Original Contributions HYPOXIA INCREASES PRODUCTION OF INTERLEUKIN-1 AND TUMOR NECROSIS FACTOR BY HUMAN MONONUCLEAR CELLS Pietro Ghezzi,‘* Charles A. Dinarello,’ Marina Bianchi,’ Mary E. Rosandich,3 John E. Repine,3 Carl W. White3 Exposure to hypoxia (PO, = 9 + 1 torr) increased human peripheral blood mononuclear cell production and secretion of interleukin-1 (IL-l)% IL-lp, and tumor necrosis factor (TNF) (percent of control = 190% for IL-l% p = 0.014; 219% for IL-lp, p = 0.014; and 243% for TNF, p = 0.037) following treatment with endotoxin (1 @ml). Hypoxia potentiated the increased production of these inflammatory cytokines at subthreshold levels of endotoxin with potentiation increasing at lower 0, concentrations. Hypoxia also increased cytokine production induced by the tumor promoter phorbol myristate acetate, suggesting a generalized biologic response. We conclude that hypoxia increases IL-1 and TNF production and speculate that this mechanism aggravates a variety of pathologic conditions involving endotoxin such as adult respiratory distress syndrome (ARDS), multiple organ failure, and septic shock. Copyright o 1991 by W.B. Saunders Company
Two macrophage-derived cytokines, interleukin-1 (IL-l) and tumor necrosis factor (TNF), play an important role in the pathogenesis of inflammation and shock. Administration of IL-1 and TNF induces a variety of responses including anorexia, fever, synthesis of hepatic acute-phase proteins, hypotension, and death.lm4 Several stimuli can trigger the synthesis of IL-l and TNF. These include bacterial endotoxin (lipopolysaccharide, LPS), complement components, the phorbol ester tumor promoter phorbol myristate acetate (PMA), and IL-l and TNF.‘.’ A few agents can modulate the production of IL-l and/or TNF. These include interferon (IFN)-y, corticosteroids, prostaglandins, and cAMP.~,~-” This modulation of cytokine synthesis may differ with the stimulus. For example, IFN-?/ enhances C5a- and LPS-induced IL-l synthesis
but reduces the amount of IL-l produced in response to PMA or IL-l?,’ Furthermore, there is evidence that gene expression and synthesis of IL-l and of TNF may be regulated differently.” The lung appears to be a primary target for the actions of IL-l and TNF. Both in vitro and within the lung in vivo these cytokines induce a generalized inflammatory response including neutrophil accumulation and activation, pulmonary endothelial cell damage, and, consequently, increased vascular permeability. 1-3~‘3-15 A recent report indicates that intratracheal administration of IL-l induces pulmonary granuloma formation.16 Our previous work demonstrated that pretreatment with small doses of IL-1 and TNF leads to increased resistance to pulmonary oxygen toxicity in
rats.” Preexposureto hypoxia also can induce toleranceto oxygentoxicity.18~20 Of current interest are the ‘Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italy. ‘Department of Medicine, Division of Infectious Disease and Geographic Medicine, Tufts University and New England Medical Center, Boston, MA, USA. ‘Departments of Pediatrics, Medicine and Surgery, The WebbWaring Lung Institute and National Jewish Center for Immunology and Respiratory Medicine, University of Colorado Health Sciences Center, Denver, CO, USA. *Address correspondence and reprint requests to: Pietro Ghezzi, Istituto di Ricerche Farmacologiche “Mario Negri,” via Eritrea 62,20157 Milano, Italy. Copyright 0 1991 by W.B. Saunders Company 1043-4666/91/0303-0006$05.00/O KEY WORDS: adult respiratory distress syndromeiendotoxinl interleukin-l/oxygen/tumor necrosis factor CYTOKINE,
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findings that, in addition to the induction of oxygen tolerance by preexposure to hypoxia, endotoxin, or the cytokines TNF and IL-l, each of these is associated with increases in superoxide dismutases (SOD), particularly of manganese (mitochondrial) SOD in lung and/or other cell types, suggesting a possible common mechanism.‘8-25 On this basis, we questioned whether the effect of hypoxia could, in part, be due to increased IL-1 and/or TNF production. Hypoxia can induce anorexia and cachexia,20’26which are also induced by IL-l and TNF.‘,*,” In addition, exposure to hypoxia potentiates the deleterious effects of complement activation in animal models for the adult respiratory 189
190 I Ghezzi et al.
distress syndrome and multiple organ failure.28zz9When present initially, hypoxemia is associated with a poor outcome in these disorders in humans.30 The purpose of this study was to investigate the effect of hypoxia on the synthesis and secretion of IL-l and TNF in vitro by human peripheral blood mononuclear cells (MNC).
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RESULTS Exposure of human peripheral blood MNC to hypoxia increased endotoxin-induced production and secretion of both IL-1 (percent of control = 190% for IL-la, p = 0.014; 219% for IL-lp, p = 0.014; n = 6) and TNF (percent of control = 243%, p = 0.037, n = 6; Fig. 1, A, B, C). Hypoxia also potentiated IL-l production by subthreshold levels of LPS. Although little detectable IL-l was generated by lower doses of LPS under normoxic conditions, severe hypoxia consistently increased IL-1 generation at levels of LPS as low as 0.1 rig/ml (Fig. 2). Dexamethasone, although fully inhibiting under normoxic conditions, only partially inhibited hypoxia-potentiated, LPS-induced IL-l production (data not shown). Detectable levels of TNF were not present under normoxic or severely hypoxic conditions at LPS doses less than 1.0 @ml. At that dosage of LPS, TNF production showed a similar pattern of inhibition by dexamethasone under normoxic or hypoxic conditions (data not shown). We then investigated the effect of varying concentrations of 0, (0, 3, 10, and 21%) on the LPSstimulated IL-1 and TNF production by mononuclear cells. In four of five donors, IL-1 production was already increased at 10% 0, compared to 21% 0, (Fig. 3A). We observed a similar effect of graded hypoxia on TNF production although potentiation of cytokine production was maximal only when MNC were exposed in chambers flushed with 0 to 3% oxygen (Fig. 3B). Since it was not clear if hypoxia specifically augmented the response to LPS or enhanced IL-l and TNF production irrespective of the stimulus, we studied the effect of hypoxia (0% 0,) on the production of IL-l and TNF induced by PMA. We found that PMA-induced IL-l and TNF (data not shown) production was also increased by hypoxia in most donors. Cell damage did not cause the effect of hypoxia; 24 h of severe hypoxia (0% 0,) was not toxic to MNC as measured by LDH release (8.4 & 0.6 in 21% 0, and 8.4 + 0.7% in 0% O,, p > 0.05, n = 4) and vital dye exclusion using erythrosin B (97.5 + 0.6% in 21% 0, and 97.0 + 0.6% in 0% O,, p > 0.05, n = 9). In addition, values for LDH release or vital dye exclusion were not different in the presence of LPS under normoxic or hypoxic conditions. This suggests that cell injury due to anoxia or anoxia-reoxygenation is not potentiating cytokine production. In fact, exposure of MNC to
C 6 ..
NORMOXIA
HYPOXIA
Figure 1. Effect of normoxia or hypoxia (chamber flushed with 95% N,, 5% CO, for 30 min) on production of the cytokines &la (A), IL-Q3 (B), or TNF (C) by peripheral blood mononuclear cells (MNC) exposed to LPS (1.0 @ml; from Escheriehiu coli 012tkB12, Sigma) over 24 h. The solid portion of each bar represents the arithmetic mean of the cell-associated cytokine fraction whereas the open portion represents that of the secreted fraction. SEM and statistical differences refer to total cytokine produced; *p < 0.05; **p < 0.02, Wilcoxon’s signed rank test for paired data. The lower limit of sensitivity for the RIA’s was 80 pg/ml (indicated by the dashed line).
severe hypoxia for 4 to 6 h followed by 18 to 20 h of normoxia (21% 0,) or hyperoxia (95% 0,) was less effective than 24 h of hypoxia (0% 0,) in potentiating cytokine synthesis (data not shown). Furthermore, acidosis did not appear to be a contributing factor as the pH of the medium did not change over 24 h of incubation. Finally, we questioned whether the increased production of IL-l during MNC exposure to hypoxia was due to an increased IL-l gene expression. Accordingly, we measured levels of IL-lp mRNA in MNC from
Hypoxia increases IL-l and TNF production
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4
Figure 2. Effect of normoxia chamber flushed 30 min with IL-lp production by MNC when rig/ml of LPS (left) or 100 @ml
(N) or hypoxia (H, 95% N,, 5% CO,) on exposed to 0.1 or 1.0 of PMA (right).
ILIp (w/ml)
In the absence of LPS, none of these donors produced detectable IL-l under normal or hypoxic conditions. Mean and SEM for total IL-1 produced are represented. *p < 0.05; **p < 0.01, Wilcoxon’s signed rank test for paired data. Data from four different donors are represented under each condition.
three donors exposed for 4 h to normoxia or hypoxia in the presence and absence of LPS (1 @ml). The Northern blot analysis of MNC RNA is shown in Fig. 4. LPS, as expected, increased the expression of IL-1 mRNA. However, in the presence of LPS, significant differences in IL-1 mRNA were not observed after 4 h under normoxic and hypoxic conditions. The finding that hypoxia did not increase cytokine mRNA suggests a posttranscriptional effect of oxygen tension on IL-l synthesis.
DISCUSSION We found that hypoxia increased production of three inflammatory cytokines, IL-lo., IL-lp, and TNF. In addition, hypoxia decreased the endotoxin dose threshold for detectable IL-1 production. Both IL-1
2
0
N
H
N
LPS 0.1 rig/ml
H
N
LPS 1.0 rig/ml
H
PMA 100 rig/ml
and TNF produced by endotoxin-stimulated mononuclear cells were inversely related to oxygen tension. Although IL-1 was optimally potentiated by 3% 0, and TNF by 0% O,, respectively, even cultures exposed to 0% 0, were not truly anoxic. This may have been due to residual dissolved oxygen present in the medium and/or to oxygen permeability of the plastic chamber. Based on Northern blot analysis, the promoting effect of hypoxia on endotoxin-induced cytokine production does not appear to occur at the mRNA level. Quantities of mRNA for IL-1 were not different from MNC exposed to endotoxin and either hypoxia or normoxia for 4 h, the time point at which maximal endotoxininduced IL-@ mRNA production occurs. The potentiation of IL-1 and TNF production reported here indicates a novel mechanism of regulation of cytokine production by hypoxia. This mechanism could be important in many pathologic condi-
DONOR A
nnm 02tension: Endotoxin:
N -
N +
H +
DONOR g N +
H +
DONOR C N +
H +
TNFa (w/ml)
4 Figure
Figure 3. Effect of increasing levels of oxygen (chambers Rushed with gas mixtures containing 21%, lo%, 3%, or 0% 0,, respectively) on (A) IL-1s or (B) TNF production by MNC exposed to LPS (1.0 &ml).
Results from six (IL-lp) represented.
or three different donors, respectively, are
4.
RNA
blot analysis
of IL-1s
mRNA.
Peripheral blood mononuclear cells from three donors were studied under normoxic (N) conditions in either the absence (-) or presence (+) of LPS (1 q/ml), or under hypoxic (H) conditions in the presence (+) of the latter. Normoxic (N) chambers were flushed with 21% 0,, 5% CO,, 74% N, and hypoxic (H) chambers with 0% 0,, 5% CO,, 95% N, for 30 min prior to incubation. In cells incubated for 4 h, the top line represents IL-1S mRNA and the middle line represents p actin mRNA. The bottom line shows total IL-lp protein in MNC after 24 h. The first lane shows RNA from a representative donor exposed to normoxia in the absence of LPS. No donor had detectable mRNA for IL-lp in the absence of LPS under normoxic or hypoxic conditions.
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/ Ghezzietal.
tions. For instance, hypoxia may occur in the center of a wound or in tumors promoting angiogenesis,31 fibrosis, or other processes through cytokine actions. Similarly hypoxia could potentiate the pulmonary fibrosis resulting from a variety of interstitial lung diseases32V33 or the pulmonary vascular remodeling due to chronic exposure to hypoxia34 through cytokine actions. Cytokine production potentiated by hypoxia might also contribute to high-altitude pulmonary edema In addition, this mechanism and/or cerebral edema. 35,36 could contribute to the worsening effect of hypoxemia on the adult respiratory distress syndrome (ARDS) and multiple organ failure,28’29particularly when considering the contribution of sepsis to the development of these disorders.30 Individual variation in cytokine production and its potentiation by hypoxia could lead to varying susceptibilities to, and severities of, these diseases. Finally, these findings suggest a mechanism by which shock, with resulting ischemia of certain vascular beds and regional hypoxemia, could potentiate itself, as well as associated inflammatory conditions, such as ARDS and multiple organ failure.
MATERIALS AND METHODS Mononuclear Cell Cultures Following informed consent, blood was obtained by venepuncture from healthy volunteers who had not used cyclooxygenase inhibitors during the previous two weeks. MNC were prepared using Ficoll-Hypaque gradients.8,37Cells were cultured in RPMI-1640 medium that had been ultrafiltered to remove LPS’* and supplemented with 1% heatinactivated autologous human serum. MNC were cultured at 2.5 x 106/ml in 96-well, flat-bottomed tissue culture plates. Cells were incubated in chambers flushed for 30 min with 5% CO, and various oxygen concentrations. Following exposure for 4 or 24 h to LPS or PMA, MNC cultures (cells with or without supernatants) were subjected to three freeze-thaw cycles to allow complete cell lysis, and the lysates were assayedfor IL-lo, IL-l& or TNF in specific radioimmunoassays (RIA, see below). In some experiments, supernatants were harvested and residual cells removed by centrifugation to allow determination of the amount of IL-1 and TNF in cells (cell-associated) and in supernatants (secreted). Oxygen concentrations measured upon termination of incubation in tissue culture medium with an 0, analyzer (Chemical Microsensor II, Diamond General Inc., Ann Arbor, MI) indicated that the 0, concentration present in normoxic (21% O,, 5% CO,, 74% NJ incubations was 101 + 4 torr (n = 5) compared to 9 + 1 torr (n = 5) in hypoxic (0% O,, 5% CO,, 95% NJ incubations. Using this instrument it was demonstrated that PO, was similarly low in tubes in which MNC were cultured for mRNA measurements and in wells of 96-well plates during hypoxic incubations for 4 or 24 h.
Radioimmunoassays RIAs were used to measure IL-la? IL-l@,4’ and TNF(Ye’and have been shown to detect lessthan 100 pg/ml of each cytokine in human MNC supernatants. Previous studies have
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demonstrated no cross-reactivity among these cytokine RIAs, nor do they detect human TNF-B, IFN-a, IFN-7, or granulocyte-macrophage colony-stimulating factor. In addition, each RIA correlated with functional bioassays(cytotoxicity, T cell proliferation) with correlation coefficients > 0.8 (p < 0.05 by paired Student’s t test).
LDH Release At termination of MNC incubations, media were aspirated and centrifuged (Microfuge 12, Beckman) for 2 min (10,500g). Cells were removed into 50 mM potassium phosphate, pH 7.4, containing 0.1% Triton X-100 and sonicated (Kontes Micro-Ultrasonic Cell Disrupter, maximum power, 2 x 10 set, 4°C). LDH activity was determined in paired supernatants and sonicates using a spectrophotometric method to measure NADH-dependent conversion of pyruvate to lactate.42 LDH release was then calculated from the formula: LDH release (percent total) = [LDH in supematant/ (LDH in supernatant + LDH in sonicate)] x 100.
mRNA Measurement MNC were lysed in guanidine isothiocyanate and RNA was prepared by centrifugation through cesium chloride.43 The concentration of RNA was determined from the absorbance at 260 nm, and A2,JAZ8,,ratios were greater than 2. RNA (2.5 kg) was analyzed by electrophoresis through a 1% agarose formaldehyde gel followed by Northern blot transfer to Gene Screen Plus membranes (New England Nuclear, Boston, MA). An IL-1B cDNA clone@ was labeled to high specific activity with [cx-“P]dCTP (3.00 Ci/mmole; Amersham) by the random priming method.45 Membranes were pretreated and hybridized in 50% formamide (Merck) with 10% dextran sulfate (Sigma) and washed twice with 2 X SSC (1 x SSC is 0.15M NaCl with 0.015M Na citrate) and 1% sodium dodecyl sulfate (SDS, Sigma) at 60°C for 30 min, and then twice more with 0.1 x SSC at room temperature for 30 min. The membranes were exposed for 4 to 8 h at -80°C using intensifying screens.
Acknowledgments The authors acknowledge the superb technical assistance of Reza Ghorbani and Gary Zerbe, Ph.D., for expert statistical consultation. We thank Jacqueline Smith and Dawnett Marples for preparing the manuscript. This work was done during Dr. White’s tenure of a Clinician-Scientist Award from the American Heart Association and with funds contributed in part by the Colorado Heart Association. Research support was also provided by National Institutes of Health Grant Al-15614, The Children’s Hospital of Denver and C. Henry Kempe Research Center, and by a Grant-in-Aid from the American Heart Association National Center.
REFERENCES 1. Dinarello 2:108-115. 2. Beutler necrosis factor.
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Hypoxia increases IL-l and TNF production 3. Tracey KJ, Beutler B, Lowry SF, Merryweather J, Wolpe S, MiIsark IW, Hariri RJ, Fahey III TJ, Zentella A, Albert JD, Shires JD, Cerami A (1986) Shock and tissue injury induced by recombinant human cachectin. Science 234:470-474. 4. Vogel SN, Kaufman EN, Tate MD, Neta R (1988) Recombinant interleukin-1 and recombinant tumor necrosis factor synergize in vivo to induce early endotoxin tolerance and associated hematopoietic changes. Infect Immun 56:2650-2657. 5. Okusawa S, Dinarello CA, Yancey KB, Endres S, Lawley TJ, Frank MM, Burke JF, and Gelfand JA (1987) C5a induction of human interleukin 1. Synergistic effect of endotoxin or interferon-y. J Immunol 139:2635-2640. 6. Okusawa S, Yancey KB, van der Meer JWM, Endres S, Lonnemann G, Hefter K, Frank MM, Burke JF, Dinarello CA, Gelfand JA (1988) C5a stimulates secretion of tumor necrosis factor from human momonuclear cells in vitro. J Exp Med 168443-448. 7. Mizel SB, Rosenstreich DL, Oppenheim JJ (1978) Phorbol myristate acetate stimulates LAF production by the macrophage cell line P388Dl. Cell Immunol40:230-235. 8. Ghezzi P, Dinarello CA (1988) Interleukin-1 induces interIeukin-1. III. Specific inhibition of interleukin-1 production by gamma-interferon. J Immunol 140:4238-4244. 9. Lee SW, Tsou A-P, Chan H, Thomas J, Petrie K, Eugui EM, Allison AC (1988) Glucocorticoids selectively inhibit the transcription of the interleukin 1 gene and decrease the stability of interleukin 1 mRNA. Proc Nat1 Acad Sci 85:1204-1208. 10. Kunkel SL, Spengler M, May MA, Spengler R, Larrick J, Remick D (1988) Prostaglandin E2 regulates macrophage-derived tumor necrosis factor gene expression. J Biol Chem 263:5380-5384. 11. Knudsen PJ, Dinarello CA, Strom TB (1986) Prostaglandins posttranscriptionally inhibit monocyte expression of interleukin 1 activity by increasing intracellular cyclic adenosine monophosphate. J Immunol 137:3189-3194. 12. Endres S, Cannon JG, Ghorbani R, Dempsey RA, Sisson SD, Lonnemann G, van der Meer JWM, Wolff SM, Dinarello CA (1989) In vitro production of IL-lp, IL-lo, TNF and IL-2 in healthy subjects: Distribution, effect of cyclooqgenase inhibition, and evidence of independent gene regulation. Eur J Immunol19:2327-2333. 13. Bevilacqua MP, Pober JS, Wheeler ME, Cotran RS, Gimbrone MA (1985) Interleukin 1 acts on cultured human vascular endothelium to increase the adhesion of polymorphonuclear leukocytes, monocytes, and related leukocyte cell lines. J Clin Invest 76:2003-2011. 14. Nathan CF (1987) Neutrophil activation on biological surfaces. Massive secretion of hydrogen peroxide in response to products of macrophages and lymphocytes. J Clin Invest 80:15501560. 15. Goldblum SE, Cohen DA, Gillespie MN, McClain CJ (1987) Interleukin-l-induced granulocytopenia and pulmonary leukostasis in rabbits. J Appl PhysioI62:122-128. 16. Kasahara K, Kobayashi K, Shikama Y, Yoneya I, Soezima K, lde H, Takahashi T (1988) Direct evidence for granulomainducing activity of interleukin-1. Am J Path01 130:629-638. 17. White CW, Ghezzi P, Dinarello CA, Caldwell SA, McMurtry IF, Repine JE (1987) Recombinant tumor necrosis factor/ cachectin and interleukin 1 pretreatment decreases lung oxidized glutathione accumulation, lung injury, and mortality in rats exposed to hyperoxia. J Clin Invest 79:1868-1873. 18. White CW, Jackson JH, McMurtry IF, Repine JE (1988). Hypoxia increases glutathione redox cycle and protects rat lungs against oxidants. J Appl Physiol65:2605-2616. 19. Sjostrom K, Crapo JD (1983) Structural and biochemical adaptive changes in rat lungs after exposure to hypoxia. Lab Invest 48:68-79. 20. Frank L (1982) Protection from 0, toxicity by preexposure to hypoxia: Lung antioxidant enzyme role. J Appl Physiol53:475-482. 21. Frank L, Summerville J, Massaro D (1980) Protection from oxygen toxicity with endotoxin: Role of the endogenous antioxidant enzymes of the lung. J Clin Invest 65:1104-1110.
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22. Shiki Y, Meyrick BO, Brigham KL, Burger IM (1987) Endotoxin increases superoxide dismutase in cultured bovine pulmonary endothelial cells. Am J Physiol252:C436-440. 23. White CW, Ghezzi P, McMahon S, Dinarello CA, Repine JE (1989) Cytokines increase rat lung antioxidant enzymes during exposure to hyperoxia. J Appl Physiol66:1003-1007. 24. Wong GHW, Goeddel DV (1988) Induction of manganous superoxide dismutase by tumor necrosis factor: Possible protective mechanism. Science 242:941-944. 25. Masuda A, Longo DL, Kobayashi Y, Appella E, Oppenheim JJ, Matshushima K (1988) Induction of mitochondrial manganese superoxide dismutase by interleukin 1. FASEB J 23087-3091. 26. Blume FD (1984) Metabolic and endocrine changes at altitude. In West JB, Lahiri S (eds) High Altitude and Man, American Physiological Society, Baltimore, pp 37-45. 27. Tracey KJ, Lowry SF, Cerami A (1988) Cachectin: A hormone that triggers acute shock and chronic cachexia. J Infect Dis 157:413-420. 28. Larsen GL, Webster RO, Worthen GS, Gumbay RS, Henson PM (1985) Additive effect of intravascular complement activation and brief episodes of hypoxia in producing increased permeability in the rabbit lung. J Clin Invest 75:902-910. 29. Nuytink JKS, Goris RJA, Weerts JGE, Schillings PHM, Stekhoven JHS (1986) Acute generalized microvascular injury by activated complement and hypoxia: The basis of the adult respiratory distress syndrome and multiple organ failure. J Exp Pathol 67:537548. 30. Bell RC, Coalson JJ, Smith JD, Johanson WG Jr (1983) Multiple organ system failure and infection in adult respiratory distress syndrome. Ann Intern Med 99:293-298. 31. Knighton DR, Hunt TK, Scheuenstuhl H, HaIIiday BJ, Werb Z, Banda MJ (1983) Oxygen tension regulates the expression of angiogenesis factor by macrophages. Science 221:1283-1285. 32. Postlethwaite AE, Raghow R, Stricklin GP, Popleton H, Seyer JM, Kang AH (1988) Modulation of fibroblast functions by interleukin 1: Increased steady-state accumulation of type 1 procollagen messenger RNAs and stimulation of other functions but not chemotaxis by human recombinant interleukin 1 alpha and beta. J Cell Biol 106:311-318. 33. Elias JA, Krol RC, Freundlich B, Sampson PM (1988) Regulation of human lung fibroblast glycosaminoglycan production by recombinant interferons, tumor necrosis factor and lymphotoxin. J Clin Invest 81:325-333. 34. Mecham RP, Whitehouse LA, Wrenn DS, Parks WC, Griffin GL, Senior RM, Crouch EC, Stenmark KR, Voelkel NF (1987) Smooth muscle-mediated connective tissue remodeling in pulmonary hypertension. Science 237:423-426. 35. Hyers TM, Scoggin CH, Will DH, Grover RF, Reeves JT (1979) Accentuated hypoxemia at high altitude in subjects susceptible to high-altitude pulmonary edema. J Appl Physiol46:41-46. 36. Stelzner TJ, O‘Brien RF, Sato K, Weil JV (1988) Hypoxiainduced increases in pulmonary transvascular protein escape in rats. Modulation by glucocorticoids. J Clin Invest 82:1840-1847. 37. Boyum A (1968) Isolation of mononuclear cells and granulocytes from human blood. Stand J Clin Lab Invest 21 (Suppl. 97):77-89. 38. Dinarello CA, Lonnemann G, Maxwell R, Shaldon S (1987) Ultrafiltration to reject human interleukin-l-inducing substances derived from bacterial cultures. J CIin Microbial 25:12331238. 39. Lonnemann G, Endres S, van der Meer JWM, Cannon JG, Dinarello CA (1988) A radioimmunoassay for human interleukin-lalpha: Measurement of IL-l-alpha produced invitro by human blood mononuclear cells stimualted with endotoxin. Lymphokine Res 7175-84. 40. Lisi PJ, Chu C-W, Koch GA, Endres S, Lonnemann G, Dinarello CA (1987) Development and use of a radioimmunoassay for human interleukin-l-beta. Lymphokine Res 6:229-235.
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41. van der Meer JWM, Endres S, Lonnemann G, Cannon JG, Ikejima T, Okusawa S, Gelfand JA, Dinarello CA (1988) Concentrations of immunoreactive human tumor necrosis factor alpha produced by human mononuclear cells in vitro. J Leukocyte Biol 43:216-223. 42. Bergmeyer HU, Garvehn K (1978) Lactate dehydrogenase. In Bergmeyer HU (ed) Methods of Enzymatic Analysis. Verlag Chemie Weinheim, Academic Press, New York, ~012, pp 574-579. 43. Chirgwin JM, Przybyla AI?, MacDonald RJ, Rutter WJ
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(1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 185294-5299. 44. Auron PE, Webb AC, Rosenwasser LJ, Mucci SF, Rich A, Wolff SM, Dinarello CA (1984) Nucleotide sequence of human monocyte interleukin 1 precursor cDNA. Proc Nat1 Acad Sci USA 81:7907-7911. 45. Feinberg AF, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13.
Two macrophage-derived cytokines, interleukin-1 (IL-l) and tumor necrosis factor (TNF), play an important role in the pathogenesis of inflammation and shock. Administration of IL-1 and TNF induces a variety of responses including anorexia, fever, synthesis of hepatic acute-phase proteins, hypotension, and death.lm4 Several stimuli can trigger the synthesis of IL-l and TNF. These include bacterial endotoxin (lipopolysaccharide, LPS), complement components, the phorbol ester tumor promoter phorbol myristate acetate (PMA), and IL-l and TNF.‘.’ A few agents can modulate the production of IL-l and/or TNF. These include interferon (IFN)-y, corticosteroids, prostaglandins, and cAMP.~,~-” This modulation of cytokine synthesis may differ with the stimulus. For example, IFN-?/ enhances C5a- and LPS-induced IL-l synthesis
but reduces the amount of IL-l produced in response to PMA or IL-l?,’ Furthermore, there is evidence that gene expression and synthesis of IL-l and of TNF may be regulated differently.” The lung appears to be a primary target for the actions of IL-l and TNF. Both in vitro and within the lung in vivo these cytokines induce a generalized inflammatory response including neutrophil accumulation and activation, pulmonary endothelial cell damage, and, consequently, increased vascular permeability. 1-3~‘3-15 A recent report indicates that intratracheal administration of IL-l induces pulmonary granuloma formation.16 Our previous work demonstrated that pretreatment with small doses of IL-1 and TNF leads to increased resistance to pulmonary oxygen toxicity in
rats.” Preexposureto hypoxia also can induce toleranceto oxygentoxicity.18~20 Of current interest are the ‘Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italy. ‘Department of Medicine, Division of Infectious Disease and Geographic Medicine, Tufts University and New England Medical Center, Boston, MA, USA. ‘Departments of Pediatrics, Medicine and Surgery, The WebbWaring Lung Institute and National Jewish Center for Immunology and Respiratory Medicine, University of Colorado Health Sciences Center, Denver, CO, USA. *Address correspondence and reprint requests to: Pietro Ghezzi, Istituto di Ricerche Farmacologiche “Mario Negri,” via Eritrea 62,20157 Milano, Italy. Copyright 0 1991 by W.B. Saunders Company 1043-4666/91/0303-0006$05.00/O KEY WORDS: adult respiratory distress syndromeiendotoxinl interleukin-l/oxygen/tumor necrosis factor CYTOKINE,
Vol. 3, No. 3 (May), 1991: pp 189-194
findings that, in addition to the induction of oxygen tolerance by preexposure to hypoxia, endotoxin, or the cytokines TNF and IL-l, each of these is associated with increases in superoxide dismutases (SOD), particularly of manganese (mitochondrial) SOD in lung and/or other cell types, suggesting a possible common mechanism.‘8-25 On this basis, we questioned whether the effect of hypoxia could, in part, be due to increased IL-1 and/or TNF production. Hypoxia can induce anorexia and cachexia,20’26which are also induced by IL-l and TNF.‘,*,” In addition, exposure to hypoxia potentiates the deleterious effects of complement activation in animal models for the adult respiratory 189
190 I Ghezzi et al.
distress syndrome and multiple organ failure.28zz9When present initially, hypoxemia is associated with a poor outcome in these disorders in humans.30 The purpose of this study was to investigate the effect of hypoxia on the synthesis and secretion of IL-l and TNF in vitro by human peripheral blood mononuclear cells (MNC).
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5 E
RESULTS Exposure of human peripheral blood MNC to hypoxia increased endotoxin-induced production and secretion of both IL-1 (percent of control = 190% for IL-la, p = 0.014; 219% for IL-lp, p = 0.014; n = 6) and TNF (percent of control = 243%, p = 0.037, n = 6; Fig. 1, A, B, C). Hypoxia also potentiated IL-l production by subthreshold levels of LPS. Although little detectable IL-l was generated by lower doses of LPS under normoxic conditions, severe hypoxia consistently increased IL-1 generation at levels of LPS as low as 0.1 rig/ml (Fig. 2). Dexamethasone, although fully inhibiting under normoxic conditions, only partially inhibited hypoxia-potentiated, LPS-induced IL-l production (data not shown). Detectable levels of TNF were not present under normoxic or severely hypoxic conditions at LPS doses less than 1.0 @ml. At that dosage of LPS, TNF production showed a similar pattern of inhibition by dexamethasone under normoxic or hypoxic conditions (data not shown). We then investigated the effect of varying concentrations of 0, (0, 3, 10, and 21%) on the LPSstimulated IL-1 and TNF production by mononuclear cells. In four of five donors, IL-1 production was already increased at 10% 0, compared to 21% 0, (Fig. 3A). We observed a similar effect of graded hypoxia on TNF production although potentiation of cytokine production was maximal only when MNC were exposed in chambers flushed with 0 to 3% oxygen (Fig. 3B). Since it was not clear if hypoxia specifically augmented the response to LPS or enhanced IL-l and TNF production irrespective of the stimulus, we studied the effect of hypoxia (0% 0,) on the production of IL-l and TNF induced by PMA. We found that PMA-induced IL-l and TNF (data not shown) production was also increased by hypoxia in most donors. Cell damage did not cause the effect of hypoxia; 24 h of severe hypoxia (0% 0,) was not toxic to MNC as measured by LDH release (8.4 & 0.6 in 21% 0, and 8.4 + 0.7% in 0% O,, p > 0.05, n = 4) and vital dye exclusion using erythrosin B (97.5 + 0.6% in 21% 0, and 97.0 + 0.6% in 0% O,, p > 0.05, n = 9). In addition, values for LDH release or vital dye exclusion were not different in the presence of LPS under normoxic or hypoxic conditions. This suggests that cell injury due to anoxia or anoxia-reoxygenation is not potentiating cytokine production. In fact, exposure of MNC to
C 6 ..
NORMOXIA
HYPOXIA
Figure 1. Effect of normoxia or hypoxia (chamber flushed with 95% N,, 5% CO, for 30 min) on production of the cytokines &la (A), IL-Q3 (B), or TNF (C) by peripheral blood mononuclear cells (MNC) exposed to LPS (1.0 @ml; from Escheriehiu coli 012tkB12, Sigma) over 24 h. The solid portion of each bar represents the arithmetic mean of the cell-associated cytokine fraction whereas the open portion represents that of the secreted fraction. SEM and statistical differences refer to total cytokine produced; *p < 0.05; **p < 0.02, Wilcoxon’s signed rank test for paired data. The lower limit of sensitivity for the RIA’s was 80 pg/ml (indicated by the dashed line).
severe hypoxia for 4 to 6 h followed by 18 to 20 h of normoxia (21% 0,) or hyperoxia (95% 0,) was less effective than 24 h of hypoxia (0% 0,) in potentiating cytokine synthesis (data not shown). Furthermore, acidosis did not appear to be a contributing factor as the pH of the medium did not change over 24 h of incubation. Finally, we questioned whether the increased production of IL-l during MNC exposure to hypoxia was due to an increased IL-l gene expression. Accordingly, we measured levels of IL-lp mRNA in MNC from
Hypoxia increases IL-l and TNF production
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4
Figure 2. Effect of normoxia chamber flushed 30 min with IL-lp production by MNC when rig/ml of LPS (left) or 100 @ml
(N) or hypoxia (H, 95% N,, 5% CO,) on exposed to 0.1 or 1.0 of PMA (right).
ILIp (w/ml)
In the absence of LPS, none of these donors produced detectable IL-l under normal or hypoxic conditions. Mean and SEM for total IL-1 produced are represented. *p < 0.05; **p < 0.01, Wilcoxon’s signed rank test for paired data. Data from four different donors are represented under each condition.
three donors exposed for 4 h to normoxia or hypoxia in the presence and absence of LPS (1 @ml). The Northern blot analysis of MNC RNA is shown in Fig. 4. LPS, as expected, increased the expression of IL-1 mRNA. However, in the presence of LPS, significant differences in IL-1 mRNA were not observed after 4 h under normoxic and hypoxic conditions. The finding that hypoxia did not increase cytokine mRNA suggests a posttranscriptional effect of oxygen tension on IL-l synthesis.
DISCUSSION We found that hypoxia increased production of three inflammatory cytokines, IL-lo., IL-lp, and TNF. In addition, hypoxia decreased the endotoxin dose threshold for detectable IL-1 production. Both IL-1
2
0
N
H
N
LPS 0.1 rig/ml
H
N
LPS 1.0 rig/ml
H
PMA 100 rig/ml
and TNF produced by endotoxin-stimulated mononuclear cells were inversely related to oxygen tension. Although IL-1 was optimally potentiated by 3% 0, and TNF by 0% O,, respectively, even cultures exposed to 0% 0, were not truly anoxic. This may have been due to residual dissolved oxygen present in the medium and/or to oxygen permeability of the plastic chamber. Based on Northern blot analysis, the promoting effect of hypoxia on endotoxin-induced cytokine production does not appear to occur at the mRNA level. Quantities of mRNA for IL-1 were not different from MNC exposed to endotoxin and either hypoxia or normoxia for 4 h, the time point at which maximal endotoxininduced IL-@ mRNA production occurs. The potentiation of IL-1 and TNF production reported here indicates a novel mechanism of regulation of cytokine production by hypoxia. This mechanism could be important in many pathologic condi-
DONOR A
nnm 02tension: Endotoxin:
N -
N +
H +
DONOR g N +
H +
DONOR C N +
H +
TNFa (w/ml)
4 Figure
Figure 3. Effect of increasing levels of oxygen (chambers Rushed with gas mixtures containing 21%, lo%, 3%, or 0% 0,, respectively) on (A) IL-1s or (B) TNF production by MNC exposed to LPS (1.0 &ml).
Results from six (IL-lp) represented.
or three different donors, respectively, are
4.
RNA
blot analysis
of IL-1s
mRNA.
Peripheral blood mononuclear cells from three donors were studied under normoxic (N) conditions in either the absence (-) or presence (+) of LPS (1 q/ml), or under hypoxic (H) conditions in the presence (+) of the latter. Normoxic (N) chambers were flushed with 21% 0,, 5% CO,, 74% N, and hypoxic (H) chambers with 0% 0,, 5% CO,, 95% N, for 30 min prior to incubation. In cells incubated for 4 h, the top line represents IL-1S mRNA and the middle line represents p actin mRNA. The bottom line shows total IL-lp protein in MNC after 24 h. The first lane shows RNA from a representative donor exposed to normoxia in the absence of LPS. No donor had detectable mRNA for IL-lp in the absence of LPS under normoxic or hypoxic conditions.
192
CYTOKINE,
/ Ghezzietal.
tions. For instance, hypoxia may occur in the center of a wound or in tumors promoting angiogenesis,31 fibrosis, or other processes through cytokine actions. Similarly hypoxia could potentiate the pulmonary fibrosis resulting from a variety of interstitial lung diseases32V33 or the pulmonary vascular remodeling due to chronic exposure to hypoxia34 through cytokine actions. Cytokine production potentiated by hypoxia might also contribute to high-altitude pulmonary edema In addition, this mechanism and/or cerebral edema. 35,36 could contribute to the worsening effect of hypoxemia on the adult respiratory distress syndrome (ARDS) and multiple organ failure,28’29particularly when considering the contribution of sepsis to the development of these disorders.30 Individual variation in cytokine production and its potentiation by hypoxia could lead to varying susceptibilities to, and severities of, these diseases. Finally, these findings suggest a mechanism by which shock, with resulting ischemia of certain vascular beds and regional hypoxemia, could potentiate itself, as well as associated inflammatory conditions, such as ARDS and multiple organ failure.
MATERIALS AND METHODS Mononuclear Cell Cultures Following informed consent, blood was obtained by venepuncture from healthy volunteers who had not used cyclooxygenase inhibitors during the previous two weeks. MNC were prepared using Ficoll-Hypaque gradients.8,37Cells were cultured in RPMI-1640 medium that had been ultrafiltered to remove LPS’* and supplemented with 1% heatinactivated autologous human serum. MNC were cultured at 2.5 x 106/ml in 96-well, flat-bottomed tissue culture plates. Cells were incubated in chambers flushed for 30 min with 5% CO, and various oxygen concentrations. Following exposure for 4 or 24 h to LPS or PMA, MNC cultures (cells with or without supernatants) were subjected to three freeze-thaw cycles to allow complete cell lysis, and the lysates were assayedfor IL-lo, IL-l& or TNF in specific radioimmunoassays (RIA, see below). In some experiments, supernatants were harvested and residual cells removed by centrifugation to allow determination of the amount of IL-1 and TNF in cells (cell-associated) and in supernatants (secreted). Oxygen concentrations measured upon termination of incubation in tissue culture medium with an 0, analyzer (Chemical Microsensor II, Diamond General Inc., Ann Arbor, MI) indicated that the 0, concentration present in normoxic (21% O,, 5% CO,, 74% NJ incubations was 101 + 4 torr (n = 5) compared to 9 + 1 torr (n = 5) in hypoxic (0% O,, 5% CO,, 95% NJ incubations. Using this instrument it was demonstrated that PO, was similarly low in tubes in which MNC were cultured for mRNA measurements and in wells of 96-well plates during hypoxic incubations for 4 or 24 h.
Radioimmunoassays RIAs were used to measure IL-la? IL-l@,4’ and TNF(Ye’and have been shown to detect lessthan 100 pg/ml of each cytokine in human MNC supernatants. Previous studies have
Vol. 3, No. 3 (May
1991: 189-194)
demonstrated no cross-reactivity among these cytokine RIAs, nor do they detect human TNF-B, IFN-a, IFN-7, or granulocyte-macrophage colony-stimulating factor. In addition, each RIA correlated with functional bioassays(cytotoxicity, T cell proliferation) with correlation coefficients > 0.8 (p < 0.05 by paired Student’s t test).
LDH Release At termination of MNC incubations, media were aspirated and centrifuged (Microfuge 12, Beckman) for 2 min (10,500g). Cells were removed into 50 mM potassium phosphate, pH 7.4, containing 0.1% Triton X-100 and sonicated (Kontes Micro-Ultrasonic Cell Disrupter, maximum power, 2 x 10 set, 4°C). LDH activity was determined in paired supernatants and sonicates using a spectrophotometric method to measure NADH-dependent conversion of pyruvate to lactate.42 LDH release was then calculated from the formula: LDH release (percent total) = [LDH in supematant/ (LDH in supernatant + LDH in sonicate)] x 100.
mRNA Measurement MNC were lysed in guanidine isothiocyanate and RNA was prepared by centrifugation through cesium chloride.43 The concentration of RNA was determined from the absorbance at 260 nm, and A2,JAZ8,,ratios were greater than 2. RNA (2.5 kg) was analyzed by electrophoresis through a 1% agarose formaldehyde gel followed by Northern blot transfer to Gene Screen Plus membranes (New England Nuclear, Boston, MA). An IL-1B cDNA clone@ was labeled to high specific activity with [cx-“P]dCTP (3.00 Ci/mmole; Amersham) by the random priming method.45 Membranes were pretreated and hybridized in 50% formamide (Merck) with 10% dextran sulfate (Sigma) and washed twice with 2 X SSC (1 x SSC is 0.15M NaCl with 0.015M Na citrate) and 1% sodium dodecyl sulfate (SDS, Sigma) at 60°C for 30 min, and then twice more with 0.1 x SSC at room temperature for 30 min. The membranes were exposed for 4 to 8 h at -80°C using intensifying screens.
Acknowledgments The authors acknowledge the superb technical assistance of Reza Ghorbani and Gary Zerbe, Ph.D., for expert statistical consultation. We thank Jacqueline Smith and Dawnett Marples for preparing the manuscript. This work was done during Dr. White’s tenure of a Clinician-Scientist Award from the American Heart Association and with funds contributed in part by the Colorado Heart Association. Research support was also provided by National Institutes of Health Grant Al-15614, The Children’s Hospital of Denver and C. Henry Kempe Research Center, and by a Grant-in-Aid from the American Heart Association National Center.
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