Objective
Amniotic fluid embolism syndrome is a fatal disease in pregnant women. The exact role of platelets and neutrophils in amniotic fluid embolism syndrome is not clear. We examined whether amniotic fluid could affect platelet-neutrophil aggregation and activation and the possible mechanisms.
Study Design
Blood samples from the pregnant women were pretreated ex vivo with their own amniotic fluid. Flow cytometry was used to measure platelet-neutrophil aggregation and activation. Neutrophil-mediated activity of p38 mitogen-activated protein kinase and extracellular signal-regulated protein kinases 1 and 2 was analyzed by Western blotting.
Results
Amniotic fluid significantly induced platelet-neutrophil aggregation, neutrophil CD11b expression, and reactive oxygen species production. Amniotic fluid induced minimal platelet P-selectin expression. The increase of intracellular calcium level of neutrophils and the activity of p38 mitogen–activated protein kinase were enhanced by amniotic fluid stimulation.
Conclusion
Amniotic fluid was able to induce neutrophil activation and platelet-neutrophil aggregation with minimal effect on platelet activation. These findings may provide a new insight in the understanding of the pathophysiologic condition of amniotic fluid embolism syndrome.
Inflammation is part of the complex biologic response of vascular tissues to protect against harmful stimuli. Inflammation is considered to be a mechanism of innate immunity that is characterized by an initial neutrophil activation response and the recruitment of various cytokines from the circulation into the injured tissues. Platelets can assist and modulate inflammatory reactions and immune responses beyond their roles in hemostasis and thrombosis. When activated, platelets coaggregate with circulating neutrophils through P-selectin glycoprotein ligand-1 and P-selectin. Platelets and neutrophils have been demonstrated to regulate their mutual functions in reciprocal ways (ie, platelet-neutrophil crosstalk). Platelet-neutrophil aggregation plays a central role in many inflammatory reactions that include sepsis, coronary artery disease, and acute lung injury.
Amniotic fluid embolism syndrome (AFES; also called anaphylactoid syndrome of pregnancy), although rare, increases maternal morbidity and mortality rates. In patients with AFES, amniotic fluid (AF) probably enters the maternal circulation, which may precipitate coagulopathy, convulsions, respiratory failure, and shock. Although the exact pathophysiologic condition of AFES is still poorly understood, the inflammatory response that is associated with AF seems to be a major contributing factor and is supported by reports of increased inflammatory markers in patients with AFES. Furthermore, anaphylactic reactions may be another potential mechanism to elicit AFES. Neutrophils recently were demonstrated to play an important role in anaphylaxis beyond the classic reactions from immunoglobulin E, mast cells, and histamine. On the contrary, platelet activation has also been considered important in AFES development, which partly exhibits thromboembolic characteristics. Therefore, it remains enigmatic how AF influences neutrophil and platelet activity. We currently hypothesize that AF could induce inflammatory responses, which in turn contribute to AFES. We investigated the effects of AF on platelet-neutrophil aggregation and neutrophil/platelet activation and the involved mechanisms.
Materials and Methods
Reagents
Anti-CD42a (platelet GP IX), anti-CD62P, anti–CD42a-FITC (fluorescein isothiocyanate), anti-CD62P-PE (phycoerythrin), and anti-CD11b (integrin alpha-M) antibodies were from Becton Dickinson (San Jose, CA). 2′, 7′-dichlorodihydrofluorescein di-acetate (H 2 DCF-DA) and fluo-3 acetoxymethyl ester (fluo-3 alpha-M) were from Molecular Probes (Eugene, OR). Adenosine 5′-diphosphate, phorbol 12-myristate 13-acetate (PMA), paraformaldehyde and dimethyl sulfoxide were purchased from Sigma Chemical (St. Louis, MO). Polyclonal antibodies against p38 mitogen-activated protein kinase (MAPK) and p44/42 (extracellular signal-regulated protein kinases 1 and 2 [Erk1/2]) MAPK were from Cell Signaling (Boston, MA).
Preparation of blood samples
This study was approved by the institutional review board of China Medical University Hospital. Patients included pregnant women scheduled for cesarean deliveries (aged 20-40 years) who had not taken any medication for at least 15 days. Informed consents were obtained before enrollment. Blood was collected from an antecubital vein with a 20-G needle without a tourniquet with a 2-syringe technique. The first 2-mL sample was discarded to avoid tissue contamination; the second sample was used for experiments. Blood samples were mixed 9:1 with anticoagulant (3.8% sodium citrate). Approximately a 5-mL blood sample from each subject was prepared for whole blood experiments. The other 20-mL blood samples were centrifuged at 200 g for 20 minutes at 25°C to obtain platelet-rich plasma (PRP). Next, PRP was washed with modified Tyrode’s solution (NaH 2 PO 4 , 0.42 mmol/L; NaCl, 136.9 mmol/L; KCl, 2.68 mmol/L; NaHCO 3 , 11.9 mmol/L; CaCl 2 , 1.85 mmol/L; MgCl 2 , 1.0 mmol/L; BSA, 0.35%; glucose. 0.1%) that contained heparin (7 U/mL) and prostaglandins E 1 (0.6 μmol/L) and was centrifuged at 600 g for 15 minutes at 25°C. After the supernatant was decanted, the pellet was washed twice with modified Tyrode’s solution that contained heparin and prostaglandins E 1 . Finally, washed platelets were resuspended to a final concentration of 3 × 10 8 platelets per milliliter in Tyrode’s solution that contained 0.35% bovine serum albumin and were incubated at 37°C. Neutrophils were isolated by a Ficoll-Paque gradient centrifugation and dextran-sedimentation method described previously. After isolation, neutrophils were resuspended in Hank’s Balanced Salt Solution. A total of 30-50 mL of AF was collected from each parturient woman by amniocentesis of the lower pole of amniotic sac during each cesarean section. After anticoagulation treatment with 3.2% sodium citrate, the AF was centrifuged at 2000 rpm for 5 minutes at 4°C. The supernatant was collected and protein quantification was performed with a bicinchoninic acid assay.
Assessment of platelet-neutrophil aggregation
Anticoagulated whole blood was preincubated at AF concentrations of 0.25, 0.5, 1.0, and 1.5 μg/μL for 20 minutes at room temperature . The blood samples were treated for 1 minute at room temperature with 10 μmol/L adenosine 5′-diphosphate in whole blood as a positive control. To determine platelet-neutrophil aggregation, whole blood was incubated for 30 minutes at room temperature with anti-CD42a-FITC antibodies. Red blood cells were lysed (FACS Lysing Solution; BD Biosciences, San Jose, CA, and the cells pelleted by centrifugation at 1800 rpm for 8 minutes and washed twice with phosphate-buffered saline solution (PBS). Stained cells were suspended in 1% paraformaldehyde in PBS for analysis with flow cytometry (FACSCanto flow cytometer; BD Biosciences). Neutrophils (granulocytes) were recognized by size (forward scatter) and granularity (side scatter). The amount of platelets attached to neutrophils was measured by the mean fluorescence intensity (MFI) of an anti-CD42a-FITC antibody.
Assessment of platelet activation
Washed platelets and PRP were preincubated with AF for 10 minutes at room temperature. Stained cells finally were suspended in 1% paraformaldehyde in PBS for flow cytometric acquisition. P-selectin expression on the surface of platelets was defined as MFI of CD62P.
Assessment of neutrophil activation
Anticoagulated whole blood was preincubated at AF concentrations of 0.25, 0.5, 1.0, and 1.5 μg/μL for 20 minutes at room temperature . Blood samples were treated for 20 minutes at room temperature with PMA (0.1 μmol/L) in whole blood and were used as a positive control. To determine neutrophil activation, isolated neutrophils and whole blood were incubated for 30 minutes at room temperature with the use of anti-CD11b-PE antibody. Red blood cells were lysed (1 × FACS Lysing Solution; BD Biosciences); the cells were pelleted by centrifugation at 1800 rpm for 8 minutes and washed twice with phosphate-buffered saline solution. Stained cells were suspended in 1% paraformaldehyde in PBS for analysis with flow cytometry. Neutrophil activation is expressed as MFI of CD11b expression.
Measurements of intracellular Ca 2+ concentration of neutrophils
Intracellular Ca 2+ levels of neutrophils were determined with the Ca 2+ -sensitive fluorochrome fluo-3 alpha-M with flow cytometry. Isolated human neutrophils (1 × 10 6 neutrophils/mL) were loaded with 5 μmol/L fluo-3 alpha-M for 30 minutes at 37°C in the dark. After being washed once, neutrophils were resuspended, and the external Ca 2+ was adjusted to 1 mmol/L. Next, the dyed neutrophils were incubated with AF concentrations of 0.25, 0.5, 1.0, and 1.5 μg/μL and were detected for 9 minutes by flow cytometry.
Determination of reactive oxygen species (ROS) formation of neutrophils
The influence of AF on ROS production of stimulated neutrophils was tested by H 2 DCF-DA and flow cytometry. Isolated neutrophils were preloaded with 10 μmol/L H 2 DCF-DA for 30 minutes at 37°C followed by stimulation with 0.25, 0.5, 1.0, 1.5 μg/μL AF for 20 minutes. ROS production was quantified by the measurement of the increase in fluorescence of DCF.
Western blot analysis
Various concentrations of AF and 0.1 μmol/L PMA were added to isolated neutrophils for 20 minutes. Proteins were extracted with lysis buffer for 30 minutes. Lysates were centrifuged, and the supernatant was subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (10%) and blotted on polyvinylidene fluoride membrane. Immunodetection was carried out using antibodies against phosphorylated and origin forms of p38 MAPK and ERK1/2. The immunoreactive band was detected by enhanced chemiluminescence.
Statistical analysis
All data were expressed as mean ± SEM. Each sample was compared with the corresponding control sample. Analysis of statistical significance was performed with a 1-way analysis of variance combined with the Tukey’s test of significance. The Student t test was used for between-group comparisons. Significance was set at a probability value of < .05.
Results
AF induces platelet-neutrophil aggregation
Ex vivo whole blood experiments investigated the effects of AF on platelet-neutrophil aggregation. There were concentration-dependent increases in platelets and CD42a-positive neutrophils after pretreatment of AF (25%, 33%, 39%, and 35% at concentrations of 0.25, 0.5, 1.0, and 1.5 μg/μL, respectively; Figure 1 ).
AF induces platelet P-selectin expression at high dose
To test the influence of AF on platelet activation, we performed ex vivo P-selectin expression studies in PRP and washed platelets. In PRP, only 1.5 μg/μL AF enhanced platelet P-selectin expression compared with control ( Figure 2 , A). There was no significant difference in P-selectin expression in washed platelets after the stimulation of AF ( Figure 2 , B), which indicates a minimal effect of AF on platelet activation.
AF induces neutrophil activation and reactive oxygen production
We next examined the effect of AF on neutrophil activation and associated ROS production. In ex vivo studies on isolated neutrophils and whole blood, CD11b expression was stimulated by PMA. In addition, AF elicited CD11b expression in a concentration-dependent manner ( Figure 3 ). The CD11b MFI in neutrophils increased significantly after AF stimulation in isolated neutrophils; this increase was not statistically significant in whole blood. Furthermore, AF was demonstrated to stimulate ROS production of isolated neutrophils ( Figure 4 ).
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