An early event in the pathogenesis of emphysema is the development of inflammation associated with accumulation of polymorphonuclear leukocytes (PMN) in small airways, and inflammatory cell recruitment from the circulation involves migration across endothelial and epithelial cell barriers. Platelet-activating factor (PAF) promotes transendothelial migration in several vascular beds, and we postulated that increased PAF production in the airways of smokers might enhance inflammatory cell recruitment and exacerbate inflammation. To examine this possibility, we incubated human lung microvascular endothelial cells (HMVEC-L) with cigarette smoke extract (CSE) and found that CSE inhibits PAF-acetylhydrolase (PAF-AH) activity. This enhances HMVEC-L PAF production and PMN adherence, and adherence is blocked by PAF receptor antagonists (CV3988 or ginkgolide B). CSE also inhibited PAF-AH activity of lung endothelial cells isolated from wild-type (WT) and iPLA(2)β knockout mice, and with WT cells, CSE enhanced PAF production and RAW 264.7 cell adherence. In contrast, CSE did not affect PAF production or RAW 264.7 cell adherence to iPLA(2)β-null cells, suggesting that iPLA(2)β plays an important role in PAF production by lung endothelial cells. These findings suggest that inhibition of PAF-AH by components of cigarette smoke may initiate or exacerbate inflammatory lung disease by enhancing PAF production and promoting accumulation of inflammatory cells in small airways. In addition, iPLA(2)β is identified as a potential target for therapeutic interventions to reduce airway inflammation and the progression of chronic lung disease.

Rastogi P, Young DM, McHowat J. Tryptase activates calciumindependent phospholipase A 2 and releases PGE2 in airway epithelial cells.

To recruit inflammatory cells to the airways, cells must move from the circulation across the endothelial and epithelial cell barriers. Platelet‐activating factor (PAF) is involved in transendothelial migration in several vascular beds. We hypothesize that an increase in PAF production and cell recruitment may exacerbate inflammatory disease in smokers. We incubated human lung microvascular endothelial cells (HMVEC‐L) with cigarette smoke extract (CSE). CSE (20 μg/ml) significantly inhibited PAF‐acetylhydrolase (PAF‐AH, hydrolyzes and inactivates PAF) activity after 12 hours of CSE incubation (2.1 ± 0.1 to 0.7 ± 0.1 nmol/mg protein/min, n=6, p<0.01). Inhibition of PAF‐AH resulted in increased PAF production (538 ± 60 to 1580 ± 241 dpm, n=6, p<0.01) and PMN adherence (14 ± 3 to 47 ± 3%, n=4, p<0.01). Pretreatment of PMN with the PAF receptor antagonist CV3988 (10μM for 10 min) resulted in complete inhibition of PMN adherence (0.4 ± 0.1, n=4, p<0.01). In the lung, PMN accumulation in the small airways represents one of the initial steps in inflammation and emphysema. In conclusion, these studies have shown that PAF likely plays a central role in the recruitment of inflammatory cells to the small airways of cigarette smokers via inactivation of PAF‐AH and may exacerbate or initiate inflammatory lung disease.

Transmigration of hematopoietic progenitor cells across the vascular endothelium and subsequent maturation in the tissue helps to maintain the pool of resident mast cells. We determined the potential of CD133+ cells isolated from umbilical cord blood to migrate across an endothelial cell monolayer. CD133+ cells possess mRNA for several adhesion molecules that are critical in the process of neutrophil transmigration. Stem cell factor & its receptor CD117 are required for homing & differentiation of mast cells in the tissue. We detected the presence of CD34, CD117, CD29, & CD18 on the surface of CD133+ cells using flow cytometry. Tryptase stimulation leads to increased endothelial cell platelet activating factor (PAF) production that may facilitate the interaction between the endothelium & the PAF receptor (PTAFR) on circulating CD133+ cells. Treatment with the PTAFR antagonist CV3988 significantly reduced the percentage of transmigrating CD133+ cells across a tryptase stimulated endothelial monolayer. This suggests that CD133+ cell migration depends on the PAF/PTAFR interaction in a manner similar to that involved in neutrophil transmigration. Following transmigration there is the potential for a subset of these cells, in the presence of SCF & IL‐6, to mature into tissue mast cells.This work was supported by AHA Heartland Affiliate Grant # 0610118Z.

The presence of polymorphonuclear leukocytes (PMN) in the airways is a hallmark of many inflammatory conditions including ARDS, COPD and cystic fibrosis. In the event of a breach in the epithelial or endothelial cell barrier, PMN can migrate from the circulation to the airway lumen. This involves a series of steps including PMN adherence and transmigration through the vascular endothelium into the interstitial tissue, followed by a similar sequence of events used to cross the airway epithelium and enter the lumen. We have determined previously that stimulation of human small airway epithelial cells (HSAEC) with thrombin results in increased iPLA2 activity that is inhibited by the iPLA2 selective inhibitor bromoenol lactone (BEL). Further, activation of iPLA2 results in increased arachidonic acid and prostacyclin release. We also found that PMN adherence to thrombin‐stimulated HSAEC is increased 25 fold over adherence to unstimulated cells. Pretreatment with of HSAEC with BEL or PMN with CV3988, a PAF receptor antagonist, leads to inhibition of thrombin‐stimulated PMN adherence. This indicates that inhibition of iPLA2 or blocking the PAF receptor may be effective tools in limiting the cascade of neutrophil adherence, inflammatory mediator production and tissue destruction in cases of acute lung inflammation.