Endotoxin, tumor necrosis factor-α and interleukin 1 induce interleukin 6 production in vivo

CLINICAL.

IMMUNOL.Ot;Y

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Tumor Necrosis Factor-a and lnterleukin lnterleukin 6 Production in Vivo

M. R. SHALABY,’

A. WAAGE,

L. AARDEN,”

1 Induce

AND T. ESPEVIK

The Institute of Cuncer Research. University of Trondheim. N-7006 Trondheim. Norway rmd *Central Laboratov of the Netherlands Red Cross Blood Trunsjiision Service, und Laborutor?, for Experimental Clinical Immunology. University of Amsterdam. Amsterdam, The Netherlands

The ability of Escherichiu co/i-derived lipopolysaccharide (LPS). recombinant (r) interleukin 1-S (rIL-IP), and r murine tumor necrosis factor-o (rMuTNF-a) to induce interleukin 6 (IL-6) production in vivo was investigated. Peak serum IL-6 concentration was attained after 2 hr of LPS injection into mice. The coinjection of antiserum against rMuTNF-o with LPS resulted in a reduction of the induced serum IL-6 level, indicating the involvement of endogenous TNF-a in LPS induction of IL-6. Recombinant IL- 1I3 and rMuTNF-a injected directly caused the production of substantial amounts of IL-6 within 30 min. The injection of a combination of rIL-1R and rTNF-cc induced a significantly greater level of IL-6 than either agent alone. The greater level of serum IL-6 was associated with hypothermia and an increased lethality among mice injected with both cytokines. These data demonstrate the abilities of IL-1R and TNF-a to induce IL-6 production in vivo and indicate that LPS induction of IL-6 may be mediated, at least partially, through TNF-a action. The data describe a new in viva biologic activity shared between IL-113 and TNF-a and suggest that IL-6 may be an important effector in the mi* 1989 Academic Prey. Inc manifestation of TNF-(r and IL- II3 actions in rive.

INTRODUCTION Interleukin 6 (IL-6) is the term suggested by Poupart and colleagues (1) to describe a molecule identical with the hybridoma growth factor, interferon-&, 26-kDa protein, and B cell stimulatory factor 2 (2). The identity of this molecule was confirmed by other investigators (2, 3), who also adopted the nomenclature “IL-6” and which will be used in this report as well. The availability of purified preparations of recombinant IL-6 (4) has facilitated studies of the biologic properties of this molecule. IL-6 has been shown to enhance the synthesis of the acute phase proteins by hepatocytes (5) and to regulate the maturation of B cells for immunoglobulin synthesis (3). Recent reports have described the ability of IL-6 to enhance the growth of myeloma cells in an autocrine fashion (6) and to act synergistically with IL-I in the activation of T cells (7). Although the precise physiologic role of IL-6 remains largely unknown at present, recent data indicate that it may have an important role in inflammatory responses. It was shown that IL-6 can induce fever in rabbits (8), and is present in elevated levels in various biologic fluids from patients with arthritis (9, lo), septic shock (ll), infectious diseases (12), and during the acute phase of renal graft rejection (13). The agent(s) responsible for this increased IL-6 level is not ’ To whom correspondence should be addressed. 488 0090-1229/89 $1.50 Copyright 6 1969 by Academic Press. Inc. All rights of reproduction in any form reserved.

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completely known at present. However, based on studies conducted in this laboratory we have postulated the relatedness of the induction mechanisms involved in the release of tumor necrosis factor-a (TNF-a), IL-l, and IL-6 in septic shock (11). In the present study, we demonstrate that endotoxin, TNF-a, and IL-1B can induce IL-6 production in viva and show that the lethal shock induced by the simultaneous injection of TNF-(Y and IL- 1B in mice is associated with a significant rise in serum IL-6 concentration. MATERIALS

AND METHODS

Animals. Eight- to lo-week-old male NMRI mice were purchased from Bomholt Got-d Breeding Research Center, Ry, Denmark. The mice were maintained at the animal care facility, University Hospital, University of Trondheim, and allowed access to purina Lab chow and water ad libitum. On the day of the study the animals weighed 2&24 g. Cytokines and other agents. The cDNA for human IL-1B (14), human IL-6 (4), and mm-me TNF-(Y (15) have been cloned and expressed in Escherichia coli. Recombinant IL-1B (rIL-1B) used (Dr. A. Shaw, Glaxo) had a specific activity of 5 x lo7 Ulmg as determined by the thymocyte proliferation assay (14); recombinant IL-6 (rIL-6) had a specific activity of lo9 U/mg as determined by the B9 assay (16); and recombinant murine TNF-ol (rMuTNF-a) used (Dr. G. R. Adolf, Boehringer Ingelheim, Austria) had a specific activity of 7 x lo7 U/mg as determined by the L929 cytotoxicity assay (17). Rabbit antiserum against rMuTNF-ol, that was used for in viva experiments, was provided by Dr. D. Remick, University of Michigan, Ann Arbor, Michigan. One milliliter of this antiserum neutralized 937 ng of rMuTNF-cx as determined in the TNF assay (see below). E. co&derived (strain 026:B6) endotoxin, also called lipopolysaccharide (LPS), was purchased from Sigma Chemical Co., St. Louis, Missouri. Experimental protocol. Experiments were carried out to determine the suitable doses of cytokines needed for optimal IL-6 production without any apparent signs of toxic effects. The injection of 0.004, 0.02, 0.1, 0.5, and 1.0 pg of rMuTNF-cu resulted in the production of 54, 169, 303, 1549, and 18000 pg/ml of IL-6, respectively, as detected in the serum 1 hr after injection (mean of four mice per treatment). These results show that an increase in serum IL-6 can be observed following the injection of as little as 0.004 pg of rMuTNF-a per mouse. Doses greater than 1 .O pg of rMuTNF-a per mouse were toxic and were not used in this study. Based on these preliminary experiments and a previous study conducted in this laboratory (18), the following protocol was performed. Doses of LPS (1 .O p,g), rMuTNF-a (0.5 p,g), or rIL-1 B (0.1 pg) were injected in each mouse intravenously (iv), in a lateral tail vein, in 0.2 ml of sterile saline. In some experiments, mice were injected iv with 1 kg of rMuTNF-o., 0.5 p,g of rIL-lB, or both. At different time points after injections, four to six mice were anesthetized using FentanyYfluanison (Janssen Pharmaceutics, Brussels, Belgium) and bled by cardiac puncture. Blood samples, collected in sterile tubes, were allowed to clot on ice and were centrifuged at 600g for 20 min to separate the serum. Individual serum samples were harvested and kept in sterile plastic tubes at -20°C until assayed for TNF and IL-6 activities.

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TNF assay. The serum TNF-a level was determined using the WEHI- 164 clone 13 cytotoxicity assay, which is capable of detecting levels as low as 0.01 pg/ml of rMuTNF-cx (19). The amount of TNF in serum samples was calculated on the basis of cytotoxicity obtained in the presence of various concentrations of a rMuTNF-a standard. The cytotoxicity induced by serum samples was completely abolished in the presence of rabbit antiserum to rMuTNF-a demonstrating the specificity of the observed activity. Data are expressed as the mean picograms of TNF per milliliter of serum + SEM. IL-6 assay. Serum IL-6 levels were determined in a bioassay using the murine hybridoma cell line B13.29 clone B9 which is dependent on IL-6 for growth (16). The cells were incubated in the presence of serially diluted serum samples and the growth was measured after 72 hr of incubation using the MTT calorimetric assay (20). The concentration of IL-6 in serum samples was calculated on the basis of cellular growth obtained in the presence of various concentrations of a human i-IL-6 standard. The data are expressed as the mean picograms of IL-6 per milliliter of serum f SEM. RESULTS

Production of TNF-a and IL-6 following injection of LPS. Levels of TNF-cx and IL-6 were measured in sera collected from mice after LPS injection and the results are presented in Fig. 1. A sharp rise in serum TNF-a was detected at 1 hr after LPS injection. This TNF-a activity was eliminated rapidly, reaching baseline levels within I hr of peak time. Although a significant amount of IL-6 was detected 1 hr after LPS injection, the peak for IL-6 was attained 1 hr after that of TNF-(r, i.e. 2 hr after LPS injection (Fig. 1). These results suggest that LPS induction of IL-6 in vivo may be mediated at least partially by LPS-induced TNF-CX. It should be noted that the calculated half-lives (t,,,) for LPS-induced TNF-(r and IL-6 were 9 and 36 min, respectively. The pospibility that LPS induction of IL-6 may be mediated through an effect by endogenously produced TNF-cx was tested and the results are presented in Fig. 2. Compared with mice injected with LPS and treated with normal rabbit serum, there is a 56% reduction in serum IL-6 among mice injected with LPS and treated simultaneously with antiserum against rMuTNF-ol (Fig. 2). These data indicate that LPS-induced IL-6 in vivo is partially through the action of endogenously produced TNF-ok. Kinetics of IL-6 production following injection of rMuTNF-cw or rIL-I. As shown in Fig. 3, the injection of rMuTNF-a into mice led to the rapid production of significant amounts of IL-6 within 30 min and reached a peak level at 1 hr after rMuTNF-a injection (t,,* = 31 min). The data in Fig. 3 show that the peak of injected rMuTNF-a was detected within 5-10 min of injection and was completely cleared from the circulation within 1 hr (t,,, = 10 min). Similar to rMuTNF-a, the injection of rIL-1B caused a sharp rise in serum IL-6 within 30 min and reached a peak at 1 hr after injection of rIL-1B (tl12 = 47 min) (Fig. 4). However, the serum IL-6 level remained elevated until the 3-hr point and was sustained at a level slightly higher than background for 24 hr. No TNF-(w activity was detected in any of the serum samples collected at the indicated time points (Fig. 4). The results of a similar experiment (not shown) demonstrated the inability of rIL-6 to induce

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FIG. 1. Production of TNF-a and IL-6 following injection of LPS: NMRI mice were each injected with 1.0 p.g of LPS in 0.2 ml of sterile saline. At the indicated time points, five mice were sacrificed and bled for isolation of serum samples. Serum from each individual mouse was tested in triplicate for TNF-a and IL-6 activities using respective bioassays as outlined under Materials and Methods. Serum samples from five untreated mice were included to determine baseline levels of TNF-a and IL-6. The data are presented as the mean ? SEM of TNF-a (0) and IL-6 (0) concentrations expressed as pg/ml of serum.

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FIG. 2. LPS induction of IL-6 following administration of antiserum against rMuTNF-a. Six mice were injected with either 0.5 kg of LPS f 50 )LI of normal rabbit serum or 0.5 ug of LPS + 50 ~1 of rabbit antiserum to rMuTNF-a. Two hours later, serum samples were collected and tested for IL-6 activity. IL-6 concentration among control mice injected with LPS + normal rabbit serum (C); among mice injected with LPS + rabbit antiserum (anti-TNF-a). TNF-a activity was present in serum of control group (590 2 139 pg/ml) but not in serum of anti-TNF-o-treated mice as monitored 1 hr after LPS injection.

IL-6 or TNF-a in mice as monitored at time points identical to those described in Figs. 3 and 4. Since a combination of IL-Il3 and TNF-o has been shown to be particularly potent in inducing lethal shock in mice (18), it was of interest to determine whether or not there is an association between the frequency of lethality and the level of serum IL-6 among mice treated with a combination of rIL-Il3 and rMuTNF-a. The results in Fig. 5 show that a combination of rMuTNF-a and rIL-ll3 induced a significantly greater level of serum IL-6 compared with either agent alone as monitored 1 hr after injection (P = 0.01, Mann-Whitney’s twosided test). Interestingly, the high concentration of serum IL-6 was concomitant with an increased mortality in the combination group where 6/10 mice died within 9 hr compared with no deaths among animals injected with either cytokine alone. In addition, the average body temperature in the combination group was 29.2 -t 2°C which was 5°C lower than the average body temperature in the other groups as monitored at 5 hr after injections. The induction of hypothermia following injection of TNF-a is not without a precedent. Similar results have been published by us (18) and also by others (21) who reported that lower doses of TNF induced fever in rats, while higher doses produced a marked fall in body temperature. It should be noted that, in the combination group, postmortem serum IL-6 concentrations ranged from 25,000 to 76,000 pg/ml which is lOO-fold greater than IL-6 concentrations recorded at similar time points in animals injected with LPS only

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or with either agent alone. These data confirm our previous observations (18) and further demonstrate that the hypothermia and the lethal shock caused by a combination of TNF-a and IL-ll3 are associated with a significant rise in the concentration of endogenously produced serum IL-6. DISCUSSION

The present data demonstrate that LPS is a potent inducer of IL-6 production in viva. The peak of serum IL-6 was observed 2 hr after injection of LPS. However, the finding that TNF-(w. production reached a maximal level 1 hr after LPS injection and prior to the attainment of the peak IL-6 level (Fig. 1) indicates that LPS-induced TNF-a may be at least partially responsible for the induction of IL-6 production. This speculation was confirmed by the finding that treatment with antibodies to rMuTNF-cx resulted in a significant reduction in LPS-induced serum IL-6 concentration (Fig. 2). The data presented in Fig. 3 show that peak IL-6 production was attained rapidly, within 1 hr, after rMuTNF-a injection. It is interesting to note that the administration of human TNF-a in cancer patients (22) leads to the rapid induction of serum IL-6 with kinetics similar to those described

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= 6).

in this paper. Similar to rMuTNF-a, rIL-ll3 induced the production of significant amounts of IL-6 within 30 min, reaching a peak level at 1 hr after t-IL-ll3 injection. These results represent a strong evidence to the involvement of endogenous TNF-a in IL-6 induction in vivo. In addition, the present findings are consistent with recent data demonstrating the ability of IL-1g and TNF-a to induce IL-6 production by endothelial cells in vitro (20). Based on the amount of serum IL-6 detected, LPS was the most potent inducer of IL-6, probably due to the involvement of LPS-induced TNF-a and perhaps LPS-induced IL-I (23) in the induction process. The ability of IL-l and TNF-a to induce IL-6 production in vivo is an additional biologic activity shared between

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Cytokines injected 5. Levels of serum IL-6 induced by a combination of rMuTNF-u and rIL-1P. Groups of mice were injected with 1 kg of rMuTNF-a, 0.5 pg of rIL-IP, or both. One hour later, 4 mice from each group were sacrificed for serum collection. Each bar represents the mean IL-6 concentration from 4 mice ? SEM. Body temperature and survival were monitored among 10 mice remaining in each group. Values in parentheses equal number of dead mice/number per group as recorded during 9 hr after injections. No more deaths occurred thereafter. FIG.

these two cytokines which have already been shown to share many of their functions (24). The fact that macrophages (4, 16), fibroblasts (23, and endothelial cells (20) can produce IL-6 may explain the presence of serum IL-6 levels as high as 0.2 &ml (Fig. 1). Although the present data demonstrate that following LPS injection into mice the peak of TNF-a production is attained prior to that of IL-6, the position of IL-1 peak in this sequence remains undetermined. However, in an independent study involving the induction of experimental shock, it was found that, respectively, TNF-c~ and IL-Ip production reached peak levels prior to

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maximal IL-6 production as measured in the spinal fluid of rabbits after the local injection of LPS (A. Waage er ul., manuscript submitted for publication). Based on these observations and the data presented here, it is tempting to speculate that TNF-a and/or IL-lp are among the agents responsible for the induction of high serum IL-6 levels observed in septic shock. This view is supported by the tinding that high serum TNF levels were detected prior to the detection of maximal IL-6 in patients with septic shock (11). Interestingly, the tliz of 9 min for LPS-induced TNF-a (Fig. 1) is in agreement with the 10 min calculated for exogenously administered rMuTNF-a (Fig. 3), and both are consistent with previously reported data (26). Further, in the present study the mean ‘- SD of the t,,: for IL-6 from all experiments performed was 38 2 8 min, which is greater than that of TNF-CY. These results are similar to our observations in the case of human septic shock (11). The finding that a combination of rIL-ll3 and rMuTNF-a induced significantly greater amounts of IL-6 than either cytokine alone is consistent with the demonstrated ability of both cytokines to interact synergistically (24, 27). Further, a key finding of this study is the presence of an association between high concentrations of induced serum IL-6 and the development of the lethal shock among animals injected with a combination of TNF-a and IL-Il3. These data indicate that IL-6 may be an important mediator of TNF-a and IL-ll3 functions in rive. However, in order to verify an association between an increased lethality and an elevated level of IL-6, it will be important to determine whether the administration of an antiIL-6 antibody could protect the mice against IL-l/TNF-a lethality. Importantly, the participation of other factors in the observed IL-lP/TNF-a-mediated lethal shock cannot be excluded. Specifically, TNF-a has been shown to stimulate the release of a platelet activating factor which can promote platelet aggregation and induce the formation of ischemic lesions (28). Additionally, both IL-l and TNF can stimulate the synthesis of prostaglandins (2 1, 24) which may further aggravate the inflammatory reaction. The presence of such multiplicity of inflammatory agents, including IL-6 (9), may cause vascular endothelial injury and lead to the development of the lethal shock. Collectively, the data in this report describe new in vivo biologic activities shared by TNF-a and IL-ll3 and suggest that endogenously induced high concentrations of IL-6 may be an important factor in the development of the lethal shock caused by a combination of TNF-a and IL- 1l3. ACKNOWLEDGMENTS We are grateful to Professor Dr. J. Lamvik for his encouragement and support. We thank Hilde Kvithyll, Mar-i Serensen, and Berit Stordal for their help; Dagmar Moholdt for preparing this manuscript; and Finn Bakke Olsen for the art work. This work was supported by grants from the Norwegian Research Council for Science and Humanities, The Norwegian Cancer Society, and Kreftfondet ved Regionsykehuset in Trondheim.

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2. Kishimoto, T., and Hirano, T., A new interleukin with pleiotropic activities. BioEssays 9, 11-15, 1988. 3. Muraguchi, A., Hirano, T., Tang, B., Matsuda, T., Horii, Y., Nakajima, K., and Kishimoto, T., The essential role of B cell stimulatory factor 2 (BSF-2/IL-6) for the terminal differentiation of B cells. J. Exp. Med. 167, 332-344, 1988. 4. Brakenhoff, .I. P., DeGroot, E. R., Evers, R. F., Pannekoek, H., and Aarden, L. A., Molecular cloning and expression of hybridoma growth factor in Escherichia coli. J. Immunol. 139, 4116 4121, 1988. 5. Gauldie. J., Richards, C., Hamish, D., Lansdorp, P., and Baumann, H., Interferon B,/B-cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells. Proc. Nat/. Acad. Sci. USA 84, 7251-7255, 1987. 6. Kawano, M., Hirano, T., Matsuda, T., Taga, T., Norii, Y ., Iwato, K., Asaoku, H., Tang, B., Tanabe, O., Tanaka, H., Kuramoto, A., and Kishimoto, T., Autocrine generation and requirement of BSF-2/IL-6 for human multiple myelomas. Nature (London) 332, 83185, 1988. 7. Houssiau, F. A., Coulie, P. G., Olive, D., and Van Snick, J., Synergistic activation of human T cells by interleukin 1 and interleukin 6. Eur. J. Immunol. 18, 653-656, 1988. 8. Helle, M., Brakenhoff, J. P. J., De Groot, E. R., and Aarden, L. A., lnterleukin 6 is involved in interleukin l-induced activities. Eur. J. Immunol. 18, 957-959, 1988. 9. Waage, A., Kaufmann, C., Espevik, T., and Husby, G., Interleukin-6 in synovial fluid from patients with arthritis. Clin. Immunol. Immunopathol. 50, 394-398, 1989. 10. Houssiau, F. A., Devogelaer, J. P., Van Damme, J., De Deuxchaisnes, C. N., and Van Snick, J.. Interleukin-6 in synovial fluid and serum of patients with rheumatoid arthritis and other inflammatory arthritides. Arthritis Rheum. 31, 784-788, 1988. It. Waage, A., Brandtzaeg, P., Halstensen, A., Kierulf, P., and Espevik. T., The complex pattern of cytokines in serum from patients with meningococcal septic shock. J. Exp. Med. 169, 333-338. 1989. 12. Houssiau, F. A., Bukasa, K., Sindic, C. J. M., Van Damme, J., and Van Snick, J., Elevated levels of the 26 K human hybridoma growth factor (interleukin-6) in cerebrospinal fluid of patients with acute infection of the central nervous system. Clin. Exp. Immunol. 71, 320-323. 1988. 13. Van Oers, M. H. J., Van der Heyden, A. A. P. A. M., and Aarden, L. A., Interleukin 6 (IL-6) in serum and urine of renal transplants recipients. Clin. Exp. Zmmunol. 71, 314-319, 1988. 14. Wingfield, P., Payton, M., Tavemier, J., Barnes, M., Shaw. A., Rose. K., Simona, M. G., Memczuk, S., Williamson, K., and Dayer, J. M., Purification and characterization of human interleukin-1 expressed in recombinant Escherichia co/i. Eur. J. Biochem. 160, 491-497, 1986. 15. Pennica, D., Hayflick, J. S., Bringman, T., Palladino, M. A., and Goeddel, D. V., Cloning and expression in E. coli of the cDNA for murine tumor necrosis factor. Proc. Natl. Acad. Sci. USA 82, 6060-6064, 1985. 16. Aarden, L. A., DeGroot, E. R., Schaap, 0. L., and Lansdorp, P. M., Production of hybridoma growth factor by human monocytes. Eur. J. Zmmunol. 17, 1411-1416, 1987. 17. Kramer, S. M., and Carver, M. E., Serum-free in vitro bioassay for the detection of tumor necrosis factor. J. Immunol. Methods 93, 201-206, 1986. 18. Waage, A., and Espevik. T., Interleukin 1 potentiates the lethal effect of tumor necrosis factor/cachectin in mice. J. Exp. Med. 167, 1987-1992, 1988. 19. Espevik, T., and Nissen-Meyer, J., A highly sensitive cell line, WEHI 164, clone 13, for measuring cytotoxic factor/tumor necrosis factor from human monocytes. J. Immunol. Methods 95, 99-105. 1986. 20. Shalaby, M. R., Waage, A., and Espevik, T., Cytokine regulation of interleukin 6 production by human endothelial cells. Ce//. Immunol. 121, 372-382, 1989. 21. Kettelhut, 1. C.. Fiers, W., and Goldberg, A. L., The toxic effects of tumor necrosis factor in vivo and their prevention by cyclooxygenase inhibitors. Proc. Natl. Acad. Sci. USA 84, 42734277, 1987. 22. Jablons, D. M., Mule, J. I., McIntosh, J. K., Sehgal. P. B., May, L. T., Huang, C. M., Rosenberg. S. D. A., and Lotze, M. T., IL-6/IFN-y-2 as a circulating hormone: Induction by cytokine administration in humans. J. Zmmunol. 142, 1542-1547. 1989.

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23. Movat. H. Z.. Tumor necrosis factor and interleukin-I: Role in acute inflammation and microvascular injury. J. Luh. C/in. Med. 110, 668-681, 1987. 24. Dinarello. C. A. The biology of interleukin I and comparison to tumor necrosis factor. Itnmrcnol. Lett. 16, 227-231, 1987. 25. Defilippi, P., Poupart, P., Tavernier. J.. Fiers. W.. and Content, J., Induction and regulation ot mRNA encoding 26-kDa protein in human cell lines treated with recombinant human tumor necrosis factor. Proc. Nutl. Acud. Sci. USA 84, 4557-4561. 1987. 26. Flick, D. A.. and Gifford. G. E.. Pharmacokinetics of murine tumor necrosis factor. J. Immunopharmacol. 8, 89-91. 1986. 27. Movat, H. Z.. Burrowes. C. E.. Cybulsky, M. I., and Dinarello, C. A., Acute inflammation and Shwartzman-like reaction induced by interleukin-1 and tumor necrosis factor: Synergistic action of the cytokines in the induction of inflammation and microvascular injury. Amer. J. Pathol. 129, 463-476, 1987. 28. Sun, X., and Hsueh, W., Bowel necrosis induced by tumor necrosis factor in rats is mediated by platelet-activating factor. /. C/in. Invest. 81, 1328-1331, 1988. Received May 24. 1989; accepted with revision August 8. 1989