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A Fatal -Induced Systemic Inflammatory Response Reveals a Critical Role for NK Cells

This information is current as William E. Carson, Haixin Yu, Julie Dierksheide, Klaus of September 28, 2021. Pfeffer, Page Bouchard, Reed Clark, Joan Durbin, Albert S. Baldwin, Jacques Peschon, Philip R. Johnson, George Ku, Heinz Baumann and Michael A. Caligiuri J Immunol 1999; 162:4943-4951; ;

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. A Fatal Cytokine-Induced Systemic Inflammatory Response Reveals a Critical Role for NK Cells1

William E. Carson,2*† Haixin Yu,† Julie Dierksheide,‡ Klaus Pfeffer,§ Page Bouchard,¶ Reed Clark,ሻ Joan Durbin,ሻ Albert S. Baldwin,** Jacques Peschon,†† Philip R. Johnson,ሻ George Ku,‡‡ Heinz Baumann,§§ and Michael A. Caligiuri†¶¶

The mechanism of cytokine-induced shock remains poorly understood. The combination of IL-2 and IL-12 has synergistic anti- tumor activity in vivo, yet has been associated with significant toxicity. We examined the effects of IL-2 plus IL-12 in a murine model and found that the daily, simultaneous administration of IL-2 and IL-12 resulted in shock and 100% mortality within 4 to 12 days depending on the strain employed. Mice treated with IL-2 plus IL-12 exhibited NK cell , pulmonary edema, degenerative lesions of the gastrointestinal tract, and elevated serum levels of proinflammatory and acute phase reac- Downloaded from tants. The actions of TNF-␣, IFN-␥, -inflammatory protein-1␣, IL-1, IL-1-converting enzyme, Fas, , inducible nitric oxide synthase, and STAT1 did not contribute to the observed toxicity, nor did B or T cells. However, toxicity and death from treatment with IL-2 plus IL-12 could be completely abrogated by elimination of NK cells. These results suggest that the fatal systemic inflammatory response induced by this cytokine treatment is critically dependent upon NK cells, but does not appear to be mediated by the known effector molecules of this cellular compartment. These data may provide insight into the pathogenesis of cytokine-induced shock in humans. The Journal of Immunology, 1999, 162: 4943–4951. http://www.jimmunol.org/

ctivation of / by bacteria, fungi, immunomodulatory effects via activation of the IL-2/15R and the , or their products results in the rapid production IL-12R expressed on NK cells has led to investigations of this A of monokines such as TNF-␣, IL-1, IL-12, IL-15, and cytokine combination for the of neoplastic disease IL-18, which in turn induce NK cell production of IFN-␥ and (11, 12). In the current study, we examined the effects of admin- TNF-␣ (1–4). IL-12 appears to be pivotal to the NK cell response, istering IL-2 or IL-15 in combination with IL-12 in a murine tox- as there is only modest NK cell cytokine production in the absence icity model. While the dose of the individual cytokines was well of this factor (5, 6). We have demonstrated previously that the tolerated, the administration of IL-2 or IL-15 in combination with

combination of IL-15 and IL-12 exerts a profound synergy upon IL-12 induced a lethal systemic inflammatory response that did not by guest on September 28, 2021 resting NK cell production of IFN-␥, TNF-␣, and MIP-1␣3 (6–8). require any of the major proinflammatory factors or signaling path- These proinflammatory cytokines and play a critical ways felt to be active in the induction of . However, role in the clearance of obligate intracellular and, in the lethal toxicity of this cytokine combination therapy was criti- some cases, the promotion of sepsis, shock, and death (9). An cally dependent upon NK cells, but not B or T cells. identical profile of NK cell cytokine and production can be induced by the combination of IL-2 and IL-12 (5, 6). This Materials and Methods reflects the fact that the heterotrimeric IL-15R and IL-2R share the Reagents IL-2R␤ and ␥ signaling subunits and differ only in the specificity of their high affinity ␣-chains (10). The ability to obtain synergistic Purified, yeast-derived rhuIL-2 (Chiron, Emeryville, CA) or rhuIL-15 (Im- munex, Seattle, WA) was administered at a dose of 3 ϫ 105 U/day via the i.p. route. rIL-12 of murine (mu) origin (Genetics Institute, Cambridge, Departments of *Surgery, ¶¶Medicine, ‡Pathology, and †Medical Microbiology and MA) was administered i.p. at a dose of 1 ␮g/day. rhuIL-2 and rmuIL-12 Immunology, Arthur G. James Comprehensive Center, Ohio State University, were administered daily until the death of the animal. rmuIL-10 was sup- Columbus, OH 43210; §Institute for Medicine, Microbiology, and Hygiene, Munich, plied by Schering-Plough (Kenilworth, NJ). Platelet-derived huTGF-␤1 ࿣ Germany; ¶Genetics Institute, Andover, MA 01810; Children’s Hospital, Columbus, (which has activity in murine systems) was purchased fromR&DSystems OH 43205; **Lineberger Comprehensive Cancer Center, Curriculum in Genetics and (Minneapolis, MN) and reconstituted according to the manufacturer’s rec- Molecular Biology, Chapel Hill, NC 27514; ††Immunex Research and Development ‡‡ ommendations in 4 mM HCl supplemented with 0.1% BSA (13). Rat IgG1 Corporation, Seattle, WA 98101; Vertex Pharmaceuticals, Cambridge, MA 02139; ␥ and §§Roswell Park Cancer Institute, Buffalo, NY 14263 anti-muIFN- mAb (Endogen, Cambridge, MA), and hamster IgG anti- muIFN-␥ mAb (Genzyme Diagnostics, Cambridge, MA) were adminis- Received for publication October 13, 1998. Accepted for publication January tered at a dose of 100 ␮g per mouse via the i.p. route 12 h before cytokine 13, 1999. therapy and then daily thereafter. Control Abs were purchased from Sigma The costs of publication of this article were defrayed in part by the payment of page (St. Louis, MO). A dimeric rhuTNF p80/IgG1 Fc fusion protein charges. This article must therefore be hereby marked advertisement in accordance (TNFR-Fc; Immunex) was used in TNF-␣ neutralization experiments (100 with 18 U.S.C. Section 1734 solely to indicate this fact. ␮g/mouse/day i.p. beginning 24 h prior cytokine treatment) (14). Human 1 This work was supported by National Institute of Health Grants CA68326 and IgG was used as a control for the fusion protein (Baxter, Glendale, CA). CA68458, and in part by Grant P30 CA16058. Dexamethasone was purchased from Moore Medical (New Britain, CT). 2 Address correspondence and reprint requests to Dr. William E. Carson III, Arthur G. Depletion of NK cells was accomplished via i.p. administration of an anti- James Comprehensive Cancer Center, Ohio State University, N924 Doan Hall, 410 asialo GM1 Ab (Wako BioProducts, Richmond, VA) every 3 days begin- W. 10th Street, Columbus, OH 43210. E-mail address: [email protected] ning 2 wk before the injection of cytokines (0.2 mg/mouse) (15). NK cell 3 Abbreviations used in this paper: MIP, macrophage-inflammatory protein; hu, hu- numbers postdepletion were evaluated via flow-cytometric analysis of mu- man; I-␬B, inhibitor of NF-␬B; ICE, IL-1␤-converting enzyme; iNOS, inducible ni- rine splenocytes using a phycoerythrin (PE)-labeled pan-NK mAb (clone tric oxide synthase; KC, ??; mu, murine; PE, phycoerythrin; rAd, recombinant DX5; PharMingen, San Diego, CA). Mice were depleted of monocytes/ adenovirus. macrophages via i.v. and i.p. injection of the F4/80 mAb (an IgG2b mAb)

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 4944 FATAL CYTOKINE-MEDIATED INFLAMMATORY RESPONSE

48 and 24 h before cytokine therapy (16). Macrophage numbers postdeple- tion were evaluated via the enumeration of plastic-adherent cells following a 2-h culture of PBMCs, splenocytes, cells, or peritoneal cells in 24-well plastic culture dishes (1 ϫ 106 cells/well in RPMI 1640 sup- plemented with 10% FBS) (7). All cytokine reagents contained less than 0.015 EU/ml endotoxin, as measured by the E-Toxate system (Sigma). Mice Female mice age 4–6 wk were utilized in all experiments. C.B-17 scid/scid (SCID) mice (BALB/c background), splenectomized C.B-17 SCID mice, sham-operated C.B-17 SCID mice, and inbred BALB/c mice were pur- chased from Taconic Farms (Germantown, NY). Perforin-deficient (Ϫ/Ϫ) mice, CD3⑀ transgenic mice, IFN-␥Ϫ/Ϫ mice, and Faslpr/lpr mice were pur- chased from The Jackson Laboratory (Bar Harbor, ME) (17–20). TNFR p55Ϫ/Ϫ mice, TNFR p75Ϫ/Ϫ mice, and TNFR p55Ϫ/Ϫ/TNFR p75Ϫ/Ϫ mice were provided by Immunex (21). Type I IL-1RϪ/Ϫ mice, type I IL-1RϪ/ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ/TNFR p55 / mice, and type I IL-1R / /TNFR p75 / mice were also provided by Immunex (22). TNFR p55Ϫ/Ϫ and Faslpr/lpr/TNFR p55Ϫ/Ϫ mice were produced by Dr. Klaus Pfeffer (Munich, Germany) (20, 23). MIP-1␣Ϫ/Ϫ mice were the gift of Dr. O. Smithies (Chapel Hill, NC) (24). Mice deficient in the enzyme-inducible nitric oxide synthase (iNOSϪ/Ϫ) FIGURE 1. Administration of IL-2 or IL-15 in combination with IL-12

were the gift of Dr. Ricardo Gazzinelli (Bethesda, MD) (25). Mice deficient Downloaded from in the IL-1␤-converting enzyme (ICEϪ/Ϫ) and STAT1Ϫ/Ϫ mice were pro- is lethal in inbred mice. Four- to six-week-old female C57BL/6 mice were duced as described (26, 27). All mice were housed in a specific - injected daily i.p. with rhuIL-2 (3 ϫ 105 IU/day) plus rmuIL-12 (1 ␮g/day), free environment and given food and water ad libitum. or rhuIL-15 (3 ϫ 105 U/day) plus IL-12 and monitored for toxicity. No deaths were observed in mice receiving IL-2, IL-15, or IL-12 alone (not Analysis of cytokine-treated mice shown). These results are representative of four separate experiments. Serum levels of IFN-␥ and TNF-␣ were measured using ELISAs obtained ␤

from Endogen (Woburn, MA). IL-1 and IL-6 levels were measured using http://www.jimmunol.org/ ELISAs obtained from Biosource International (Camarillo, CA). KC and tion (data not shown). Indeed, SCID mice, which lack B and T MIP-2 levels were measured using ELISAs fromR&DSystems. Serum chemistries were performed on mouse serum using a Vitros 500 analyzer cells (34), exhibited 100% mortality within 3 to 5 days of the (Johnson and Johnson, Raritan, NJ). Histopathologic examination of cyto- initiation of treatment and were utilized extensively in the majority kine-treated SCID mice and determination of mean wet-to-dry lung ratios of experiments. were performed as described (28). NK cells were isolated from the of cytokine-treated C.B-17 SCID mice and analyzed for endonucleolytic Histopathology cleavage of cellular DNA via a flow-cytometric assay using propidium iodide and DNA gel electrophoresis, as described (12, 29). SCID mice receiving IL-2, IL-12, or IL-2 plus IL-12 were sub- jected to histopathologic evaluations. IL-2-associated findings in- Measurement of acute phase proteins cluded mononuclear cell infiltrates in the portal areas of the liver, by guest on September 28, 2021 Serially diluted serum was analyzed for haptoglobin and ␣1-acid glyco- splenic extramedullary hemopoiesis, and pulmonary interstitial protein by immunoelectrophoresis (30). The area under the precipitation mononuclear cell infiltrates. IL-12-associated changes included peak was quantitated in arbitrary units using the National Institute of mild hyperplastic and degenerative changes of the gastrointestinal Health Image program 1.61. The data for each peak were then converted into mg/ml values by comparison with the values obtained with calibrated mucosa, and scattered foci of apoptotic lymphoid cells within the mouse acute phase plasma. lymphoid organs. These IL-2- and IL-12-induced lesions were mild, of late onset (72–96 h), and consistent with those previously ␬ Inhibition of the NF- B signaling pathways in mice receiving described for these cytokines (35, 36). Changes in SCID mice IL-2 and IL-12 treated with IL-2 plus IL-12 included both significant exacerbation An E1A-deleted recombinant adenovirus (rAd) expressing a dominant- of IL-2- and IL-12-associated lesions as well as novel changes negative form of the I-␬B␣ protein was constructed (31). A rAd engineered such as fibrinoid of lymphoid tissue in the and 9 to express the lacZ gene served as a control. A total of 10 PFUs of rAd/ lymph nodes, confluent foci of necrosis in pancreatic exocrine tis- I-␬B or rAd/lacZ was administered to mice via tail vein injection 48 h before treatment with IL-2 plus IL-12 (32). In vivo protein expression sue, macrophage activation and proliferation, and fibrinoid necro- derived from these adenoviral vectors was confirmed via immunohisto- sis of pulmonary arterioles. Apoptosis of SCID (i.e., chemistry, as described (7). NK cells) within the lymphoid tissues (spleen and lymph nodes) Statistical analysis was markedly enhanced (Fig. 2A). Analysis of nonadherent splenic NK cells from cytokine-treated SCID mice by DNA gel electro- Statistical significance was analyzed by the Student’s t test. phoresis and propidium iodide staining (12) confirmed this obser- vation (Fig. 2B and data not shown). Lesions of the gastrointestinal Results tract associated with IL-12 administration were also markedly ex- Concomitant administration of IL-2 or IL-15 with IL-12 is lethal acerbated by the combination of IL-2 and IL-12 (Fig. 2C). Pul- in mice monary pathology was prominent and included perivascular and Intraperitoneal administration of rhuIL-2 (3 ϫ 105 U/day) plus septal mononuclear cell infiltrates associated with multifocal hem- rmuIL-12 (1 ␮g/day) was lethal to C57BL/6 mice within 4 to 6 orrhage and alveolar edema. The formation of pulmonary edema at days (Fig. 1). The combination of IL-15 plus IL-12 elicited iden- the 72-h time point was significantly greater in mice receiving the tical results, which was expected given that IL-15 signals through combination of IL-2 plus IL-12 ( p Ͻ 0.05, Fig. 2D) (28). Analysis components of the IL-2R (6, 33). No deaths were observed in of serum chemistries revealed significant increases in the liver en- control mice receiving daily injections of IL-2, IL-15, or IL-12 zymes ALT, AST, and LDH (5.3-, 9.9-, and 2.4-fold increases over alone. Similar results were obtained with IL-2 or IL-15 plus IL-12 baseline, respectively) as well as acute phase proteins beginning in several different species of mice, including BALB/c, 129, B6 ϫ 24–48 h after treatment with IL-2 plus IL-12, but not following 129, CD-1, and C.B-17 mice bearing the scid/scid (SCID) muta- the administration of IL-2 or IL-12 alone (Fig. 2E) (37). The Journal of Immunology 4945

A

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D E by guest on September 28, 2021

FIGURE 2. Histopathology and serum chemistries. A, Hemotoxylin/eosin-stained sections of spleens obtained from SCID mice receiving PBS (left panel)or IL-2 plus IL-12 (right panel) for 72 h. Apoptotic splenocytes were noted throughout the spleens of mice receiving IL-2 plus IL-12 (white arrow). Magnification, ϫ100. B, Nonadherent splenocytes (85–90% NK cells by FACS) from cytokine-treated SCID mice were harvested at 72 h and analyzed for the endonucleolytic cleavage of DNA via gel electrophoresis. Lane 1, ladder; lane 2, PBS-treated SCID mice; lane 3, IL-2- plus IL-12-treated mice; lane 4, IL-2-treated mice; lane 5, IL-12-treated mice. This experiment was repeated twice with similar results. C, Photomicrographs of colon obtained from SCID mice treated with PBS (A), IL-12 alone (B), or IL-2 plus IL-12 (C) for 72 h. Animals treated with IL-12 alone exhibited thinning of the mucosa, depletion of goblet cells, and multifocal single cell necrosis of colonic epithelial cells (arrow). Animals treated with IL-2 plus IL-12 exhibited atrophy of the mucosa, marked depletion of goblet cells, flattening of the surface epithelium, and cystic dilation of glands. D, SCID mice (n ϭ 5 per group) received daily injections of PBS, IL-2 alone, IL-12 alone, or IL-2 plus IL-12 for 72 h. Mouse lungs were harvested, weighed, thoroughly dried, and then reweighed. Mean wet-to-dry lung ratios (ϮSEM) were calculated for each condition. Denotes a significant increase in the wet-to-dry lung ratio for mice receiving IL-2 plus IL-12 as compared with mice receiving PBS, IL-2 alone, or IL-12 alone ,ء (p Ͻ 0.05). E, SCID mice received daily injections of IL-2 alone, IL-12 alone, or IL-2 plus IL-12 for a period of 72 h. Serum was harvested at the indicated time points and analyzed for the presence of haptoglobin and ␣1-acid glycoprotein via immunoelectrophoresis. Induction of serum haptoglobin by IL-2 plus IL-12 was significantly greater than PBS, IL-2, or IL-12 alone at the 48-h time point (p Ͻ 0.002). Induction of serum of ␣1-acid glycoprotein by IL-2 plus IL-12 was significantly greater than PBS, IL-2, or IL-12 alone at the 48- and 72-h time points (p Ͻ 0.02).

Cytokine levels in SCID mice treated with IL-2 and IL-12 IL-1␤, IL-6, KC, and MIP-2 were also found to be elevated during Serum levels of IFN-␥ and TNF-␣ rose rapidly in SCID mice combined administration of IL-2 with IL-12 (summarized in Fig. treated with IL-2 plus IL-12, peaked at approximately 24 h, and 3C). The elevated levels of proinflammatory cytokines seen with remained elevated until death (Fig. 3, A and B). Anti-asialo GM1 the combination of IL-2 plus IL-12 were not the result of a simple Ab-treated SCID mice did not exhibit elevated serum levels of additive effect, since administration of IL-2 or IL-12 alone did not IFN-␥ or TNF-␣ after receiving IL-2 plus IL-12, suggesting that elicit significant cytokine production in SCID mice (with the ex- this cytokine combination acted directly on NK cells to induce ception of IFN-␥ production in mice receiving IL-12). Of note, production of IFN-␥ and TNF-␣ (not shown). Serum levels of elevations in TNF-␣ and IL-1␤ occurred early in the course of 4946 FATAL CYTOKINE-MEDIATED INFLAMMATORY RESPONSE

(p55 and/or p75). We noted somewhat prolonged survival within both the control and experimental groups in our initial experiments with TNFR p55Ϫ/Ϫ mice. However, in subsequent experiments with the identical strain of TNFR p55Ϫ/Ϫ mouse and an independently generated strain, we found that all back- ground mice and TNFR p55Ϫ/Ϫ mice died within 5 to 7 days (Table I and data not shown). These data in conjunction with the results of our TNF-␣ neutralization experiments led us to con- clude that TNF-␣ was not solely responsible for the toxicity seen in this model. STAT1Ϫ/Ϫ mice were also susceptible to the toxicity of IL-2 plus IL-12, which is significant because STAT1 is critical for IFN-␥-induced gene regulation and also for the induction of apoptosis by TNF-␣ (39, 40). IFN-␥ and TNF-␣ were simultaneously neutralized using both an anti-IFN-␥ mAb and a TNFR-Fc soluble receptor construct (14, 41). Mortality rates of the experimental and control groups were identical. TNF-␣ was neutralized in IFN-␥Ϫ/Ϫ mice, and IFN-␥ was neu- tralized in TNFR p55Ϫ/Ϫ/TNFR p75Ϫ/Ϫ mice, without effect. Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Type I IL-1R / mice, IL-1R / /TNFR p55 / mice, and IL- Downloaded from 1RϪ/Ϫ/TNFR p75Ϫ/Ϫ mice all succumbed to the lethal effects of IL-2 plus IL-12, whereas mice receiving IL-2 alone or IL-12 alone exhibited minimal toxicity (not shown). In addition, IL- 1RϪ/Ϫ/TNFR p55Ϫ/Ϫ mice were treated with a neutralizing Ab to muIFN-␥ during treatment with IL-2 plus IL-12, yet this

intervention did not ameliorate toxicity or prolong survival (not http://www.jimmunol.org/ shown). Several alternative effector molecules were considered as possible mediators of toxicity; however, mice with targeted genetic deficiencies in MIP-1␣, ICE, perforin, Fas, and iNOS remained completely susceptible to the toxic effects of IL-2 plus IL-12. Inhibition of the inflammatory response to IL-2 plus IL-12 Dexamethasone, TGF-␤1, and ibuprofen have been used effec- tively to prevent morbidity and death in animal models of septic by guest on September 28, 2021 FIGURE 3. Serum cytokine levels in SCID mice receiving daily injec- shock and other inflammatory processes (42–44). These agents tions of IL-2 plus IL-12. Four- to six-week-old female C.B-17 SCID mice were administered in pharmacologically relevant doses before the received daily i.p. injections of PBS, IL-2, IL-12, or IL-2 plus IL-12. Se- start of cytokine treatment and then daily thereafter; however, none rum was obtained from cytokine-treated mice at the indicated times and was capable of ameliorating the toxicity of IL-2 plus IL-12 analyzed for the presence of muIFN-␥ (A) and muTNF-␣ (B) by ELISA. (Table II). Inhibition of NF-␬B signaling in vivo via overex- These results are representative of three separate experiments and represent ␬ the mean Ϯ SEM of duplicate wells. C, C.B-17 SCID mice received daily pression of I- B in the liver was also ineffective in preventing i.p. injections of PBS, IL-2, IL-12, or IL-2 plus IL-12. Serum was obtained mortality in this model, in contrast to its ability to protect mice from cytokine-treated mice at the indicated times and analyzed for the from the lethal effects of endotoxin (45). Indeed, mice express- presence of IL-1␤, IL-6, KC, and MIP-2 via ELISA. Values are presented ing the I-␬B protein were actually more susceptible to the toxic as a percentage of maximal cytokine production (the following peak values effects of IL-2 plus IL-12 than were mice treated with the con- were obtained: IL-1␤ ϭ 101 pg/ml, IL-6 ϭ 1037 pg/ml, KC ϭ 5007 pg/ml, trol vector ( p Ͻ 0.05, Table II). This experiment was repeated and MIP-2 ϭ 59 pg/ml). Peak cytokine levels were less than 5% of max- in TNFR p55Ϫ/Ϫ mice because of the role I-␬B has in potentiating imum in mice receiving IL-2 or IL-12 alone (not shown). These results are TNF-mediated apoptosis (31, 46) (M. Karin, unpublished observa- Ϯ representative of three separate experiments and represent the mean tion). The toxicity of IL-2 plus IL-12 in TNFR p55Ϫ/Ϫ mice overex- SEM of duplicate wells. pressing I-␬B was essentially identical in the experimental and control groups (Table II). treatment (i.e., within 1–3 h), whereas peak levels of IL-6, KC, and The role of NK cells MIP-2 appeared later in the disease course, as is observed in an- SCID mice that lack T and B lymphocytes undergo massive NK imal models of septic shock (Fig. 3, B and C, and data not cell apoptosis following administration of IL-2 plus IL-12 (Fig. 2, shown) (9, 38). A and B). To determine whether the toxicity of this model was mediated by NK cells, we administered IL-2 plus IL-12 to SCID ␣ ␥ Role of TNF- , IFN- , and IL-1 in death induced by IL-2 plus mice depleted of NK cells by pretreatment with an anti-asialo IL-12 GM1 Ab (Fig. 4A). IL-2 (or IL-15) plus IL-12 elicited minimal We investigated the mechanism of this fatal cytokine-induced toxicity when administered to SCID mice depleted of NK cells, inflammatory response treatment using cytokine neutralization and 100% of mice in this group survived, whereas control mice strategies and genetically altered mouse strains. Results are receiving IL-2 plus IL-12 all died within 5 days of the initiation of summarized in Table I. Neutralization of IFN-␥ or TNF-␣ did treatment (Fig. 5A). To confirm this observation, we administered not afford protection to SCID mice treated with IL-2 plus IL-12, IL-2 and IL-12 to SCID mice that had been depleted of NK cells nor did genetic deficiencies in IFN-␥ or the TNFR complex by splenectomy and observed 100% survival (47). Sham-operated The Journal of Immunology 4947

Table I. Role of IFN-␥, TNF-␣, and IL-1␤ in the toxicity of IL-2 and IL-12a

Expt. Strain Ab/Construct Mortality Time to Death (days)

1 C.B-17 SCID Anti-IFN-␥ Abb 12/12 2–5 C.B-17 SCID Control Ab 5/5 3–4 2 IFN-␥Ϫ/Ϫ 10/10 3–4 C57BL/6c 10/10 4–5 3 C.B-17 SCID Sol. TNFR-Fc 6/6 2–5 C.B-17 SCID HuIgG 6/6 2–5 4 TNFR p55Ϫ/Ϫ 5/5 10–11 TNFR p75Ϫ/Ϫ 16/16 8–10 TNFR p55p75Ϫ/Ϫ 16/16 5–9 129c 8/8 6–12 C57BL/6c 8/8 5–11 5 TNFR p55Ϫ/Ϫd 5/5 3–5 C57BL/6c 5/5 5–7 6 C.B-17 SCID Anti-IFN-␥ Ab & Sol. TNFR-Fc 5/5 2–3 C.B-17 SCID Control Ab & HuIgG 5/5 3–4 7 INF-␥Ϫ/Ϫ Sol. TNFR-Fc 6/6 4–5 IFN-␥Ϫ/Ϫ HuIgG 6/6 4–6 8 TNFR p55p75Ϫ/Ϫ Anti-IFN-␥ Ab 8/8 4–7 Ϫ/Ϫ

TNFR p55p75 Control Ab 8/8 5–9 Downloaded from 9 Type I IL-1RϪ/Ϫ 5/5 5 IL-1R/TNFR p55Ϫ/Ϫ 5/5 3–7 IL-1R/TNFR p75Ϫ/Ϫ 4/4 6 C57L/6c 5/5 5 10 MIP-1␣Ϫ/Ϫ 3/3 4–9 C57BL/6c 3/3 9–12 11 ICEϪ/Ϫ 8/8 3–6 c

B6 ϫ 129 8/8 6–8 http://www.jimmunol.org/ 12 PerforinϪ/Ϫ 8/8 5–9 C57BL/6c 8/8 6–12 13 Fas/TNFR p55Ϫ/Ϫ 5/5 3–5 FasϪ/Ϫ 5/5 3–7 TNFR p55Ϫ/Ϫ 5/5 3–5 C57BL/6c 5/5 5–7 14 iNOSϪ/Ϫ 6/6 7–10 B6 ϫ 129c 6/6 7–8 15 STAT1Ϫ/Ϫ 6/6 5–6 C57BL/6c 6/6 5–6

a Mice received daily injections of rhuIL-2 (3 ϫ 105 IU/day) and rmuIL-12 (1 ␮g/day) via the i.p. route. by guest on September 28, 2021 b Similar results were obtained with a neutralizing Ab from a different commercial source. c Background strain. d Independently generated TNFR p55Ϫ/Ϫ strain (Ref. 23).

SCID mice treated with IL-2 plus IL-12 exhibited 100% mortality block (18) showed absolutely no toxicity when treated with IL-2 at 5 days (Fig. 5B). Furthermore, CD3⑀ transgenic mice that com- plus IL-12. Control mice of the appropriate background all died pletely lack mature NK cells (and T cells) due to a developmental between 4 and 8 days (Fig. 5C).

Table II. Effects of various treatments on the toxicity of IL-2 and IL-12 in SCID mice

Expt. Strain Pretreatment Mortality Time to Death (days)

1 C.B-17 SCID Dexa (2 mg/kg) 6/6 3–5 C.B-17 SCID Dex (6 mg/kg) 5/5 3–5 C.B-17 SCID PBS 5/5 3–5 2 C.B-17 SCID TGF-␤b (50 ng) 5/5 3–5 C.B-17 SCID TGF-␤ (100 ng) 5/5 3–5 C.B-17 SCID PBS 5/5 3–5 3 C.B-17 SCID Ibuprofenc 6/6 3–5 C.B-17 SCID PBS 6/6 3–5 4 C.B-17 SCID rAd/I␬Bd 8/8 2 C.B-17 SCID rAd/lacZ 8/8 4–5 5 TNFR p55Ϫ/Ϫ rAd/I␬B 8/8 4–8 TNFR p55Ϫ/Ϫ rAd/lacZ 8/8 5–9 6 C.B-17 SCID muIL-10e (5 ␮g) 2/6 6–7 (n ϭ 2) C.B-17 SCID muIL-10 (20 ␮g) 2/6 6–7 (n ϭ 2) C.B-17 SCID PBS 6/6 3–4

a Mice received daily i.p. injections of dexamethsone beginning 2 days prior to treatment with rhuIL-2 plus rmuIL-12. b Mice received daily i.p. injections of TGF-␤1 beginning 12 h prior to treatment with IL-2 plus IL-12. c Ibuprofen was administered via dietary supplementation (ϳ2 mg/day) beginning 3 days prior to treatment with IL-2 plus IL-12. d rAd was administered via tail vein injeciton 48 h prior to treatment with IL-2 plus IL-12. e Mice received daily i.p. injections of muIL-10 beginning 2 days prior to treatment with IL-2 plus IL-12. 4948 FATAL CYTOKINE-MEDIATED INFLAMMATORY RESPONSE

Discussion Administration of IL-12 in combination with IL-2 or IL-15 in- duced a systemic inflammatory response that rapidly progressed to a fatal shocklike state. The NK cell compartment was the only population responsible for mediating this toxicity be- cause mice that underwent depletion of NK cells or had congenital absence of NK cells were completely protected from the lethal effects of this cytokine combination. Treatment of SCID mice with IL-2 plus IL-12 resulted in high serum levels of IFN-␥ and TNF-␣, and production of these cytokines was dependent upon the pres- ence of NK cells. However, neither IFN-␥ nor TNF-␣ was required for the fatal inflammatory reaction induced by IL-2 plus IL-12. Other effector molecules of the NK cell and macrophage compart- ments, namely MIP-1␣, IL-1, ICE, Fas, perforin, iNOS, and the STAT1 pathway of signal transduction, were eliminated as critical mediators of toxicity in this model. Our histopathologic findings and analysis of serum cytokine lev-

els indicated that the administration of IL-2 plus IL-12 had induced Downloaded from a severe systemic inflammatory response. High circulating levels of IL-1␤, IL-6, and the IL-8 homologues (KC and MIP-2) ap- peared only in the serum of mice receiving the combination of IL-2 plus IL-12, which is significant in that the production of these cytokines is limited to periods of inflammation, tissue injury, and

immunologic challenge (38). Indeed, the sequence of cytokine in- http://www.jimmunol.org/ duction was highly reminiscent of that observed in experimental models of endotoxemia and in humans diagnosed with septic shock (9, 38). The presence of high serum levels of IL-6 at the time of death is particularly significant in that they represent the net FIGURE 4. Analysis of NK cell and macrophage depletion following effect of biologically active IL-1␤ and TNF-␣, and have been the administration of Ab. A, Splenocytes were isolated from the spleens of found to correlate inversely with survival in patients with septic SCID mice that had received injections of PBS (left-hand panel)oran shock (50). Thus, treatment of mice with IL-2 plus IL-12 induces anti-asialo GM1 Ab (right-hand panel). NK cell numbers were evaluated many of the same proinflammatory mediators that are active in

via flow-cytometric analysis using a PE-labeled pan-NK mAb. The fluo- sepsis. The presence of increased pulmonary edema, multiple or- by guest on September 28, 2021 rescence of splenocytes stained with a PE-labeled control Ab fell gan system toxicities, and an acute phase response in mice receiv- within the first log (not shown). These results are representative of three separate determinations. B, PBMCs, splenocytes, bone marrow cells, and ing the combination of IL-2 and IL-12 lends additional support to peritoneal cells were isolated from SCID mice that had received injections this observation (9, 51). Induction of proinflammatory mediators of the F4/80 mAb or a control Ab. These cell preparations were resus- has also been implicated in the pathogenesis of the shocklike states pended in RPMI 1640 media supplemented with 10% FBS and plated at a associated with high dose cytokine therapy in humans, and we density of 1 ϫ 106 cells/well in 24-well plastic culture dishes. Following initially hypothesized that the lethal reaction to IL-2 plus IL-12 a 2-h culture, nonadherent cells were removed and the number of adherent might be the result of additive toxicities induced by the overlap- cells was determined (7). ping actions of known proinflammatory factors (52, 53). However, no protection was afforded by simultaneous neutralization of TNF-␣ and IFN-␥ or utilization of IL-1RϪ/Ϫ/TNFR p55Ϫ/Ϫ and IL-1RϪ/Ϫ/TNFR p75Ϫ/Ϫ mice. The data also suggest that the fatal Depletion/Deactivation of monocytes/macrophages toxicity of IL-2 plus IL-12 was not mediated via NF-␬B signaling ␬ Given the ability of NK cell-derived cytokines to potentiate mac- within the liver; however, it is possible that NF- B was only par- rophage effector functions (1–3, 48), the role of macrophages in tially inhibited and therefore still able to activate the transcription ␬ the toxicity of this model was investigated. SCID mice were de- of NF- B-responsive genes during this intense inflammatory re- sponse (45). We cannot exclude the possibility that other cyto- pleted of monocytes and macrophages by approximately 50% in kines, cytokine receptors, or effector molecules might combine the peripheral , spleen, and bone marrow, and by Ͼ95% in with IL-1, TNF-␣, or IFN-␥ following administration of IL-2 plus the peritoneal cavity by injecting the F4/80 mAb (16) via the i.v. IL-12 to induce a lethal inflammatory response. However, the data and i.p. routes 48 and 24 h before the administration of IL-2 plus suggest that stimulation of the NK cell compartment with IL-2 plus IL-12 (Fig. 4B). Mice receiving the F4/80 mAb tolerated the ad- IL-12 might result in the production of novel factors or factors that ministration of IL-2 plus IL-12 significantly better than mice re- have yet to be characterized as proinflammatory. Such a hypothesis is Ͻ ceiving the control Ab and exhibited a 50% survival rate ( p supported by our characterization of several unique pathologic lesions 0.05). Control mice all died within 3 to 4 days (Fig. 5D). IL-10 is that have not been observed in other models of shock (9, 51). a potent macrophage deactivator (49). Pretreatment of SCID mice The appearance of IL-1␤, IL-6, and other macrophage-derived with rmuIL-10 afforded significant protection from the toxicity of cytokines in the circulation, and the presence of activated and pro- IL-2 plus IL-12 (66% survival overall, p Ͻ 0.02, Table II). Taken liferating macrophages in the splenic bed following the adminis- together, these data suggest that monocytes/macrophages, in addi- tration of IL-2 plus IL-12 implied that this treatment had directly tion to NK cells, have a role in mediating the lethal toxicity of IL-2 or indirectly activated the macrophage compartment (54). The impor- plus IL-12. tance of macrophages in the toxicity of this model was confirmed by The Journal of Immunology 4949 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. Death induced by administration of IL-2 plus IL-12 is critically dependent upon the NK cell compartment. A, C.B-17 SCID mice were depleted of NK cells by pretreatment with an anti-asialo GM1 Ab (see Materials and Methods). Control mice were pretreated with injections of PBS. Mice in both groups subsequently received daily i.p. injections of rhuIL-2 (3 ϫ 105 IU/day) plus rmuIL-12 (1 ␮g/day) and were monitored for survival. This experiment was repeated three times with similar results. B, Splenectomized SCID mice or sham-operated controls received daily i.p. injections of IL-2 and IL-12. This experiment was repeated twice with similar results. C, Transgenic mice expressing the CD3⑀ subunit of the human TCR received daily injections of IL-2 and IL-12 via the i.p. route. Normal mice of the identical background served as controls. This experiment was repeated twice with similar results. D, SCID mice were partially depleted of macrophages by pretreatment with the F4/80 mAb. Control mice were pretreated with injections of a control mAb. Mice in both groups subsequently received daily i.p. injections of IL-2 plus IL-12 and were monitored for survival. This experiment was repeated twice with similar results. All experiments depicted in this utilized at least six mice per group. studies in which partial depletion of the macrophage compartment the macrophage compartment in the toxicity of this model; however, resulted in a 50% survival rate for mice receiving IL-2 plus IL-12. The we cannot rule out the possibility that IL-10 may exert its protective protection afforded by IL-10 pretreatments also suggested a role for effects via some other pathway (49). The prevention of death by the 4950 FATAL CYTOKINE-MEDIATED INFLAMMATORY RESPONSE congenital absence or depletion of NK cells and the improved survival the induction of programmed cell death at approximately 48–72 h by the partial depletion of macrophages suggested that the toxicity of (12). Activation-induced apoptosis appears to be a common strat- this model is the result of interactions between these two cell popu- egy for the removal of activated effector cells and attenuation of lations. NK cells constitutively express the IL-2/15R, and activation the immune response to specific pathogens (65). The ability of of this receptor complex results in further up-regulation of NK cell IL-2 plus IL-12 to induce NK cell apoptosis in vivo is further IL-12R expression (55). Thus, we suspect that the toxicity of this evidence for the central role of the NK cell compartment in the model first involves stimulation of NK cells by IL-2/15 plus IL-12, toxicity of this treatment. The existence of this suicide pathway followed by an NK cell-dependent activation of macrophages. The following NK cell activation further suggests that unbridled am- inability of IL-2 plus IL-12 administration to induce TNF-␣ in NK- plification of NK cell effector functions may be deleterious to the depleted SCID mice also supports our proposed sequence of events, organism. This apoptotic regulatory mechanism was apparently as NK cells and macrophages are both sources of this cytokine. Thus, insufficient to limit toxicity in the present model, possibly due to mobilization of macrophage effector function may occur as a result of the intensity and rapidity of the NK cell response to administration continuous and uncontrolled production of NK cell factors in response of IL-2/15 plus IL-12. Activation-induced apoptosis associated to IL-2 plus IL-12 (2), although the precise cellular source of these with the induction of a shocklike state has also been described proinflammatory cytokines cannot be determined based upon mea- following experimental activation of T cells with staphylococcal surements of serum cytokines. Our postulate that chronic stimulation enterotoxins () or anti-CD3 Ab (66, 67). However, in of the NK cell compartment via daily injection of IL-2 plus IL-12 contrast to our model, the toxicity of these -mediated shock results in macrophage activation is not an unprecedented one (1, 2, 56, syndromes is mediated primarily by TNF-␣. 57). However, the inability of IFN-␥ and TNF-␣ neutralization strat- In summary, we have demonstrated that the administration of Downloaded from egies to prevent death was unexpected and suggests that NK cells may IL-2 or IL-15 in combination with IL-12 results in a fatal systemic be able to activate macrophage effector functions via alternate inflammatory response that is critically dependent upon the NK pathways. cell compartment. This lethal shocklike reaction does not appear to To the best of our knowledge, the present model represents the first be mediated by any of the known cytokine products of the NK cell example of a cytokine-induced shock syndrome that is mediated by compartment, nor any of the effector molecules associated with components of the innate . Our current understanding NK cell cytotoxic activity. This is the first time that the NK cell (or http://www.jimmunol.org/ of the role of the NK cell in other models of shock is quite limited, but any cellular compartment) has been identified as an important there is evidence that NK cells can be activated during septic events component of cytokine-induced systemic inflammation. Elucida- and may contribute to the pathogenesis of this condition via the se- tion of the factors involved in this novel inflammatory pathway cretion of IFN-␥, which acts primarily to augment macrophage func- may therefore have relevance for understanding the complications tion. One well-studied example of this phenomena is the Shwartzman of high dose cytokine therapy as well as other forms of shock. reaction in which an intradermal priming dose of LPS is followed 24 h later by an i.v. LPS challenge (58). The first LPS dose induces the Acknowledgments production of IL-12, which stimulates the release of IFN-␥ (presum-

We thank Dr. Ricardo Gazzinelli for his assistance with experiments in- by guest on September 28, 2021 ably by NK cells) and permits the priming of macrophages. Upon volving the inducible nitric oxide synthase-deficient murine strain, and Dr. subsequent LPS challenge, sensitized macrophages release massive Peter Kantor for analysis of serum chemistries. amounts of TNF-␣ and IL-1␤, which mediate the lethal effects of this treatment (58). Heremans et al. have demonstrated that depletion of References NK cells before the induction of the generalized Shwartzman reaction 1. Bancroft, G. J. 1993. The role of natural killer cells in innate resistance to in- leads to a 70% reduction in mortality and significantly lower levels of fection. Curr. Opin. Immunol. 5:503. IFN-␥ and TNF-␣ following the systemic injection of LPS (59). In 2. Orange, J. S., B. Wang, C. Terhorst, and C. A. Biron. 1995. Requirement for contrast, mice depleted of CD4ϩ or CD8ϩ T cells were still highly natural killer cell-produced ␥ in defense against murine cytomegalo- and enhancement of this defense pathway by 12 ad- susceptible to the lethal effects of the Shwartzman reaction. Ozmen et ministration. J. Exp. Med. 182:1045. al. analyzed the Shwartzman reaction in detail and found that simple 3. Fearon, D. T., and R. M. Locksley. 1996. The instructive role of innate coinjection of TNF-␣ plus IL-1␤ or TNF-␣ plus IFN-␥ was sufficient in the acquired immune response. Science 272:50. 4. Ahn, H. J., S. Maruo, M. Tomura, J. Mu, T. Hamaoka, K. Nakanishi, S. Clark, to induce lethality following priming of mice with IL-12 or IFN-␥ M. Kurimoto, H. Okamura, and H. Fujiwara. 1997. A mechanism underlying alone (60). However, regardless of the priming event, no mortality synergy between IL-12 and IFN-␥-inducing factor in enhanced production of ␤ ␥ IFN-␥. J. Immunol. 159:2125. was observed if IL-1 and IFN- were administered in the absence of 5. Chan, S. H., B. Perusia, J. W. Gupta, M. Kobayashi, M. Popisil, H. A. Young, TNF-␣. Thus, in distinct contrast to our model, TNF-␣ is crucial for S. F. Wolf, D. Young, S. C. Clark, and G. Trinchieri. 1991. Induction of IFN-␥ the lethality of the Shwartzman reaction. Furthermore, our experi- production by NK cell stimulatory factor (NKSF): characterization of the re- ␥Ϫ/Ϫ Ϫ/Ϫ ␥ sponder cells and synergy with other inducers. J. Exp. Med. 173:869. ments with IFN- and STAT1 mice would suggest that IFN- 6. Carson, W. E., J. G. Giri, M. J. Lindemann, M. L. Linett, M. Ahdieh, priming (following IL-12 administration) is not a critical event in our D. Anderson, J. Eisenmann, K. Grabstein, and M. A. Caligiuri. 1994. Interleu- model. kin-15 is a novel cytokine which activates human natural killer cells via com- ponents of the interleukin-2 receptor. J. Exp. Med. 180:1395. The induction of NK cell apoptosis in the present model was 7. Carson, W. E., M. E. Ross, R. A. Baiocchi, M. J. Marien, N. Boiani, K. Grabstein, dependent upon the coadministration of IL-2 with IL-12. Nonad- and M. A. Caligiuri. 1995. Endogenous production of by activated human monocytes is critical for optimal production of interferon-␥ by natural herent splenocytes from cytokine-treated SCID mice did not ex- killer cells in vitro. J. Clin. Invest. 96:2578. hibit endonucleosomal DNA degradation or the morphologic fea- 8. Bluman, E., K. J. Bartynski, B. R. Avalos, and M. A. Caligiuri. 1996. Human tures of apoptosis unless mice had received both cytokines, results natural killer cells produce abundant macrophage inflammatory protein-1␣ in response to -derived cytokines. J. Clin. Invest. 97:2722. that were confirmed in vitro (not shown). Apoptosis of NK cells 9. Hack, C. E., L. A. Aarden, and L. G. Thijs. 1997. Role of cytokines in sepsis. Adv. has also been observed following coactivation with IL-2 and en- Immunol. 66:101. gagement of the low affinity FcR␥ (or cross-linking of CD94), and 10. Anderson, D. A., S. Kumaki, M. Ahdieh, J. Bertles, M. Tometsko, A. Loomis, J. Giri, N. G. Copeland, D. J. Gilbert, N. A. Jenkins, V. Valentine, D. N. Shapiro, after cell-mediated of leukemic targets cells in the presence of S. W. Morris, L. S. Park, and D. Cosman. 1995. Functional characterization of the IL-2 (61–64). We have demonstrated previously that in vitro stim- human interleukin-15 receptor chain and close linkage of IL15RA and IL2RA genes. J. Biol. Chem. 270:29862. ulation of resting human NK cells with IL-2/15 and IL-12 leads to 11. Wigginton, J. M., K. L. Komschlies, T. C. Back, J. L. Franco, M. J. Brunda, and the production of large amounts of IFN-␥ and TNF-␣, followed by R. H. Wiltrout. 1996. Administration of with pulse and The Journal of Immunology 4951

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