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Cyclooxygenase-2-deficient mice are resistant to endotoxin-induced inflammation and death¹

KUNIAKI EJIMA^*, MATTHEW D. LAYNE^*,, IRVITH M. CARVAJAL^*, PATRICIA A. KRITEK^*, REBECCA M. BARON^*,, YEN-HSU CHEN^*,, JEFFREY VOM SAAL^*, BRUCE D. LEVY^*,, SHAW-FANG YET^*, and MARK A. PERRELLA^*,,2

^* Pulmonary and Critical Care Medicine, Department of Internal Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA;
Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA; and
Division of Infectious Diseases, Department of Internal Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

²Correspondence: Brigham and Women’s Hospital, Pulmonary and Critical Care Medicine, 75 Francis St., Boston, MA 02115, USA. E-mail: mperrella{at}rics.bwh.harvard.edu

SPECIFIC AIM

The present study was designed to elucidate the role of COX-2 in host responses to bacterial endotoxemia. We assessed the effect of Escherichia coli LPS on survival and acute inflammatory responses in wild-type and COX-2-deficient mice.

PRINCIPAL FINDINGS

1. Absence of COX-2 improves survival in mice exposed to high doses of endotoxin
COX-2^–/– mice and their COX-2^+/+ littermates were exposed to LPS at a dose of 100 mg/kg i.p. and followed over 96 h. COX-2^+/+ mice began to die by 48 h (Fig. 1 ). Survival rate for COX-2^+/+ mice was 64% after 48 h and 54% after 72 and 96 h. COX-2^–/– mice were resistant to LPS with 100% survival throughout the study and significantly improved survival (P<0.05) at 72 and 96 h. COX-2^–/– mice given LPS at a dose of 40 mg/kg i.p. x3 consecutive days also exhibited the same resistance to endotoxin and experienced improved survival.

View larger version (15K): [in this window] [in a new window]   Figure 1. Absence of COX-2 improves survival after LPS administration in mice. Wild type (+/+) or homozygous (–/–) mice targeted for disruption of COX-2 were administered E. coli LPS 100 mg/kg i.p. in a single dose (+/+ n=8, -/- n=7) and survival was monitored over 96 h. Data are expressed as % of mice alive at each time point. *P< 0.05 compared with COX-2–/– mice.

2. Absence of COX-2 leads to a decreased inflammatory response after endotoxin administration
To further determine the effect of COX-2 on endotoxin-induced inflammatory responses, LPS (10 mg/kg i.p.) was administered to COX-2^+/+ and COX-2^–/– mice. Because damage to kidneys and lungs during sepsis often contributes to the associated morbidity and mortality, these organs were harvested 4 h later and leukocyte infiltrates were assessed by immunohistochemical staining for CD45. After LPS administration to COX-2^+/+ mice, inflammatory cell infiltrates were increased in kidney and lung tissue. Quantitative analysis of staining for CD45 revealed a 4.3-fold increase in kidney tissue and a 31-fold increase in lung tissue after LPS stimulation in COX-2^+/+ mice. In contrast, COX-2^–/– mice displayed striking reductions in LPS-induced CD45 staining in kidney (1.8-fold increase) and lung (1.5-fold increase) tissue. These data suggest that a marked reduction in tissue inflammation may contribute to the improved outcome after endotoxin administration in the absence of COX-2.

3. Alterations in cytokine-inducible gene expression in the absence of COX-2
HO-1 and NOS2 are genes known to be induced by proinflammatory cytokines and during endotoxemia. To further assess the inflammatory responses that occur in the absence of COX-2, tissue levels of HO-1 and NOS2 mRNA were analyzed. In wild-type mice, peak induction of HO-1 (50-fold in kidney and 13-fold in lung) and NOS2 (50-fold in kidney and 7-fold in lung) mRNA levels occurred 4 h after LPS administration (10 mg/kg i.p.). This peak induction of HO-1 and NOS2 corresponded to the peak induction of COX-2. Peak induction of HO-1 and NOS2 mRNA levels was delayed (12 h) in COX-2^–/– mice. In COX-2^–/– mice, induction of HO-1 (3-fold in kidney and lung) and NOS2 (13-fold in kidney and 4-fold in lung) mRNA levels were markedly blunted. By 12 h after LPS stimulation, peak induction of HO-1 and NOS2 mRNA levels in COX-2^–/– mice was comparable to COX-2^+/+ mice, whose levels were returning toward baseline in kidney and lung tissues.

4. Transcription factor binding and translocation is altered in the absence of COX-2
Transcription factors such as NF-B and AP-1 play a critical role in signaling events that occur during the inflammatory response to endotoxin and in regulating NOS2 and HO-1 gene expression, respectively. Thus, we investigated the effect of COX-2 deficiency on the binding of transcription factors to the 5'-flanking sequences of NOS2 and HO-1 genes. Nuclear protein binding to the NOS2 and HO-1 probes was increased after 2 and 4 h of LPS stimulation (10 mg/kg i.p.) in tissue from COX-2^+/+ mice. However, nuclear protein binding was reduced and peak binding was delayed in tissue from COX-2^–/– mice, similar to the mRNA response for these genes. Incubation of the nuclear extracts with antibodies specific for p50 and p65 demonstrated that these NF-B subunits comprise the DNA–protein complex binding to the NOS2 probe. Similar supershift experiments using antibodies against c-Jun and c-Fos revealed these AP-1 subunits are present in the DNA–protein complex binding to the HO-1 probe.

To determine whether nuclear translocation of NF-B was altered in the absence of COX-2, immunostaining for p65 was performed. In wild-type mice, LPS (10 mg/kg i.p.) produced a striking translocation of p65 from the cytoplasm to the nucleus in kidney and lung tissue. In contrast, COX-2^–/– mice displayed marked reductions in LPS-induced nuclear translocation of p65 in kidney and lung tissue. Together, these data indicate that nuclear translocation and DNA binding of transcription factors important for the induction of NOS2 and HO-1 genes by inflammatory stimuli were also reduced, similar to mRNA levels, in the absence of COX-2.

5. Alteration in anti-inflammatory but not proinflammatory cytokine expression in the absence of COX-2
To further investigate the mechanism(s) for COX-2 regulation of the inflammatory response to endotoxemia, peritoneal macrophages were harvested from COX-2^+/+ and COX-2^–/– mice and treated with LPS (Fig. 2 ). mRNA levels for proinflammatory cytokines (IL-6, IL-1ß, and TNF-) were induced by LPS stimulation (500 ng/mL) at 4, 12, and 24 h without significant differences in expression in macrophages from COX-2^+/+ and COX-2^–/– mice (Fig. 2A ). The induction of mRNA levels for the anti-inflammatory cytokine IL-10 was markedly increased in COX-2^–/– cells (Fig. 2A ). Release of IL-10 protein by the LPS-exposed macrophages was also increased in the supernatant from COX-2^–/– cells vs. COX-2^+/+ cells (Fig. 2B ). These data suggest that the reduced inflammatory response in COX-2^–/– mice may reflect an augmented induction of the anti-inflammatory cytokine IL-10 in response to endotoxemia rather than an altered expression of proinflammatory cytokines (IL-6, IL-1ß, and TNF-).

View larger version (35K): [in this window] [in a new window]   Figure 2. LPS-induced expression of pro- and anti-inflammatory cytokines in the presence or absence of COX-2. Peritoneal macrophages were harvested from COX-2+/+ and COX-2–/– mice. A) The macrophages were exposed to LPS (500 ng/mL) and total RNA was harvested after 4 h of stimulation. Northern blot was performed using 2 µg/lane of RNA. The membranes were hybridized with 32P-labeled cDNA probes for COX-2, IL-10, IL-6, IL-1ß, and TNF-. RNA loading was assessed by hybridization with an 18S ribosomal probe. Data show a representative experiment performed in duplicate. B) The macrophages were exposed to LPS (500 ng/mL) and after 48 h an ELISA for IL-10 was performed on aliquots of supernatant from the cells. For vehicle controls n=3 and for LPS stimulated groups n= 6. *P< 0.05 compared with vehicle control, P< 0.05 compared with COX-2+/+ cells receiving LPS.

6. Binding of NF-B to DNA is decreased when IL-10 is administered in vivo
To understand the importance of this additional increase in IL-10 levels during endotoxemia, IL-10 was given to wild-type mice (100 and 500 µg/kg i.p.) 30 min before a single administration of LPS (10 mg/kg i.p.). After 2 h, nuclear extracts were prepared from kidney tissue and EMSAs were performed. NF-B binding to the NOS2 5'-flanking sequence was reduced by IL-10 in a concentration-dependent manner.

CONCLUSIONS AND SIGNIFICANCE

Sepsis is a frequent and severe systemic inflammatory response to infection associated with excess morbidity and mortality. Despite intense efforts to improve therapeutic strategies, the incidence of sepsis continues to rise. Greater understanding of the endogenous counter-regulatory pathways dysregulated during sepsis may yield insight into new therapeutic strategies. In the present study, we determined that a deficiency in COX-2 has a dramatic protective effect, improving survival in endotoxemic mice (Fig. 1) and reducing leukocyte infiltration into vital organs and activation of transcription factors important for signaling events during endotoxemia.

Previous studies have shown that nonspecific inhibition of COX-1 and COX-2 by nonsteroidal anti-inflammatory drugs (NSAIDs) improves survival in animal models of endotoxemia, yet selective inhibition of COX-2 by NS-398 in a recent study did not show late protection during endotoxemia (LPS) and bacterial peritonitis-induced sepsis (cecal ligation and puncture). These data led to the speculation that COX-1, and not COX-2, may have been primarily responsible for prostanoid-mediated pathophysiologic consequences in endotoxemia and sepsis. We assessed the effect of E. coli LPS on survival and the acute inflammatory response in COX-2-deficient mice. Results from our experiments provide several lines of evidence demonstrating that COX-2 is a critical component of the lethal response associated with endotoxemia in mice. Besides a role in LPS responses, our data suggest a link between COX-2 products and gene regulation of HO-1 and NOS2. Although the blunted and delayed induction of HO-1 and NOS2 by LPS in the absence of COX-2 may result indirectly from an overall reduction in the systemic inflammatory response, studies are needed to determine whether regulation of HO-1 and NOS2 in COX-2^–/– mice has a direct effect on the improved survival. Earlier studies demonstrated that an absolute deficiency in HO-1 or NOS2 has detrimental consequences during endotoxemia. However, we cannot exclude the possibility that a reduction (instead of an absence) in HO-1 and NOS2 during the early stages of endotoxemia may have contributed to the beneficial outcome in these COX-2^–/– mice.

Finally, we assessed the macrophage production of pro- and anti-inflammatory cytokines. We found no difference in the levels of proinflammatory cytokines (TNF-, IL-1ß, and IL-6) in cells from COX-2^–/– mice compared with wild-type mice. However, we found an exaggerated induction of IL-10 mRNA and protein levels in the absence of COX-2 in these inflammatory cells (Fig. 2) . Earlier studies had shown that exogenous administration of recombinant IL-10 reduces lethality in endotoxemic mice and that IL-10-deficient mice can be rescued from endotoxin-induced death by gene therapy using adeno-associated virus encoding IL-10. We also found that exogenous administration of IL-10 to wild-type mice markedly decreased NF-B activation. Together, our data suggest that COX-2-deficient mice are resistant to the detrimental consequences of endotoxemia (including inflammatory cell infiltration of tissues and NF-B activation) and that this beneficial effect occurs in part by a compensatory IL-10 anti-inflammatory response that counterbalances the proinflammatory responses to endotoxemia (Fig. 3 ).

View larger version (24K): [in this window] [in a new window]   Figure 3. Schematic diagram of the role of COX-2 in the response to endotoxemia in mice. Our data suggest that a deficiency in COX-2 and the resulting blockade (X) in prostaglandin (PG) and thromboxane (Tx) production improve survival in endotoxemic mice and reduce several parameters of the inflammatory response, including translocation and DNA binding of transcription factors (NF-B and AP-1, ), important for signaling events during endotoxemia. LPS induction of proinflammatory cytokines (IL-6, IL-1ß, and TNF-, ) was not different between COX-2+/+ and COX-2–/– mice; however, induction of the anti-inflammatory cytokine IL-10 (•) was exaggerated in the absence of COX-2. This compensatory IL-10 anti-inflammatory response counterbalances the proinflammatory responses to endotoxemia and helps restore homeostasis in COX-2–/– mice.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1078fje; doi: 10.1096/fj.02-1078fje

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