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mRNA and Protein Stability Regulate the Differential Expression of Pro- and Anti-inflammatory Genes in Endotoxin-tolerant THP-1 Cells

2000; Elsevier BV; Volume: 275; Issue: 16 Linguagem: Inglês

10.1074/jbc.275.16.12185

1083-351X

Chris A. Learn, Steven B. Mizel, Charles E. McCall,

S100 Proteins and Annexins

The products of proinflammatory genes such as interleukin-1β (IL-1β) and cyclooxygenase-2 (COX-2) initiate many of the events associated with sepsis. Transcription of these genes is subsequently down-regulated, whereas expression of anti-inflammatory genes such as secretory interleukin-1 receptor antagonist (sIL-1 RA) is maintained. Differential expression is associated with endotoxin tolerance, a cellular phenomenon common to sepsis and characterized by reduced proinflammatory gene expression after repeated exposure to lipopolysaccharide. As a model for endotoxin tolerance, we examined the expression of COX-2 and sIL-1 RA in a human promonocyte cell line, THP-1. We observed a 5-fold decrease in COX-2 protein in endotoxin-tolerant cells relative to control cells. In contrast, sIL-1 RA protein increased 5-fold in control and tolerant cells and remained elevated. Decreased COX-2 production is due to repressed transcription and not enhanced mRNA degradation. In addition, COX-2 protein is turned over rapidly. Transcription of sIL-1 RA is also repressed during tolerance. However, sIL-1 RA mRNA is degraded more slowly than COX-2 mRNA, allowing continued synthesis of sIL-1 RA protein that is very stable. These results indicate that differential expression during endotoxin tolerance occurs by transcriptional repression of COX-2 and by protein and mRNA stabilization of sIL-1 RA. The products of proinflammatory genes such as interleukin-1β (IL-1β) and cyclooxygenase-2 (COX-2) initiate many of the events associated with sepsis. Transcription of these genes is subsequently down-regulated, whereas expression of anti-inflammatory genes such as secretory interleukin-1 receptor antagonist (sIL-1 RA) is maintained. Differential expression is associated with endotoxin tolerance, a cellular phenomenon common to sepsis and characterized by reduced proinflammatory gene expression after repeated exposure to lipopolysaccharide. As a model for endotoxin tolerance, we examined the expression of COX-2 and sIL-1 RA in a human promonocyte cell line, THP-1. We observed a 5-fold decrease in COX-2 protein in endotoxin-tolerant cells relative to control cells. In contrast, sIL-1 RA protein increased 5-fold in control and tolerant cells and remained elevated. Decreased COX-2 production is due to repressed transcription and not enhanced mRNA degradation. In addition, COX-2 protein is turned over rapidly. Transcription of sIL-1 RA is also repressed during tolerance. However, sIL-1 RA mRNA is degraded more slowly than COX-2 mRNA, allowing continued synthesis of sIL-1 RA protein that is very stable. These results indicate that differential expression during endotoxin tolerance occurs by transcriptional repression of COX-2 and by protein and mRNA stabilization of sIL-1 RA. lipopolysaccharide secretory interleukin-1 receptor antagonist cyclooxygenase-2 interleukin-1 type II receptor glyceraldehyde-3-phosphate dehydrogenase chloramphenicol acetyltransferase interleukin tumor necrosis factor-α enzyme-linked immunosorbent assay polyacrylamide gel electrophoresis Tris-buffered saline Severe sepsis with septic shock is the major cause of death in critical care units in the United States, killing over 100,000 people each year (1.McCall C. Grosso-Wilmoth L. LaRue K. Guzman R. Cousart S. J. Clin. Invest. 1993; 91: 853-861Crossref PubMed Scopus (170) Google Scholar). It is a disease characterized by acute disseminated intravascular inflammation with multiple organ failure and hypotension due to infection by various bacteria, viruses, and fungi. Gram-negative bacterial infections are the most common causative agent of septic shock (2.Bone R.C. Moellering Jr., R.C. Infectious Disease Clinics of America. 5. W. B. Saunders, Philadelphia1991: 739-753Google Scholar). Bacterial endotoxin lipopolysaccharide (LPS),1 a component of Gram-negative bacterial cell walls, is a potent inducer of monocyte, macrophage and polymorphonuclear leukocyte inflammatory gene expression during infection and a common inducer of septic shock (3.LaRue K. McCall C. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (99) Google Scholar).COX-2 is a 70-kDa dimeric inducible enzyme that has a vital role in modulating inflammation through production of prostaglandin H2, a precursor of several potent eicosanoid mediators (4.Inoue H. Yokoyama C. Hara S. Tone Y. Tanabe T. J. Biol. Chem. 1995; 270: 24965-24971Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar,5.Durando M. Fernando L. Ashton S. Halushka P. Cook J. Shock. 1998; 9: 359-363Crossref PubMed Scopus (5) Google Scholar). COX-2 expression is clearly associated with proinflammatory activity in inflammatory diseases and the COX-2 gene responds like other proinflammatory genes during sepsis (6.Shoup M. He L.K. Liu H. Shankar R. Gamelli R. J. Trauma. 1998; 45: 215-220Crossref PubMed Scopus (57) Google Scholar, 7.Ogura S. Umegaki O. Genovese F. Fernando L.P. Cochran J.B. Romeo C.M. Cook J.A. Shock. 1999; 12: 105-110Crossref PubMed Scopus (16) Google Scholar). The induction of proinflammatory genes such as IL-1β, tumor necrosis factor-α (TNF-α), and COX-2 by bacterial products such as LPS is essential and necessary to initiate the septic response (2.Bone R.C. Moellering Jr., R.C. Infectious Disease Clinics of America. 5. W. B. Saunders, Philadelphia1991: 739-753Google Scholar). However, these genes are rapidly and continuously down-regulated soon after the sepsis syndrome is initiated (2.Bone R.C. Moellering Jr., R.C. Infectious Disease Clinics of America. 5. W. B. Saunders, Philadelphia1991: 739-753Google Scholar, 8.Zuckerman S. Evans G. Butler L. Infect. Immun. 1991; 59: 2774-2780Crossref PubMed Google Scholar, 9.Ziegler-Heitbrock H.W.L. Blumenstein M. Kafferlein E. Keiper D. Petersmann L. Endres S. Flegel W.A. Northoff H. Riethmuller G. Haas J.G. Immunology. 1992; 75: 264-268PubMed Google Scholar). In contrast to COX-2, regulatory factors, such as secretory interleukin-1 receptor antagonist (sIL-1 RA) (10.Arend W.P. Smith M.F. Janson R. Joslin F. J. Immunol. 1991; 147: 1530-1536PubMed Google Scholar), serve to neutralize the activity and overproduction of inflammatory defenses (11.Smith M. Eidlen D. Arend W. Gutierrez-Hartmann A. J. Immunol. 1994; 153: 3584-3593PubMed Google Scholar, 12.Porreca E. Reale R. Febbo C.D. Gioacchino M.D. Barbacane R. Castellani M. Baccante G. Conti P. Cuccurullo F. Immunology. 1996; 89: 424-429Crossref PubMed Scopus (24) Google Scholar). Anti-inflammatory genes such as interleukin-1 type II receptor (IL-1 R2), IL-10, and sIL-1 RA are also induced early in sepsis. However, the products encoded by anti-inflammatory genes are persistently elevated during the course of sepsis (13.Rogy M.A. J. Am. Coll. Surg. 1994; 178: 132-138PubMed Google Scholar).In 1947, Beeson first documented that cells exposed to LPS become refractory to further challenge with this stimulus, a process termed “endotoxin tolerance” (14.Beeson P. J. Exp. Med. 1947; 86: 29-45Crossref PubMed Scopus (201) Google Scholar). This adaptive response to LPS is a cellular phenomenon that is associated with reduced levels of inflammatory mediators after a second exposure to LPS when compared with the levels induced by an initial exposure. Adaptation to stimulation by LPS is thought to have evolved as a mechanism to down-regulate the continuous and often injurious inflammatory response the immune system sustains during severe sepsis, presumably to reduce the potentially lethal autotoxic effects brought about by overproduction of inflammatory mediators such as IL-1β, TNF-α, and COX-2 (1.McCall C. Grosso-Wilmoth L. LaRue K. Guzman R. Cousart S. J. Clin. Invest. 1993; 91: 853-861Crossref PubMed Scopus (170) Google Scholar). This differential expression of pro- and anti-inflammatory genes leads to a state of immunosuppression. Although adaptation to LPS is protective against the potentially autotoxic effects of LPS when it exists prior to initiation, its presence during sepsis can compromise innate immunity (15.Olszyna D.P. Prins J.M. Buis B. Van Deventer S.J.H. Speelman P. Van Der Poll T. Infect. Immun. 1998; 66 (2534): 3527Crossref PubMed Google Scholar). sIL-1 RA protein is expressed at persistently elevated levels in patients suffering from sepsis, while COX-2, IL-1β, and TNF-α protein levels are decreased, thus prolonging an immunosuppressed state (13.Rogy M.A. J. Am. Coll. Surg. 1994; 178: 132-138PubMed Google Scholar, 16.DeBont E. DeLeij L. Okken A. Baarsma R. Kimpen J. Pediatr. Res. 1995; 37: 626-629Crossref PubMed Scopus (33) Google Scholar, 17.Fisher C.J. Circ. Shock. 1994; 44: 1-8PubMed Google Scholar). Currently, it is unclear how the differential regulation of pro- and anti-inflammatory genes occurs.THP-1 cells respond to repeated exposure to LPS in a fashion similar to leukocytes obtained from patients with sepsis, exhibiting repressed IL-1β and TNF-α expression (3.LaRue K. McCall C. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (99) Google Scholar). Suppression of these genes is under the control of a labile protein(s) (3.LaRue K. McCall C. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (99) Google Scholar). In the present study, THP-1 cells were used to determine the molecular mechanisms responsible for the differential expression of COX-2 and sIL-1 RA in endotoxin tolerance.DISCUSSIONIn endotoxin-tolerant THP-1 cells, the level of COX-2 protein is decreased, whereas the level of sIL-1 RA protein is not. This differential expression results from repressed transcription of both COX-2 and sIL-1 RA genes combined with stabilization of sIL-1 RA protein and mRNA. The mechanism(s) that decrease transcription and stabilize protein and mRNA in tolerant cells are not yet known, but may involve a labile repressor(s) of transcription as well as protein and mRNA stabilizing/destabilizing elements (3.LaRue K. McCall C. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (99) Google Scholar, 24.Ziegler-Heitbrock H. Wedel A. Schraut W. Strobel M. Wendelgass P. Sternsdorf T. Bauerle P. Haas J. Riethmuller G. J. Biol. Chem. 1994; 269: 17001-17004Abstract Full Text PDF PubMed Google Scholar). The resulting steady state mRNA levels are amplified at the level of protein with dramatic differences in COX-2 and sIL-1 RA expression in endotoxin-tolerant THP-1 cells.Given the observation that COX-2 and sIL-1 RA were differentially expressed at the protein level, we examined their transcriptional regulation in THP-1 cells. COX-2 promoter fragments exhibit 3–5.5-fold decreases in reporter gene activity in transient transfection assays (Fig. 4 A). Similarly, transcription of the endogenous COX-2 gene exhibits an equivalent decrease in tolerant cells upon stimulation with LPS in comparison to control cells (data not shown). Based on these data and mRNA levels (Fig. 3 B), we find that COX-2 transcription is induced early and then quickly repressed. In addition, PGE2 lipid levels are decreased approximately 3-fold in tolerant cells when compared to control cells stimulated with LPS, possibly due to lower levels of COX-2 protein or available substrate present in tolerant cells. Similar to COX-2, sIL-1 RA transcription is also markedly reduced in tolerant cells (Fig. 4 B). This transcriptional repression may involve negative regulatory element(s) similar to those required for silencer activity in interferon-A gene promoters (25.Lopez S. Reeves R. Island M.-L. Bandu M.-T. Christeff N. Doly J. Navarro S. J. Biol. Chem. 1997; 272: 22788-22799Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). These findings are consistent with repressed transcription of the two genes, possibly by the same type of labile repressor that is thought to act on IL-1β (3.LaRue K. McCall C. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (99) Google Scholar). The large increase in sIL-1 RA protein in the presence of repressed transcriptional activity and mRNA levels (Fig. 3 A) indicates that other regulatory mechanisms must contribute to the elevated protein levels in endotoxin-tolerant THP-1 cells. We propose two potential mechanisms. First, differences in mRNA stability may account for the increase in sIL-1 RA protein levels in tolerant cells. Second, enhanced translational efficiency of declining pools of mRNA may potentiate sIL-1 RA expression in tolerance. We believe that it most likely is a combination of these two potential mechanisms. In support of this notion, Cassatella (26.Cassatella M. Meda L. Gasperini S. Calzetti F. Bonora S. J. Exp. Med. 1994; 179: 1695-1699Crossref PubMed Scopus (269) Google Scholar) has shown that anti-inflammatory IL-10 up-regulates IL1-RA production in LPS-stimulated human polymorphonuclear leukocytes by delaying mRNA degradation. We have also shown that delaying mRNA degradation in THP-1 cells tolerant to IL-1β overcomes repression of IL-1β synthesis (27.Yoza B.K. Wells J.D. McCall C.E. Clin. Diag. Lab. Immunol. 1998; 5: 281-287Crossref PubMed Google Scholar). These studies and others (28.Colotta F. Saccani S. Giri J. Dower M. Sims J. Introna M. Mantovani A. J. Immunol. 1996; 156: 2534-2541PubMed Google Scholar) suggest that increased anti-inflammatory expression may result from positive feedback mechanisms that are in place to help counterbalance proinflammatory expression. In addition, Beutler (29.Beutler B. Brown T. J. Clin. Invest. 1991; 87: 1336-1344Crossref PubMed Scopus (46) Google Scholar) has shown that cis-acting elements from the 3′-untranslated region of TNF-α are required for efficient translational activation of the gene. A similar form of translational regulation by cis-acting elements within the IL-1 RA 3′-untranslated region is therefore plausible.Post-transcriptional mechanisms of gene regulation have important roles in the expression of pro- and anti-inflammatory genes (26.Cassatella M. Meda L. Gasperini S. Calzetti F. Bonora S. J. Exp. Med. 1994; 179: 1695-1699Crossref PubMed Scopus (269) Google Scholar, 29.Beutler B. Brown T. J. Clin. Invest. 1991; 87: 1336-1344Crossref PubMed Scopus (46) Google Scholar). It was unknown whether similar mechanisms regulated COX-2 and sIL-1 RA expression. We investigated this issue by mRNA decay analysis. Our findings demonstrated that the turnover rate for the biphasic decay of COX-2 mRNA (Fig. 6 A) in tolerant cells (t 1/2 = 6 h) is 6 times as long as in control cells (t 1/2 = 1 h). In addition, we believe that even at lower levels in tolerant cells, COX-2 mRNA is efficiently translated (Fig. 1 A). The biphasic decay of COX-2 in control cells may be important in that elevated levels of proinflammatory mRNAs are rapidly turned over, whereas lower levels are subject to different turnover mechanisms and kinetics. Similarly, mRNA half-life analysis (Fig. 6 B) demonstrated that the turnover rate for sIL-1 RA in tolerant cells (t 1/2 = 12 h) is doubled in comparison to control cells (t 1/2 = 6 h), indicating that sIL-1 RA mRNA is relatively stable in tolerant cells. This mRNA stability may permit continued translation of sIL-1 RA. Additionally, mRNA stabilization may serve as a general mechanism of regulating expression for certain pro- and anti-inflammatory genes, potentially as a negative feedback loop (i.e. COX-2) or as means for continued expression (i.e. sIL-1 RA). Brooks et al. have shown that IL-1-induced JunB mRNA levels are not directly correlated with the level of JunB protein synthesis (30.Brooks J.W. Yoza B.K. Mizel S.B. Mol. Immunol. 1995; 32: 779-788Crossref PubMed Scopus (4) Google Scholar). Rather, JunB protein levels remain elevated as a result of enhanced translational efficiency. Our findings with sIL-1 RA in endotoxin-tolerant THP-1 cells are consistent with this mechanism. In addition, Cassatella and others have shown that the sIL-1 RA 3′-untranslated region does not contain AU-rich destabilizing sequences that are characteristic of rapidly turned over proinflammatory mRNA, such as COX-2 and IL-1 (26.Cassatella M. Meda L. Gasperini S. Calzetti F. Bonora S. J. Exp. Med. 1994; 179: 1695-1699Crossref PubMed Scopus (269) Google Scholar, 31.Kastelic T. Schnyder J. Leutwiler A. Traber R. Streit B. Niggli H. Mackenzie A. Cheneval D. Cytokine. 1996; 8: 751-761Crossref PubMed Scopus (48) Google Scholar).With the data supporting the notion that mRNA stability may in part explain differential expression of COX-2 and sIL-1 RA, we investigated protein stability by half-life analysis. Protein levels for COX-2 in both control and tolerant cells reached 50% of their maximal level within 2 h of cycloheximide addition (Fig. 5 A). This relatively short turnover time for COX-2 is consistent with what is known for the rapid decay of proinflammatory proteins in vitro (3.LaRue K. McCall C. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (99) Google Scholar) and their relative absence clinically in sepsis (17.Fisher C.J. Circ. Shock. 1994; 44: 1-8PubMed Google Scholar). In marked contrast, sIL-1 RA protein is very stable (Fig. 5 B). In both control and tolerant cells, sIL-1 RA protein is stable over 24 h. Interestingly, this stability is still maintained at 96 h (data not shown). These findings are supportive of clinical studies in sepsis in which anti-inflammatory protein levels remain elevated for several days (13.Rogy M.A. J. Am. Coll. Surg. 1994; 178: 132-138PubMed Google Scholar). Although elevated levels of anti-inflammatory proteins, such as sIL-1 RA, often help to decrease the potentially lethal inflammatory response in sepsis, the stability of these proteins may in part promote the severe immunosuppression commonly seen in sepsis patients.Taken collectively, these data are consistent with the notion that there is no significant difference in protein stability between control and endotoxin-tolerant cells for COX-2 or sIL-1 RA. However, the corresponding mRNAs are more stable in tolerant cells than in control cells, delaying degradation. In addition, due to undefined differences between pro- and anti-inflammatory genes, sIL-1 RA mRNA and protein are significantly more stable than those of COX-2, resulting in a pronounced difference at the level of protein in endotoxin tolerance.In summary, the results of this study demonstrate differential regulation and expression of COX-2 and sIL-1 RA in endotoxin-tolerant THP-1 cells. COX-2 protein levels are decreased, yet sIL-1 RA protein levels remain elevated. Differential expression is consistent with repressed transcription and protein turnover for COX-2, whereas sIL-1 RA mRNA and protein are stabilized. We believe that increased stability of sIL-1 RA mRNA may be coupled with enhanced translational efficiency of sIL-1 RA protein in tolerant cells. These data demonstrate that COX-2 and sIL-1 RA proteins are differentially expressed in endotoxin tolerant THP-1 cells, similar to differential expression observed during sepsis (13.Rogy M.A. J. Am. Coll. Surg. 1994; 178: 132-138PubMed Google Scholar, 17.Fisher C.J. Circ. Shock. 1994; 44: 1-8PubMed Google Scholar). In addition, the time courses for the THP-1 expression of COX-2 (rapid and transient) and sIL-1 RA (slow and constitutive) proteins are similar to the pattern of expression of pro- and anti-inflammatory genes observed during clinical sepsis (23.van der Poll T. Coyle S.M. Moldawer L.L. Lowry S.F. J. Infect. Dis. 1996; 174: 1356-1360Crossref PubMed Scopus (71) Google Scholar). These results indicate that the THP-1 cell line is as a useful system to dissect endotoxin responsiveness and the regulation of pro- and anti-inflammatory genes. Our results not only establish mechanisms for the differential regulation of pro- and anti-inflammatory genes and their protein products, but also serve as a foundation for future studies on the factors that are responsible for repressing and destabilizing COX-2 mRNA and protein and that promote sIL-1 RA expression. Severe sepsis with septic shock is the major cause of death in critical care units in the United States, killing over 100,000 people each year (1.McCall C. Grosso-Wilmoth L. LaRue K. Guzman R. Cousart S. J. Clin. Invest. 1993; 91: 853-861Crossref PubMed Scopus (170) Google Scholar). It is a disease characterized by acute disseminated intravascular inflammation with multiple organ failure and hypotension due to infection by various bacteria, viruses, and fungi. Gram-negative bacterial infections are the most common causative agent of septic shock (2.Bone R.C. Moellering Jr., R.C. Infectious Disease Clinics of America. 5. W. B. Saunders, Philadelphia1991: 739-753Google Scholar). Bacterial endotoxin lipopolysaccharide (LPS),1 a component of Gram-negative bacterial cell walls, is a potent inducer of monocyte, macrophage and polymorphonuclear leukocyte inflammatory gene expression during infection and a common inducer of septic shock (3.LaRue K. McCall C. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (99) Google Scholar). COX-2 is a 70-kDa dimeric inducible enzyme that has a vital role in modulating inflammation through production of prostaglandin H2, a precursor of several potent eicosanoid mediators (4.Inoue H. Yokoyama C. Hara S. Tone Y. Tanabe T. J. Biol. Chem. 1995; 270: 24965-24971Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar,5.Durando M. Fernando L. Ashton S. Halushka P. Cook J. Shock. 1998; 9: 359-363Crossref PubMed Scopus (5) Google Scholar). COX-2 expression is clearly associated with proinflammatory activity in inflammatory diseases and the COX-2 gene responds like other proinflammatory genes during sepsis (6.Shoup M. He L.K. Liu H. Shankar R. Gamelli R. J. Trauma. 1998; 45: 215-220Crossref PubMed Scopus (57) Google Scholar, 7.Ogura S. Umegaki O. Genovese F. Fernando L.P. Cochran J.B. Romeo C.M. Cook J.A. Shock. 1999; 12: 105-110Crossref PubMed Scopus (16) Google Scholar). The induction of proinflammatory genes such as IL-1β, tumor necrosis factor-α (TNF-α), and COX-2 by bacterial products such as LPS is essential and necessary to initiate the septic response (2.Bone R.C. Moellering Jr., R.C. Infectious Disease Clinics of America. 5. W. B. Saunders, Philadelphia1991: 739-753Google Scholar). However, these genes are rapidly and continuously down-regulated soon after the sepsis syndrome is initiated (2.Bone R.C. Moellering Jr., R.C. Infectious Disease Clinics of America. 5. W. B. Saunders, Philadelphia1991: 739-753Google Scholar, 8.Zuckerman S. Evans G. Butler L. Infect. Immun. 1991; 59: 2774-2780Crossref PubMed Google Scholar, 9.Ziegler-Heitbrock H.W.L. Blumenstein M. Kafferlein E. Keiper D. Petersmann L. Endres S. Flegel W.A. Northoff H. Riethmuller G. Haas J.G. Immunology. 1992; 75: 264-268PubMed Google Scholar). In contrast to COX-2, regulatory factors, such as secretory interleukin-1 receptor antagonist (sIL-1 RA) (10.Arend W.P. Smith M.F. Janson R. Joslin F. J. Immunol. 1991; 147: 1530-1536PubMed Google Scholar), serve to neutralize the activity and overproduction of inflammatory defenses (11.Smith M. Eidlen D. Arend W. Gutierrez-Hartmann A. J. Immunol. 1994; 153: 3584-3593PubMed Google Scholar, 12.Porreca E. Reale R. Febbo C.D. Gioacchino M.D. Barbacane R. Castellani M. Baccante G. Conti P. Cuccurullo F. Immunology. 1996; 89: 424-429Crossref PubMed Scopus (24) Google Scholar). Anti-inflammatory genes such as interleukin-1 type II receptor (IL-1 R2), IL-10, and sIL-1 RA are also induced early in sepsis. However, the products encoded by anti-inflammatory genes are persistently elevated during the course of sepsis (13.Rogy M.A. J. Am. Coll. Surg. 1994; 178: 132-138PubMed Google Scholar). In 1947, Beeson first documented that cells exposed to LPS become refractory to further challenge with this stimulus, a process termed “endotoxin tolerance” (14.Beeson P. J. Exp. Med. 1947; 86: 29-45Crossref PubMed Scopus (201) Google Scholar). This adaptive response to LPS is a cellular phenomenon that is associated with reduced levels of inflammatory mediators after a second exposure to LPS when compared with the levels induced by an initial exposure. Adaptation to stimulation by LPS is thought to have evolved as a mechanism to down-regulate the continuous and often injurious inflammatory response the immune system sustains during severe sepsis, presumably to reduce the potentially lethal autotoxic effects brought about by overproduction of inflammatory mediators such as IL-1β, TNF-α, and COX-2 (1.McCall C. Grosso-Wilmoth L. LaRue K. Guzman R. Cousart S. J. Clin. Invest. 1993; 91: 853-861Crossref PubMed Scopus (170) Google Scholar). This differential expression of pro- and anti-inflammatory genes leads to a state of immunosuppression. Although adaptation to LPS is protective against the potentially autotoxic effects of LPS when it exists prior to initiation, its presence during sepsis can compromise innate immunity (15.Olszyna D.P. Prins J.M. Buis B. Van Deventer S.J.H. Speelman P. Van Der Poll T. Infect. Immun. 1998; 66 (2534): 3527Crossref PubMed Google Scholar). sIL-1 RA protein is expressed at persistently elevated levels in patients suffering from sepsis, while COX-2, IL-1β, and TNF-α protein levels are decreased, thus prolonging an immunosuppressed state (13.Rogy M.A. J. Am. Coll. Surg. 1994; 178: 132-138PubMed Google Scholar, 16.DeBont E. DeLeij L. Okken A. Baarsma R. Kimpen J. Pediatr. Res. 1995; 37: 626-629Crossref PubMed Scopus (33) Google Scholar, 17.Fisher C.J. Circ. Shock. 1994; 44: 1-8PubMed Google Scholar). Currently, it is unclear how the differential regulation of pro- and anti-inflammatory genes occurs. THP-1 cells respond to repeated exposure to LPS in a fashion similar to leukocytes obtained from patients with sepsis, exhibiting repressed IL-1β and TNF-α expression (3.LaRue K. McCall C. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (99) Google Scholar). Suppression of these genes is under the control of a labile protein(s) (3.LaRue K. McCall C. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (99) Google Scholar). In the present study, THP-1 cells were used to determine the molecular mechanisms responsible for the differential expression of COX-2 and sIL-1 RA in endotoxin tolerance. DISCUSSIONIn endotoxin-tolerant THP-1 cells, the level of COX-2 protein is decreased, whereas the level of sIL-1 RA protein is not. This differential expression results from repressed transcription of both COX-2 and sIL-1 RA genes combined with stabilization of sIL-1 RA protein and mRNA. The mechanism(s) that decrease transcription and stabilize protein and mRNA in tolerant cells are not yet known, but may involve a labile repressor(s) of transcription as well as protein and mRNA stabilizing/destabilizing elements (3.LaRue K. McCall C. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (99) Google Scholar, 24.Ziegler-Heitbrock H. Wedel A. Schraut W. Strobel M. Wendelgass P. Sternsdorf T. Bauerle P. Haas J. Riethmuller G. J. Biol. Chem. 1994; 269: 17001-17004Abstract Full Text PDF PubMed Google Scholar). The resulting steady state mRNA levels are amplified at the level of protein with dramatic differences in COX-2 and sIL-1 RA expression in endotoxin-tolerant THP-1 cells.Given the observation that COX-2 and sIL-1 RA were differentially expressed at the protein level, we examined their transcriptional regulation in THP-1 cells. COX-2 promoter fragments exhibit 3–5.5-fold decreases in reporter gene activity in transient transfection assays (Fig. 4 A). Similarly, transcription of the endogenous COX-2 gene exhibits an equivalent decrease in tolerant cells upon stimulation with LPS in comparison to control cells (data not shown). Based on these data and mRNA levels (Fig. 3 B), we find that COX-2 transcription is induced early and then quickly repressed. In addition, PGE2 lipid levels are decreased approximately 3-fold in tolerant cells when compared to control cells stimulated with LPS, possibly due to lower levels of COX-2 protein or available substrate present in tolerant cells. Similar to COX-2, sIL-1 RA transcription is also markedly reduced in tolerant cells (Fig. 4 B). This transcriptional repression may involve negative regulatory element(s) similar to those required for silencer activity in interferon-A gene promoters (25.Lopez S. Reeves R. Island M.-L. Bandu M.-T. Christeff N. Doly J. Navarro S. J. Biol. Chem. 1997; 272: 22788-22799Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). These findings are consistent with repressed transcription of the two genes, possibly by the same type of labile repressor that is thought to act on IL-1β (3.LaRue K. McCall C. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (99) Google Scholar). The large increase in sIL-1 RA protein in the presence of repressed transcriptional activity and mRNA levels (Fig. 3 A) indicates that other regulatory mechanisms must contribute to the elevated protein levels in endotoxin-tolerant THP-1 cells. We propose two potential mechanisms. First, differences in mRNA stability may account for the increase in sIL-1 RA protein levels in tolerant cells. Second, enhanced translational efficiency of declining pools of mRNA may potentiate sIL-1 RA expression in tolerance. We believe that it most likely is a combination of these two potential mechanisms. In support of this notion, Cassatella (26.Cassatella M. Meda L. Gasperini S. Calzetti F. Bonora S. J. Exp. Med. 1994; 179: 1695-1699Crossref PubMed Scopus (269) Google Scholar) has shown that anti-inflammatory IL-10 up-regulates IL1-RA production in LPS-stimulated human polymorphonuclear leukocytes by delaying mRNA degradation. We have also shown that delaying mRNA degradation in THP-1 cells tolerant to IL-1β overcomes repression of IL-1β synthesis (27.Yoza B.K. Wells J.D. McCall C.E. Clin. Diag. Lab. Immunol. 1998; 5: 281-287Crossref PubMed Google Scholar). These studies and others (28.Colotta F. Saccani S. Giri J. Dower M. Sims J. Introna M. Mantovani A. J. Immunol. 1996; 156: 2534-2541PubMed Google Scholar) suggest that increased anti-inflammatory expression may result from positive feedback mechanisms that are in place to help counterbalance proinflammatory expression. In addition, Beutler (29.Beutler B. Brown T. J. Clin. Invest. 1991; 87: 1336-1344Crossref PubMed Scopus (46) Google Scholar) has shown that cis-acting elements from the 3′-untranslated region of TNF-α are required for efficient translational activation of the gene. A similar form of translational regulation by cis-acting elements within the IL-1 RA 3′-untranslated region is therefore plausible.Post-transcriptional mechanisms of gene regulation have important roles in the expression of pro- and anti-inflammatory genes (26.Cassatella M. Meda L. Gasperini S. Calzetti F. Bonora S. J. Exp. Med. 1994; 179: 1695-1699Crossref PubMed Scopus (269) Google Scholar, 29.Beutler B. Brown T. J. Clin. Invest. 1991; 87: 1336-1344Crossref PubMed Scopus (46) Google Scholar). It was unknown whether similar mechanisms regulated COX-2 and sIL-1 RA expression. We investigated this issue by mRNA decay analysis. Our findings demonstrated that the turnover rate for the biphasic decay of COX-2 mRNA (Fig. 6 A) in tolerant cells (t 1/2 = 6 h) is 6 times as long as in control cells (t 1/2 = 1 h). In addition, we believe that even at lower levels in tolerant cells, COX-2 mRNA is efficiently translated (Fig. 1 A). The biphasic decay of COX-2 in control cells may be important in that elevated levels of proinflammatory mRNAs are rapidly turned over, whereas lower levels are subject to different turnover mechanisms and kinetics. Similarly, mRNA half-life analysis (Fig. 6 B) demonstrated that the turnover rate for sIL-1 RA in tolerant cells (t 1/2 = 12 h) is doubled in comparison to control cells (t 1/2 = 6 h), indicating that sIL-1 RA mRNA is relatively stable in tolerant cells. This mRNA stability may permit continued translation of sIL-1 RA. Additionally, mRNA stabilization may serve as a general mechanism of regulating expression for certain pro- and anti-inflammatory genes, potentially as a negative feedback loop (i.e. COX-2) or as means for continued expression (i.e. sIL-1 RA). Brooks et al. have shown that IL-1-induced JunB mRNA levels are not directly correlated with the level of JunB protein synthesis (30.Brooks J.W. Yoza B.K. Mizel S.B. Mol. Immunol. 1995; 32: 779-788Crossref PubMed Scopus (4) Google Scholar). Rather, JunB protein levels remain elevated as a result of enhanced translational efficiency. Our findings with sIL-1 RA in endotoxin-tolerant THP-1 cells are consistent with this mechanism. In addition, Cassatella and others have shown that the sIL-1 RA 3′-untranslated region does not contain AU-rich destabilizing sequences that are characteristic of rapidly turned over proinflammatory mRNA, such as COX-2 and IL-1 (26.Cassatella M. Meda L. Gasperini S. Calzetti F. Bonora S. J. Exp. Med. 1994; 179: 1695-1699Crossref PubMed Scopus (269) Google Scholar, 31.Kastelic T. Schnyder J. Leutwiler A. Traber R. Streit B. Niggli H. Mackenzie A. Cheneval D. Cytokine. 1996; 8: 751-761Crossref PubMed Scopus (48) Google Scholar).With the data supporting the notion that mRNA stability may in part explain differential expression of COX-2 and sIL-1 RA, we investigated protein stability by half-life analysis. Protein levels for COX-2 in both control and tolerant cells reached 50% of their maximal level within 2 h of cycloheximide addition (Fig. 5 A). This relatively short turnover time for COX-2 is consistent with what is known for the rapid decay of proinflammatory proteins in vitro (3.LaRue K. McCall C. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (99) Google Scholar) and their relative absence clinically in sepsis (17.Fisher C.J. Circ. Shock. 1994; 44: 1-8PubMed Google Scholar). In marked contrast, sIL-1 RA protein is very stable (Fig. 5 B). In both control and tolerant cells, sIL-1 RA protein is stable over 24 h. Interestingly, this stability is still maintained at 96 h (data not shown). These findings are supportive of clinical studies in sepsis in which anti-inflammatory protein levels remain elevated for several days (13.Rogy M.A. J. Am. Coll. Surg. 1994; 178: 132-138PubMed Google Scholar). Although elevated levels of anti-inflammatory proteins, such as sIL-1 RA, often help to decrease the potentially lethal inflammatory response in sepsis, the stability of these proteins may in part promote the severe immunosuppression commonly seen in sepsis patients.Taken collectively, these data are consistent with the notion that there is no significant difference in protein stability between control and endotoxin-tolerant cells for COX-2 or sIL-1 RA. However, the corresponding mRNAs are more stable in tolerant cells than in control cells, delaying degradation. In addition, due to undefined differences between pro- and anti-inflammatory genes, sIL-1 RA mRNA and protein are significantly more stable than those of COX-2, resulting in a pronounced difference at the level of protein in endotoxin tolerance.In summary, the results of this study demonstrate differential regulation and expression of COX-2 and sIL-1 RA in endotoxin-tolerant THP-1 cells. COX-2 protein levels are decreased, yet sIL-1 RA protein levels remain elevated. Differential expression is consistent with repressed transcription and protein turnover for COX-2, whereas sIL-1 RA mRNA and protein are stabilized. We believe that increased stability of sIL-1 RA mRNA may be coupled with enhanced translational efficiency of sIL-1 RA protein in tolerant cells. These data demonstrate that COX-2 and sIL-1 RA proteins are differentially expressed in endotoxin tolerant THP-1 cells, similar to differential expression observed during sepsis (13.Rogy M.A. J. Am. Coll. Surg. 1994; 178: 132-138PubMed Google Scholar, 17.Fisher C.J. Circ. Shock. 1994; 44: 1-8PubMed Google Scholar). In addition, the time courses for the THP-1 expression of COX-2 (rapid and transient) and sIL-1 RA (slow and constitutive) proteins are similar to the pattern of expression of pro- and anti-inflammatory genes observed during clinical sepsis (23.van der Poll T. Coyle S.M. Moldawer L.L. Lowry S.F. J. Infect. Dis. 1996; 174: 1356-1360Crossref PubMed Scopus (71) Google Scholar). These results indicate that the THP-1 cell line is as a useful system to dissect endotoxin responsiveness and the regulation of pro- and anti-inflammatory genes. Our results not only establish mechanisms for the differential regulation of pro- and anti-inflammatory genes and their protein products, but also serve as a foundation for future studies on the factors that are responsible for repressing and destabilizing COX-2 mRNA and protein and that promote sIL-1 RA expression. In endotoxin-tolerant THP-1 cells, the level of COX-2 protein is decreased, whereas the level of sIL-1 RA protein is not. This differential expression results from repressed transcription of both COX-2 and sIL-1 RA genes combined with stabilization of sIL-1 RA protein and mRNA. The mechanism(s) that decrease transcription and stabilize protein and mRNA in tolerant cells are not yet known, but may involve a labile repressor(s) of transcription as well as protein and mRNA stabilizing/destabilizing elements (3.LaRue K. McCall C. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (99) Google Scholar, 24.Ziegler-Heitbrock H. Wedel A. Schraut W. Strobel M. Wendelgass P. Sternsdorf T. Bauerle P. Haas J. Riethmuller G. J. Biol. Chem. 1994; 269: 17001-17004Abstract Full Text PDF PubMed Google Scholar). The resulting steady state mRNA levels are amplified at the level of protein with dramatic differences in COX-2 and sIL-1 RA expression in endotoxin-tolerant THP-1 cells. Given the observation that COX-2 and sIL-1 RA were differentially expressed at the protein level, we examined their transcriptional regulation in THP-1 cells. COX-2 promoter fragments exhibit 3–5.5-fold decreases in reporter gene activity in transient transfection assays (Fig. 4 A). Similarly, transcription of the endogenous COX-2 gene exhibits an equivalent decrease in tolerant cells upon stimulation with LPS in comparison to control cells (data not shown). Based on these data and mRNA levels (Fig. 3 B), we find that COX-2 transcription is induced early and then quickly repressed. In addition, PGE2 lipid levels are decreased approximately 3-fold in tolerant cells when compared to control cells stimulated with LPS, possibly due to lower levels of COX-2 protein or available substrate present in tolerant cells. Similar to COX-2, sIL-1 RA transcription is also markedly reduced in tolerant cells (Fig. 4 B). This transcriptional repression may involve negative regulatory element(s) similar to those required for silencer activity in interferon-A gene promoters (25.Lopez S. Reeves R. Island M.-L. Bandu M.-T. Christeff N. Doly J. Navarro S. J. Biol. Chem. 1997; 272: 22788-22799Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). These findings are consistent with repressed transcription of the two genes, possibly by the same type of labile repressor that is thought to act on IL-1β (3.LaRue K. McCall C. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (99) Google Scholar). The large increase in sIL-1 RA protein in the presence of repressed transcriptional activity and mRNA levels (Fig. 3 A) indicates that other regulatory mechanisms must contribute to the elevated protein levels in endotoxin-tolerant THP-1 cells. We propose two potential mechanisms. First, differences in mRNA stability may account for the increase in sIL-1 RA protein levels in tolerant cells. Second, enhanced translational efficiency of declining pools of mRNA may potentiate sIL-1 RA expression in tolerance. We believe that it most likely is a combination of these two potential mechanisms. In support of this notion, Cassatella (26.Cassatella M. Meda L. Gasperini S. Calzetti F. Bonora S. J. Exp. Med. 1994; 179: 1695-1699Crossref PubMed Scopus (269) Google Scholar) has shown that anti-inflammatory IL-10 up-regulates IL1-RA production in LPS-stimulated human polymorphonuclear leukocytes by delaying mRNA degradation. We have also shown that delaying mRNA degradation in THP-1 cells tolerant to IL-1β overcomes repression of IL-1β synthesis (27.Yoza B.K. Wells J.D. McCall C.E. Clin. Diag. Lab. Immunol. 1998; 5: 281-287Crossref PubMed Google Scholar). These studies and others (28.Colotta F. Saccani S. Giri J. Dower M. Sims J. Introna M. Mantovani A. J. Immunol. 1996; 156: 2534-2541PubMed Google Scholar) suggest that increased anti-inflammatory expression may result from positive feedback mechanisms that are in place to help counterbalance proinflammatory expression. In addition, Beutler (29.Beutler B. Brown T. J. Clin. Invest. 1991; 87: 1336-1344Crossref PubMed Scopus (46) Google Scholar) has shown that cis-acting elements from the 3′-untranslated region of TNF-α are required for efficient translational activation of the gene. A similar form of translational regulation by cis-acting elements within the IL-1 RA 3′-untranslated region is therefore plausible. Post-transcriptional mechanisms of gene regulation have important roles in the expression of pro- and anti-inflammatory genes (26.Cassatella M. Meda L. Gasperini S. Calzetti F. Bonora S. J. Exp. Med. 1994; 179: 1695-1699Crossref PubMed Scopus (269) Google Scholar, 29.Beutler B. Brown T. J. Clin. Invest. 1991; 87: 1336-1344Crossref PubMed Scopus (46) Google Scholar). It was unknown whether similar mechanisms regulated COX-2 and sIL-1 RA expression. We investigated this issue by mRNA decay analysis. Our findings demonstrated that the turnover rate for the biphasic decay of COX-2 mRNA (Fig. 6 A) in tolerant cells (t 1/2 = 6 h) is 6 times as long as in control cells (t 1/2 = 1 h). In addition, we believe that even at lower levels in tolerant cells, COX-2 mRNA is efficiently translated (Fig. 1 A). The biphasic decay of COX-2 in control cells may be important in that elevated levels of proinflammatory mRNAs are rapidly turned over, whereas lower levels are subject to different turnover mechanisms and kinetics. Similarly, mRNA half-life analysis (Fig. 6 B) demonstrated that the turnover rate for sIL-1 RA in tolerant cells (t 1/2 = 12 h) is doubled in comparison to control cells (t 1/2 = 6 h), indicating that sIL-1 RA mRNA is relatively stable in tolerant cells. This mRNA stability may permit continued translation of sIL-1 RA. Additionally, mRNA stabilization may serve as a general mechanism of regulating expression for certain pro- and anti-inflammatory genes, potentially as a negative feedback loop (i.e. COX-2) or as means for continued expression (i.e. sIL-1 RA). Brooks et al. have shown that IL-1-induced JunB mRNA levels are not directly correlated with the level of JunB protein synthesis (30.Brooks J.W. Yoza B.K. Mizel S.B. Mol. Immunol. 1995; 32: 779-788Crossref PubMed Scopus (4) Google Scholar). Rather, JunB protein levels remain elevated as a result of enhanced translational efficiency. Our findings with sIL-1 RA in endotoxin-tolerant THP-1 cells are consistent with this mechanism. In addition, Cassatella and others have shown that the sIL-1 RA 3′-untranslated region does not contain AU-rich destabilizing sequences that are characteristic of rapidly turned over proinflammatory mRNA, such as COX-2 and IL-1 (26.Cassatella M. Meda L. Gasperini S. Calzetti F. Bonora S. J. Exp. Med. 1994; 179: 1695-1699Crossref PubMed Scopus (269) Google Scholar, 31.Kastelic T. Schnyder J. Leutwiler A. Traber R. Streit B. Niggli H. Mackenzie A. Cheneval D. Cytokine. 1996; 8: 751-761Crossref PubMed Scopus (48) Google Scholar). With the data supporting the notion that mRNA stability may in part explain differential expression of COX-2 and sIL-1 RA, we investigated protein stability by half-life analysis. Protein levels for COX-2 in both control and tolerant cells reached 50% of their maximal level within 2 h of cycloheximide addition (Fig. 5 A). This relatively short turnover time for COX-2 is consistent with what is known for the rapid decay of proinflammatory proteins in vitro (3.LaRue K. McCall C. J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (99) Google Scholar) and their relative absence clinically in sepsis (17.Fisher C.J. Circ. Shock. 1994; 44: 1-8PubMed Google Scholar). In marked contrast, sIL-1 RA protein is very stable (Fig. 5 B). In both control and tolerant cells, sIL-1 RA protein is stable over 24 h. Interestingly, this stability is still maintained at 96 h (data not shown). These findings are supportive of clinical studies in sepsis in which anti-inflammatory protein levels remain elevated for several days (13.Rogy M.A. J. Am. Coll. Surg. 1994; 178: 132-138PubMed Google Scholar). Although elevated levels of anti-inflammatory proteins, such as sIL-1 RA, often help to decrease the potentially lethal inflammatory response in sepsis, the stability of these proteins may in part promote the severe immunosuppression commonly seen in sepsis patients. Taken collectively, these data are consistent with the notion that there is no significant difference in protein stability between control and endotoxin-tolerant cells for COX-2 or sIL-1 RA. However, the corresponding mRNAs are more stable in tolerant cells than in control cells, delaying degradation. In addition, due to undefined differences between pro- and anti-inflammatory genes, sIL-1 RA mRNA and protein are significantly more stable than those of COX-2, resulting in a pronounced difference at the level of protein in endotoxin tolerance. In summary, the results of this study demonstrate differential regulation and expression of COX-2 and sIL-1 RA in endotoxin-tolerant THP-1 cells. COX-2 protein levels are decreased, yet sIL-1 RA protein levels remain elevated. Differential expression is consistent with repressed transcription and protein turnover for COX-2, whereas sIL-1 RA mRNA and protein are stabilized. We believe that increased stability of sIL-1 RA mRNA may be coupled with enhanced translational efficiency of sIL-1 RA protein in tolerant cells. These data demonstrate that COX-2 and sIL-1 RA proteins are differentially expressed in endotoxin tolerant THP-1 cells, similar to differential expression observed during sepsis (13.Rogy M.A. J. Am. Coll. Surg. 1994; 178: 132-138PubMed Google Scholar, 17.Fisher C.J. Circ. Shock. 1994; 44: 1-8PubMed Google Scholar). In addition, the time courses for the THP-1 expression of COX-2 (rapid and transient) and sIL-1 RA (slow and constitutive) proteins are similar to the pattern of expression of pro- and anti-inflammatory genes observed during clinical sepsis (23.van der Poll T. Coyle S.M. Moldawer L.L. Lowry S.F. J. Infect. Dis. 1996; 174: 1356-1360Crossref PubMed Scopus (71) Google Scholar). These results indicate that the THP-1 cell line is as a useful system to dissect endotoxin responsiveness and the regulation of pro- and anti-inflammatory genes. Our results not only establish mechanisms for the differential regulation of pro- and anti-inflammatory genes and their protein products, but also serve as a foundation for future studies on the factors that are responsible for repressing and destabilizing COX-2 mRNA and protein and that promote sIL-1 RA expression. We thank Jean Hu, Sue Cousart, and Jon Wells for excellent technical support. We are especially grateful to Dr. Michael F. Smith, Dr. Steve Prescott, Dr. Eugene O'Neill, and Dr. Timothy Hla, who supplied us with reagents as identified in the text; Dr. Barbara K. Yoza for insightful discussion; and Dr. Douglas Lyles for critical reading of the manuscript. Oligonucelotide synthesis was performed in the DNA Synthesis Core Laboratory of the Cancer Center of Wake Forest University.