Platelet-activating-factor-hydrolyzing phospholipase C in the salivary glands and saliva of the mosquito Culex quinquefasciatus
Section of Medical Entomology, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Building 4, Room 126, 4 Center Drive, MSC 0425, Bethesda, MD 20892-0425, USA
*Author for correspondence (e-mail: JRibeiro{at}NIH.gov)
Accepted August 9, 2001
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Summary |
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Key words: Platelet activating factor, saliva, hematophagy, phospholipase C, mosquito, Culex quinquefasciatus.
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Introduction |
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PAF is an ether phospholipid (Fig. 1) involved in inflammatory and allergic reactions (Braquet et al., 1987). It is a potent agonist in rabbit platelets and the responses induced have been shown to be independent of thromboxane A2 production and secreted ADP (Cazenave et al., 1979
; Chignard et al., 1979
). Although it has been suggested that platelet responses to PAF were mediated by a third pathway independent of these mechanisms (Vargaftig et al., 1980
), studies on the role of thromboxane A2 and ADP in the responses of human platelets to PAF have yielded controversial conclusions. A few studies have shown that ADP scavengers and indomethacin do not inhibit platelet secretion induced by PAF in human platelets (McManus et al., 1981
). In contrast, most studies have shown that PAF-induced full aggregation and secretion in human platelets is dependent on secreted ADP and on thromboxane synthesis (Marcus et al., 1981
; Chesney et al., 1982
; Rao et al., 1982
; Kloprogge et al., 1983
). In addition, platelet responses to PAF are clearly impaired in patients with congenital secretion defects, providing additional evidence that PAF is a weak platelet agonist in human platelets (Rao et al., 1984
). It thus appears that platelet response to PAF is highly dependent on the species in which platelets originate.
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Materials and methods |
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Insects
Mosquitoes were reared in the Section of Medical Entomology (LPD/NIAID/NIH) under the expert supervision of Mr André Laughinghouse. Insectary rooms were kept at 26±0.5°C, 70 % relative humidity and a 16 h:8 h L:D cycle.
The strains of mosquito used were the Liverpool black eye strain of Aedes aegypti, the G3 strain of Anopheles gambiae and the Vero Beach strain of Culex quinquefasciatus. The most recent strain (Culex quinquefasciatus) has been in continuous culture for 8 years. Mosquito female adults used in the experiments were between 3 and 7 days old, took no blood meals and were maintained on a diet of 10 % Karo syrup solution.
Salivary glands from adult female mosquitoes were dissected and transferred to 10 or 20 µl Hepes saline (HS: 0.15 mol l1 NaCl, 10 mmol l1 Hepes, pH 7.0) in 1.5 ml polypropylene vials, in groups of 20 pairs of glands in 20 µl of HS or as individual glands in 10 µl of HS. Salivary glands were kept at 75°C until needed, when they were disrupted by sonication using a Branson Sonifier 450 homogenizer (Branson Ultrasonics, Danbury, CT, USA). Salivary homogenates were centrifuged at 10 000 g for 2 min and the supernatants used for experiments. To obtain saliva, female mosquitoes were lightly anesthetised by exposure to 0°C for 1 min. After removal of their wings and legs, 0.1 µl of 1 mmol l1 serotonin in HS was injected into the coelomic cavity using a heat-pulled glass capillary micropipette operated by mouth pressure (Ribeiro et al., 1984a). After the injection, the feeding fascicle was exposed from within the sheath and inserted into a 2 cm piece of polyethylene tubing (PE-10, i.d. 0.28 mm, o.d. 0.61 mm; Clay Adams, Parsippany, NJ, USA) containing a column of mineral oil of approximately 0.5 cm. This operation was done under a stereoscope with two fine-pointed tweezers. After salivating in the oil at room temperature for 10 min, the mouthparts were removed from the tubing, a #30 needle was inserted in the end of the tubing not containing oil and the oil was blown into 20 µl of HS with the help of a 20 µl syringe (Hamilton, Reno, NV, USA) containing a Luer-lock fitting. After accumulating 10 samples per 20 µl of solution, the tube was spun at 10 000 g for 5 min and the water solution removed without the small amount of the oil phase.
Platelet aggregation assays
To prepare PAF solutions for platelet aggregation assays, 26 µl of PAF (1.9 mmol l1 in ethanol) was evaporated under gentle helium atmosphere. PBS (100 µl, pH 7.4) was added and the sample (500 µmol l1 PAF) was sonicated at room temperature at a duty cycle of 100 % and output control set at 10 using a Branson Sonifier. Finally, the sample was diluted to 500 nmol l1 in tyrode-BSA (137 mmol l1 NaCl, 27 mmol l1 KCl, 12 mmol l1 NaHCO3, 0.42 mmol l1 NaH2PO4, 1 mmol l1 MgCl2, 5.55 mmol l1 glucose, 0.25 % bovine serum albumin, pH 7.4).
Fresh platelet-rich plasma (PRP) in acid citric/dextrose (ACD, 1:10) anticoagulant was obtained by plateletpheresis from medication-free donors (Department of Transfusion Medicine/NIH Blood Bank, under the direction of S. Leitman). Platelet aggregation assays were performed with a Thermomax microplate reader (Molecular Devices, Menlo Park, CA, USA). Briefly, in a flat-bottomed 96-well plate, 50 µl of PAF (250 nmol l1, final concentration) was incubated at 37°C with 0, 0.025, 0.1, 0.25 and 0.5 pairs of Culex quinquefasciatus or Aedes aegypti SGH per assay, yielding a final volume of 85 µl. After 15 µl of PRP was added to start platelet aggregation, the plate was stirred for 5 s in a microplate mixer before being transferred to the microplate reader, where the turbidity change at 650 nm was measured in units of absorbance every 11 s with agitation between readings.
Enzymatic assays
Enzymatic assays were performed using 50 µl of 0.1 mol l1 ammonium acetate adjusted to pH 7.2 and containing 100 µmol l1 of the indicated substrate (stock solutions at 10 mmol l1 or higher were prepared in ethanol) and the indicated amounts of enzyme, usually one or two pairs of homogenised salivary glands or oil-collected saliva, as indicated. After addition of the substrate but before addition of the enzyme, the mixture was sonicated using a Branson sonicator as described above. The reaction, started by adding the enzyme source, proceeded at 37°C. At the indicated time points, 5 µl of the reaction mixture was transferred to a tube containing 50 µl of 80 % methanol plus 0.1 % acetic acid, mixed and injected into the mass spectrometer. Areas corresponding to the substrate masses were integrated using the instrument software (see next paragraph). For pH dependence studies, the 100 mmol l1 ammonium acetate buffer was adjusted with NH4OH or acetic acid to the desired pH. Although most pH values were far from the pK of the buffers, pH did not change by more than 0.1 pH units during the incubation. For these pH measurements, a 50 µl sample was diluted to 100 µl with water and the pH measured using a glass electrode.
Mass spectrometric experiments
Mass spectrometry was performed with an LCQ-Duo ion trap mass spectrometer (ThermoFinnegan, San José, CA, USA) equipped with an electron spray interface. A solution of 80 % methanol in water containing 0.1 % acetic acid was pumped into the interface at 50 µl per minute, using a Spectra System P400 pump from ThermoSeparation Products (Rivera Beach, FL, USA). Samples were injected through a model 7125 loop injector (Rheodyne, Rohnert Park, CA, USA). The instrument was tuned using authentic PAF or other standards, as indicated. These conditions were: sheath gas flow rate, 20 arbitrary units; spray voltage, 4.5 kV; capillary temperature, 200°C; capillary voltage, 27 V. Samples were injected in volumes of 50 µl. To estimate the amount of particular masses of interest, the area under the curve determined by the desired range of ion intensity over time was estimated using the instruments XCalibur software, and compared with a standard curve performed under the same conditions. The instrument response to PAF was linear in the range of 30600 fmol injected. It is important to have the methanol concentration at or higher than 80 % to achieve a good signal intensity. Additionally, to avoid erratic results, which are presumably due to the separation of the lipid micelles from the suspension and/or adsorption to the tube wall, the samples should not be cooled after enzymatic assays or prior to injection on the mass spectrometer.
Although the output of the mass spectrometer reports the intensity of ions with mass m divided by their charge z (m/z), inspection of the m/z ions just above the peak of interest reveals whether the z value is equal to 1, 2 or larger. For example, a singly charged peak at 272 will display a peak at 273, usually with 815 % of the intensity of the 272 mass due to heavier isotopes of C, N and H. A higher intensity at 272.5 would be indicative of a doubly charged ion. For this reason, the Results section refers to masses, as all ions reported in this paper are singly ionized.
Chromatographic experiments
Molecular sieving chromatography was done with a Superdex-75 column (3.2 mmx300 mm) (Amersham/Pharmacia Biotech, Piscataway, NJ, USA) perfused with 150 mmol l1 ammonium acetate, pH 7.2, at 50 µl min1. A Spectra System P-400 pump (ThermoSeparation Products) was used. The eluate was monitored at 220 nm and samples collected at 1-min intervals. Portions (5 µl) of these samples were incubated for 30 min at 37°C with 5 µl of 0.2 mmol l1 PAF in 0.1 mol l1 ammonium acetate, pH 7.2 (previously sonicated). The reaction was stopped by dilution to 100 µl with 80 % methanol containing 0.1 % acetic acid. 50 µl reaction samples were injected into the mass spectrometer to determine substrate disappearance and product formation. The column was calibrated with bovine serum albumin, ovalbumin, carbonic anhydrase, myoglobin and cytochrome c.
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Results |
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Discussion |
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To our knowledge, this is also the first description of a phospholipase C in the saliva of any blood-sucking arthropod, although phospholipase A2 and PAF acetylhydrolase have been described in the salivary glands of ticks and fleas, respectively (Bowman et al., 1997; Cheeseman et al., 2001
). While abundant in the saliva of the human-feeding Culex quinquefasciatus mosquito, such salivary activity is lacking in two other anthropophilic mosquitoes (Aedes aegypti and Anopheles gambiae). PAF is an important physiologic agonist in aggregation of blood of some mammals such as the rabbit (Cazenave et al., 1979
), and also for bird thrombocytes (Cox, 1985
). Indeed, PAF is considered a strong agonist in rabbit platelets, where it induces platelet activation at picomolar concentrations, independently of other mediators (Braquet et al., 1984
). PAF also participates as a pro-aggregatory molecule after platelet stimulation with collagen, thrombin and other inducers (Braquet et al., 1987
). In contrast, the role of PAF as an aggregating agent in human platelets is less remarkable. In fact, platelet aggregation by PAF in these cell types requires the concomitant production of TXA2 or secretion of ADP (Marcus et al., 1981
; Chesney et al., 1982
; Rao et al., 1982
; Kloprogge et al., 1983
; Rao et al., 1984
). In addition, the aggregatory response of human platelets activated by other ligands is unaffected by specific PAF receptor antagonists (Ostermann et al., 1990
). Moreover, most (87 %) of PAF released by collagen and thrombin-stimulated platelets are not in the soluble form, but associated with pro-coagulant microparticles (Iwamoto et al., 1996
). However, the participation of PAF in platelet physiology turns out to be relevant in the context of platelet-neutrophil interactions (Li et al., 2000
). At the site of injury where thrombogenic molecules are exposed (such as collagen), platelet-released adenine nucleotides may promote neutrophil activation, leading to the release of superoxide anion (O2), elastase (Si-Tahar et al., 1997
), cathepsin G (Si-Tahar et al., 1996
) and PAF itself which, in turn, activates platelets and endothelial cells (Braquet et al., 1984
). Accordingly, the primary role of PAF in humans has been characterized as a mediator of intercellular interactions (Prescott et al., 2000
). We suggest that the presence of a Culex PAF-hydrolyzing enzyme, together with other anti-hemostatic Culex salivary molecules, is likely to promote a negative modulation of the inflammatory response in the microenvironments where such interactions occur.
While the genus Aedes is mostly associated with primates and Anopheles with mammals (Horsfall, 1955), all close relatives of Culex quinquefasciatus are bird feeders, indicating that this mosquito is only recently associated with humans (Chevillon et al., 1995
). Indeed, Culex quinquefasciatus has less salivary antiplatelet activity than either Aedes aegypti or Anopheles albimanus and takes longer to find blood on mammals than on chicks (Ribeiro, 2000
). Culex quinquefasciatus salivary PAF phosphorylcholine hydrolase may thus reflect the adaptation to a previous non-human host where PAF is an important physiological agonist of platelet or thrombocyte aggregation.
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Acknowledgments |
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