Barnett C C, Moore E E, Moore F A, Biffl W L, Partrick D A
Department of Surgery, Denver General Hospital, Colo.
Surgery. 1996 Aug;120(2):395-401; discussion 401-2. doi: 10.1016/s0039-6060(96)80315-1.
Elevated levels of soluble intercellular adhesion molecule-1 (sICAM-1) correlate with the development of postinjury multiple organ failure. Soluble ICAM-1 secretion is known to be induced in endothelial cells and monocytes by diverse inflammatory stimuli. We have found that incubation of quiescent polymorphonuclear leukocytes (PMNs) with sICAM-1 elicits elastase release and, more recently, that cross-linking CD18 receptors on PMNs also produces elastase release. Consequently, our study hypothesis was that sICAM-1 provokes PMN elastase release through its interaction with CD18.
To obtain sICAM-1, Chinese hamster ovarian cells transfected with human ICAM-1 were lysed and centrifuged at 150,000 g for 1 hour; the supernatant was passed over an ICAM-1 affinity column, eluted with 0.1 mmol/L glycine HCl, and concentrated with dialysis filter. Human PMNs (2.5 x 10(5)) were saturated with specific monoclonal antibodies for the beta 2 subunits (CD11a, CD11b, CD18) or nonspecific monoclonal antibodies for 30 minutes on ice before a 1-hour incubation with sICAM-1 (75 ng/ml) at 37 degrees C. Elastase activity was measured by the cleavage of n-methoxysuccinyl-A-A-P-V-p-nitroanilide.
Neutrophil incubation with sICAM-1 resulted in 19.2% +/- 2.8% of total PMN elastase, compared with 2.4% +/- 0.5% in the controls. Blockade of CD18 abrogated sICAM-1 provoked elastase release with monoclonal antibodies to CD18 (TS1/18, 31H8) resulting in 4.3% +/- 1.0% and 5.5% +/- 1.4% elastase release, respectively. Blockade of CD11a, CD11b, and nonspecific antibody controls had no effect on sICAM-1 induced elastase release.
In vitro, sICAM-1 provokes PMN elastase release through CD18. This may represent a mechanism by which elevated levels of circulating sICAM-1, released from local injury sites, provoke distal organ dysfunction.
可溶性细胞间黏附分子-1（sICAM-1）水平升高与损伤后多器官功能衰竭的发生相关。已知多种炎症刺激可诱导内皮细胞和单核细胞分泌可溶性ICAM-1。我们发现，将静止的多形核白细胞（PMN）与sICAM-1孵育可引发弹性蛋白酶释放，最近还发现，PMN上的CD18受体交联也会产生弹性蛋白酶释放。因此，我们的研究假设是，sICAM-1通过与CD18相互作用引发PMN弹性蛋白酶释放。
为获得sICAM-1，将转染了人ICAM-1的中国仓鼠卵巢细胞裂解，并在150,000 g下离心1小时；将上清液通过ICAM-1亲和柱，用0.1 mmol/L甘氨酸盐酸盐洗脱，并用透析滤器浓缩。人PMN（2.5×10⁵）在冰上用针对β2亚基（CD11a、CD11b、CD18）的特异性单克隆抗体或非特异性单克隆抗体饱和30分钟，然后在37℃下与sICAM-1（75 ng/ml）孵育1小时。通过裂解n-甲氧基琥珀酰-A-A-P-V-对硝基苯胺来测量弹性蛋白酶活性。
与对照组的2.4%±0.5%相比，PMN与sICAM-1孵育导致总PMN弹性蛋白酶释放19.2%±2.8%。用针对CD18的单克隆抗体（TS1/18、31H8）阻断CD18可消除sICAM-1引发的弹性蛋白酶释放，分别导致弹性蛋白酶释放4.3%±1.0%和5.5%±1.4%。阻断CD11a、CD11b和非特异性抗体对照对sICAM-1诱导的弹性蛋白酶释放无影响。
在体外，sICAM-1通过CD18引发PMN弹性蛋白酶释放。这可能代表了一种机制，通过该机制，从局部损伤部位释放的循环sICAM-1水平升高会引发远端器官功能障碍。
