Annexin I is a 36 kilodalton (kD) calcium-dependent phospholipid-binding protein which may have anti-inflammatory properties. Previous investigations which sampled lower respiratory tract epithelial lining fluid (ELF) via bronchoalveolar lavage (BAL) have demonstrated that annexin I can be degraded in inflammatory lung disease. We analyzed BAL fluid from patients with cystic fibrosis (CF) to determine the effects of lung inflammation on the structure and activity of annexin I. Intact annexin I was absent in 17 out of 20 BAL fluid samples from patients with CF, due largely to degradation to a 33 kD protein. The three CF BAL fluids in which annexin I was detectable had very little or no unopposed neutrophil elastase activity in contrast to the 17 in which no annexin I was detectable. Annexin I was present in all BAL fluid samples from 10 normal volunteer (NV) subjects and 12 patients with interstitial lung disease (ILD). The 33 kD annexin I breakdown product was not detectable in samples from NV, but was detectable only in ILD patients with relatively high percentages of neutrophils on BAL differential cell counts. Annexin I appeared to be cleaved by neutrophil elastase at the N-terminal portion between Val-36 and Ser-37 to yield the 33 kD protein. Cleavage of the N-terminal portion of annexin I was accompanied by a marked change in the annexin I isoelectric point (pI) value (from 6.0 to 8.5-9.0) and greatly diminished annexin I functional activity. Our findings demonstrate that annexin I degradation in epithelial lining fluid is closely related to lung inflammation.

Phosphatidylcholine (PtdCho) is a major membrane phospholipid, and its loss is sufficient in itself to induce cell death. PtdCho homeostasis is regulated by the balance between hydrolysis and synthesis. PtdCho is hydrolyzed by phospholipase A 2 (PLA 2 ), PtdChospecific phospholipase C (PtdCho-PLC), and phospholipase D (PLD). PtdCho synthesis is rate-limited by CTP:phosphocholine cytidylyltransferase (CCT), which makes CDP-choline. The final step of PtdCho synthesis is catalyzed by CDP-choline:1,2-diacylglycerol cholinephosphotransferase. PtdCho synthesis in the brain is predominantly through the CDP-choline pathway. Transient middle cerebral artery occlusion (tMCAO) significantly increased PLA 2 activity, secretory PLA 2 (sPLA 2 )-IIA mRNA and protein levels, PtdCho-PLC activity, and PLD2 protein expression following reperfusion. CDP-choline treatment significantly attenuated PLA 2 activity, sPLA 2 -IIA mRNA and protein levels, and PtdCho-PLC activity, but did not affect PLD2 protein expression. tMCAO also resulted in loss of CCT activity and CCT␣ protein, which were partially restored by CDP-choline. No changes were observed in cytosolic PLA 2 or calcium-independent PLA 2 protein levels after tMCAO. Up-regulation of PLA 2 , PtdCho-PLC, and PLD and downregulation of CCT collectively resulted in loss of PtdCho, which was significantly restored by CDP-choline treatment. CDP-choline treatment significantly attenuated the infarction volume by 55 ؎ 5% after 1 h of tMCAO and 1 day of reperfusion. Taken together, these results suggest that CDP-choline significantly restores PtdCho levels by differentially affecting sPLA 2 -IIA, PtdCho-PLC, and CCT␣ after transient focal cerebral ischemia. A hypothetical scheme is proposed integrating results from this study and from other reports in the literature.Focal cerebral ischemia or stroke is characterized by an obstruction of blood flow to the brain, resulting in disruption of the glucose and oxygen that supply the brain's energy needs. Energy failure results in rapid loss of ATP and uncontrolled leakage of ions across the cell membrane, causing membrane depolarization and release of neurotransmitters such as glutamate and dopamine (1, 2). Excess glutamate release and stimulation of its receptors result in activation of phospholipases (3)(4)(5)

Background The function of sPLA2 is site dependent. In tissue, sPLA2 regulates eicosanoid production; in the blood, sPLA2 primes neutrophils; and in the intestinal lumen sPLA2 provides innate bactericidal immunity as a defensin-related protein. Since parenteral nutrition (PN) with lack of enteral stimulation primes leukocytes while suppressing intra-luminal mucosal immunity, we hypothesized that 1) PN would diminish luminal sPLA2 activity, but increase sPLA2 activity in small intestinal (SI) tissue and serum and 2) stress would accentuate these changes. Methods Mice received Chow, Complex Enteral Diet (CED), intragastric PN (IG-PN), or PN in Experiment 1, and Chow, Chow + Stress, PN, and PN + Stress in Experiment 2. Tissue, intestinal luminal fluid, and portal and systemic serum were analyzed for sPLA2 activity. IgA was measured in luminal fluid as a marker of acquired mucosal immunity. Results Expt1 Luminal fluid sPLA2 activity was greatest in Chow and decreased in CED (p=0.0001), IG-PN (p=0.0002), and PN (p=0.0001) with PN lower than CED (p<0.002) or IG-PN (p<0.0001). Compared to Chow, serum sPLA2 activity dropped after CED (p = 0.042), IG-PN (p<0.0001), and PN (p=0.0004). Serum sPLA2 was higher in portal than systemic serum (p=0.04). Expt2 PN lowered luminal fluid sPLA2 activity vs Chow (p<0.0001). Stress lowered luminal sPLA2 activity in Chow (p<0.0001), without a change with PN. Following stress, luminal IgA increased in Chow (p=0.0025) but not PN (p=0.18). Serum sPLA2 activity was unchanged after Chow but increased in PN (p<0.03). Conclusions Parenteral nutrition with lack of enteral stimulation attenuates sPLA2 activity in intestinal fluid consistent with a suppressed innate mucosal defense. Stress suppresses luminal fluid sPLA2 activity in Chow, but not the IgA response: PN impairs both. Stress significantly elevates serum sPLA2 in PN fed mice consistent with the known increased neutrophil priming with PN. PN reduces innate bactericidal immunity of the gut but up-regulates serum pro-inflammatory products after stress.

No abstract