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1University of South Alabama Comprehensive Sickle Cell Center, and Departments of 2Medicine, 3Pharmacology, 4Pathology, 5Microbiology, and 6Cell Biology and Neuroscience, University of South Alabama College of Medicine, Mobile, Alabama
Submitted 13 March 2006 ; accepted in final form 11 May 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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zileuton; microvascular occlusion; pulmonary circulation
Adhesion molecules and their ligands on the RBC membrane surface, on EC, and in plasma have been implicated in the process of increased SRBC adhesion to vascular endothelium. Receptors on the RBC membrane implicated in this process include the integrin 
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1 and the thrombospondin receptor CD36 (5, 13, 22, 37, 38). EC receptors involved are the integrin V3, VCAM-1, glycoprotein Ib, and CD36 (13, 18, 25, 26, 35, 37, 38). Potential ligands in plasma and in the endothelial matrix are thrombospondin, laminin, fibronectin, fibrinogen, and von Willebrand factor (4, 15, 25, 41). Another potentially significant membrane property that contributes to SRBC adhesion to vascular endothelium is the presence of phosphatidylserine (PS) on the external surface of the SRBC membrane (PS-exposed) (30, 34). Measurement of erythrocyte membrane PS asymmetry in sickle and normal erythrocytes using the calcium-dependent phospholipid-binding protein annexin V is well established (7, 27, 30, 39, 43). Setty et al. (34) have reported that PS-exposed SRBCs exhibit increased adhesion to EC monolayers that can be blocked by annexin V. The PS role in SRBC-EC adhesion is of particular interest because every erythrocyte could potentially be involved. This is in contrast to 41, which is expressed on stress reticulocytes, and CD36, which is expressed on reticulocytes and in low levels on mature RBCs (36).
Turhan et al. (40) have recently introduced a new model of SRBC-leukocyte adhesion in vasoocclusion. With the use of intravital microscopy in mice expressing human sickle hemoglobin, vasoocclusion was demonstrated when SRBCs transiently bound leukocytes that were adherent to inflamed venules. The mechanism by which SRBCs bind to leukocytes was not elucidated, but mice expressing human sickle hemoglobin deficient in P- and E-selectins were protected from vasoocclusion.
In this study, we demonstrate that ANs increase SRBC retention/adhesion in the pulmonary circulation through a mechanism that involves increasing the number of PS-exposed SRBCs. We also show that this effect can be blocked with the 5-lipoxygenase inhibitor zileuton, suggesting involvement of the 5-lipoxygenase pathway in this phenomenon.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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For flow cytometry analyses, phycoerythrin (PE)-conjugated, anti-glycophorin A (anti-GPA) antibody and the isotypic negative control antibody were purchased from Immunotec (Beckman-Coulter, Miami, FL). Annexin V-FITC was purchased from R&D Systems (Minneapolis, MN). In studies performed in isolated lung, [51Cr]sodium chromate was purchased from New England Nuclear (Boston, MA). Unlabeled annexin V (used to cloak erythrocyte phosphytidylserine), PMA (used to stimulate neutrophils), and BSA (66,000 mol wt) were purchased from Sigma Immunochemicals (St. Louis, MO). Zileuton was purchased from the University of South Alabama Pharmacy (Mobile, AL). PMA was dissolved in DMSO, and zileuton was dissolved in 95% ethanol.
Blood Collection
All participants gave informed consent and the Institutional Review Board approved the protocol. During steady state, blood samples (
40 ml) were obtained from normal (hemoglobin A) human volunteers and from individuals with homozygous sickle cell anemia in the sickle cell clinics at the University of South Alabama. Samples were collected in syringes containing heparin sodium and utilized within 24 h of collection. Individuals who had received blood transfusions within the previous 6 wk were excluded from this study. Sickle cell anemia was documented by hemoglobin electrophoresis in each subject.
Flow Cytometric Analysis of PS on Erythrocytes
The presence of cell surface PS was quantitated on RBCs with a FACSVantage SE flow cytometer (BD Biosciences, San Jose, CA) using 110 mW of the 488-nm line of an air-cooled argon laser (Spectra-Physics, Mountain View, CA). A 560-nm short pass dichroic mirror is placed in front of the photomultiplier tubes (PMT) PMT-1 and PMT-2 that detect fluorescence from FITC and PE. Annexin V-FITC fluorescence is detected with PMT-1 using a 530/30-nm bandpass filter. RBCs were identified with PE-conjugated, anti-GPA antibody. PE-anti-GPA fluorescence was detected with a 585/32-nm bandpass filter in front of PMT-2.
A minimum of 50,000 cells were analyzed, and gates for viable cells were determined by forward and right angle light scatter. Approximately 98% of the cells within this gate were RBCs by GPA staining. Cells positive for both GPA and annexin V-FITC in a two-color bivariate dot plot were classified as PS-exposed RBCs. The percentage of annexin V positive RBCs in each sample was determined by dividing the number of double-positive (GPA and annexin V) cells by the total number of GPA-positive cells. CELLQuest (BD Biosciences) software was used for data acquisition and analysis.
RBC Isolation and 51Cr Labeling
Each whole blood sample was centrifuged at 4,100 rpm for 5 min, followed by the removal of the plasma and buffy coat. Packed RBCs were then mixed at a 1:3 dilution with normal saline and washed three times. With each wash, the RBC suspension was centrifuged for 10 min at 3,000 rpm, followed by the removal of the supernatant. After the third wash, complete removal of platelets and polymorphonuclear neutrophils was verified by microscopic examination of stained smears. Packed RBCs were resuspended in PBS (1 ml PBS and 1 ml packed RBCs) and incubated with 100 µCi 51Cr for 60 min in a shaker water bath at 37°C. After incubation, the 51Cr-labeled RBCs were washed once with normal saline and resuspended in physiological salt solution (PSS)-BSA to obtain a hematocrit of 10%.
Neutrophil Isolation and Activation
Heparinized whole blood (20 ml) from individuals with homozygous sickle cell anemia was diluted 1:1 with normal saline. Neutrophils were then isolated after dextran sedimentation and centrifugation on histopaque 1077 cushions as described previously (3). Briefly, Dextran 500 was added to the 1:1 mix at one-fourth the volume of the 1:1 mix. This was allowed to incubate for 45 min at 37°C. The supernatant (containing neutrophils) was aspirated into a 50-ml centrifuge tube and underlayered with 10-ml histopaque 1077. This was centrifuged at 1,250 rpm for 30 min. The supernatant was aspirated and discarded, leaving the neutrophil pellet. Sterile H2O (3 ml) was added to the neutrophil pellet and agitated for 30 s to lyse any residual RBCs. PBS (45 ml) was added immediately thereafter to stop the reaction. This was centrifuged at 1,250 rpm for 10 min. The supernatant was aspirated and discarded. This process of RBC lysis, followed by PBS, was repeated until the neutrophil pellet was without visual evidence of RBC contamination. The pellet was suspended in 1 ml of sterile HBSS. A total neutrophil count was obtained by using the Cell-DYN 900 Coulter counter. Final preparations contained 95% neutrophils, and viability was >95% as assessed by trypan blue exclusion. In studies utilizing ANs, PMA (20 ng/ml) of cells in 1 ml of HBSS for 30 min was used to activate neutrophils in the test tube before being added to RBC suspensions used in flow cytometric analysis and in the isolated lung. In all studies, the AN concentration was 200,000/ml.
Isolated Perfused Lung
Male Sprague-Dawley rats (275–350 g) were anesthetized with pentobarbital sodium (25 mg ip; Nembutal), and lungs were removed for extracorporeal perfusion as previously described (17, 31). A tracheostomy was performed that permitted ventilation with a Harvard rodent ventilator (model 683) at 55 breaths/min with a tidal volume of 2.5 ml and 2.0 cmH2O-positive end-expiratory pressure. The inspired gas mixture was 21% O2-5% CO2-balance N2 (room air gas). A median sternotomy was performed, heparin sodium (100 IU) was injected in the right ventricle, and cannulas were placed in the pulmonary artery and left ventricle. Heart, lungs, and mediastinal structures were removed en bloc and placed into a humidified chamber. Lungs were perfused with the use of a Gilson Minipuls 2 peristaltic pump at a constant flow of 0.03 ml·g body wt–1·min–1. Lungs were perfused with a PSS containing BSA. The PSS-BSA perfusate contained (in mM) 119 NaC1, 4.7 KC1, 1.17 MgSO4, 22.5 NaHCO3, 1.18 KH2PO4, 3.2 CaC12, and 5.5 glucose and 4 g/100 ml BSA. PSS-BSA (100 ml) was perfused through the lungs in a nonrecirculating fashion to remove residual blood cells and plasma. The perfusate was then changed to a PSS-BSA perfusate that contained RBCs from individuals homozygous for sickle cell anemia. The perfusate hematocrit was 10%. Pulmonary arterial pressure and pulmonary venous pressure were continuously monitored with pressure transducers (model 041–500-503, Cope) and recorded on a polygraph recorder (model 7E, Grass Instruments). Zone 3 flow conditions (arterial > venous > alveolar pressures) were maintained throughout all experiments.
RBC Retention
Cell counts of the 51Cr-labeled RBC suspension were obtained in a ZM Coulter counter. The specific activity of the 51Cr-labeled RBC perfusate was calculated after gamma counts at 162–176 Kev for 3 min and expressed as counts per minute (cpm)/RBC. The isolated rat lung was then perfused with the 51Cr-labeled RBC perfusate for 30 min. After this, the lung was washed with 50 ml of RBC-free PSS-BSA under identical hemodynamic conditions. Perfusion was stopped, and the lung was dissected into lobes and weighed. Each lobe was gamma counted for 3 min. Retention (R) was calculated as previously described (16): R = (specific activity of lung)/(specific activity of perfusate); R = (cpm/gram of lung)/(cpm/RBC); and R = (retained RBC)/(gram of lung). In the isolated perfused lung, the increased retention of SRBCs can occur from increased adhesion to vascular endothelium or secondary to mechanical obstruction. Thus, throughout this study, the number of retained SRBCs per gram per lung is referred to as retention/adherence.
Light Microscopy
Isolated rat lungs were perfused as described above [SRBC alone, SRBC + unstimulated neutrophils (UN), SRBC + AN, SRBC + zileuton (50 µM) pretreated-AN, or SRBC + AN + Annexin V; see Specific Protocols]. Lungs were then washed with 50 ml of RBC-free PSS-BSA under zone 3 conditions, followed by perfusion fixation with 3% glutaraldehyde in 0.1 M cacodylate buffer, and immersed in fixative. Lungs were next cut into 1-mm slices, rinsed in cacodylate buffer, postfixed for 1 h with 1% osmium tetroxide, dehydrated through a graded alcohol series, and embedded in PolyBed 812 resin (Polysciences, Warrington, PA). Thick sections (1 µm) from the embedded tissue were cut with glass knives and stained with 1% toluidine blue. Eight thick sections from each rat were examined by light microscopy. RBCs and neutrophils in a x40 field (Olympus light microscope) on each thick section were counted and recorded. Cells in the microvasculature (septal capillaries) were counted. Fields with large vessels were excluded.
Statistics
All results are presented as means ± SE. Statistical analyses were performed using the unpaired t-test and one-way ANOVA. Tukey's test (44) was used for multiple comparisons when ANOVA indicated statistically significant differences between or within groups. Differences were considered to be significant when P < 0.05.
Specific Protocols
Comparison of PS-exposed normal and sickle RBCs in the absence and presence of ANs. With the use of flow cytometry as described in MATERIALS AND METHODS, double-labeling studies were performed with the calcium-dependent phospholipid-binding protein annexin V-FITC (25 µg/ml) fluorescence and the erythroid-specific marker GPA (12 µg/ml) to assess the number of PS-exposed RBCs relative to the total RBC population (expressed as %positive RBC) in the absence and presence of ANs. The following comparisons were made: 1) normal RBC alone versus normal RBC + AN (n = 4 each) and 2) SRBC alone versus SRBC + AN versus SRBC + zileuton pretreated-AN (2.5 µM; n = 11 each) versus SRBC + zileuton pretreated-AN (50 µM; n = 7). In studies using zileuton, neutrophils were incubated with zileuton for 30 min before activation with PMA in the test tube. The zileuton concentrations used were based on previous findings reported by Guidot et al. (14), Haynes and Obiako (16), and studies performed in our laboratory demonstrating a 50% reduction of leukotriene B4 at 2.5 µM and a 95% reduction of leukotriene B4 at the 50 µM in A-23187-stimulated whole blood from individuals with sickle cell anemia. The effect of zileuton on SRBC PS externalization was assessed based on a previous study (16), which demonstrated that zileuton attenuates AN mediated retention/adherence of SRBCs in isolated perfused lung.
Effects of annexin V cloaking on SRBC retention/adherence in isolated perfused rat lung.
51Cr-labeled SRBCs from nine donors were individually pretreated with annexin V (to cloak cell surface PS), 40 µg/ml of packed SRBCs, for 45 min at 37°C. This concentration of annexin V was based on previous findings reported by Setty et al. (34). After the incubation period, packed SRBCs were washed once with normal saline and then resuspended in PSS-BSA to obtain a hematocrit of 10%. SRBC suspensions from five donors were used to compare retention/adherence in perfused lung at baseline (SRBC alone) to retention/adherence of SRBCs treated with annexin V. SRBC suspensions from four other donors were used to compare retention/adherence in lung perfused with SRBCs alone, SRBC + AN, and SRBC + AN + annexin V. Retention was determined as described in MATERIALS AND METHODS, and comparisons were made.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The retention/adherence of SRBCs in isolated perfused rat lung is 3.5 times greater than that seen in lung perfused with normal (hemoglobin A) human RBCs under basal conditions (16). Therefore, we looked at whether differences in PS externalization between sickle and normal RBCs could serve as the bases for the differences in retention/adherence previously reported. The number of PS-exposed SRBCs was significantly higher than that seen in normal RBCs at baseline (before being exposed to ANs). The percentage of PS-exposed normal RBCs and SRBCs was 1.4 ± 0.4% and 5.8 ± 0.6%, respectively (P = 0.006).
Neutrophil Activation Results in Increased PS Externalization in Normal and SRBCs
Infections are known triggers of sickle cell vasoocclusive events, and neutrophil activation occurs with infection. Furthermore, neutrophils activated with PMA and supernatant from ANs increase SRBC retention/adherence in isolated perfused lung compared with SRBC control (16). This led to the question: does neutrophil activation and/or its soluble products increase the externalization of RBC PS? Coincubation of normal RBCs with ANs resulted in an approximate threefold increase in the percentage of PS-exposed cells (Fig. 1). Note that the percentage of PS-exposed normal RBC + AN group (4.7 ± 0.04%) does not differ significantly from that seen with the SRBC control (5.8 ± 0.6%).
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PS-exposed SRBCs exhibit increased adhesion to EC monolayers. This process can be blocked by the calcium-dependent phospholipid-binding protein annexin V (34). This along with the previous experiments suggested PS may play a role in AN-mediated increases in SRBC retention/adherence previously reported in isolated perfused lung (16). In lungs perfused with SRBCs alone compared with lungs perfused with SRBCs cloaked with annexin V, the number of retained SRBCs as expressed in RBC per gram of lung was 4.2 ± 0.1 x 108 and 2.0 ± 0.4 x 108, respectively (Fig. 3). Cloaking the PS-exposed SRBCs resulted in a 52% reduction in retained cells. The magnitude of retention seen with cloaked SRBCs is comparable to findings we have previously reported that assessed normal human RBC retention in perfused isolated lung (16).
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Retention of SRBCs in the isolated perfused rat lung was analyzed by histological sections in the following treatment groups: SRBC alone (n = 6; 48 sections); SRBC + UN (n = 3; 24 sections); SRBC + AN (n = 8; 64 sections); SRBC + zileuton-pretreated AN (n = 4; 32 sections); and SRBC + AN + annexin V (n = 4; 32 sections). No significant difference in SRBC retention was seen between lungs perfused with SRBCs alone and SRBCs + UN. In the presence of ANs, SRBC retention in isolated perfused rat lung was 2.2 times greater than that seen in lungs perfused with SRBCs alone (P < 0.001) and 1.9 times greater than that seen in lungs perfused with UN (P < 0.001; Fig. 5A). Pretreatment of neutrophils with zileuton before activation with a phorbol ester significantly decreased in the number of RBCs retained compared with that of the SRBC + AN group (P < 0.001). Similarly, the addition of annexin V (binding phosphatidylserine exposed cells) to the perfusate significantly decreased AN-mediated increased retention of SRBCs in isolated perfused rat lung compared with that of the SRBC + AN group (P < 0.01; Fig. 5A).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The major findings in this study are the following: 1) ANs increase PS externalization in normal RBCs and SRBCs; 2) the 5-lipoxygenase inhibitor zileuton attenuates AN-mediated increases in PS-exposed SRBCs and decreases SRBC retention in lung on histological evaluation; 3) retention/adherence of SRBCs, cloaked with annexin V in isolated perfused lungs, is attenuated under basal conditions and in association with neutrophil activation; and 4) annexin V attenuates AN-mediated SRBC retention when assessed using histological lung sections.
The previous study by Haynes and Obiako (16), which demonstrated that ANs increase SRBC retention/adherence in isolated perfused rat lung, provides proof of principle that AN-SRBC-EC interactions could play a major role in furthering our understanding of events that initiate microvascular occlusion. As opposed to the systemic circulation, the role that neutrophils play may be of even greater importance because the concentration of neutrophils in pulmonary capillary blood is 40–80 times than that seen in the blood of large vessels (1, 9). Whereas soluble products in the supernatant of ANs mimic increased SRBC retention/adherence seen with neutrophils activation (16), the end effect on the SRBCs, which renders the cell more adhesive to vascular endothelium, has not been elucidated. In the current study, we demonstrated that activated neutrophils increase the number of PS-exposed cells in normal and SRBCs. This is a potentially important observation because PS externalization on SRBCs enhances their ability to adhere to vascular endothelium (6, 30, 34). In this study, the percentage of PS-exposed RBCs reported from normal patients and individuals with sickle cell anemia closely parallels findings previously reported by Wood et al. (42). Wood et al. point out that the percentage of PS-exposed cells may vary substantially over time in a patient and between patients studied. Of note in this study is that under basal conditions, the percentage of PS-exposed SRBCs was approximately four times that seen in normal RBCs and that the mean increase in PS-exposed normal RBCs after coincubation with ANs was not different from that seen under basal conditions in SRBCs. This finding clearly demonstrates that differences exist in the susceptibility of the SRBC membrane to the ANs and/or its products compared with normal RBCs. Although the exact cause for the perturbation of phospholipid asymmetry in the SRBC membrane was not assessed, possible explanations include an increase in cytosolic calcium, which induces phospholipid scrambling (8); oxidative cross-linking of membrane proteins (32); or in deoxygenated SRBCs, an uncoupling of the lipid bilayer from the underlying skeleton at the tip of the SRBC spicules (11, 12, 29).
In this study, histological evaluation of lung sections revealed increased numbers of retained neutrophils in all groups perfused with ANs compared with lung sections from the SRBC control group. Interestingly, neutrophil retention in lung sections from studies using ANs did not differ significantly from lungs perfused with UN. This suggests that in the UN group, the neutrophils were activated to some degree before treatment with PMA. It is highly probable that the neutrophils were activated somewhat during isolation. Furthermore, Lard et al. (28) have demonstrated that the basal state of activation of the neutrophils from individuals with sickle cell anemia is higher than that seen in normal controls. In addition, Boghossian et al. (2) and Fadlon et al. (10) have demonstrated that during sickle cell pain crisis, the neutrophil is often more adherent to vascular endothelium than during steady state. These observations suggest that varying states of neutrophil activation occur in sickle cell anemia and support the histological findings reported in this study.
During neutrophil activation, phospholipase A2 and 5-lipoxygenase activities are increased (2, 33), which results in membrane phospholipid metabolism. Of the potential products of membrane phospholipid metabolism in the human neutrophils, the 5-lipoxygenase product leukotriene B4 is the principal product (14). Our previous work demonstrated that 5-lipoxygenase inhibition in neutrophils treated with zileuton before stimulation with PMA attenuates AN-mediated retention of SRBCs in perfused lung (16). Thus we asked the questions: does zileuton inhibit AN-mediated PS externalization on SRBC membranes, and does zileuton decrease AN-mediated SRBC retention when assessed by direct histological assessment of lung sections? What we found was that zileuton (50 µM) does attenuate AN-mediated SRBC PS externalization, but only a downward trend was seen at the 2.5 µM concentration. Zileuton (50 µM) also attenuated AN-mediated SRBC retention, assessed by using lung slices. This finding supports previous work (16) using 51Cr-labeled SRBC in the isolated perfused lung.
After the demonstration that PS-exposed SRBCs were present under basal conditions and the percentage of PS-exposed SRBCs increased by 122% above baseline after coincubation with ANs, studies were designed to assess the functional significance of PS-exposed erythrocytes by evaluating SRBC retention/adherence in isolated perfused rat lung (see MATERIALS AND METHODS). Blocking studies were performed by using SRBCs pretreated with annexin V to cloak externalized PS. In isolated perfused lung, retention/adherence of annexin V-treated SRBCs under basal conditions resulted in a 52% reduction in retained SRBCs (Fig. 3). This reduction in retention/adherence is most likely a result of subpopulations of PS-exposed SRBCs, of which their PS was cloaked by annexin V (27, 30, 43). A second possible explanation is that even during steady state, some basal level of neutrophil activation exists as suggested by Lard et al. (28), resulting in higher than normal numbers of PS-exposed cells. Similar to studies done under basal conditions, increased retention/adherence of SRBCs mediated by ANs was completely blocked when SRBCs were treated with annexin V, and the decrease in SRBC retention/adherence also fell below the SRBC control (Fig. 4). Direct histological evaluation of lung slices confirmed that SRBC retention/adherence was significantly reduced below that seen in the SRBC + AN group when SRBCs were pretreated with annexin V (Fig. 5A). Unlike studies comparing retention/adherence in isolated perfused lung, the number of retained SRBCs in lung tissue did not decrease below SRBC control when pretreated with annexin V. A probable explanation for the difference seen in perfused lung versus lung tissue is that the use of 51Cr-labeled SRBCs to measure cell retention is more sensitive and reflects whole lung SRBC retention in contrast to light microscopy that reflects simply the average number of retained cells observed from the sampling of lung slices. What also must be considered is that ANs may increase SRBC adhesion to vascular endothelium through mechanisms not involving PS-exposed SRBCs (5, 13, 22, 37, 38, 40). The reported findings support our hypothesis that ANs mediate increased SRBC adhesion to EC at least in part through increasing PS externalization on the RBC membrane outer leaflet.
In conclusion, this study provides strong evidence supporting the hypothesis that ANs mediate SRBC adhesion to vascular endothelium by altering erythrocyte membrane phospholipid asymmetry with resultant PS externalization. In addition, this study provides a plausible common pathway as to how infectious and inflammatory insults in the lung may trigger vasoocclusion and the acute chest syndrome. Although no clinical data are available to support that 5-lipoxygenase inhibitors are of clinical importance in sickle cell disease, this and a previous study (16) suggest 5-lipoxygenase inhibitors may be therapeutically beneficial in the prevention and treatment of sickle vasoocclusive disease.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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41 and glycoprotein IV (CD36) are expressed on circulating reticulocytes in sickle cell anemia. Blood 82: 3548–3555, 1993.v3 (7E3 and LM609) inhibit sickle red blood cell-endothelium ineractions induced by platelet-activating factor. Blood 95: 368–374, 2000.41 Integrin expression on sickle erythrocytes: vascular cell adhesion molecular-1 dependent binding to endothelium. Blood 82: 1891–1899, 1993.This article has been cited by other articles:
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  J. Haynes Jr., B. Obiako, R. B. Hester, B. S. Baliga, and T. Stevens Hydroxyurea attenuates activated neutrophil-mediated sickle erythrocyte membrane phosphatidylserine exposure and adhesion to pulmonary vascular endothelium Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H379 - H385. [Abstract] [Full Text] [PDF]
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P < 0.05 compared with SRBC group; 
P < 0.01 compared with SRBC control; 
P < 0.01 compared with SRBC + AN. B: number of retained neutrophils in a x40 field from same treatment groups defined in A. Number of retained neutrophils in the SRBC + AN, SRBC + ZL-pretreated AN, and SRBC + AN + AnV treatment groups was significantly increased compared with SRBC control (*P < 0.01). No difference in number of retained neutrophils was seen between any of the treatment groups associated with the addition of neutrophils.