1. Combined activities of secretory phospholipases and eosinophil lysophospholipases induce pulmonary surfactant dysfunction by phospholipid hydrolysis 
Mark A. Kwatia, MS, Christine B. Doyle, BA, Wonwha Cho, PhD, Goran Enhorning, MD, PhD, Steven J. Ackerman, PhD 
Journal of Allergy and Clinical Immunology 
Volume 119, Issue 4, Pages (April 2007)
DOI: /j.jaci
Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2. Fig 1
Mechanism of surfactant DPPC hydrolysis by the sequential actions of eosinophil-expressed phospholipases and lysophospholipases. Western blot analyses of human and murine eosinophil expression of the 25-kd LPLase (A) and group IIA sPLA2 enzymes (B). The soluble fraction of whole cell sonicates of purified human blood and AML14.3D10 eosinophils, purified blood eosinophils from IL-5 transgenic mice, along with recombinant human 25-kd LPLase, group IIA sPLA2, and synovial fluid as positive controls, were analyzed by SDS-PAGE and Western blotting. C, DPPC is initially cleaved to LPC and free palmitic acid by sPLA2's expressed by eosinophils or other inflammatory or resident cells in the airways, for example, epithelial cells. The LPC product is then hydrolyzed by eosinophil LPLases, generating glycerophosphocholine (GPC) and free palmitic acid, cleavage products incapable of maintaining minimal surfactant surface tension and surfactant function at the air-liquid interface in the small terminal airways.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3. Fig 2
Effect of the combined hydrolytic activities of PLA2 and LPLase on surfactant function. CLSE (4 mg/mL) was incubated with bee venom PLA2, PLB Vibrio sp (LPLase), or both at 37°C for the indicated reaction times. Surfactant activity was measured in triplicate using the capillary surfactometer. Mean surfactant function (± SD, n = 3), expressed as % openness, is plotted.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4. Fig 3
Whole cell lysates of AML14 eosinophil lines and blood eosinophils induce dose-dependent losses in surfactant function that are enhanced by the addition of exogenous PLA2. A, Dilutions of whole cell lysates prepared from equivalent numbers (1 × 107 cells) of AML14 myeloblasts and AML14.3D10 eosinophil myelocytes, along with U/mL PLA2, were added to 4 mg/mL CLSE and incubated at 37°C for 1 hour. Samples were assayed for surfactant activity by CS and mean surfactant function (±SD, n = 3), expressed as % openness, is plotted. AML14.3D10 (B and C) or normal donor blood eosinophil (D and E) whole cell lysates were added to 4 mg/mL of CLSE containing 200 μg/mL BSA and incubated at 37°C for 1 hour. Controls contained additional BSA, added at a protein concentration equivalent to the AML14.3D10 or blood eosinophil lysate total protein added. The CLSE samples were incubated without (B and D) or with (C and E) the addition of bee venom PLA2 (0.003 U). Mean surfactant function (±SD, n = 5) as measured by CS is plotted (∗∗∗P ≤ .001).
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5. Fig 4
Human 25-kd LPLase requires exogenous PLA2 to induce maximal loss of surfactant function. Human 25-kd LPLase expressed in Escherichia coli was purified and tested for its ability to induce surfactant dysfunction alone or in combination with PLA2. Recombinant hLPLase (100 μg/mL) was incubated with 4 mg/mL CLSE and 100 μg/mL BSA, without or with the addition of bee venom PLA2 (0.003 U) as indicated. Controls contained additional BSA added at a concentration equivalent to the amount of rLPLase and PLA2 protein added to the CLSE samples. Surfactant function was measured by CS. Mean surfactant function is shown as the % of the BSA control at each time point ± SD (n = 5; ∗P ≤ .05; ∗∗P ≤ .01).
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6. Fig 5
The combined activities of PLA2 and 25-kd eosinophil LPLase lead to essentially complete hydrolysis of surfactant PC and loss of surfactant function: analysis by TLC and CS. CLSE (4 mg/mL) was incubated without or with the addition of PLA2 and/or recombinant 25-kd LPLase. Samples were analyzed for lipid composition by TLC, including PA, PC, and LPC, after 15-minute or 4-hour reaction times at 37°C. The mobility of PA, PC, and LPC as resolved by TLC is indicated by the arrows, and the relative amount of [PC] for each sample is indicated above the PC bands. Samples were also analyzed for surfactant activity by CS at 15 minutes and 4 hours, and the mean surfactant function (% openness, n = 3) for the various reaction mixtures at these time points is at the top above each lane.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7. Fig 6
Proposed model for the role and functions of secretory phospholipases and LPLases in surfactant dysfunction and airway pathophysiology in asthma. Activated eosinophils recruited to the small peripheral airways secrete or induce secretion of phospholipases and LPLases whose combined hydrolytic activities are capable of efficiently hydrolyzing surfactant phospholipids to GPC and FA, leading to increased surfactant surface tension, loss of surfactant function, and loss of airway patency (closure/collapse of terminal bronchioles/alveoli). Type II epithelial cells lining the smallest airways likely contribute significant secretory phospholipase activity. The arrows with question marks (?) indicate potential collateral effects of surfactant phospholipid hydrolysis on airway function.
Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci )
Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions