Role Of Interleukin 18 In Acute Lung Injury Induced By Intestinal Ischemia/reperfusion And Characterization Of A New Isoform Of The Mouse Interleukin 18

Intestinal ischemia/reperfusion is a critical and triggering event in thedevelopment of distal organ dysfunction where the lung is the first organ affected andmay progress to acute respiratory dysfunction syndrome(ARDS). Respiratory failureis a common cause of death and complications after intestinal I/R. This study wasdesigned to investigate the effect of IL-18 on lung injury and possible mechanismfollowing intestinal I/R. A superior mesenteric artery occlusion(SMAO) model wasselected for this research. The mice were randomly divided into 3 groups: normalgroup, Sham operation(sham), and 30 min of superior mesenteric artery occlusionfollowed by different times(0.5 h to 3 h) of reperfusion(I/R group). For exogenousIL-18 studies or for IL-18 neutralization studies, animals were pretreated with eitherIL-18 or monoclonal anti-IL-18 antibody 15 min before ischemia. For the study onpulmonary microvascular leakage, Evans blue dye(EBD) was administrated justbefore ischemia. For the effects of inducible nitric oxide synthase(iNOS) on the roleof IL-18, the selective inhibitor of iNOS, L-NIL, was injected 15 min before ischemia.Serum IL-18 levels were detected by Western blot and ELISA, and the levels of IL-18in lung tissue were evaluated by immunohistochemical staining. The expression ofTNF-α was detected by RT-PCR and ELISA. Lung injury was evaluated by Evansblue dye(EBD) concentration, myeloperoxidase(MPO) activity and morphologicalanalysis. One hour after declamping and reperfusion, serum IL-18 concentrationsincreased to 660±153 pg/mL compared with 174±18 pg/mL before ischemia(p<0.01),and then declined to baseline after 3 h of reperfusion. In sham operated group, serumIL-18 concentrations did not increase from baseline concentrations. The levels ofIL-18 in lung tissue as shown by immunohistochemical staining were graduallyenhanced as the progress of reperfusion in I/R model. Compared with I/R model,exogenous administration of IL-18(I/R+IL-18) further remarkably enhanced thepulmonary MPO activity, pulmonary microvascular leakage and inflammatory cellinfiltration. Furthermore, exogenous administration of IL-18 in sham operated but notin normal animals, also induced severe lung injury. Treatment with antibodies toIL-18 before ischemia attenuated the pulmonary neutrophil sequestration, pulmonaryvascular leakage and lung injury. Further study revealed that IFN-γ may be notinvolved in the pathogenesis of lung injury following intestinal I/R and TNF-α maybe the mediator downstream the IL-18: exogeneous IL-18 obviously enhanced theexpression of TNF-α in sham operated and I/R model mice at both mRNA and proteinlevels, and inhibition of the activity of IL-18 down-regulated the serum levels ofTNF-α in I/R group. Selective inhibition of inducible nitric oxide synthase(iNOS)inhibited plasma extravasation and pulmonary injury without affecting the MPOactivity in all treated animals. These finding suggests that intestinalischemia/reperfusion induces the increase of IL-18 expression, which may makeIL-18 act as an important proinflammatory cytokine and contribute to intestinal I/Rinduced lung injury. Our results further suggested a role of IL-18 on the activation andsequestration of neutrophils in lung, and were consistent with the hypothesis thatpulmonary neutrophils sequestration probably in part via IL-18 and elevated iNOSactivity which mediating the increased microvascular leakage might synergicallycontributed to intestinal I/R-induced pulmonary dysfunction.Interleukin-18 is a novel proinflammatory cytokine with potentinterferon-γ inducing activity and plays an important biological role in theenhancement of the activity of natural killer cells and cytotoxic T lymphocytes. In thisstudy, we have identified a novel short form of IL-18 in mouse, termed mIL-18s.IL-18s may be an alternative splicing variant of IL-18 and its cDNA contains a 57bpin-frame deletion. Like IL-18, IL-18s is also widely expressed in mouse tissues. Intransient transfection assay, it was suggested that IL-18s experienced Caspase-1dependent mechanism for maturation and secretion similar to that of IL-18: whentransfected in COS-7 cells, a 22KDa proIL-18s could be detected, and the 16KDamature IL-18s could be detected in the cells and in the supernatants when combinedwith Caspase-1. Preliminary studies on the biological activities of IL-18s proteinswere performed. Recombinant mouse IL-18s did not show any IL-18-like function. Intesting whether IL-18s affects the activities of IL-18, we observed that IL-18s couldenhance the ability of IL-18 to induce IFN-γ by about 30% in mouse splenocytes. Thiseffect was observed primarily at relative low concentrations of IL-18(less than50ng/ml) suggesting that IL-18s may regulate the activity of IL-18 in thephysiological conditions.