulated for three days with the indicated cytokines, as described in Materials and Methods. *Cells proliferating in response to IL-12 and IL-18 were sorted by flow cytometry on the basis of their FSC/SSC pattern (upper-right region in Fig. 1B). E/T: effector/target. N + ; nylon wool-filtered. These data are representative of four and two distinct experiments performed on total N + and sorted cells, respectively.
also indicated that cultures supplemented with IL-2 contained a population of CD3 + T cell blasts (Fig. 2D), that were absent in cultures stimulated with IL-12 and IL-18. We also investigated the effects of IL-15 in our experimental setting. IL-15 strongly stimulated the proliferation of nylon-wool filtered splenocytes (stimulation index: 101 and 142 in two experiments) and FACS analyses indicated that cells proliferating in response to IL-15 were phenotypically similar to those responding to IL-2, with three distinct populations being identified, namely NK cells (Ly-49C + CD3 ), T cells (Ly-49C CD3 + ) and T-NK cells (Ly-49C + CD3 + ). Again, most (89%) Ly-49C + cells induced by IL-15 were NK1.1 + (data not shown).
Cytokine production in cultures stimulated with IL-12 and IL-18, IL-2 or IL-15 We measured the concentrations of various cytokines in the supernatants from cells stimulated with IL-12 and/or IL-18 and compared them to those determined in cultures supplemented with IL-2 or IL-15. As indicated in Figure 3, IL-12 and IL-18 were essentially inactive on their own but strongly synergized, not only for IFN- production but also for IL-3, IL-6 and TNF induction. By contrast, IL-2 and IL-9 were not detected in cultures stimulated with IL-12 and IL-18. Interestingly, addition of IL-2 or IL-15 had no significant effect on the production of IL-3, IL-6, IL-9, IFN- and TNF. In order to determine whether IFN- detected in cultures supplemented with IL-12 and IL-18 was produced by cells proliferating in response to these
cytokines, we performed cytoplasmic IFN- staining experiments by FACS analyses. These experiments confirmed that large granular cells appearing in response to IL-12 and IL-18, and not those proliferating in response to IL-2 or IL-15, produced IFN- (Fig. 4). More specifically, double-labelling experiments indicated that Ly-49C + blasts produced IFN- in response to IL-12 and IL-18 (data not shown).
IFN- does not mediate the proliferation and activation of NK cells induced by IL-12 and IL-18 Next, we evaluated whether IFN- was involved in the proliferation of NK cells in response to IL-12 and IL-18. Although large amounts of IFN- were produced by cells stimulated with IL-12 and IL-18, two lines of evidence argued against the possibility that IFN- was involved in the proliferation of NK cells induced by the two cytokines. First, addition of IFN- to spleen cells did not induce NK-cell proliferation. Second, splenocytes isolated from IFN-R knock-out 129 mice responded similarly to IL-12 and IL-18 compared to wild type 129 mice, both in terms of proliferation and cytotoxicity (Fig. 5).
IL-12R and IL-18R gene expression studies We wondered whether the synergy between IL-12 and IL-18 for the proliferation and activation of NK cells could be explained by reciprocal induction of their receptor. We addressed this issue by RT-PCR analyses performed on cDNA synthesized after RNA extraction from cells cultured in the absence or presence of IL-12 and/or IL-18. Specific murine IL-12R1, IL-12R2 and IL-18R oligonucleotides were used as primers. For the latter, we assumed that the murine IL-18R gene was identical to the previously cloned murine IL-1R related protein (IL-1Rrp) gene, as recently described for human cells.9 As indicated in Figure 6, IL-12R1, IL-12R2 and IL-18R gene expression was detected in unstimulated cells. Addition of IL-12 and IL-18 increased IL-12R, IL-12R1 and IL-12R2 gene expression but did not influence that of IL-18R. These results did not support the hypothesis that the mechanism underlying the synergy between IL-12 and IL-18 involved reciprocal induction of their receptors.
DISCUSSION NK cells play a critical role in innate immunity against pathogens, virus-infected cells and tumors through MHC-unrestricted cytotoxicity and their capacity to produce cytokines. Their function is a tightly regulated process, involving activating (CD16, CD2, CD28 and NKR-P1) and inhibitory (Ly-49,
Synergistic activation of NK cells by IL-12 and IL-18 / 825
B
A 1
45 12
2
40
3
NK1.1
60 37
Ly-49C
C
3
3
56 11 28
5
CD3
53 41
D
Ly-49C Figure 2.
Surface markers analysis.
Nylon wool-filtered C57BL/6 splenocytes were cultured with IL-12 and IL-18 (A and C) or with IL-2 (B and D), as described in Materials and Methods. After 3 days of culture, cells were double-labelled with fluorochrome-conjugated mAb (anti-NK1.1 and anti-Ly-49C in Panels A and B; anti-CD3 and anti-Ly-49C in Panels C and D). FACS analyses were gated on large granular cells appearing in response to IL-12 and IL-18 or to IL-2. Isotype-matched control mAb, using the same cut-off fluorescence values, stained no more than 0.5% of the cells (data not shown). The data are representative of two distinct experiments.
KIR) membrane receptors, the latter recognizing polymorphic MHC class I molecules.10,11 Several cytokines stimulate NK cell proliferation and activation, in particular IL-2,12 IL-12,13,14 IL-15,15,16 and IL-18.3,17–19 The experiments presented here demonstrate the existence of a striking synergy between IL-12 and IL-18, not only for the proliferation but also for the activation of NK cells, in particular for cytotoxicity and cytokine production. Functional comparison of cells appearing in response to the combination of IL-12 and IL-18 on the one hand, and in response to IL-2 or IL-15 on the other hand, revealed several differences. First, Ly-49 + NK cells appearing in response to IL-12 and IL-18 were NK1.1 , while most Ly-49 + NK cells detected in IL-2- or IL-15-supplemented cultures were NK1.1 + . NK1.1, the prototypical mouse NK cell antigen,20,21 is
a member of the family of NKR-P1 NK cell receptors.22 Although its function is currently unknown, some data indicate that NK1.1 plays a role in target recognition23 and functions as an activating signal. Regarding NK1.1 expression, our results contrast with those published recently by Tomura et al., suggesting that IL-18 plays an obligatory role in inducing NK1.1 + NK cells from murine CD4 CD8 sIg Ia splenocytes.19 In our experimental setting, IL-2 and IL-15 were capable, on their own, without addition of IL-18, to stimulate the proliferation of NK1.1 + NK cells. The reasons for this discrepancy are unclear but might be related to differences in the splenic cell preparations used to derive NK cells. Another phenotypical difference between cultures derived in the presence of IL-12 and IL-18 and those derived with IL-2 or IL-15 was the presence, in the
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CYTOKINE, Vol 11, No. 11 (November, 1999: 822–830)
A
B
– IL-12 IL-18 IL-12 + IL-18
Cytokines added
IL-2 IL-15 0
1
2
4 3 µg/ml
5
6
0
C
100
200 ng/ml
300
400
200
400 pg/ml
600
800
D
– IL-12 IL-18 IL-12 + IL-18 IL-2 IL-15 0 Figure 3.
10
20 pg/ml
30
40
0
Cytokine production.
Nylon wool-filtered Balb/c splenocytes were cultured with the indicated cytokine(s), as described in Materials and Methods. (A) IFN-; (B) IL-3; (C) IL-6; (D) TNF concentrations (meanSEM) were measured in supernatants from duplicate or triplicate cultures by specific bioassays performed in quadruplicate cultures. The data are representative of 3 distinct experiments for IFN- and IL-6 and 2 experiments for IL-3 and TNF.
latter, of a population of T-NK cells (Ly-49 + CD3 + ) that was not detected amongst cells responding to IL-12 and IL-18. T-NK cells represent a small percentage of T cells expressing both T cell (TCR, CD3) and NK cell (Ly-49) antigens.24 Their tissue distribution, i.e. mainly in the bone marrow and the liver, where T-NK cells represent roughly one third of mature T cells, is puzzling and raises some questions regarding their function. T-NK cells have a restricted TCR repertoire, are cytotoxic and secrete large amounts of cytokines, in particular IL-4,25 which may contribute to skew the immune response towards a Th2 phenotype. A third difference between cells derived with IL-12 and IL-18 and those grown in IL-2 or IL-15 relates to their pattern of cytokine production, as only the former produced IFN-, IL-3, IL-6 and TNF. The role of IL-12 and IL-18 as inducers of cytokine production by NK cells is likely to be of critical importance in innate immune responses against tumour cells, pathogens and virus-infected cells, a process in which cytokine production is thought to be a pivotal physiological event. We examined several possible mechanisms that could mediate the effects of IL-12 and IL-18 on NK
cells. The experiments performed in IFN-R-knockout mice clearly demonstrated that IFN- is not involved in the synergy between the two cytokines. We also tested the possibility that IL-2 or IL-6 mediates NK activation induced by IL-12 and IL-18. Addition of blocking anti-IL2R (CD25) or anti-IL6 mAb to cultures stimulated by IL-12 and IL-18 failed to block the proliferative responses induced by the two cytokines (data not shown), thereby indicating that IL-2 and IL-6 were not likely to be involved in the process. We next investigated whether the synergy between IL-12 and IL-18 was related to the induction by IL-12 of IL-18R gene expression, as recently demonstrated by Ahn et al. for IFN- production by an IL-12responsive murine T cell line (2D6) and by naive murine T cells stimulated with anti-CD3 and antiCD28 mAb, that were shown to express IL-18R after exposure to IL-12.26 by RT-PCR analyses, we observed that IL-12R1, IL-12R2 and IL-18R were constitutively expressed on unstimulated nylon woolfiltered splenocytes, and not reciprocally induced by IL-12 and IL-18. It should be stressed, however, that this issue is difficult to address in our experimental setting, as NK cell precursors represent only a small
Synergistic activation of NK cells by IL-12 and IL-18 / 827
tion factors, in particular regarding cytokine production. These observations might be relevant not only for anti-tumour responses but also in autoimmune diseases. In the latter, an immunoregulatory role for NK cells has been recently postulated, e.g. through up-regulation of TGF- production and, thereby, downregulation of inappropriate B-cell immune responses.28
A
B
Cell counts
MATERIALS AND METHODS Cytokines Murine rIL-12 and rIL-18 were purchased from R&D Systems Europe Ltd. (Abingdon, UK), as was human rIL-15 (which is active on murine cells). In some experiments, we used rIL-18 obtained from Peprotech EC Ltd. (London, UK). Murine rIFN- was a kind gift from Dr W. Fiers (University of Ghent, Belgium) and human rIL-2 was purchased from Eurocetus (Chiron Corporation, Amsterdam, The Netherlands). The following cytokine concentrations were used: 1 ng/ml for IL-12, 400 ng/ml for IL-18 obtained from R&D Systems Europe Ltd., 100 ng/ml for IL-18 obtained from Peprotech EC Ltd., 50 ng/ml for IL-15, 50 U/ ml for IL-2, and 100 ng/ml for IFN-.
C
D
Purification and stimulation of splenocytes Log fluorescence Figure 4.
Intracytoplasmic IFN- staining.
Nylon wool-filtered Balb/c splenocytes were cultured with IL-12 and IL-18 (A and B), IL-2 (C) or IL-15 (D), as described in Materials and Methods, before being fixed, permeabilized with saponine and labelled with an FITC-conjugated anti-IFN- mAb. FACS analyses were gated on large granular cells appearing in response to the corresponding cytokine(s). (B) illustrates the reversion of intracytoplasmic IFN- staining in cells cultured with IL-12 and IL-18, by addition of exogenous IFN- during labelling procedure. Shaded areas correspond to autofluorescence. The data are representative of 5 distinct experiments.
percentage of splenocyte populations, thereby preventing correct interpretation of the results at the single cell level. An alternative explanation for the synergy between the two cytokines might be related to ‘‘postreceptor’’ events. In this respect, IL-12 and IL-18 were recently found to differentially regulate the transcriptional activity of the human IFN- promoter in primary CD4 + lymphocytes, with IL-18 causing direct activation of AP-1 whereas both AP-1 and STAT4 are required for IL-12-dependent IFN- promotor activation.27 Taken together, the results reported here demonstrate that the combination of IL-12 and IL-18 displays unique NK-cell activating properties, distinct from those exerted by other NK-cell growth and differentia-
Balb/c, C57BL/6, 129 and IFN-R-knock-out 129 mice (G129) were bred in our animal facility. The latter, initially derived by Dr S. Huang and Dr M. Aguet29 were obtained through the courtesy of Dr F. Brombacher (Max Planck Institute for Immunobiology, Freiburg, Germany). Splenocytes from 12-week-old female mice were prepared by Lymphoprep (Nycomed AS, Oslo, Norway) density gradient centrifugation. Adherent cells were removed by nylon wool filtration and the remaining cells were seeded at a density of 3106 cells/well in 24-well plates in DMEM supplemented with 10% FCS, 50 M 2-ME, 0.55 mM -arginine, 0.24 mM -asparagine and 1.25 mM -glutamine. Cells were stimulated with various additives and harvested three days later for FACS analyses, cytotoxicity assays, and/or RNA extraction. Cytokine concentrations were measured in their supernatants. For proliferation assays, nylon wool-filtered splenocytes were stimulated in triplicate cultures for 48 h at a density of 2105 cells/well in microtitre plates and pulsed overnight with [3H]-thymidine (0.5 Ci/well).
FACS analyses Cells were washed and incubated first with an antiCD16/32 (FcRIII) mAb (Pharmingen, San Diego, CA; 10 /ml) (to prevent aspecific binding of mAb to FcR) in a sodium phosphate (1 mM, pH=7.4) buffer containing 137 mM NaCl, 5 mM KCl, 0.4 mM MgSO4, 0.3 mM MgCl2, 5 mM glucose, 4 mM NaHCO3, 1 mM EDTA, 3% FCS and 20 mM sodium azide. Cells were further stained with a PEand/or a FITC-conjugated mAb directed against surface antigens (or with a biotinylated mAb followed by streptavidine-PE; Pharmigen, San Diego, CA). Cells were
828 / Lauwerys et al.
CYTOKINE, Vol 11, No. 11 (November, 1999: 822–830)
Specific lysis (%)
75
75
A
60
60
45
45
30
30
15
15
0
100/1
30/1
10/1
3/1
0
1/1
B
100/1
Effector/Target ratio
30/1
10/1
3/1
1/1
Effector/Target ratio
C
D
Cytokines added
– IL-12 IL-18 IL-12 + IL-18 IFN-γ IL-2 0
10
20
30
40
3
H-Thymidine incorporation (kcpm)
50
0
10
20
30
40
50
3
H-Thymidine incorporation (kcpm)
Figure 5. Effects of IL-12 and IL-18 on IFN-R-knock-out splenocytes. Nylon wool-filtered 129 (A and C) or IFN-R-knock-out 129 (B and D) splenocytes were cultured with the indicated cytokines(s), as described in Materials and Methods. Cytotoxic activity against Yac-1 cells was determined at different effector/target (E/T) ratios by 51Cr-release assays. ( ), IL-12+IL-18; ( ), IL-2; ( ), control (A and B). Proliferations (meanSEM) were measured by [3H]-thymidine incorporations (C and D).
fixed in paraformaldehyde (0.6%) before being analysed by flow cytometry (Becton Dickinson, Mountain View, CA). The following fluorochrome-conjugated mAb used for FACS analyses were purchased from Pharmingen (San Diego, CA): anti-DX5, anti-Ly-49C, anti-NK1.1, anti-CD3, anti-CD19, and anti-IFN-. Anti-Mac-1 was purified from culture supernatants of ATCC clone M1/70 and biotinylated in our laboratory. Fluorochrome-conjugated unrelevant isotype-matched mAbs (Pharmingen, San Diego, CA) were used as controls. NK1.1 labelling experiments were performed on C57BL/6 splenocytes, as Balb/c cells do not transcribe any of the known NKR-P1 genes, including NK1.1.22 For intracellular IFN- staining experiments, cells were fixed in a 4% paraformaldehyde solution for 20 min, before being permeabilized in ice-cold PBS containing 0.1% saponine and 1% FCS. A PE-conjugated anti-IFN- mAb (1.25 g/ml) was added for 30 min. To confirm the specificity of the intracellular labelling, similar experiments were performed in the presence of exogenous IFN- (1.5 g/ml).
Cytotoxicity assays Yac-1 cells (1103 cells/well), a murine NK cell target, were 51Cr-labelled and incubated for 4 h in U-shaped micro-
titre wells with effector populations at various effector/target ratios in quadruplicate cultures. 51Cr release was determined in supernatants and specific lysis was calculated as the ratio: [measured 51Cr release minus minimal 51Cr release (targets alone)]/[maximal 51Cr release (targets in 1% triton) minus minimal 51Cr release].
Cytokine assays IFN- titres were measured by PACIA (particle counting immunoassay)30 using latex particles coated with two rat anti-mouse IFN- mAbs (R46A2 and XMG1.2), both purchased from Endogen (Woburn, USA). IL-2, IL-3, IL-6, IL-9 and TNF titres were measured on CTLL-2, Ba/F3, 7TD1,31] TS132 and WEHI 164 cells, respectively.
RNA extraction and reverse-transcriptase PCR analyses Cells were lysed in TriPur (Bœhringer Mannheim GmbH, Mannheim, Germany) and total RNA was purified by chloroform extraction. cDNA was synthesized by using oligo(dT) primers (Bœhringer Mannheim GmbH) and murine Moloney leukaemia virus reverse-transcriptase (Life Technologie Inc., Grand Island, NY). Diluted cDNA was
Synergistic activation of NK cells by IL-12 and IL-18 / 829
REFERENCES
Figure 6.
IL-12R and IL-18R gene expression.
Nylon wool-filtered Balb/c splenocytes were cultured with the indicated cytokines(s). RT-PCR analyses were performed with primers specific for the indicated receptors, as described in Materials and Methods. -actin, IL-12R2 and IL-18R RT-PCR products were analysed by agarose gel electrophoresis while IL-12R1 RT-PCR products were blotted onto nylon filters before hybridization with an 32 P-labelled IL-12R1 internal oligonucleotide. cDNA synthesized from con A-stimulated DBA/2 splenocytes was used as a positive control (Pos), while the negative control PCR (Neg) was performed without addition of cDNA. The data are representative of 4, 2 and 3 experiments for IL-18R, IL-12R1 and IL-12R, respectively.
amplified by PCR using recombinant Taq DNA polymerase (Takara, Shiga, Japan) and specific primers for m-actin (5 -ATGGATGACATATCGCTGC-3 ; 3 -GAGTGACAG GTGGAAGGTCG-5 ), mIL-12R1 (5 -CTGCGAGGCTG AAGACGG-3 ; 3 -GTCTCCGTCTCCGTCCAC-5 ), mIL12R2 (5 -CATCACGAAGTTTCCCCCAC-3 ; 3 -CGTAG CGATAGTAGTGCCAC-5 ), and mIL-18R (5 -AGAAGC CATAGACACCAAGA-3 ; 3 -TATGAAAACGACACCT CTGC-5 ). PCR was performed as follows: 1 min at 94C, followed by 1 min at 60C for -actin, 55C for IL-12R1, 55C for IL-12R2, 50C for IL-18R and by 2 min at 72C for 25 ( actin) or 35 (IL-12R1, IL-12R2 and IL-18R) cycles. The -actin, IL-12R2 and IL-18R PCR products were analysed by agarose gel electrophoresis. The IL-12R1 PCR products were blotted on nylon filters and hybridized with a 32P-labelled IL-12R1 internal oligonucleotide (5 -ATTTCCCGTTTATCCATCATT-3 ).
Acknowledgements This work was supported in part by grants from the Centre de Recherche Interuniversitaire en Vaccinologie, the Fonds National de la Recherche Scientifique et Me´ dicale, the Association Lupus Erythe´ mateux, and by the Belgian Federal Service for Scientific, Technical and Cultural Affairs. J.-C. R. is Research Associate with the Fonds National de la Recherche Scientifique.
1. Gately MK, Renzetti LM, Magram J, Stern AS, Adorini L, Gubler U, Presky DH (1998) The interleukin-12/interleukin-12receptor system; role in normal and pathologic immunes responses. Annu Rev Immunol 16:495–521. 2. Okamura H, Tsutsui H, Komatsu T, Yutsudo M, Hakura A, Tanimoto T, Torigoe K, Okura T, Nukada Y, Hattori K, Akita K, Namba M, Tanabe F, Konishi K, Fukuda S, Kurimoto M (1995) Cloning of a new cytokine that induces IFN- production by T cells. Nature 378:88–91. 3. Ushio S, Namba M, Okura T, Hattori K, Nukada Y, Akita K, Tanabe F, Konishi K, Micallef M, Fujii M, Torigoe K, Tanimoto T, Fukuda S, Ikeda M, Okamura H, Kurimoto M (1996) Cloning of the cDNA for human IFN-gamma-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein. J Immunol 156:4274–4279. 4. Micallef MJ, Ohtuski T, Kohno K, Tanabe F, Ushio S, Namba M, Tanimoto T, Torigoe K, Fujii M, Ikeda M, Fukuda S, Kurimoto M (1996) Interferon--inducing factor enhances T helper 1 cytokine production by stimulated human T cells: synergism with interleukin-12 for interferon- production. Eur J Immunol 26: 1647–1651. 5. Robinson D, Shibuya K, Mui A, Zonin F, Murphy E, Sana T, Hartley SB, Menon S, Kastelein R, Bazan F, O’Garra A (1997) IGIF does not drive Th1 development but synergizes with IL-12 for interferon- production and activates IRAK and NF-B. Immunity 7:571–581. 6. Okamura H, Kashiwamura S-I, Tsutsui H, Yoshimoto T, Nakanishi K (1998) Regulation of interferon- production by IL-12 and IL-18. Curr Opin Immunol 10:259–264. 7. Yoshimoto T, Okamura H, Tagawa Y-I, Nakanishi K (1997) Interleukin 18 together with interleukin 12 inhibits IgE production by induction of interferon- production from activated B cells. Proc Natl Acad Sci USA 94:3948–3953. 8. Lauwerys BR, Renauld J-C, Houssiau FA (1998) Inhibition of in vitro immunoglobulin production by IL-12 in murine chronic graft-vs.-host disease; synergism with IL-18. Eur J Immunol 28:2017–2024. 9. Torigoe K, Ushio S, Okura T, Kobayashi S, Taniai M, Kunikata T, Murakami T, Sanou O, Kojima H, Fujii M, Ohta T, Ikeda M, Ikegami H, Kurimoto M (1997) Purification and characterization of human interleukin-18 receptor. J Biol Chem 272: 25737–25742. 10. Moretta A, Bottino C, Vitale M, Bende D, Biassoni R, Mingari MC, Moretta L (1996) Receptors for HLA class-I molecules in human natural killer cells. Annu Rev Immunol 14:619–648. 11. Lanier LL (1998) NK cell receptors. Annu Rev Immunol 16:359–393. 12. Siegel JP, Sharon M, Smith PL, Leonard WJ (1987) The IL-2 receptor chain (p70): role in mediating signals for LAK, NK, and proliferative activities. Science 238:75–78. 13. Kobayashi M, Fitz L, Ryan M, Hewick RM, Clark SC, Chan S, Loudon R, Sherman F, Perussia B, Trinchieri G (1989) Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biological effects on human lymphocytes. J Exp Med 170:827–845. 14. Schoenhaut DS, Chua AO, Wolitzky AG Quinn PM, Dwyer CM, McComas W, Familletti PC, Gately MK, Gubler U (1992) Cloning and expression of murine IL-12. J Immunol 148:3433–3440. 15. Mingari MC, Vitale C, Cantoni C, Bellomo R, Ponte M, Schiavetti F, Bertone S, Moretta A, Moretta L (1997) Interleukin15-induced maturation of human natural killer cells from early thymic precursors: selective expression of CD94/NKG2-A as the only HLA class I-specific inhibitory receptor. Eur J Immunol 27:1374–1380. 16. Carson WE, Fehniger TA, Haldar S, Eckhert K, Lindemann MJ, Lai CF, Croce CM, Baumann H, Caligiuri MA (1997) A potential role for interleukin-15 in the regulation of human natural killer cell survival. J Clin Invest 99:937–943. 17. Tsutsui H, Nakanishi K, Matsui K, Higashino K, Okamura H, Miyazawa Y, Kaneda K (1996) IFN--inducing factor
830 / Lauwerys et al. up-regulates fas ligand-mediated cytotoxic activity or murine natural killer cell clones. J. Immunol 157:3967–3973. 18. Hunter CA, Timans J, Pisacane P, Menon S, Cai G, Walker W, Aste-Amezaga M, Chizzonite R, Bazan JF, Kastelein RA (1997) Comparison of the effects of interleukin-1, interleukin-1 and interferon--inducing factor on the production of interferon- by natural killer cells. Eur J Immunol 27:2787–2792. 19. Tomura M, Zhou XY, Maruo S, Ahn H-J, Hamaoka T, Okamura H, Nakanishi K, Tanimoto T, Kurimoto M, Fujiwara H (1998) A critical role for IL-18 in the proliferation and activation of NK1.1 + CD3 cells. J Immunol 160:4738–4746. 20. Glimcher L, Shen FW, Cantor H (1977) Identification of a cell-surface antigen selectively expressed on the natural killer cell. J Exp Med 145:1–9. 21. Ballas ZK, Rasmussen W (1990) NK1.1 + thymocytes. Adult murine CD4 , CD8 thymocytes contain an NK1.1 + , CD3 + , CD5hi, CD44hi, TCR-V beta 8 + subset. J Immunol 145:1039–1045. 22. Giorda R, Weisberg EP, Ip TK, Trucco M (1992) Genomic structure and strain-specific expression of the natural killer cell receptor NKR-P1. J Immunol 149:1957–1963. 23. Kung SKP, Miller RG (1995) The NK1.1 antigen in NK-mediated F1 antiparent killing in vitro. J Immunol 154: 1624–1633. 24. Vicari AP, Zlotnik A (1996) Mouse NK1.1 + T cells: a new family of T cells. Immunol Today 17:71–76. 25. Yoshimoto T, Paul WE (1994) CD4 + , NK1.1 + T cells promptly produce interleukin 4 in response to in vivo challenge with anti-CD3. J Exp Med 179:1285–1295.
CYTOKINE, Vol 11, No. 11 (November, 1999: 822–830) 26. Ahn H-J, Maruo S, Tomura M, Mu J, Hamaoka T, Nakanishi K, Clark S, Kurimoto M, Okamura H, Fujiwara H (1997) A mechanism underlying synergy between IL-12 and IFN- inducing factor in enhanced production of IFN-. J Immunol 159:2125–2131. 27. Barbulescu K, Becker C, Schlaak JF, Schmitt E, Meyer zum Bu¨ schenfelde K-H, Neurath MF (1998) Cutting edge: IL-12 and IL-18 differentially regulate the transcriptional activity of the human IFN- promoter in primary CD4 + T lymphocytes. J Immunol 160:3642–3647. 28. Horwitz DA, Gray JD, Ohtsuka K, Hirokawa M, Takahashi T (1997) The immunoregulatory effects of NK cells: the role of TGF- and implications for autoimmunity. Immunol Today 18:538–542. 29. Huang S, Hendriks W, Althage A, Hemmi S, Bluethmann H, Kamijo R, Vilcek J, Zinkernagel RM, Aguet M (1993) Immune response in mice that lack the interferon- receptor. Science 259:1742–1745. 30. Masson PL, Cambiaso CL, Collet-Cassart D, Magnusson CG, Richards CB, Sindic CJ (1981) Particle counting immunoassay (PACIA). Methods Enzymol 74:106–139. 31. Van Snick J, Cayphas S, Vink A, Uyttenhove C, Coulie PG, Rubira MR, Simpson RJ (1986) Purification and NH2-terminal amino acid sequence of a T-cell-derived lymphokine with growth factor activity for B-cell hybridomas. Proc Natl Acad Sci USA 83:9679–9683. 32. Uyttenhove C, Simpson RJ, Van Snick J (1988) Functional and structural characterization of P40, a mouse glycoprotein with T-cell growth factor activity. Proc Natl Acad Sci USA 85:6934–6938.
SYNERGISTIC PROLIFERATION AND ACTIVATION OF NATURAL KILLER CELLS BY INTERLEUKIN 12 AND INTERLEUKIN 18 Bernard R. Lauwerys,1 Jean-Christophe Renauld,2,3 Fre´de´ric A. Houssiau1 We investigated the effects of IL-12 and IL-18 on unstimulated murine splenocytes and observed that the two cytokines strongly synergized for their proliferation, whereas IL-12 and IL-18 alone were essentially inactive in this respect. Phenotypical and functional analyses of cells proliferating in response to IL-12 and IL-18 revealed that large granular Ly-49C + DX5 + CD3 NK blasts were expanded in these cultures and that they displayed cytotoxic activity against Yac-1 cells, a murine NK cell target. Further analyses indicated three major differences between NK cells appearing in response to IL-12 and IL-18 and those derived in the presence of other NK cell growth factors, such as IL-2 or IL-15. First, a population of T-NK cells, i.e. expressing T cell (TCR, CD3) and NK cell (Ly-49) markers, was detected amongst cells growing in IL-2 or IL-15 but not in cultures supplemented with IL-12 and IL-18. Second, most NK cells derived with IL-2 or IL-15 expressed the NK1.1 antigen, while those derived with IL-12 and IL-18 did not. Finally, striking differences were observed regarding cytokine production. Cells stimulated with IL-12 and IL-18 in combination, but not with IL-2 or IL-15, produced IFN-, IL-3, IL-6 and TNF. IFN- was not involved in the response of NK cells to IL-12 and IL-18, as indicated by experiments demonstrating that the combination of the two cytokines displayed similar effects on spleen cells from IFN-R-knock-out mice. Receptor (IL-12R1, IL-12R2 and IL-18R) gene expression studies did not indicate that the mechanism underlying the synergy between IL-12 and IL-18 involved reciprocal induction of their receptors. Taken together, our results demonstrate that IL-12 and IL-18 exert striking synergistic activities for NK cell proliferation and activation, distinct from those induced by IL-2 or IL-15. 1999 Academic Press
Interleukin (IL)12 is a heterodimeric cytokine produced by B cells, macrophages and dendritic cells that plays a well-known immunoregulatory role by promoting cell-mediated immunity, in particular through upregulation of IFN- production.1 Another phagocyte-derived cytokine displaying a potent IFN-inducing activity, provisionally called IGIF (IFN-inducing factor), was recently cloned2 and re-named IL-18.3. Interestingly, IL-12 and IL-18 were found to synergize for IFN- production by human and murine T cells.4–6 We and others unmasked a similar synergy between the two cytokines for IFN- production by B From the 1Rheumatology and 2Experimental Medicine Units, Christian de Duve Institute of Cellular Pathology, Universite´ catholique de Louvain, Bruxelles, Belgium; and 3Ludwig Institute for Cancer Research, Brussels Branch, Belgium Correspondence to: Fre´de´ric A. Houssiau, Rheumatology Unit, UCL 5390, Avenue Hippocrate 10, B-1200 Bruxelles, Belgium; E-mail: [email protected] Received 24 September 1998; accepted for publication 11 January 1999 1999 Academic Press 1043–4666/99/110822+09 $30.00/0 KEY WORDS: Interleukin 12/Interleukin 18/NK cells 822
cells. Thus, murine naive B cells stimulated with antiCD40 mAb and IL-4 produce huge amounts of IFN- in response to the combination of IL-12 and IL-18.7 Similarly, activated B cells isolated from mice with chronic graft-vs-host disease produce IFN- after stimulation with both cytokines.8 In these two experimental settings, the synergy between IL-12 and IL-18 for IFN- induction accounted for the strong inhibition of immunoglobulin (Ig) production exerted by the two cytokines.7,8 These results, by indicating that the synergy between IL-12 and IL-18 was operating on at least two distinct lymphocyte subsets, prompted us to investigate further the effects of IL-12 and IL-18 on normal murine splenocytes. This approach led to the intriguing observation that the two cytokines strongly synergized for the proliferation of unstimulated murine spleen cells, whereas IL-12 and IL-18 alone were inactive in this respect. Characterization of splenocytes proliferating in response to IL-12 and IL-18 indicated that they belonged to the natural killer (NK) cell lineage and CYTOKINE, Vol. 11, No. 11 (November), 1999: pp 822–830
Synergistic activation of NK cells by IL-12 and IL-18 / 823
Surface markers analysis of cells proliferating in response to IL-12 and IL-18, IL-2 or IL-15 The phenotype of cells proliferating in response to the combination of IL-12 and IL-18 was further studied and compared to that of cells cultured in the presence of IL-2 or IL-15, two other well-known NK cell growth factors. Double-labelling experiments revealed that Ly-49C + cells proliferating in response to IL-12 and IL-18 consisted of a homogeneous population of Ly-49C + NK1.1 CD3 NK cells (Figs 2A and 2C). While a population of Ly-49C + blasts was also detected in cultures supplemented with IL-2, these cells differed from those appearing in cultures supplemented with IL-12 and IL-18 regarding CD3 and
IL-2 IL-12 IL-18
IL-12 + IL-18 0
5
10
15
20
25
30
35
40
45
3 [ H]Thymidine incorporation (kcpm)
B
No stimulation
IL-12 + IL-18
SSC
Nylon wool-filtered splenocytes were cultured in the presence or absence of IL-12 and/or IL-18. As indicated in Figure 1A, addition of IL-12 and IL-18 alone had no effect on cell proliferation. By contrast, the combination of the two cytokines induced as strong a proliferation as that observed with IL-2 (50 U/ml), with half-maximal stimulation being obtained with 10 pg/ml of IL-12 and 50 ng/ml of IL-18. To characterize the cells responding to IL-12 and IL-18, we performed FACS analyses with T cell (CD3), B cell (CD19), monocyte (Mac-1) and NK cell (Ly-49C, DX-5) markers. These experiments revealed that a population of large (high FSC), granular (high SSC), Ly-49C + DX5 + CD3 NK blasts was present in cultures stimulated with IL-12 and IL-18 (Fig. 1B and 1C). Similar results were obtained with nylon woolfiltered splenocytes prepared from C57BL/6 and 129 mice. In order to confirm that cells proliferating in response to the combination of IL-12 and IL-18 belonged to the NK cell lineage, we performed cytotoxity assays using NK-sensitive Yac-1 cells as targets and nylon wool-filtered splenocytes as effectors. Spleen cells cultured with IL-12 and IL-18 in combination, exerted a strong cytotoxic activity, comparable to that observed with cells supplemented with IL-2. Again, a strong synergy between IL-12 and IL-18 was unmasked (Table 1). Cells proliferating in response to IL-12 and IL-18 were sorted by flow cytometry on the basis of their FSC/SSC pattern (upper-right region in Figure 1B) and they also displayed a potent cytotoxic activity on Yac-1 targets (Table 1).
Cytokines added
Synergistic proliferation and activation of NK cells induced by IL-12 and IL-18
–
SSC
RESULTS
A
FSC
FSC
C
DX5
Cell counts
that these NK cells were phenotypically and functionally different from those derived in the presence of IL-2 or IL-15.
Ly-49C
Log fluorescence Figure 1.
Proliferation of NK cells.
Nylon wool-filtered Balb/c splenocytes were cultured in the presence of the indicated cytokine(s), as described in Materials and Methods. Proliferations (mean kcpmSEM) were measured by [3H]thymidine incorporation (A). The plots shown in (B) indicate the distribution of cells (cultured without or with IL-12 and IL-18) along the forward scatter channel (FSC) and the side scatter channel (SSC) on flow cytometric analysis. FACS analyses (DX5, Ly-49C) shown in (C) were gated on large granular cells appearing in response to IL-12 and IL-18, as defined by the area depicted in (B). Shaded areas correspond to labelling obtained with an isotype-matched control mAb. The data presented in A, B and C are representative of 12, 10 and 8 experiments, respectively.
NK1.1 expression. Thus, most (80%) Ly-49C + cells present in IL-2 cultures were NK1.1 + (Fig. 2B) and could be categorized according to CD3 expression into a Ly-49C + CD3 + subset of T-NK cells and a Ly-49C + CD3 subset of NK cells (Fig. 2D). These experiments
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TABLE 1.
CYTOKINE, Vol 11, No. 11 (November, 1999: 822–830)
Lytic activity of NK cell populations Cytokines added
Cells Total N + cells
Sorted cells*
E/T ratio
None
IL-12
IL-18
IL-12 IL-18
IL-2
100 30 10 3 1 30 10 3 1
0 0 0 0 0 ND ND ND ND
19 4 0 0 0 ND ND ND ND
0 0 0 0 0 ND ND ND ND
58 41 18 3 2 69 27 17 6
62 36 33 16 6 61 52 27 13
Figures represent percentage of specific lysis (SD<10%) of 51Cr-labelled Yac-1 cells (1103 cells) measured in quadruplicate (for total N + cells) or triplicate (for sorted cells) experiments; effector cells used in the cytotoxity assays have been previously stimulated for three days with the indicated cytokines, as described in Materials and Methods. *Cells proliferating in response to IL-12 and IL-18 were sorted by flow cytometry on the basis of their FSC/SSC pattern (upper-right region in Fig. 1B). E/T: effector/target. N + ; nylon wool-filtered. These data are representative of four and two distinct experiments performed on total N + and sorted cells, respectively.
also indicated that cultures supplemented with IL-2 contained a population of CD3 + T cell blasts (Fig. 2D), that were absent in cultures stimulated with IL-12 and IL-18. We also investigated the effects of IL-15 in our experimental setting. IL-15 strongly stimulated the proliferation of nylon-wool filtered splenocytes (stimulation index: 101 and 142 in two experiments) and FACS analyses indicated that cells proliferating in response to IL-15 were phenotypically similar to those responding to IL-2, with three distinct populations being identified, namely NK cells (Ly-49C + CD3 ), T cells (Ly-49C CD3 + ) and T-NK cells (Ly-49C + CD3 + ). Again, most (89%) Ly-49C + cells induced by IL-15 were NK1.1 + (data not shown).
Cytokine production in cultures stimulated with IL-12 and IL-18, IL-2 or IL-15 We measured the concentrations of various cytokines in the supernatants from cells stimulated with IL-12 and/or IL-18 and compared them to those determined in cultures supplemented with IL-2 or IL-15. As indicated in Figure 3, IL-12 and IL-18 were essentially inactive on their own but strongly synergized, not only for IFN- production but also for IL-3, IL-6 and TNF induction. By contrast, IL-2 and IL-9 were not detected in cultures stimulated with IL-12 and IL-18. Interestingly, addition of IL-2 or IL-15 had no significant effect on the production of IL-3, IL-6, IL-9, IFN- and TNF. In order to determine whether IFN- detected in cultures supplemented with IL-12 and IL-18 was produced by cells proliferating in response to these
cytokines, we performed cytoplasmic IFN- staining experiments by FACS analyses. These experiments confirmed that large granular cells appearing in response to IL-12 and IL-18, and not those proliferating in response to IL-2 or IL-15, produced IFN- (Fig. 4). More specifically, double-labelling experiments indicated that Ly-49C + blasts produced IFN- in response to IL-12 and IL-18 (data not shown).
IFN- does not mediate the proliferation and activation of NK cells induced by IL-12 and IL-18 Next, we evaluated whether IFN- was involved in the proliferation of NK cells in response to IL-12 and IL-18. Although large amounts of IFN- were produced by cells stimulated with IL-12 and IL-18, two lines of evidence argued against the possibility that IFN- was involved in the proliferation of NK cells induced by the two cytokines. First, addition of IFN- to spleen cells did not induce NK-cell proliferation. Second, splenocytes isolated from IFN-R knock-out 129 mice responded similarly to IL-12 and IL-18 compared to wild type 129 mice, both in terms of proliferation and cytotoxicity (Fig. 5).
IL-12R and IL-18R gene expression studies We wondered whether the synergy between IL-12 and IL-18 for the proliferation and activation of NK cells could be explained by reciprocal induction of their receptor. We addressed this issue by RT-PCR analyses performed on cDNA synthesized after RNA extraction from cells cultured in the absence or presence of IL-12 and/or IL-18. Specific murine IL-12R1, IL-12R2 and IL-18R oligonucleotides were used as primers. For the latter, we assumed that the murine IL-18R gene was identical to the previously cloned murine IL-1R related protein (IL-1Rrp) gene, as recently described for human cells.9 As indicated in Figure 6, IL-12R1, IL-12R2 and IL-18R gene expression was detected in unstimulated cells. Addition of IL-12 and IL-18 increased IL-12R, IL-12R1 and IL-12R2 gene expression but did not influence that of IL-18R. These results did not support the hypothesis that the mechanism underlying the synergy between IL-12 and IL-18 involved reciprocal induction of their receptors.
DISCUSSION NK cells play a critical role in innate immunity against pathogens, virus-infected cells and tumors through MHC-unrestricted cytotoxicity and their capacity to produce cytokines. Their function is a tightly regulated process, involving activating (CD16, CD2, CD28 and NKR-P1) and inhibitory (Ly-49,
Synergistic activation of NK cells by IL-12 and IL-18 / 825
B
A 1
45 12
2
40
3
NK1.1
60 37
Ly-49C
C
3
3
56 11 28
5
CD3
53 41
D
Ly-49C Figure 2.
Surface markers analysis.
Nylon wool-filtered C57BL/6 splenocytes were cultured with IL-12 and IL-18 (A and C) or with IL-2 (B and D), as described in Materials and Methods. After 3 days of culture, cells were double-labelled with fluorochrome-conjugated mAb (anti-NK1.1 and anti-Ly-49C in Panels A and B; anti-CD3 and anti-Ly-49C in Panels C and D). FACS analyses were gated on large granular cells appearing in response to IL-12 and IL-18 or to IL-2. Isotype-matched control mAb, using the same cut-off fluorescence values, stained no more than 0.5% of the cells (data not shown). The data are representative of two distinct experiments.
KIR) membrane receptors, the latter recognizing polymorphic MHC class I molecules.10,11 Several cytokines stimulate NK cell proliferation and activation, in particular IL-2,12 IL-12,13,14 IL-15,15,16 and IL-18.3,17–19 The experiments presented here demonstrate the existence of a striking synergy between IL-12 and IL-18, not only for the proliferation but also for the activation of NK cells, in particular for cytotoxicity and cytokine production. Functional comparison of cells appearing in response to the combination of IL-12 and IL-18 on the one hand, and in response to IL-2 or IL-15 on the other hand, revealed several differences. First, Ly-49 + NK cells appearing in response to IL-12 and IL-18 were NK1.1 , while most Ly-49 + NK cells detected in IL-2- or IL-15-supplemented cultures were NK1.1 + . NK1.1, the prototypical mouse NK cell antigen,20,21 is
a member of the family of NKR-P1 NK cell receptors.22 Although its function is currently unknown, some data indicate that NK1.1 plays a role in target recognition23 and functions as an activating signal. Regarding NK1.1 expression, our results contrast with those published recently by Tomura et al., suggesting that IL-18 plays an obligatory role in inducing NK1.1 + NK cells from murine CD4 CD8 sIg Ia splenocytes.19 In our experimental setting, IL-2 and IL-15 were capable, on their own, without addition of IL-18, to stimulate the proliferation of NK1.1 + NK cells. The reasons for this discrepancy are unclear but might be related to differences in the splenic cell preparations used to derive NK cells. Another phenotypical difference between cultures derived in the presence of IL-12 and IL-18 and those derived with IL-2 or IL-15 was the presence, in the
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CYTOKINE, Vol 11, No. 11 (November, 1999: 822–830)
A
B
– IL-12 IL-18 IL-12 + IL-18
Cytokines added
IL-2 IL-15 0
1
2
4 3 µg/ml
5
6
0
C
100
200 ng/ml
300
400
200
400 pg/ml
600
800
D
– IL-12 IL-18 IL-12 + IL-18 IL-2 IL-15 0 Figure 3.
10
20 pg/ml
30
40
0
Cytokine production.
Nylon wool-filtered Balb/c splenocytes were cultured with the indicated cytokine(s), as described in Materials and Methods. (A) IFN-; (B) IL-3; (C) IL-6; (D) TNF concentrations (meanSEM) were measured in supernatants from duplicate or triplicate cultures by specific bioassays performed in quadruplicate cultures. The data are representative of 3 distinct experiments for IFN- and IL-6 and 2 experiments for IL-3 and TNF.
latter, of a population of T-NK cells (Ly-49 + CD3 + ) that was not detected amongst cells responding to IL-12 and IL-18. T-NK cells represent a small percentage of T cells expressing both T cell (TCR, CD3) and NK cell (Ly-49) antigens.24 Their tissue distribution, i.e. mainly in the bone marrow and the liver, where T-NK cells represent roughly one third of mature T cells, is puzzling and raises some questions regarding their function. T-NK cells have a restricted TCR repertoire, are cytotoxic and secrete large amounts of cytokines, in particular IL-4,25 which may contribute to skew the immune response towards a Th2 phenotype. A third difference between cells derived with IL-12 and IL-18 and those grown in IL-2 or IL-15 relates to their pattern of cytokine production, as only the former produced IFN-, IL-3, IL-6 and TNF. The role of IL-12 and IL-18 as inducers of cytokine production by NK cells is likely to be of critical importance in innate immune responses against tumour cells, pathogens and virus-infected cells, a process in which cytokine production is thought to be a pivotal physiological event. We examined several possible mechanisms that could mediate the effects of IL-12 and IL-18 on NK
cells. The experiments performed in IFN-R-knockout mice clearly demonstrated that IFN- is not involved in the synergy between the two cytokines. We also tested the possibility that IL-2 or IL-6 mediates NK activation induced by IL-12 and IL-18. Addition of blocking anti-IL2R (CD25) or anti-IL6 mAb to cultures stimulated by IL-12 and IL-18 failed to block the proliferative responses induced by the two cytokines (data not shown), thereby indicating that IL-2 and IL-6 were not likely to be involved in the process. We next investigated whether the synergy between IL-12 and IL-18 was related to the induction by IL-12 of IL-18R gene expression, as recently demonstrated by Ahn et al. for IFN- production by an IL-12responsive murine T cell line (2D6) and by naive murine T cells stimulated with anti-CD3 and antiCD28 mAb, that were shown to express IL-18R after exposure to IL-12.26 by RT-PCR analyses, we observed that IL-12R1, IL-12R2 and IL-18R were constitutively expressed on unstimulated nylon woolfiltered splenocytes, and not reciprocally induced by IL-12 and IL-18. It should be stressed, however, that this issue is difficult to address in our experimental setting, as NK cell precursors represent only a small
Synergistic activation of NK cells by IL-12 and IL-18 / 827
tion factors, in particular regarding cytokine production. These observations might be relevant not only for anti-tumour responses but also in autoimmune diseases. In the latter, an immunoregulatory role for NK cells has been recently postulated, e.g. through up-regulation of TGF- production and, thereby, downregulation of inappropriate B-cell immune responses.28
A
B
Cell counts
MATERIALS AND METHODS Cytokines Murine rIL-12 and rIL-18 were purchased from R&D Systems Europe Ltd. (Abingdon, UK), as was human rIL-15 (which is active on murine cells). In some experiments, we used rIL-18 obtained from Peprotech EC Ltd. (London, UK). Murine rIFN- was a kind gift from Dr W. Fiers (University of Ghent, Belgium) and human rIL-2 was purchased from Eurocetus (Chiron Corporation, Amsterdam, The Netherlands). The following cytokine concentrations were used: 1 ng/ml for IL-12, 400 ng/ml for IL-18 obtained from R&D Systems Europe Ltd., 100 ng/ml for IL-18 obtained from Peprotech EC Ltd., 50 ng/ml for IL-15, 50 U/ ml for IL-2, and 100 ng/ml for IFN-.
C
D
Purification and stimulation of splenocytes Log fluorescence Figure 4.
Intracytoplasmic IFN- staining.
Nylon wool-filtered Balb/c splenocytes were cultured with IL-12 and IL-18 (A and B), IL-2 (C) or IL-15 (D), as described in Materials and Methods, before being fixed, permeabilized with saponine and labelled with an FITC-conjugated anti-IFN- mAb. FACS analyses were gated on large granular cells appearing in response to the corresponding cytokine(s). (B) illustrates the reversion of intracytoplasmic IFN- staining in cells cultured with IL-12 and IL-18, by addition of exogenous IFN- during labelling procedure. Shaded areas correspond to autofluorescence. The data are representative of 5 distinct experiments.
percentage of splenocyte populations, thereby preventing correct interpretation of the results at the single cell level. An alternative explanation for the synergy between the two cytokines might be related to ‘‘postreceptor’’ events. In this respect, IL-12 and IL-18 were recently found to differentially regulate the transcriptional activity of the human IFN- promoter in primary CD4 + lymphocytes, with IL-18 causing direct activation of AP-1 whereas both AP-1 and STAT4 are required for IL-12-dependent IFN- promotor activation.27 Taken together, the results reported here demonstrate that the combination of IL-12 and IL-18 displays unique NK-cell activating properties, distinct from those exerted by other NK-cell growth and differentia-
Balb/c, C57BL/6, 129 and IFN-R-knock-out 129 mice (G129) were bred in our animal facility. The latter, initially derived by Dr S. Huang and Dr M. Aguet29 were obtained through the courtesy of Dr F. Brombacher (Max Planck Institute for Immunobiology, Freiburg, Germany). Splenocytes from 12-week-old female mice were prepared by Lymphoprep (Nycomed AS, Oslo, Norway) density gradient centrifugation. Adherent cells were removed by nylon wool filtration and the remaining cells were seeded at a density of 3106 cells/well in 24-well plates in DMEM supplemented with 10% FCS, 50 M 2-ME, 0.55 mM -arginine, 0.24 mM -asparagine and 1.25 mM -glutamine. Cells were stimulated with various additives and harvested three days later for FACS analyses, cytotoxicity assays, and/or RNA extraction. Cytokine concentrations were measured in their supernatants. For proliferation assays, nylon wool-filtered splenocytes were stimulated in triplicate cultures for 48 h at a density of 2105 cells/well in microtitre plates and pulsed overnight with [3H]-thymidine (0.5 Ci/well).
FACS analyses Cells were washed and incubated first with an antiCD16/32 (FcRIII) mAb (Pharmingen, San Diego, CA; 10 /ml) (to prevent aspecific binding of mAb to FcR) in a sodium phosphate (1 mM, pH=7.4) buffer containing 137 mM NaCl, 5 mM KCl, 0.4 mM MgSO4, 0.3 mM MgCl2, 5 mM glucose, 4 mM NaHCO3, 1 mM EDTA, 3% FCS and 20 mM sodium azide. Cells were further stained with a PEand/or a FITC-conjugated mAb directed against surface antigens (or with a biotinylated mAb followed by streptavidine-PE; Pharmigen, San Diego, CA). Cells were
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CYTOKINE, Vol 11, No. 11 (November, 1999: 822–830)
Specific lysis (%)
75
75
A
60
60
45
45
30
30
15
15
0
100/1
30/1
10/1
3/1
0
1/1
B
100/1
Effector/Target ratio
30/1
10/1
3/1
1/1
Effector/Target ratio
C
D
Cytokines added
– IL-12 IL-18 IL-12 + IL-18 IFN-γ IL-2 0
10
20
30
40
3
H-Thymidine incorporation (kcpm)
50
0
10
20
30
40
50
3
H-Thymidine incorporation (kcpm)
Figure 5. Effects of IL-12 and IL-18 on IFN-R-knock-out splenocytes. Nylon wool-filtered 129 (A and C) or IFN-R-knock-out 129 (B and D) splenocytes were cultured with the indicated cytokines(s), as described in Materials and Methods. Cytotoxic activity against Yac-1 cells was determined at different effector/target (E/T) ratios by 51Cr-release assays. ( ), IL-12+IL-18; ( ), IL-2; ( ), control (A and B). Proliferations (meanSEM) were measured by [3H]-thymidine incorporations (C and D).
fixed in paraformaldehyde (0.6%) before being analysed by flow cytometry (Becton Dickinson, Mountain View, CA). The following fluorochrome-conjugated mAb used for FACS analyses were purchased from Pharmingen (San Diego, CA): anti-DX5, anti-Ly-49C, anti-NK1.1, anti-CD3, anti-CD19, and anti-IFN-. Anti-Mac-1 was purified from culture supernatants of ATCC clone M1/70 and biotinylated in our laboratory. Fluorochrome-conjugated unrelevant isotype-matched mAbs (Pharmingen, San Diego, CA) were used as controls. NK1.1 labelling experiments were performed on C57BL/6 splenocytes, as Balb/c cells do not transcribe any of the known NKR-P1 genes, including NK1.1.22 For intracellular IFN- staining experiments, cells were fixed in a 4% paraformaldehyde solution for 20 min, before being permeabilized in ice-cold PBS containing 0.1% saponine and 1% FCS. A PE-conjugated anti-IFN- mAb (1.25 g/ml) was added for 30 min. To confirm the specificity of the intracellular labelling, similar experiments were performed in the presence of exogenous IFN- (1.5 g/ml).
Cytotoxicity assays Yac-1 cells (1103 cells/well), a murine NK cell target, were 51Cr-labelled and incubated for 4 h in U-shaped micro-
titre wells with effector populations at various effector/target ratios in quadruplicate cultures. 51Cr release was determined in supernatants and specific lysis was calculated as the ratio: [measured 51Cr release minus minimal 51Cr release (targets alone)]/[maximal 51Cr release (targets in 1% triton) minus minimal 51Cr release].
Cytokine assays IFN- titres were measured by PACIA (particle counting immunoassay)30 using latex particles coated with two rat anti-mouse IFN- mAbs (R46A2 and XMG1.2), both purchased from Endogen (Woburn, USA). IL-2, IL-3, IL-6, IL-9 and TNF titres were measured on CTLL-2, Ba/F3, 7TD1,31] TS132 and WEHI 164 cells, respectively.
RNA extraction and reverse-transcriptase PCR analyses Cells were lysed in TriPur (Bœhringer Mannheim GmbH, Mannheim, Germany) and total RNA was purified by chloroform extraction. cDNA was synthesized by using oligo(dT) primers (Bœhringer Mannheim GmbH) and murine Moloney leukaemia virus reverse-transcriptase (Life Technologie Inc., Grand Island, NY). Diluted cDNA was
Synergistic activation of NK cells by IL-12 and IL-18 / 829
REFERENCES
Figure 6.
IL-12R and IL-18R gene expression.
Nylon wool-filtered Balb/c splenocytes were cultured with the indicated cytokines(s). RT-PCR analyses were performed with primers specific for the indicated receptors, as described in Materials and Methods. -actin, IL-12R2 and IL-18R RT-PCR products were analysed by agarose gel electrophoresis while IL-12R1 RT-PCR products were blotted onto nylon filters before hybridization with an 32 P-labelled IL-12R1 internal oligonucleotide. cDNA synthesized from con A-stimulated DBA/2 splenocytes was used as a positive control (Pos), while the negative control PCR (Neg) was performed without addition of cDNA. The data are representative of 4, 2 and 3 experiments for IL-18R, IL-12R1 and IL-12R, respectively.
amplified by PCR using recombinant Taq DNA polymerase (Takara, Shiga, Japan) and specific primers for m-actin (5 -ATGGATGACATATCGCTGC-3 ; 3 -GAGTGACAG GTGGAAGGTCG-5 ), mIL-12R1 (5 -CTGCGAGGCTG AAGACGG-3 ; 3 -GTCTCCGTCTCCGTCCAC-5 ), mIL12R2 (5 -CATCACGAAGTTTCCCCCAC-3 ; 3 -CGTAG CGATAGTAGTGCCAC-5 ), and mIL-18R (5 -AGAAGC CATAGACACCAAGA-3 ; 3 -TATGAAAACGACACCT CTGC-5 ). PCR was performed as follows: 1 min at 94C, followed by 1 min at 60C for -actin, 55C for IL-12R1, 55C for IL-12R2, 50C for IL-18R and by 2 min at 72C for 25 ( actin) or 35 (IL-12R1, IL-12R2 and IL-18R) cycles. The -actin, IL-12R2 and IL-18R PCR products were analysed by agarose gel electrophoresis. The IL-12R1 PCR products were blotted on nylon filters and hybridized with a 32P-labelled IL-12R1 internal oligonucleotide (5 -ATTTCCCGTTTATCCATCATT-3 ).
Acknowledgements This work was supported in part by grants from the Centre de Recherche Interuniversitaire en Vaccinologie, the Fonds National de la Recherche Scientifique et Me´ dicale, the Association Lupus Erythe´ mateux, and by the Belgian Federal Service for Scientific, Technical and Cultural Affairs. J.-C. R. is Research Associate with the Fonds National de la Recherche Scientifique.
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