行政院國家科學委員會補助專題研究計畫成果報告
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敗血症造成不同族群的單核吞噬細胞凋亡與其花生四烯酸
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代謝調節：治療敗血症動物使用抗發炎藥物退燒合理否？
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計畫類別：■個別型計畫
□整合型計畫
計畫編號：NSC90－2314－B－039－013－
執行期間：90 年 08 月 01 日至 91 年 07 月 31 日
計畫主持人：盧敏吉 eMail: [email protected]
執行單位：中國醫藥學院醫學系
中
華
民
國
91
年
11
月
25
日
行政院國家科學委員會專題研究計畫成果報告
國科會專題研究計畫成果報告撰寫格式說明
Pr epar ation of NSC Pr oject Repor ts
計畫編號：NSC 89－2314－B－039－025
執行期限：89 年 8 月 1 日至 90 年 7 月 31 日
主持人：盧敏吉 中國醫藥學院醫學系
一、中文摘要 敗血症及敗血性休克是急症與重症加 護病人的死亡及長期醫療最重要原因之 一，也是社會醫療資源的重大負擔。敗血 症候群最常伴隨急性肺傷害如肺血管滲 漏、肺炎、及成人呼吸窘迫症候群﹐其病 理機轉與治療至今仍未有突破。已知花生 四烯酸代謝產物﹐包括前列腺素及白烯 素，是肺炎症反應與免疫的重要細胞介 質；其缺乏或過多常伴隨發炎與免疫疾 病，包括肺間質纖維化，且是其致病機轉 之一；而肺泡巨噬細胞是肺第一線的防禦 機制，也是花生四烯酸代謝產物的重要來 源。近年的研究更發現花生四烯酸代謝酵 素 COX-2 及白烯素具有抗細胞死亡作用， 其作用會被臨床上常用來退燒止痛的非類 固醇類抗發炎藥或其它類固醇所阻斷，而 可能造成細胞死亡。我們先前的研究顯 示，肺泡巨噬細胞的存活及其白烯素產量 隨著敗血症的嚴重度增加而降低；這個結 果指出花生四烯酸代謝及肺細胞的凋亡可 能參與敗血症肺傷害的病因機制。另一方 面，我們也發現不同族群的肺泡巨噬細胞 在早期與晚期敗血症時，會有不同程度且 不同類型的凋亡。本研究顯示，敗血症肺 泡巨噬細胞的死亡及急性肺傷害的嚴重度 是與退燒止痛的非類固醇類抗發炎藥的使 用相關的；在敗血症後早(3 小時)或晚 (9 小時)給 COX-2-specific 抑制劑均使肺傷害 的嚴重度增加，但在敗血症後早(3 小時) 給 COX-1/COX-2-nonspecific 抑制劑並不 會使肺傷害更嚴重。 本研究提供的訊息是，敗血症的動物 在感染發生及進行時，使用抗發炎藥退燒 止痛會使得肺傷害惡化，可能進一步造成 呼吸窘迫及肺炎；因此，敗血症及敗血性 休克時，應少用或小心使用抗發炎退燒 藥，尤其是 COX-2-specific 抑制劑。 改變的機制；也可能提供敗血症候群 及其伴隨肺傷害的預防及不同的臨床治療 方向。 關鍵詞：敗血症，休克，肺炎，成人呼吸窘 迫症候群，花生四烯酸，前列腺素，白烯 素，肺泡巨噬細胞，非類固醇類抗發炎藥 AbstractAcute lung injury (ALI) is a major complication of sepsis, which could leads to the increased susceptibility to pulmonary infection. Nonsteroid anti-inflammatory drugs (NSAIDs) are commonly used
clinically for the management of
sepsis-related manifestations, including pain, fever, and inflammation. However, their effects on septic lung injury are not recognized. Recent studies revealed that NSAIDs cause apoptosis in several cell types. Our hypothesis was that, during the process of sepsis, the usage of NSAIDs may enhance AM apoptosis and, thereafter, decrease the ability of the lung to clear bacteria. In this study, SD rats were cecal ligated and punctured (CLP). At 3 and 9 h after CLP, septic rats were treated with traditional NSAIDs (indomrthacin or ibuprofen) or COX-2-seclective blockers (rofecoxib or NS398). The rats were scarified at 20 h and the lungs and AM were obtained to investigate bacterial burden and apoptosis, respectively. The results showed a deteriorated AM apoptosis and a parallel increased lung bacterial burden in the
COX-2-selective blocker-treated septic animals, but not in the early traditional NSAIDs-treated ones. Consequently, the
utilization of NSAIDs, especially
COX-2-seclective blockers, should be careful or limited in septic subjects to prevent further damages and secondary infections of septic lungs
Keywords ： sepsis, lung, alveolar
macrophages, ARDS, arachidonic acid, prostaglandins, leukotrienes, 5-lipoxygenase, cyclooxygenase, apoptosis, NSAIDs
二、計畫緣由與目的
Sepsis has been one of the major causes of illness and death in the elderly, the
neonate, and the patients in the intensive care unit (ICU; Luce, 1987). The incidence of sepsis is continuing to increase, for which the contributing factors might be the increasing longevity of patients with chronic diseases and the widespread use of antimicrobial agents and glucocorticoids (Munford, 1998). The successful treatment of sepsis in humans continues to be a substantial clinical challenge and the expensive costs of treatment and the high mortality rate for septic patients impose a heavy burden on our health care system (Bone, 1996). There are quite a few clinical manifestations, including fever, tachycardia, and tachypnea, signifying the systemic inflammatory responses to microbial invasion, i.e., sepsis. The septic response often intensifies over time from mild to extremely severe (septic shock). It has been demonstrated both experimentally and clinically that sepsis causes the appearance in plasma of a series of cytokines, such as interleukin (IL)-1, tumor necrosis factor (TNF)- , or IL-6 (Ertel et al, 1991).
In spite of early intervention and aggressive treatment, sepsis still represents a major threat to the hospitalized patients with
a mortality rate exceeding 50%. In addition, for patients who survive the initial episodes, complications may occur and result in a devastating condition (Luce, 1987). In ICUs, a major complication of septic patients is impaired respiration, often leading to adult respiratory distress syndrome (ARDS), the most severe form of acute lung injury (ALI), and onset of multiple organ failure (Moore et al, 1993; Polk and Shields, 1977). ARDS develops in 20 to 50 percent of patients with sepsis, which results in a mortality rate more than 50 to 80 percent (Munford, 1998).
ROLES OF NONSTEROIDAL
ANTIINFLAMMTORY DRUGS IN
SEPSIS MANAGEMENT
Fever is a prevailing manifestation of sepsis. Except for a few proportions of
patients exhibiting depressed body
temperature, nearly 90% of severe septic patients are febrile (Clemmer et al., 1992; Bernard et al., 1997). Fever has been considered as a body reaction to infection and inflammation, that is, an adaptive response. Not only fever could inhibit the growth of invading microorganisms, but also it was demonstrated to improve the survival of
patients with polymicrobial sepsis
(Mackowiak et al., 1980), Pseudomonas aeruginosa sepsis (Kuikka and Valtonen,
1998), and E. coli bacteremia (Kuikka and
Sivonen, 1997). Although antipyretic therapy is commonly administered to septic patients, there are few data to support this practice. Some studies showed that nonsteroidal anti-inflammatory drugs (NSAIDs), with their anti-inflammatory properties, caused reductions in both body temperature and metabolic rate, however, no improvement in the survival of septic patient was conferred (Bernard et al., 1997).
The effects of NSAIDs on the outcome of sepsis are unclear due to the lack of
definitive clinical studies. In some experimental models, however, elevations of
core temperature have been associated with an increased survival rate and antipyretic agents with a higher death rate during bacterial infection (Jiang et al., 2000; Esposito, 1984). Fever was also shown to reduce plasma TNFá levels in mice with
experimental Klebsiella pneumoniae
peritonitis (Jiang et al., 2000) and to augment macrophage functions, including expression of Fc receptors, phagocytosis, pinocytosis, and killing of intracellular bacteria, in several animal models (Bruggen et al., 1991; Berman et al., 1981). TNFá and IL-1â expression by macrophages was attenuated with an elevation of temperatures (Jiang et al., 1999; Klostergaard et al., 1989). Febrile temperatures appear to influence the biological activities of cytokines, for example, enhancing the cytotoxity of human TNFá (Tomasobic et al., 1992) as well.
A common rationale for reducing fever is to prevent tissue injury caused by elevated core temperatures and to reduce the metabolic demands of the febrile response. This concept is important for sepsis patient since the host’s ability to meet the increased metabolic demands of fever may be limited due to disturbances in cardiac and pulmonary functions. As a result, it is crucial to develop rational protocols for treating fever in septic patients.
PREVIOUS AND CURRENT STUDIES
The regulation of AA metabolism in AM is altered in various clinical situations. The
Principle Investigator has previously shown that neonatal AM (nAM) and adult AM (aAM) exhibited different patterns of arachidonate metabolism in response to either A23187 or zymosan stimulation (Lu et al., 1996). In aAM, endogenously released AA was converted predominantly to LTB4 and 5-HETE, with only a minimal degree of conversion to CO products. This “adult pattern” of AM AA metabolism is consistent
with reports from cattle as well as other species (Balter et al.,1989; Peters-Golden et al., 1990). In nAM, by contrast, the major AA metabolites released were prostanoids rather than 5-LO metabolites (the “juvenile pattern”). The age-related enhancement of AM 5-LO metabolic capacity was paralleled by increases in both 5-LO and FLAP protein expression of a similar magnitude. It is plausible that these changes in protein expression explain most of the changes in 5-LO metabolic capacity. The changes in neonatal AM AA metabolism might explain the decreased host lung defense in neonatal
animal. Another study from Dr.
Peters-Golden’s laboratory showed that AM from HIV-infected patients has a decreased 5-LO metabolism (Coffey et al., 1996), i.e., a AA metabolic pattern similar to the juvenile pattern.
The AM number and AM 5-LO metabolites decrease during sepsis. Our
previous experiments showed that the rats undergoing cecal ligation and puncture (CLP) with a 18-G needle has a mortality rate of about 90%, which is consistent with previous reports (Wichterman et al., 1980). The data from CLP-rat showed that, in late septic stage, the number of AM obtained from BAL fluid was less than that in control and in the early septic rats (Figure not shown). This phenomenon has not been documented, and the underlying mechanism will be studied. Furthermore, AM from early and late septic rats are less capable of releasing LTB4 as the pro-inflammatory mediator (Figures not shown), which was accompanied by a parallel lower expression of 5-LO protein (Figure not shown). Since PLA2 metabolic capacities are similar among all groups of animals (Figure not shown), it is highly suggested that the released AA will be metabolized via the COX pathway for prostanoid production.
Subsets of AM underwent different degree and pattern of apoptosis during early and late phases of apoptosis. RB (big cell with more granularity) and RS (small cell with less granularity) subsets of AM represent 2 distinct populations of in situ alveolar mononuclear phagocytes (Figure not shown). RS-AM likely represents young and newcomer of AM. Their baseline apoptotic percentage was low, but increased rapidly as sepsis progressed from early (3h and 9h) to late (20h) stages (Figures not shown). On the other hand, RB-AM stands for older macrophages. Though their level of programmed cell death was high at the very early stage (3h) of Sham-operated (“minor trauma”) rats, it decreased at later stages (9h and 20h). Whereas in the septic animals, the apopoptic rate of RB-AM was maintained at a relatively constant level. This indicated that RB-AM could “repair” or “prevent” damage caused by minor injury. When confronted with major septic incidence, the alveolar microenviroment or RB-AM themselves may as well provide RB-AM ways of escaping apoptotic stress and keep protecting the lungs.
From the damage by invading
microorganisms.
In the present study, we has shown that
cox-1/cox2-nonspecific inhibitors
indomethacin (Fig 1) and ibuprofen (Fig 2) tended to decrease the sepsis-induced AM apoptosis when given at 3 h after CLP. This effect is not present when given at 9 hr after CLP (Fig 3).
Along with the increase in number of viable AM with indomethacin (Fig 4) and ibuprofen (Fig 5) treatment at 3 h, the bacterial burdens decreased. On the contrary at 9 h, the treatment caused an increased in bacterial burden (Fig 6) Fig 1 A p o p to si s( % ) 0 5 10 15 20 Vehicle Indocin Fig 2 0 5 10 A p o p to si s % Vehicle Ibuprofen Fig 3 0 5 10 15 20 A pop to sis % Vehicle Ibuprofen Fig 4
3.4 3.45 3.5 3.55 3.6 3.65 3.7 0 0.5 1 1.5 2 2.5 vehicle indocin B ac ter ial co un t ( lo g1 0 cf u/ g) Fig 5 0 0.5 1 1.5 2 2.5 3 3.5 4 b ac ter ia l co u n t (l o g 1 0 c fu /g ) Vehicle Ibuprofe n Fig6 2.75 2.8 2.85 2.9 2.95 3 3.05 vehicle indocin B ac te ria l cou nt ( lo g1 0 c fu /g )
Furthermore, we tested the effects of
COX-2-specific NSAIDS rofecoxib or
NS398. When given at 3 h or 9 h, all deteriorated AM apoptosis and increased
lung bacterial burdens (Fig not shown).
三、結果與討論
The long-term goals of my research
have been to elucidate the pathogenic mechanisms and the pathophysiological changes of sepsis-induced lung injury. I expect that the ongoing rigorous studies in my laboratory will contribute to the establishment of a rational strategy to treat sepsis syndrome, to prevent the development of ALI/ARDS, and to maintain lung functions during the process of sepsis. This proposed study is, therefore, a continuing effort of my previous researches, including NSC89-2314-B-039-025 (“Apoptosis and Regulation of Arachidonic Acid Metabolism in Lung Cells from Rats at the Early and the Late Stages of Sepsis”) and CMC87-M-02 (“Decrement in the number and alterations in phospholipid metabolism of AM from rats with septic lung injury”).
In this study, we have shown that in the progression of sepsis from early to late phase, apoptosis of mononuclear phagocytes occurs. This not only results in a poor lung defense due to the apoptosis of competent AM as well as alveolar epithelial and vascular endothelial cells, but also in the subsequent destruction of “alveolar-capillary membrane” causing vascular leakage. The decreased 5-LO capacity probably serves as an anti-inflammatory mechanism to prevent overwhelming lung cell damages by the addition of inflammatory cells. Furthermore, the released arachidonic acid likely served as a substrate for the surrounding cells to release prostanoids, which together with COX-2, maintain the homeostasis of lung microenvironment and prevent cellular
apoptosis. Late administration
NSAIDs ,especially COX-2 inhibitors, enhanced lung injuries in septic animals since
mononuclear phagocytes are differentially regulated at early and late phases of sepsis. Bacterial burden also increased with administration of NSAIDs.
四、計畫成果自評
Since the rat CLP model relates closely to the pathogenesis of events occurring in humans during sepsis, the data in the current study may have clinical relevance. Acute lung injury, as demonstrated by alterations of septic AM arachidonate metabolism in previous study, developed very early in hyperdynamic phase and progressively worsened through late hypodynamic phase. Prostanoids and LTB4 are important cellular mediators for normal organ functions. AM suffered from further apoptosis when given
NSAIDs during the processes of
sepsis-induced ALI. This likely contributes to the enhanced susceptibility of septic lung to further infection and the development of adult respiratory distress syndrome in sepsis subjects.
With the completion of this project (NSC90 － 2314 － B － 039 － 013), sepsis-induced acute lung injury, in terms of AM apoptosis and lung bacterial burden alterations, has been documented to deteriorate with the administration of cox-2
specific drugs and late
cox-1/cox-2-nonspecific drugs. Most of the work in my laboratory has been performed in accordance with the design set in the NSC proposal and the results will be helpful for the management of clinical patients and used for further studies. Furthermore, a paper is in preparation.
