Vol. 59, No. 5
INFECTION AND IMMUNITY, May 1991, p. 1709-1715 0019-9567/91/051709-07$02.00/0 Copyright © 1991, American Society for Microbiology
Regulation of Gamma Interferon Production by Natural Killer Cells in scid Mice: Roles of Tumor Necrosis Factor and Bacterial Stimuli JANICE C. WHERRY,t ROBERT D. SCHREIBER, AND EMIL R. UNANUE* Department of Pathology, Washington University School of Medicine, 660 S. Euclid, St. Louis, Missouri 63110 Received 11 October 1990/Accepted 6 February 1991
CB-17 scid mice exhibit a T-cell-independent but gamma interferon (IFN--y)-dependent immunity to Listeria monocytogenes. In this study, we analyzed the specific cellular interactions involved in this process. scid mouse-derived natural killer (NK) cells cultured with heat-killed (HK) L. monocytogenes and macrophages secreted IFN-'y. No IFN--y was produced in cultures containing HK L. monocytogenes but lacking macrophages. However, medium derived from macrophages incubated with HK L. monocytogenes or other microorganisms stimulated IFN--y production by isolated NK cells. Treatment of macrophage-conditioned supernatants with neutralizing monoclonal anti-tumor necrosis factor (TNF) significantly reduced their capacity to stimulate NK cells to produce IFN-y. Yet, purified recombinant TNF-a by itself was unable to stimulate NK cells. Thus, TNF was necessary but not sufficient to induce maximal IFN--y production by NK cells. Sonicated L. monocytogenes stimulated production of IFN--y by NK cells that was resistant to anti-TNF. Stimulation was markedly enhanced by the addition of recombinant TNF-a. These studies demonstrated that activation of scid NK cells for secretion of IFN--y requires two signals: TNF-a and a second product which may be of bacterial origin and may require processing by mononuclear phagocytes. We suggest that the T-celi-independent production of IFN--y by NK cells provides the host with a rapid mechanism to temporarily heighten nonspecific resistance to infection until such time as T-cell-dependent sterilizing immune responses can be generated.
We previously characterized a T-cell-independent process of resistance to Listeria monocytogenes in mice with the scid mutation (1-3). This process provides the scid host with partial protection from microbial infection and leads to profound macrophage activation in vivo in the complete absence of T cells. We have identified two cell types that participate in this process: macrophages that ingest the L. monocytogenes and cells with characteristics of natural killer (NK) cells. Other investigators had identified NK cells in scid mice (7, 10, 15). We also showed that at least two cytokines were involved: tumor necrosis factor (TNF) and gamma interferon (IFN--y) (1-3). In vivo and in vitro experiments showed that uptake of L. monocytogenes by macrophages resulted in TNF production which in turn caused the release of IFN--y by cells with characteristics of NK cells. While TNF appeared to be an essential component in inducing IFN-y production in cultures of scid spleen cells with L. monocytogenes, it was not sufficient to induce IFN--y production by spleen cells. Here we further characterize the cellular interactions involved in this process and demonstrate a role for microbial products that act in concert with TNF to affect IFN--y production by purified scid NK cells. MATERIALS AND METHODS Mice. CB-17/Icr (CB-17) and the CB-17/Icr (scid) mutant strain, originally obtained from Melvin Bosma (Institute for Cancer Research, Philadelphia, Pa.), were bred in specific pathogen-free conditions at Washington University. NK cells. NK cells were produced by a 5- to 7-day culture of scid bone marrow cells with recombinant human interleukin-2 (IL-2). Bone marrow cells were plated at Corresponding author. t Present address: Clinical Research B47, Cutter Laboratories, Miles Inc., 4th and Parker St., P.O. Box 1986, Berkeley, CA 94701. *
1.5 x 107/100-_um-diameter dishes in 10 ml of RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 50 units of penicillin per ml, 50 ,uzg of streptomycin (GIBCO Laboratories, Grand Island, N.Y.) per ml, 10 mM HEPES (N-2-hydroxethylpiperazine-N'-2-ethanesulfonic acid) (GIBCO), 5 x 10-5 M 2-mercaptoethanol (Bio-Rad Laboratories, Richmond, Calif.), 1 mM sodium pyruvate (GIBCO), 1% (vol/vol) nonessential amino acids (GIBCO), and 300 U of recombinant human IL-2 (Hoffmann-La Roche Inc., Nutley, N.J.) per ml. Nonviable cells were removed, usually from 6-day-old cultures, by passage over FicollPaque gradients (Pharmacia LKB Biotechnology Inc., Piscataway, N.J.). Most of the cells recovered from the gradients had the appearance of medium-sized lymphocytes, with reniform nuclei and variable numbers of cytoplasmic vacuoli. Such preparations were devoid of CD4 and CD8 markers. Contamination by macrophages was negligible and not appreciable by functional criteria (i.e., production of TNF after exposure to L. monocytogenes). The NK cell nature of the purified cells was confirmed by standard cytotoxicity assays with 51Cr-labeled YAC-1 cells as targets. Macrophages. Macrophages were obtained by lavage of scid mouse peritoneal cavities with RPMI 1640 medium as described previously (2), resuspension of cells in medium containing 5 ,ug of indomethacin (Sigma Chemical Co., St. Louis, Mo.) per ml, and adhesion in 96- or 24-well plates for 4 h. Wells were washed three times with room temperature medium to remove nonadherent cells. Macrophage-conditioned media were prepared by exposure of macrophage monolayers to bacterial stimuli for 3 to 48 h. The supernatants were cleared by centrifugation, filtered through a 0.2-,um-pore-size filter, and stored at 4°C until assayed. Indomethacin was used in all experiments to ablate inhibiting effects of prostaglandins. However, control experiments revealed no differences when medium either containing or lacking indomethacin was used. 1709
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TABLE 1. IFN-y production requires both NK cells and macrophagesa Cells
IFN-y (IRUb/ml) in expt no.:
Stimulus 98
NK cells Macrophages NK cells Macrophages Macrophages + NK cells
117
122
121
135
152
0 HK L. monocytogenes HK L. monocytogenes HK L. monocytogenes
0 51 0 1,080
72 0 1,404
24 0 4,617
0 0 2,349
0 57 531
172
0
0 0
15 17 1,782
51
0 3,645
a This table shows the results of seven different experiments. Macrophages (3 x 105) were plated and incubated for 4 h at 37'C, and nonadherent cells were removed. Bone marrow-derived NK cells were added to macrophage cultures at 2 x 105 cells per well. HK L. monocytogenes was added at 6 x 105/ml. All were incubated for 48 h, and then the supernatants were assayed for IFN-y. b IRU, Interferon reference units.
Cytokine production and quantitation. NK cells were cultured with or without activating stimuli in 200 ,ul of medium at 2 x 105 cells per well in 96-well flat-bottomed plates for 48 h. The plates were centrifuged, and the supernatants were collected and stored at -20°C. In all experiments, the NK cells were maintained in recombinant human IL-2 (300 U/ml) during the 48-h stimulation since removal of IL-2 resulted in loss of cell viability. In selected cases, Transwell chambers (Costar, Cambridge, Mass.) were used to culture cells such that macrophages and NK cells were on different sides of a filter during the incubation. Listeria stimuli included heatkilled (HK) L. monocytogenes prepared as previously described (1) and sonicated L. monocytogenes prepared as follows. A log-phase culture of approximately 107 L. monocytogenes per ml was washed in phosphate-buffered saline and sonicated for eight bursts of 15 s each at 4°C in a sonicator. The sonicates were filtered through a 0.22-jimpore-size filter, concentrated in a Centricon concentrator containing a YM 30 filter, and stored at -20°C. Other bacterial stimuli included Salmonella typhimurium (Ribi Research Laboratory, Hamilton, Mont.), Corynebacterium parvum (Ribi Research Laboratory), lipopolysaccharide from Salmonella typhi (Ribi Research Laboratory), purified protein derivative of Mycobacterium tuberculosis (kindly provided by P. M. Allen), and muramyl dipepetide (Sigma). Other stimuli included concanavalin A (2.5 pLg/ml; Sigma) and latex beads (2.9-,um diameter; Polyscience, Warrington, Pa.). IFN--y and TNF were measured by enzyme-linked immunosorbent assays (ELISAs) (5, 21), using standard curves employing the appropriate purified recombinant cytokine. Those assays showed a minimum sensitivity of 1 to 10 units/ml. Cytokines and cytokine-specific antibodies. We used monoclonal antibodies directed against murine TNF (TN3-19.12) (19), IL-lot (161.1) (8), IFN-y (H22.1) (5), and IL-6 (MP520F3.11 rat immunoglobulin Gl; courtesy of John Abrams, DNAX, and Marvin Siegel, Schering-Plough Corp.). Polyclonal rabbit anti-murine IL-lp was provided by David Chaplin (Washington University School of Medicine, St. Louis, Mo.). Plates coated with anti-TNF were prepared by diluting the TN3-19.12 to 2 ,ug/ml in pH 9.6 carbonate buffer, placing 0.2-ml aliquots into Immulon plates (Dynatech Laboratories, Chantilly, Va.), and storing them at 4°C until use. For antibody addition to NK cultures, anti-TNF was diluted to 0.2 jig/ml, anti-IL-la was diluted to 10 ,ug/ml, anti-IL-1p was diluted to 10 ,ug/ml, and anti-IL-6 was diluted to 50 jLg/ml. Recombinant murine TNF-a (specific activity, 2.9 x 107 U/mg) and recombinant murine IFN-y (specific activity, 7 x 106 U/mg) were kindly provided by Susan Kramer, Genentech Inc., So. San Francisco, Calif.
Statistics and experimental reproducibility. All experiments were performed at least three times, with standard deviations from the mean exceeding no more than 10%.
RESULTS IFN--y production requires both macrophages and NK cells. In a previous study, we demonstrated the production of IFN-y from a mixture of scid spleen cells that contained macrophages and NK cells (1). The NK cells were characterized as cells that did not adhere to plastic dishes, reacted with antibodies to asialo-GM1, and killed YAC-1 cells. To better analyze the cellular constituents in this system, we tested purified scid NK cells grown from bone marrow precursors by culture in IL-2 and macrophages obtained from the peritoneal cavity. NK cells, macrophages, or both were incubated with HK L. monocytogenes, and the production of IFN--y was determined by ELISA after 48 h of culture (Table 1). NK cells or macrophages cultured by themselves with or without HK L. monocytogenes produced little, if any, IFN-y. However, addition of HK L. monocytogenes to cocultures of NK cells plus macrophages resulted in secretion of large amounts of IFN-y. The same results were obtained with live L. monocytogenes (data not shown). This effect was not exclusive to L. monocytogenes; addition of two other microorganisms, S. typhimurium and C. parvum, to cocultures of NK cells and macrophages resulted in production of IFN--y (Fig. 1). NK cells incubated alone with these bacteria failed to secrete IFN--y (data not shown). S. typhi
Listeria
C. parvum
3500 3000
2500a:
2000
>. 1500-
z
0
107
106
i05
100
20
4
100
20
4
mg/ml mg/ml FIG. 1. Effects of bacterial stimuli on IFN--y production. NK cells were cultured with the indicated stimuli in the presence of macrophages, and the supernatants were assayed for IFN--y at 48 h. In the absence of macrophages, NK cells produced less than 20 interferon reference units (IRU) per ml. Bacteria/ml
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ROLE OF TNF-ot IN INDUCING IFN-y PRODUCTION BY NK CELLS
Lower chamber Upper chamber Macrophages Macrophages NK ( 10 5) NK ( 10 5)
(a) (b) (c) (d)
(e) (f)
(g)
(h) (i)
1.8 1.8 1.8 4.6 4.6 4.6 9.2 9.2 9.2
IFNy
TNF
(IRU/ml)
(U/ml)
1711
+ +
0
8
16
0
80
6
mm~
+ +
+ +
(i) 0 800 1600 0 800 1600 FIG. 2. Cell-cell contact between macrophages and NK cells is not absolutely required for stimulation of NK cells to produce IFN--y. Transwell chambers were used to culture macrophages and NK cells without allowing cell-cell contact between the two cell types. Macrophages were cultured at the indicated concentrations. NK cells were at 106 per well. All the wells with macrophages received 2 x 105 HK L. monocytogenes.
Thus, IFN--y production by NK cells required the presence of macrophages. Macrophage-conditioned medium stimulates NK cells to produce IFN-y. From our previous studies, we knew that TNF was involved in the process of induction of IFN--y by NK cells. To evaluate the contribution of products released by macrophages in stimulating NK cells to produce fFN-y, we performed two types of experiments. The first involved the use of Transwell chambers in which macrophages and HK L. monocytogenes were placed on one side of a filter and NK cells were placed on the other side. IFN--y production was determined after 48 h of incubation (Fig. 2). As expected, NK cells or macrophages separately incubated with HK L. monocytogenes produced minimal IFN--y (Fig. 2, rows a, d, g, and j). TNF was produced only when macrophages were present in the wells (rows a to i). The culture of NK cells, macrophages, and HK L. monocytogenes in the same well resulted in high levels of both TNF
and IFN--y (rows b, e, and h). When macrophages and NK cells were separated by the Transwell filter, significant amounts of IFN-y were produced, although not to the same extent as when the two cell types were present in the same chamber (rows c, f, and i). In the second type of experiment, filtered conditioned medium from macrophages incubated with HK L. monocytogenes was added to NK cells and the secretion of IFN--y was determined 48 h later (Table 2). The macrophageconditioned medium was active in inducing NK cells to produce IFN-y. In most experiments, the most active conditioned medium was obtained after 8 h of culture of mac-
Experiment 1 E
TABLE 2. Effect of macrophage supematants on NK cell IFN-y secretiona
1U-
z
IFN-y (IRUb/ml) Expt 137 146 149 152 155 162 164 172 180
NK cells
NK cells + HK L. monocytogenes
0 0 0 0 0 0
33 15
0 0
0 0
NK cells +
2,997 1,040 2,916 1,391 1,269 1,269 2,673 1,782 1,026
a This table shows the results of nine different experiments. Macrophages were cultured with HK L. monocytogenes for 8 h, and the supematants were recovered. NK cells were cultured alone, with HK L. monocytogenes (6 x 105/ml), or with a 50% (vol/vol) concentration of the macrophage-conditioned medium. After 48 h of incubation, the supernatants were tested for IFN--y
production. b IRU, Interferon reference units.
Experiment 2
conditioned medium E U-
z
Time (hours)
Time (hours)
FIG. 3. Activity of macrophage-conditioned media obtained at different times. Macrophages were cultured with HK L. monocytogenes for the times indicated in the figure, and the supernatants were recovered and tested for TNF activity. Stimulation of IFN-y secretion was determined by adding a 50% (vol/vol) concentration of each supernatant to NK cells and measuring IFN--y production. IRU, Interferon reference units.
1712
WHERRY ET AL.
INFECT. IMMUN.
180 160 0o140
E
120
z
100
Antibodies
IL-1
IL-1a
IL-6
IFN y
(IRU/ml)
TNF
'0~~~~
>. 80 E 60 40'0 20i0 z
'0~~~ +
0
0
10 20 30 40 50 60 70 80 Macrophage Conditioned Media (%)
FIG. 4. Dose response of NK cells to macrophage-conditioned media. NK cells (2 x 105) were cultured in medium containing different percentages (vol/vol) of an 8-h supernatant from macrophages cultured with HK L. monocytogenes. After 48 h of incubation, the NK cell supernatants were tested for IFN--y production. IRU, Interferon reference units.
0
S. typhi
1600
FIG. 6. Effects of neutralizing cytokine-specific antibodies on macrophage supernatant stimulation of IFN--y. An 8-h macrophage supernatant was incubated with NK cells in the presence of antiIL-la (10 ,ug/ml), anti-IL-1B (10 ,ug/ml), anti-IL-6 (50 pg/ml), or anti-TNF (140 ,ug/ml), and the supernatants were assayed for IFN--y secretion. IRU, Interferon reference units.
rophages with HK L. monocytogenes (Fig. 3). Longer incubation periods resulted in conditioned media that were less active in subsequently stimulating NK cells to produce IFN--y. The amount of IFN--y produced by NK cells depended on the concentration of macrophage supernatant used (Fig. 4). Active supernatants were also obtained when macrophages were cocultured with other bacteria or their products but not when they were exposed to latex beads (Fig. 5) or concanavalin A (data not shown). As noted above, the stimulating activity of macrophageconditioned media decayed with longer times of culture. However, the extent of decay varied from experiment to experiment, ranging from an 87% to a 66% decrease. The decreased activity in 48-h supernatants was not due to lack of TNF since similar amounts of TNF protein were found in
Listeria
800
8-h and 48-h macrophage supernatants. However, it is possible that the decay in activity was due to the presence of a TNF inhibitor in the 48-h supernatant. Experiments are currenitly under way to examine this issue. Role of TNF in production of IFN-y by NK cells. NK cells were cultured with macrophage-conditioned medium in the presence of antibodies against IL-la, IL-1,, IL-6, or TNF. IFN-y secretion was not affected by the presence of antibodies to IL-1 or IL-6 but was suppressed about 80% by anti-TNF (Fig. 6). Addition of purified recombinant murine TNF-(x to the culture supernatant restored NK cell secretion
C. parvum
PPD
LPS
No Latex stimuli
1400
1200 1000
E 800 D
600 400
200 0 100
20 4
100
20 4
50 5
Stimuli (Bacteria or
:
Mg/ml)
FIG. 5. Production of active macrophage-conditioned media by various bacterial stimuli. Macrophages were cultured with the indicated materials for 8 h, and the supernatants were recovered. TNF in each supernatant was tested (0). NK cells were cultured for 48 h at 50% (vol/vol) with each of them, and IFN--y was measured (-). NK cells cultured directly with the indicated stimuli produced less than 50 interferon reference units (IRU) of IFN--y per ml.
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ROLE OF TNF-a IN INDUCING IFN--y PRODUCTION BY NK CELLS
rTNF (U/ml)
Anti-TNF
IFNy
TNF
Listeria Sonicate
(IRU/mI)
(U/ml)
(jig/ml)
TNF (240 U/mI)
1713
IFNy (IRU/ml)
375 375 +
3843
+
960
+
240
+
60
0
1750
3500
30 6 6 0
900
1800
FIG. 7. Reconstitution of anti-TNF-treated macrophage-conditioned supernatant by purified recombinant murine TNF-a. An 8-h macrophage-conditioned medium was incubated with NK cells in the presence of 0.2 ,ug of anti-TNF per ml and various concentrations of TNF; the supernatants were assayed for IFN--y secretion at 48 h. IRU, Interferon reference units; rTNF, recombinant TNF-a.
of IFN--y (Fig. 7). Similar inactivation of supernatant stimulatory ability occurred when the conditioned medium was incubated in plates containing surface-bound anti-TNF. Activity of the depleted supernatants could be completely reconstituted with purified recombinant murine TNF-a (data not shown). To test whether TNF alone or with HK L. monocytogenes could stimulate NK cell release of IFN-y, we incubated NK cells with recombinant TNF-ot and/or HK L. monocytogenes (Table 3). Minimal IFN--y secretion was found. This result agrees with our previous observation that TNF alone could not stimulate IFN--y secretion by unfractionated scid spleen cells (1). Thus, TNF is necessary but not sufficient to stimulate NK cell-dependent IFN-y production. Effect of bacterial products on NK cell IFN--y production. Since macrophage-derived cytokines were unable to directly stimulate production of NK cell IFN--y, we evaluated the TABLE 3. Effects of TNF and/or HK L. monocytogenes on IFN--y production by NK cellsa Stimulus
(U/mi)
0 3,843 960 240
960 960 960
IFN-a
HK L. monocytogenes
Mac-CMc
Macrophages
0
-
-
_
-
+
-
-
0 81 0 0 51 3,645 0 153 63
+
-
1,782
-
6x107 6X 106 6 x 105 6 x 107 6 x 106 6 x 105
150 150 30
-
(IRUb/ml) 0
a A representative experiment is shown. NK cells were cultured as indicated, and IFN--y was measured after 48 h. b IRU, Interferon reference units. c Mac-CM, Conditioned medium from macrophages cultured with HK L. monocytogenes for 8 h.
=
+
0 800 1600 FIG. 8. Effects of Listeria sonicates on IFN--y secretion by NK cells. This is representative of five different experiments with three different preparations of sonicated L. monocytogenes. Sonicated L. monocytogenes preparations were incubated with NK cells in the presence or absence of 960 U of exogenous recombinant TNF-a per ml. IRU, Interferon reference units.
role of bacterial products in this process. NK cells were incubated with either intact bacteria or L. monocytogenes sonicates in the presence or absence of exogenous TNF-a, and IFN--y production was determined (Fig. 8). Whereas no IFN-y was produced with intact L. monocytogenes, soluble L. monocytogenes products caused significant IFN--y production by the NK cell culture. Similar levels of IFN-y were induced by L. monocytogenes sonicates added with or without the neutralizing anti-TNF monoclonal antibody (in four different experiments, no inhibition by anti-TNF was found). Thus, the soluble bacterial product could directly stimulate NK cells to produce IFN--y in the absence of TNF. However, addition of TNF-a to NK cell cultures treated with bacterial products resulted in marked enhancement of IFN--y secretion. Thus, two stimuli can synergize to induce IFN--y production: macrophage-derived TNF and a soluble Listeria product. DISCUSSION The present study adds to our previous report (1) the finding that purified NK cells can release IFN--y upon interaction with a soluble conditioned medium from macrophages that have taken up L. monocytogenes. The conditioned medium must contain TNF and some other unrecognized product. We confirmed that TNF by itself is not able to stimulate NK cells, although some stimulation could be induced by a soluble Listeria sonicate. However, TNF and soluble bacterial products acted synergistically to induce maximal secretion of IFN--y. We do not know whether the additional active moiety in the macrophage-conditioned medium is a soluble Listeria product or some other factor derived from the macrophages. If it is a macrophage-derived cytokine, it is not IL-la, IL-,, or IL-6. Not all the activity in the macrophage-conditioned media was abolished by antiTNF. This suggests that the activity may well be a product equivalent to the sonicate, which was not inhibited by anti-TNF antibodies. Work is now in progress to resolve this issue. A new contribution made here is the clear identification of the cellular protagonists in this non-T-cell-dependent pathway of IFN--y production (1-3). Undoubtedly they involve
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INFECT. IMMUN.
WHERRY ET AL.
macrophages and NK cells as surmised from the use of purified macrophages and NK cells grown from bone marrow. In our previous report (1), the evidence for NK cells was quite strong but required further steps of cellular purification to be definitive. Another important feature demonstrated in the current study is that the responsiveness of NK cells to macrophage-conditioned media depends on their state of activation. While activated NK cells grown in IL-2 responded to this medium, NK cells freshly harvested from scid spleens did not. Our interpretation is that (i) the activation state of NK cells is critical for a response to occur and (ii) the activation state of NK cells dictates whether cell contact between NK cells and macrophages is required. These items will be addressed in future experiments. Activation of NK cells to a cytotoxic state or for IFN-y secretion has been reported in a number of in vitro and in vivo situations. These include responses to some bacteria (4, 13, 16, 28), viruses (27), fungi (23), and tumors (6, 12, 20, 25). Although IFN (9) and IL-2 (11, 14, 17, 18, 21, 22, 24, 26, 29) are known NK cell activators, our results in the scid mouse imply that more than one cellular or soluble component is necessary to achieve maximal IFN--y secretion by NK cells. Our studies with the scid cells clearly document a pathway for production of IFN--y which is induced by some facultative intracellular bacteria and does not involve the participation of CD4+ or CD8+ T cells (1-3). We speculate that this pathway provides the initial defense against microorganisms that are then partially eliminated by target cells (such as macrophages) activated by NK cell production of IFN-y. Thus, this pathway could be operative before T-cell immunity has developed. It is possible that in an immunologically intact host, this pathway can also participate synergistically with the usual antigen-presenting cell-CD4/CD8 T-cell interactions that result in the production of IFN-y and other cytokines. We therefore believe that this T-cell-independent production of IFN--y plays an important physiologic role in initiating and propagating host responses to infectious agents. ACKNOWLEDGMENTS We thank Susan Kramer of Genentech, Inc., for providing purified recombinant murine TNF-ot and Maury Gately and Grace Ju of Hoffmann-La Roche for providing purified recombinant human IL-2. We are also grateful to Karen Larimore for expert technical assistance and Marian Simandl Florea for secretarial assistance. This work was supported by Public Health Service grants A115322, A124854, and CA43059 from the National Institutes of Health. REFERENCES 1. Bancroft, G. J., M. J. Bosma, G. C. Bosma, and E. R. Unanue. 1986. Regulation of macrophage Ia expression in mice with severe combined immunodeficiency: induction of Ia expression by a T cell-independent mechanism. J. Immunol. 137:4-9. 2. Bancroft, G. J., R. D. Schreiber, G. C. Bosma, M. J. Bosma, and E. R. Unanue. 1987. A T cell-independent mechanism of macrophage activation by interferon-gamma. J. Immunol. 139:11041107. 3. Bancroft, G. J., K. C. F. Sheehan, R. D. Schreiber, and E. R. Unanue. 1989. Tumor necrosis factor is involved in the T cell-independent pathway of macrophage activation in scid mice. J. Immunol. 143:127-130. 4. Blanchard, D. K., H. Friedmann, W. E. Stewart II, T. W. Klein, and J. Y. Djeu. 1988. Role of gamma-interferon in induction of natural killer activity by Legionella pneumophila in vitro and in an experimental murine infection model. Infect. Immun. 56: 1187-1193. 5. Buchmeier, N. A., and R. D. Schreiber. 1985. Requirement of
endogenous interferon-gamma production for resolution of Listeria monocytogenes infection. Proc. Natl. Acad. Sci. USA
82:7404-7408. 6. Djeu, J. Y., K. Y. Huang, and R. B. Herberman. 1980. Augmentation of mouse NK activity and induction of interferon by
tumor cells in vivo. J. Exp. Med. 151:781-789. 7. Dorschkind, K., S. B. Pollack, M. J. Bosma, and R. A. Phillips.
1985. Natural killer (NK) cells are present in mice with severe combined immunodeficiency (scid). J. Immunol. 134:3798-3801. 8. Fuhlbrigge, R. D., K. C. F. Sheehan, R. D. Schreiber, D. D. Chaplin, and E. R. Unanue. 1988. Monoclonal antibodies to murine interleukin-la: production, characterization, and inhibition of membrane-associated interleukin-1 activity. J. Immunol. 141:2643-2650. 9. Gidlund, M., A. Orn, H. Wigzell, A. Senik, and I. Gresser. 1978. Enhanced NK cell activity in mice injected with interferon and interferon inducers. Nature (London) 273:759-761.
10. Hackett, J., Jr., G. C. Bosma, M. J. Bosma, M. Bennett, and V. Kumar. 1986. Transplantable progenitors of natural killer cells are distinct from those of T and B lymphocytes. Proc. Natl. Acad. Sci. USA 83:3427-3431. 11. Handa, K., R. Suzuki, H. Matsui, Y. Shimizu, and K. Kumagai. 1983. NK cells as a responder to IL-2. II. IL-2-induced interferon-gamma production. J. Immunol. 130:988-992. 12. Herberman, R. B., M. E. Nunn, H. T. Holden, S. Staal, and J. Y. Djeu. 1977. Augmentation of natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic target cells. Int. J. Cancer 19:555-564. 13. Holmberg, L. A., T. A. Springer, and K. A. Ault. 1981. Natural killer activity in the peritoneal exudates of mice infected with Listeria monocytogenes: characterization of the natural killer cells by using a monoclonal rat anti-murine macrophage antibody (M1/70). J. Immunol. 127:1792-1799. 14. Kasahara, T., J. J. Hooks, S. Dougherty, and J. J. Oppenheim. 1983. Interleukin 2-mediated immune interferon-gamma production by human T cells and T cell subsets. J. Immunol. 130:17841789. 15. Lauzon, R. J., K. A. Siminovitch, G. M. Fulop, R. A. Phillips, and J. C. Roder. 1986. An expanded population of natural killer cells in mice with severe combined immunodeficiency (scid) lack rearrangement and expression of T cell receptor genes. J. Exp. Med. 164:1797-1802. 16. Ojo, E., 0. Haller, and H. Wigzell. 1978. Corynebacterium
17.
18. 19.
20.
21.
22.
23.
parvum-induced peritoneal exudate cells with rapid cytolytic activity against tumour cells are non-phagocytic cells with characteristic of NK cells. Scand. J. Immunol. 8:215-222. Ortaldo, J. R., A. T. Mason, J. P. Gerard, L. E. Henderson, W. Farrar, R. F. Hopkins, R. B. Herberman, and H. Rabin. 1984. Effects of natural and recombinant IL-2 on regulation of interferon-gamma production and NK activity: lack of involvement of the Tac antigen for these immunoregulatory effects. J. Immunol. 133:779-783. Reem, G. H., and N.-H. Yeh. 1984. IL-2 regulates expression of its receptor and synthesis of gamma-interferon by human T lymphocytes. Science 225:429-430. Sheehan, K. C. F., N. Ruddle, and R. D. Schreiber. 1989. Generation and characterization of hamster monoclonal antibodies that neutralize murine tumor necrosis factor. J. Immunol. 142:3884-3893. Shellam, G. R., V. Winterfourn, and H. J. S. Dawkins. 1980. Augmentation of cell-mediated cytotoxicity to a rat lymphoma. III. In vitro stimulation of NK cells by a soluble factor. Int. J. Cancer 25:331-339. Shiiba, K., K. Iton, U. Shimizu, and K. Kumagai. 1984. Interleukin-2 (IL-2)-dependent proliferation of human NK cells accompanied by interferon-gamma production, p. 187-192. In T. Hoshina, H. S. Koren, and A. Uchida (ed.), Natural killer activity and its regulation. Excerpta Medica, Amsterdam. Suzuki, R., K. Handa, K. Itoh, and K. Kumagai. 1983. Natural killer (NK) cells as a responder to interleukin-2 (IL-2). I. Proliferative response and establishment of cloned cells. J. Immunol. 130:981-987. Tartof, D., I. J. Check, A. Matutis, R. L. Hunter, and F. W.
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Fitch. 1980. Studies on stimulation of cell-mediated cytotoxicity by skin test antigens. I. Candida antigen stimulation of cellmediated cytotoxicity in vitro correlated with the skin test response to candida antigen in vivo. J. Immunol. 125:2790-2796. 24. Trinchieri, G., M. Matsumota-Kobayashi, S. C. Clark, J. Seehra, L. London, and B. Perussia. 1984. Response of resting human peripheral blood NK cells to IL-2. J. Exp. Med. 160: 1147-1169. 25. Trinchieri, G., D. Santoli, R. R. Dee, and B. Knowles. 1978. Anti-viral activity induced by culturing lymphocytes with tumor-derived or virus-infected cells. Identification of the antiviral activity as interferon and characterization of the human effector lymphocyte subpopulation. J. Exp. Med. 147:12991313. 26. Vilcek, J., D. Henriksen-Destefano, D. Siegel, A. Klion, R. J.
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Robb, and J. Le. 1985. Regulation of interferon-gamma induction in human peripheral blood cells by exogenous and endog-
enously produced interleukin 2. J. Immunol. 135:1851-1856. 27. Welsh, R. M., Jr. 1978. Cytotoxic cells induced during lymphocyte choriomeningitic virus infection of mice.
I. Characterization of NK cell induction. J. Exp. Med. 146:163-181. 28. Wolfe, S. A., D. E. Tracey, and C. S. Henney. 1977. BCGinduced murine effector cells. II. Characterization of NK cells in peritoneal exudates. J. Immunol. 119:1152-1158. 29. Ythier, A., L. Delmon, E. Reinherz, A. Nowill, P. Moingeon, Z. Mishal, C. Bohuon, and T. Hercend. 1985. Proliferative responses of circulating human NK cells: delineation of a unique pathway involving both direct and helper signals. Eur. J. Immunol. 15:1209-1215.
INFECTION AND IMMUNITY, May 1991, p. 1709-1715 0019-9567/91/051709-07$02.00/0 Copyright © 1991, American Society for Microbiology
Regulation of Gamma Interferon Production by Natural Killer Cells in scid Mice: Roles of Tumor Necrosis Factor and Bacterial Stimuli JANICE C. WHERRY,t ROBERT D. SCHREIBER, AND EMIL R. UNANUE* Department of Pathology, Washington University School of Medicine, 660 S. Euclid, St. Louis, Missouri 63110 Received 11 October 1990/Accepted 6 February 1991
CB-17 scid mice exhibit a T-cell-independent but gamma interferon (IFN--y)-dependent immunity to Listeria monocytogenes. In this study, we analyzed the specific cellular interactions involved in this process. scid mouse-derived natural killer (NK) cells cultured with heat-killed (HK) L. monocytogenes and macrophages secreted IFN-'y. No IFN--y was produced in cultures containing HK L. monocytogenes but lacking macrophages. However, medium derived from macrophages incubated with HK L. monocytogenes or other microorganisms stimulated IFN--y production by isolated NK cells. Treatment of macrophage-conditioned supernatants with neutralizing monoclonal anti-tumor necrosis factor (TNF) significantly reduced their capacity to stimulate NK cells to produce IFN-y. Yet, purified recombinant TNF-a by itself was unable to stimulate NK cells. Thus, TNF was necessary but not sufficient to induce maximal IFN--y production by NK cells. Sonicated L. monocytogenes stimulated production of IFN--y by NK cells that was resistant to anti-TNF. Stimulation was markedly enhanced by the addition of recombinant TNF-a. These studies demonstrated that activation of scid NK cells for secretion of IFN--y requires two signals: TNF-a and a second product which may be of bacterial origin and may require processing by mononuclear phagocytes. We suggest that the T-celi-independent production of IFN--y by NK cells provides the host with a rapid mechanism to temporarily heighten nonspecific resistance to infection until such time as T-cell-dependent sterilizing immune responses can be generated.
We previously characterized a T-cell-independent process of resistance to Listeria monocytogenes in mice with the scid mutation (1-3). This process provides the scid host with partial protection from microbial infection and leads to profound macrophage activation in vivo in the complete absence of T cells. We have identified two cell types that participate in this process: macrophages that ingest the L. monocytogenes and cells with characteristics of natural killer (NK) cells. Other investigators had identified NK cells in scid mice (7, 10, 15). We also showed that at least two cytokines were involved: tumor necrosis factor (TNF) and gamma interferon (IFN--y) (1-3). In vivo and in vitro experiments showed that uptake of L. monocytogenes by macrophages resulted in TNF production which in turn caused the release of IFN--y by cells with characteristics of NK cells. While TNF appeared to be an essential component in inducing IFN-y production in cultures of scid spleen cells with L. monocytogenes, it was not sufficient to induce IFN--y production by spleen cells. Here we further characterize the cellular interactions involved in this process and demonstrate a role for microbial products that act in concert with TNF to affect IFN--y production by purified scid NK cells. MATERIALS AND METHODS Mice. CB-17/Icr (CB-17) and the CB-17/Icr (scid) mutant strain, originally obtained from Melvin Bosma (Institute for Cancer Research, Philadelphia, Pa.), were bred in specific pathogen-free conditions at Washington University. NK cells. NK cells were produced by a 5- to 7-day culture of scid bone marrow cells with recombinant human interleukin-2 (IL-2). Bone marrow cells were plated at Corresponding author. t Present address: Clinical Research B47, Cutter Laboratories, Miles Inc., 4th and Parker St., P.O. Box 1986, Berkeley, CA 94701. *
1.5 x 107/100-_um-diameter dishes in 10 ml of RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 50 units of penicillin per ml, 50 ,uzg of streptomycin (GIBCO Laboratories, Grand Island, N.Y.) per ml, 10 mM HEPES (N-2-hydroxethylpiperazine-N'-2-ethanesulfonic acid) (GIBCO), 5 x 10-5 M 2-mercaptoethanol (Bio-Rad Laboratories, Richmond, Calif.), 1 mM sodium pyruvate (GIBCO), 1% (vol/vol) nonessential amino acids (GIBCO), and 300 U of recombinant human IL-2 (Hoffmann-La Roche Inc., Nutley, N.J.) per ml. Nonviable cells were removed, usually from 6-day-old cultures, by passage over FicollPaque gradients (Pharmacia LKB Biotechnology Inc., Piscataway, N.J.). Most of the cells recovered from the gradients had the appearance of medium-sized lymphocytes, with reniform nuclei and variable numbers of cytoplasmic vacuoli. Such preparations were devoid of CD4 and CD8 markers. Contamination by macrophages was negligible and not appreciable by functional criteria (i.e., production of TNF after exposure to L. monocytogenes). The NK cell nature of the purified cells was confirmed by standard cytotoxicity assays with 51Cr-labeled YAC-1 cells as targets. Macrophages. Macrophages were obtained by lavage of scid mouse peritoneal cavities with RPMI 1640 medium as described previously (2), resuspension of cells in medium containing 5 ,ug of indomethacin (Sigma Chemical Co., St. Louis, Mo.) per ml, and adhesion in 96- or 24-well plates for 4 h. Wells were washed three times with room temperature medium to remove nonadherent cells. Macrophage-conditioned media were prepared by exposure of macrophage monolayers to bacterial stimuli for 3 to 48 h. The supernatants were cleared by centrifugation, filtered through a 0.2-,um-pore-size filter, and stored at 4°C until assayed. Indomethacin was used in all experiments to ablate inhibiting effects of prostaglandins. However, control experiments revealed no differences when medium either containing or lacking indomethacin was used. 1709
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WHERRY ET AL.
TABLE 1. IFN-y production requires both NK cells and macrophagesa Cells
IFN-y (IRUb/ml) in expt no.:
Stimulus 98
NK cells Macrophages NK cells Macrophages Macrophages + NK cells
117
122
121
135
152
0 HK L. monocytogenes HK L. monocytogenes HK L. monocytogenes
0 51 0 1,080
72 0 1,404
24 0 4,617
0 0 2,349
0 57 531
172
0
0 0
15 17 1,782
51
0 3,645
a This table shows the results of seven different experiments. Macrophages (3 x 105) were plated and incubated for 4 h at 37'C, and nonadherent cells were removed. Bone marrow-derived NK cells were added to macrophage cultures at 2 x 105 cells per well. HK L. monocytogenes was added at 6 x 105/ml. All were incubated for 48 h, and then the supernatants were assayed for IFN-y. b IRU, Interferon reference units.
Cytokine production and quantitation. NK cells were cultured with or without activating stimuli in 200 ,ul of medium at 2 x 105 cells per well in 96-well flat-bottomed plates for 48 h. The plates were centrifuged, and the supernatants were collected and stored at -20°C. In all experiments, the NK cells were maintained in recombinant human IL-2 (300 U/ml) during the 48-h stimulation since removal of IL-2 resulted in loss of cell viability. In selected cases, Transwell chambers (Costar, Cambridge, Mass.) were used to culture cells such that macrophages and NK cells were on different sides of a filter during the incubation. Listeria stimuli included heatkilled (HK) L. monocytogenes prepared as previously described (1) and sonicated L. monocytogenes prepared as follows. A log-phase culture of approximately 107 L. monocytogenes per ml was washed in phosphate-buffered saline and sonicated for eight bursts of 15 s each at 4°C in a sonicator. The sonicates were filtered through a 0.22-jimpore-size filter, concentrated in a Centricon concentrator containing a YM 30 filter, and stored at -20°C. Other bacterial stimuli included Salmonella typhimurium (Ribi Research Laboratory, Hamilton, Mont.), Corynebacterium parvum (Ribi Research Laboratory), lipopolysaccharide from Salmonella typhi (Ribi Research Laboratory), purified protein derivative of Mycobacterium tuberculosis (kindly provided by P. M. Allen), and muramyl dipepetide (Sigma). Other stimuli included concanavalin A (2.5 pLg/ml; Sigma) and latex beads (2.9-,um diameter; Polyscience, Warrington, Pa.). IFN--y and TNF were measured by enzyme-linked immunosorbent assays (ELISAs) (5, 21), using standard curves employing the appropriate purified recombinant cytokine. Those assays showed a minimum sensitivity of 1 to 10 units/ml. Cytokines and cytokine-specific antibodies. We used monoclonal antibodies directed against murine TNF (TN3-19.12) (19), IL-lot (161.1) (8), IFN-y (H22.1) (5), and IL-6 (MP520F3.11 rat immunoglobulin Gl; courtesy of John Abrams, DNAX, and Marvin Siegel, Schering-Plough Corp.). Polyclonal rabbit anti-murine IL-lp was provided by David Chaplin (Washington University School of Medicine, St. Louis, Mo.). Plates coated with anti-TNF were prepared by diluting the TN3-19.12 to 2 ,ug/ml in pH 9.6 carbonate buffer, placing 0.2-ml aliquots into Immulon plates (Dynatech Laboratories, Chantilly, Va.), and storing them at 4°C until use. For antibody addition to NK cultures, anti-TNF was diluted to 0.2 jig/ml, anti-IL-la was diluted to 10 ,ug/ml, anti-IL-1p was diluted to 10 ,ug/ml, and anti-IL-6 was diluted to 50 jLg/ml. Recombinant murine TNF-a (specific activity, 2.9 x 107 U/mg) and recombinant murine IFN-y (specific activity, 7 x 106 U/mg) were kindly provided by Susan Kramer, Genentech Inc., So. San Francisco, Calif.
Statistics and experimental reproducibility. All experiments were performed at least three times, with standard deviations from the mean exceeding no more than 10%.
RESULTS IFN--y production requires both macrophages and NK cells. In a previous study, we demonstrated the production of IFN-y from a mixture of scid spleen cells that contained macrophages and NK cells (1). The NK cells were characterized as cells that did not adhere to plastic dishes, reacted with antibodies to asialo-GM1, and killed YAC-1 cells. To better analyze the cellular constituents in this system, we tested purified scid NK cells grown from bone marrow precursors by culture in IL-2 and macrophages obtained from the peritoneal cavity. NK cells, macrophages, or both were incubated with HK L. monocytogenes, and the production of IFN--y was determined by ELISA after 48 h of culture (Table 1). NK cells or macrophages cultured by themselves with or without HK L. monocytogenes produced little, if any, IFN-y. However, addition of HK L. monocytogenes to cocultures of NK cells plus macrophages resulted in secretion of large amounts of IFN-y. The same results were obtained with live L. monocytogenes (data not shown). This effect was not exclusive to L. monocytogenes; addition of two other microorganisms, S. typhimurium and C. parvum, to cocultures of NK cells and macrophages resulted in production of IFN--y (Fig. 1). NK cells incubated alone with these bacteria failed to secrete IFN--y (data not shown). S. typhi
Listeria
C. parvum
3500 3000
2500a:
2000
>. 1500-
z
0
107
106
i05
100
20
4
100
20
4
mg/ml mg/ml FIG. 1. Effects of bacterial stimuli on IFN--y production. NK cells were cultured with the indicated stimuli in the presence of macrophages, and the supernatants were assayed for IFN--y at 48 h. In the absence of macrophages, NK cells produced less than 20 interferon reference units (IRU) per ml. Bacteria/ml
VOL. 59, 1991
ROLE OF TNF-ot IN INDUCING IFN-y PRODUCTION BY NK CELLS
Lower chamber Upper chamber Macrophages Macrophages NK ( 10 5) NK ( 10 5)
(a) (b) (c) (d)
(e) (f)
(g)
(h) (i)
1.8 1.8 1.8 4.6 4.6 4.6 9.2 9.2 9.2
IFNy
TNF
(IRU/ml)
(U/ml)
1711
+ +
0
8
16
0
80
6
mm~
+ +
+ +
(i) 0 800 1600 0 800 1600 FIG. 2. Cell-cell contact between macrophages and NK cells is not absolutely required for stimulation of NK cells to produce IFN--y. Transwell chambers were used to culture macrophages and NK cells without allowing cell-cell contact between the two cell types. Macrophages were cultured at the indicated concentrations. NK cells were at 106 per well. All the wells with macrophages received 2 x 105 HK L. monocytogenes.
Thus, IFN--y production by NK cells required the presence of macrophages. Macrophage-conditioned medium stimulates NK cells to produce IFN-y. From our previous studies, we knew that TNF was involved in the process of induction of IFN--y by NK cells. To evaluate the contribution of products released by macrophages in stimulating NK cells to produce fFN-y, we performed two types of experiments. The first involved the use of Transwell chambers in which macrophages and HK L. monocytogenes were placed on one side of a filter and NK cells were placed on the other side. IFN--y production was determined after 48 h of incubation (Fig. 2). As expected, NK cells or macrophages separately incubated with HK L. monocytogenes produced minimal IFN--y (Fig. 2, rows a, d, g, and j). TNF was produced only when macrophages were present in the wells (rows a to i). The culture of NK cells, macrophages, and HK L. monocytogenes in the same well resulted in high levels of both TNF
and IFN--y (rows b, e, and h). When macrophages and NK cells were separated by the Transwell filter, significant amounts of IFN-y were produced, although not to the same extent as when the two cell types were present in the same chamber (rows c, f, and i). In the second type of experiment, filtered conditioned medium from macrophages incubated with HK L. monocytogenes was added to NK cells and the secretion of IFN--y was determined 48 h later (Table 2). The macrophageconditioned medium was active in inducing NK cells to produce IFN-y. In most experiments, the most active conditioned medium was obtained after 8 h of culture of mac-
Experiment 1 E
TABLE 2. Effect of macrophage supematants on NK cell IFN-y secretiona
1U-
z
IFN-y (IRUb/ml) Expt 137 146 149 152 155 162 164 172 180
NK cells
NK cells + HK L. monocytogenes
0 0 0 0 0 0
33 15
0 0
0 0
NK cells +
2,997 1,040 2,916 1,391 1,269 1,269 2,673 1,782 1,026
a This table shows the results of nine different experiments. Macrophages were cultured with HK L. monocytogenes for 8 h, and the supematants were recovered. NK cells were cultured alone, with HK L. monocytogenes (6 x 105/ml), or with a 50% (vol/vol) concentration of the macrophage-conditioned medium. After 48 h of incubation, the supernatants were tested for IFN--y
production. b IRU, Interferon reference units.
Experiment 2
conditioned medium E U-
z
Time (hours)
Time (hours)
FIG. 3. Activity of macrophage-conditioned media obtained at different times. Macrophages were cultured with HK L. monocytogenes for the times indicated in the figure, and the supernatants were recovered and tested for TNF activity. Stimulation of IFN-y secretion was determined by adding a 50% (vol/vol) concentration of each supernatant to NK cells and measuring IFN--y production. IRU, Interferon reference units.
1712
WHERRY ET AL.
INFECT. IMMUN.
180 160 0o140
E
120
z
100
Antibodies
IL-1
IL-1a
IL-6
IFN y
(IRU/ml)
TNF
'0~~~~
>. 80 E 60 40'0 20i0 z
'0~~~ +
0
0
10 20 30 40 50 60 70 80 Macrophage Conditioned Media (%)
FIG. 4. Dose response of NK cells to macrophage-conditioned media. NK cells (2 x 105) were cultured in medium containing different percentages (vol/vol) of an 8-h supernatant from macrophages cultured with HK L. monocytogenes. After 48 h of incubation, the NK cell supernatants were tested for IFN--y production. IRU, Interferon reference units.
0
S. typhi
1600
FIG. 6. Effects of neutralizing cytokine-specific antibodies on macrophage supernatant stimulation of IFN--y. An 8-h macrophage supernatant was incubated with NK cells in the presence of antiIL-la (10 ,ug/ml), anti-IL-1B (10 ,ug/ml), anti-IL-6 (50 pg/ml), or anti-TNF (140 ,ug/ml), and the supernatants were assayed for IFN--y secretion. IRU, Interferon reference units.
rophages with HK L. monocytogenes (Fig. 3). Longer incubation periods resulted in conditioned media that were less active in subsequently stimulating NK cells to produce IFN--y. The amount of IFN--y produced by NK cells depended on the concentration of macrophage supernatant used (Fig. 4). Active supernatants were also obtained when macrophages were cocultured with other bacteria or their products but not when they were exposed to latex beads (Fig. 5) or concanavalin A (data not shown). As noted above, the stimulating activity of macrophageconditioned media decayed with longer times of culture. However, the extent of decay varied from experiment to experiment, ranging from an 87% to a 66% decrease. The decreased activity in 48-h supernatants was not due to lack of TNF since similar amounts of TNF protein were found in
Listeria
800
8-h and 48-h macrophage supernatants. However, it is possible that the decay in activity was due to the presence of a TNF inhibitor in the 48-h supernatant. Experiments are currenitly under way to examine this issue. Role of TNF in production of IFN-y by NK cells. NK cells were cultured with macrophage-conditioned medium in the presence of antibodies against IL-la, IL-1,, IL-6, or TNF. IFN-y secretion was not affected by the presence of antibodies to IL-1 or IL-6 but was suppressed about 80% by anti-TNF (Fig. 6). Addition of purified recombinant murine TNF-(x to the culture supernatant restored NK cell secretion
C. parvum
PPD
LPS
No Latex stimuli
1400
1200 1000
E 800 D
600 400
200 0 100
20 4
100
20 4
50 5
Stimuli (Bacteria or
:
Mg/ml)
FIG. 5. Production of active macrophage-conditioned media by various bacterial stimuli. Macrophages were cultured with the indicated materials for 8 h, and the supernatants were recovered. TNF in each supernatant was tested (0). NK cells were cultured for 48 h at 50% (vol/vol) with each of them, and IFN--y was measured (-). NK cells cultured directly with the indicated stimuli produced less than 50 interferon reference units (IRU) of IFN--y per ml.
VOL. 59, 1991
ROLE OF TNF-a IN INDUCING IFN--y PRODUCTION BY NK CELLS
rTNF (U/ml)
Anti-TNF
IFNy
TNF
Listeria Sonicate
(IRU/mI)
(U/ml)
(jig/ml)
TNF (240 U/mI)
1713
IFNy (IRU/ml)
375 375 +
3843
+
960
+
240
+
60
0
1750
3500
30 6 6 0
900
1800
FIG. 7. Reconstitution of anti-TNF-treated macrophage-conditioned supernatant by purified recombinant murine TNF-a. An 8-h macrophage-conditioned medium was incubated with NK cells in the presence of 0.2 ,ug of anti-TNF per ml and various concentrations of TNF; the supernatants were assayed for IFN--y secretion at 48 h. IRU, Interferon reference units; rTNF, recombinant TNF-a.
of IFN--y (Fig. 7). Similar inactivation of supernatant stimulatory ability occurred when the conditioned medium was incubated in plates containing surface-bound anti-TNF. Activity of the depleted supernatants could be completely reconstituted with purified recombinant murine TNF-a (data not shown). To test whether TNF alone or with HK L. monocytogenes could stimulate NK cell release of IFN-y, we incubated NK cells with recombinant TNF-ot and/or HK L. monocytogenes (Table 3). Minimal IFN--y secretion was found. This result agrees with our previous observation that TNF alone could not stimulate IFN--y secretion by unfractionated scid spleen cells (1). Thus, TNF is necessary but not sufficient to stimulate NK cell-dependent IFN-y production. Effect of bacterial products on NK cell IFN--y production. Since macrophage-derived cytokines were unable to directly stimulate production of NK cell IFN--y, we evaluated the TABLE 3. Effects of TNF and/or HK L. monocytogenes on IFN--y production by NK cellsa Stimulus
(U/mi)
0 3,843 960 240
960 960 960
IFN-a
HK L. monocytogenes
Mac-CMc
Macrophages
0
-
-
_
-
+
-
-
0 81 0 0 51 3,645 0 153 63
+
-
1,782
-
6x107 6X 106 6 x 105 6 x 107 6 x 106 6 x 105
150 150 30
-
(IRUb/ml) 0
a A representative experiment is shown. NK cells were cultured as indicated, and IFN--y was measured after 48 h. b IRU, Interferon reference units. c Mac-CM, Conditioned medium from macrophages cultured with HK L. monocytogenes for 8 h.
=
+
0 800 1600 FIG. 8. Effects of Listeria sonicates on IFN--y secretion by NK cells. This is representative of five different experiments with three different preparations of sonicated L. monocytogenes. Sonicated L. monocytogenes preparations were incubated with NK cells in the presence or absence of 960 U of exogenous recombinant TNF-a per ml. IRU, Interferon reference units.
role of bacterial products in this process. NK cells were incubated with either intact bacteria or L. monocytogenes sonicates in the presence or absence of exogenous TNF-a, and IFN--y production was determined (Fig. 8). Whereas no IFN-y was produced with intact L. monocytogenes, soluble L. monocytogenes products caused significant IFN--y production by the NK cell culture. Similar levels of IFN-y were induced by L. monocytogenes sonicates added with or without the neutralizing anti-TNF monoclonal antibody (in four different experiments, no inhibition by anti-TNF was found). Thus, the soluble bacterial product could directly stimulate NK cells to produce IFN--y in the absence of TNF. However, addition of TNF-a to NK cell cultures treated with bacterial products resulted in marked enhancement of IFN--y secretion. Thus, two stimuli can synergize to induce IFN--y production: macrophage-derived TNF and a soluble Listeria product. DISCUSSION The present study adds to our previous report (1) the finding that purified NK cells can release IFN--y upon interaction with a soluble conditioned medium from macrophages that have taken up L. monocytogenes. The conditioned medium must contain TNF and some other unrecognized product. We confirmed that TNF by itself is not able to stimulate NK cells, although some stimulation could be induced by a soluble Listeria sonicate. However, TNF and soluble bacterial products acted synergistically to induce maximal secretion of IFN--y. We do not know whether the additional active moiety in the macrophage-conditioned medium is a soluble Listeria product or some other factor derived from the macrophages. If it is a macrophage-derived cytokine, it is not IL-la, IL-,, or IL-6. Not all the activity in the macrophage-conditioned media was abolished by antiTNF. This suggests that the activity may well be a product equivalent to the sonicate, which was not inhibited by anti-TNF antibodies. Work is now in progress to resolve this issue. A new contribution made here is the clear identification of the cellular protagonists in this non-T-cell-dependent pathway of IFN--y production (1-3). Undoubtedly they involve
1714
INFECT. IMMUN.
WHERRY ET AL.
macrophages and NK cells as surmised from the use of purified macrophages and NK cells grown from bone marrow. In our previous report (1), the evidence for NK cells was quite strong but required further steps of cellular purification to be definitive. Another important feature demonstrated in the current study is that the responsiveness of NK cells to macrophage-conditioned media depends on their state of activation. While activated NK cells grown in IL-2 responded to this medium, NK cells freshly harvested from scid spleens did not. Our interpretation is that (i) the activation state of NK cells is critical for a response to occur and (ii) the activation state of NK cells dictates whether cell contact between NK cells and macrophages is required. These items will be addressed in future experiments. Activation of NK cells to a cytotoxic state or for IFN-y secretion has been reported in a number of in vitro and in vivo situations. These include responses to some bacteria (4, 13, 16, 28), viruses (27), fungi (23), and tumors (6, 12, 20, 25). Although IFN (9) and IL-2 (11, 14, 17, 18, 21, 22, 24, 26, 29) are known NK cell activators, our results in the scid mouse imply that more than one cellular or soluble component is necessary to achieve maximal IFN--y secretion by NK cells. Our studies with the scid cells clearly document a pathway for production of IFN--y which is induced by some facultative intracellular bacteria and does not involve the participation of CD4+ or CD8+ T cells (1-3). We speculate that this pathway provides the initial defense against microorganisms that are then partially eliminated by target cells (such as macrophages) activated by NK cell production of IFN-y. Thus, this pathway could be operative before T-cell immunity has developed. It is possible that in an immunologically intact host, this pathway can also participate synergistically with the usual antigen-presenting cell-CD4/CD8 T-cell interactions that result in the production of IFN-y and other cytokines. We therefore believe that this T-cell-independent production of IFN--y plays an important physiologic role in initiating and propagating host responses to infectious agents. ACKNOWLEDGMENTS We thank Susan Kramer of Genentech, Inc., for providing purified recombinant murine TNF-ot and Maury Gately and Grace Ju of Hoffmann-La Roche for providing purified recombinant human IL-2. We are also grateful to Karen Larimore for expert technical assistance and Marian Simandl Florea for secretarial assistance. This work was supported by Public Health Service grants A115322, A124854, and CA43059 from the National Institutes of Health. REFERENCES 1. Bancroft, G. J., M. J. Bosma, G. C. Bosma, and E. R. Unanue. 1986. Regulation of macrophage Ia expression in mice with severe combined immunodeficiency: induction of Ia expression by a T cell-independent mechanism. J. Immunol. 137:4-9. 2. Bancroft, G. J., R. D. Schreiber, G. C. Bosma, M. J. Bosma, and E. R. Unanue. 1987. A T cell-independent mechanism of macrophage activation by interferon-gamma. J. Immunol. 139:11041107. 3. Bancroft, G. J., K. C. F. Sheehan, R. D. Schreiber, and E. R. Unanue. 1989. Tumor necrosis factor is involved in the T cell-independent pathway of macrophage activation in scid mice. J. Immunol. 143:127-130. 4. Blanchard, D. K., H. Friedmann, W. E. Stewart II, T. W. Klein, and J. Y. Djeu. 1988. Role of gamma-interferon in induction of natural killer activity by Legionella pneumophila in vitro and in an experimental murine infection model. Infect. Immun. 56: 1187-1193. 5. Buchmeier, N. A., and R. D. Schreiber. 1985. Requirement of
endogenous interferon-gamma production for resolution of Listeria monocytogenes infection. Proc. Natl. Acad. Sci. USA
82:7404-7408. 6. Djeu, J. Y., K. Y. Huang, and R. B. Herberman. 1980. Augmentation of mouse NK activity and induction of interferon by
tumor cells in vivo. J. Exp. Med. 151:781-789. 7. Dorschkind, K., S. B. Pollack, M. J. Bosma, and R. A. Phillips.
1985. Natural killer (NK) cells are present in mice with severe combined immunodeficiency (scid). J. Immunol. 134:3798-3801. 8. Fuhlbrigge, R. D., K. C. F. Sheehan, R. D. Schreiber, D. D. Chaplin, and E. R. Unanue. 1988. Monoclonal antibodies to murine interleukin-la: production, characterization, and inhibition of membrane-associated interleukin-1 activity. J. Immunol. 141:2643-2650. 9. Gidlund, M., A. Orn, H. Wigzell, A. Senik, and I. Gresser. 1978. Enhanced NK cell activity in mice injected with interferon and interferon inducers. Nature (London) 273:759-761.
10. Hackett, J., Jr., G. C. Bosma, M. J. Bosma, M. Bennett, and V. Kumar. 1986. Transplantable progenitors of natural killer cells are distinct from those of T and B lymphocytes. Proc. Natl. Acad. Sci. USA 83:3427-3431. 11. Handa, K., R. Suzuki, H. Matsui, Y. Shimizu, and K. Kumagai. 1983. NK cells as a responder to IL-2. II. IL-2-induced interferon-gamma production. J. Immunol. 130:988-992. 12. Herberman, R. B., M. E. Nunn, H. T. Holden, S. Staal, and J. Y. Djeu. 1977. Augmentation of natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic target cells. Int. J. Cancer 19:555-564. 13. Holmberg, L. A., T. A. Springer, and K. A. Ault. 1981. Natural killer activity in the peritoneal exudates of mice infected with Listeria monocytogenes: characterization of the natural killer cells by using a monoclonal rat anti-murine macrophage antibody (M1/70). J. Immunol. 127:1792-1799. 14. Kasahara, T., J. J. Hooks, S. Dougherty, and J. J. Oppenheim. 1983. Interleukin 2-mediated immune interferon-gamma production by human T cells and T cell subsets. J. Immunol. 130:17841789. 15. Lauzon, R. J., K. A. Siminovitch, G. M. Fulop, R. A. Phillips, and J. C. Roder. 1986. An expanded population of natural killer cells in mice with severe combined immunodeficiency (scid) lack rearrangement and expression of T cell receptor genes. J. Exp. Med. 164:1797-1802. 16. Ojo, E., 0. Haller, and H. Wigzell. 1978. Corynebacterium
17.
18. 19.
20.
21.
22.
23.
parvum-induced peritoneal exudate cells with rapid cytolytic activity against tumour cells are non-phagocytic cells with characteristic of NK cells. Scand. J. Immunol. 8:215-222. Ortaldo, J. R., A. T. Mason, J. P. Gerard, L. E. Henderson, W. Farrar, R. F. Hopkins, R. B. Herberman, and H. Rabin. 1984. Effects of natural and recombinant IL-2 on regulation of interferon-gamma production and NK activity: lack of involvement of the Tac antigen for these immunoregulatory effects. J. Immunol. 133:779-783. Reem, G. H., and N.-H. Yeh. 1984. IL-2 regulates expression of its receptor and synthesis of gamma-interferon by human T lymphocytes. Science 225:429-430. Sheehan, K. C. F., N. Ruddle, and R. D. Schreiber. 1989. Generation and characterization of hamster monoclonal antibodies that neutralize murine tumor necrosis factor. J. Immunol. 142:3884-3893. Shellam, G. R., V. Winterfourn, and H. J. S. Dawkins. 1980. Augmentation of cell-mediated cytotoxicity to a rat lymphoma. III. In vitro stimulation of NK cells by a soluble factor. Int. J. Cancer 25:331-339. Shiiba, K., K. Iton, U. Shimizu, and K. Kumagai. 1984. Interleukin-2 (IL-2)-dependent proliferation of human NK cells accompanied by interferon-gamma production, p. 187-192. In T. Hoshina, H. S. Koren, and A. Uchida (ed.), Natural killer activity and its regulation. Excerpta Medica, Amsterdam. Suzuki, R., K. Handa, K. Itoh, and K. Kumagai. 1983. Natural killer (NK) cells as a responder to interleukin-2 (IL-2). I. Proliferative response and establishment of cloned cells. J. Immunol. 130:981-987. Tartof, D., I. J. Check, A. Matutis, R. L. Hunter, and F. W.
VOL. 59, 1991
ROLE OF TNF-a IN INDUCING IFN--y PRODUCTION BY NK CELLS
Fitch. 1980. Studies on stimulation of cell-mediated cytotoxicity by skin test antigens. I. Candida antigen stimulation of cellmediated cytotoxicity in vitro correlated with the skin test response to candida antigen in vivo. J. Immunol. 125:2790-2796. 24. Trinchieri, G., M. Matsumota-Kobayashi, S. C. Clark, J. Seehra, L. London, and B. Perussia. 1984. Response of resting human peripheral blood NK cells to IL-2. J. Exp. Med. 160: 1147-1169. 25. Trinchieri, G., D. Santoli, R. R. Dee, and B. Knowles. 1978. Anti-viral activity induced by culturing lymphocytes with tumor-derived or virus-infected cells. Identification of the antiviral activity as interferon and characterization of the human effector lymphocyte subpopulation. J. Exp. Med. 147:12991313. 26. Vilcek, J., D. Henriksen-Destefano, D. Siegel, A. Klion, R. J.
1715
Robb, and J. Le. 1985. Regulation of interferon-gamma induction in human peripheral blood cells by exogenous and endog-
enously produced interleukin 2. J. Immunol. 135:1851-1856. 27. Welsh, R. M., Jr. 1978. Cytotoxic cells induced during lymphocyte choriomeningitic virus infection of mice.
I. Characterization of NK cell induction. J. Exp. Med. 146:163-181. 28. Wolfe, S. A., D. E. Tracey, and C. S. Henney. 1977. BCGinduced murine effector cells. II. Characterization of NK cells in peritoneal exudates. J. Immunol. 119:1152-1158. 29. Ythier, A., L. Delmon, E. Reinherz, A. Nowill, P. Moingeon, Z. Mishal, C. Bohuon, and T. Hercend. 1985. Proliferative responses of circulating human NK cells: delineation of a unique pathway involving both direct and helper signals. Eur. J. Immunol. 15:1209-1215.
