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Address correspondence to Andrew S. Weyrich, Program in Human Molecular Biology and Genetics, University of Utah, 15 North 2030 East, Bldg. 533, Rm. 4220, Salt Lake City, UT 84112. Tel.: (801) 585-0702. Fax: (801) 585-0701. E-mail: andy.weyrich{at}hmbg.utah.edu
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Abstract |
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Key Words: adhesion; cytokines; integrins; platelets; translation
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Introduction |
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Platelets are thought to play a minor role, if any, in ongoing inflammation or vascular injury and repair beyond initial adhesion, secretion, and eicosanoid synthesis. However, activation of platelets does not appear to decrease their life span (Reimers et al., 1976; Michelson et al., 1996; Berger et al., 1998), and thrombogenic adhesive platelets remain functional (Berger et al., 1998). This observation suggests that activated platelets may contribute to thrombotic and inflammatory cascades for much longer periods than previously believed. Consistent with this notion, we recently demonstrated that activated platelets translate constitutive mRNAs into proteins, and that this process continues for hours after stimulation with thrombin- or integrin-mediated adhesion (Weyrich et al., 1998; Pabla et al., 1999). In this report, we use a model of plateletfibrin clot formation to demonstrate for the first time that activated platelets synthesize an inflammatory cytokine, interleukin (IL)*-1ß. Accumulation of IL-1ß is prolonged, attenuated by blocking engagement of ß3 integrins, and mediates signaling of endothelial cells with consequent neutrophil (polymorphonuclear leukocyte [PMN]) adhesion. It was reported earlier that stimulated platelets possess IL-1ß activity, but they were not thought to actively synthesize this inflammatory protein (Hawrylowicz et al., 1989; Loppnow et al., 1998). Regulated cytokine synthesis establishes the potential for previously unrecognized biologic roles for platelets in inflammation and vascular injury.
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Results and discussion |
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IL-1ß mRNA is translated into protein by activated platelets during fibrin clot formation
Next, we examined expression of the IL-1ß gene product in resting platelets by immunocytochemistry, and found neither the precursor nor the mature form (Fig. 2, A
, top left, B, and Fig. 3)
. In contrast, IL-1ß was robustly detected in complexes of platelets and fibrin after activation with thrombin in the presence of fibrinogen, a model of clot formation and retraction (Fig. 2 A, top right). The result is consistent with previous reports that IL-1ß activity is present in stimulated platelets (Hawrylowicz et al., 1989; Loppnow et al., 1998). Pretreatment of platelets with puromycin, a translational inhibitor, completely inhibited IL-1ß synthesis (Fig. 2, A, bottom left, B, and C). However, plateletfibrin clumps formed in response to thrombin, indicating that puromycin was not nonspecifically toxic. Similarly, staining of parallel samples for P-selectin, which is constitutively stored in -granules, was unchanged by pretreatment with puromycin, indicating that this translational inhibitor did not alter the detection of resident proteins (Fig. 2 A, bottom right). When platelets were stimulated with thrombin in the presence of fibrinogen, IL-1ß and P-selectin were associated with the fibrin lattice (Fig. 2 A), consistent with a previous report for P-selectin distribution (Siljander et al., 1996) (also see below).
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We next measured IL-1ß levels in resting and activated platelets over an extended time period. Accumulation of proIL-1ß was sustained over hours and was followed by processing of the precursor into its mature, active form (Fig. 3 A). ProIL-1ß was detectable in thrombin-stimulated platelets within 30 min (unpublished data), a rapid synthetic response that may result from localization of IL-1ß transcripts in polysomes in resting platelets (see above) (Clark et al., 2000). Platelets synthesized IL-1ß in every experiment (n > 30), although the magnitude was variable among donors (unpublished data). This variability was not likely to be due to contaminating leukocytes, as we did not detect macrophage colony stimulating factor (M-CSF) (60 ± 13 vs. 69 ± 1 pg/ml in control vs. activated cells [18 h], respectively), a monocyte-derived cytokine. In addition, we detected no increase in IL-1ß protein levels when lymphocytes or monocytes were added to isolated platelets, concentrations of which were 10-fold greater than the trace numbers of leukocytes that were sometimes detected in our preparations (unpublished data). Platelet-activating factor (PAF) also induced IL-1ß synthesis (Fig. 3 B) as did ADP, collagen, and epinephrine (Fig. 3 C). Together, Figs. 13 demonstrate that platelets synthesize an important cytokine, IL-1ß, and have the potential to influence inflammatory events over several hours by activation-induced translation of a cytokine from constitutive mRNA.
IL-1ß synthesized by activated platelets signals endothelial cells and induces PMN adhesion
Approximately 60% of mature IL-1ß is retained within the platelets or the fibrin mesh that surrounds aggregated platelets (Fig. 2 A). The remaining IL-1ß is found in the extracellular milieu. Fractionation of the supernatant revealed that half of the IL-1ß was associated with platelet microvesicles, a finding confirmed by assay of ß3 integrins and P-selectin in the same samples (Fig. 4
A and unpublished data). The remaining IL-1ß in the supernatant fraction was completely soluble (Fig. 4 A). Soluble and microvesicle-associated IL-1ß delivered outside-in signals to human endothelial cells and induced their adhesiveness for neutrophils (Fig. 4 B and unpublished data), a response dependent on synthesis of E-selectin, intercellular adhesion molecule 1, and chemokines (McEver et al., 1998). However, IL-1ß exported in microvesicles was at least fivefold more potent than soluble IL-1ß when equivalent volumes of sample were tested (unpublished data), suggesting that other inflammatory mediators packaged in microvesicles (Barry et al., 1999) may act in concert with IL-1ß to induce endothelial adhesiveness. In this regard, we recently found that IL-1ß acts in concert with P-selectin to induce expression of cyclooxygenase (COX)-2 when human monocytes interact with activated platelets (unpublished data). Recombinant IL-1ß receptor antagonist inhibited the response to platelet-derived IL-1ß and purified IL-1ß, but did not inhibit adhesiveness induced by tumor necrosis factor (Fig. 4 B and unpublished data). These data indicate that IL-1ß synthesized by activated platelets induces inflammatory responses of target endothelial cells.
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Although the exact mechanisms are not yet clear, integrins may regulate translation in platelets at checkpoints independent of initial ribosomal mRNA interactions, as IL-1ß transcripts are constitutively present in platelet polysomes (see above). In parallel experiments we have found that translational control proteins are physically separate from mRNAs in resting platelets, and that integrin engagement redistributes these proteins to mRNA-rich areas (Lindemann et al., 2001). Dissection of mechanisms of signal-dependent synthesis of specific gene products such as IL-1ß by platelets may yield new insights into translational control in specialized cells (Weyrich et al., 1998; Clark et al., 2000) and reveal new activities of platelets in atherogenesis, tissue remodeling, and tumor angiogenesis (Weyrich et al., 1996; Barry et al., 1997; Mannaioni et al., 1997; Pinedo et al., 1998).
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Materials and methods |
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Polysome analysis
Polysomes were isolated from platelets using minor modifications of published protocols (Kaspar and Gehrke, 1994). In brief, platelets (7.5 x 109) were lysed in a high-salt buffer to remove polysomes from the cytoskeleton (200 mM Tris, 520 mM KCl, 30 mM MgCl2, 4% Triton X-100, and 200 mM sucrose, pH 9.5). The cells were passed three times through a 21-gauge tuberculin syringe, followed by a brief centrifugation to remove all insoluble material and heparin (10 mg/ml), and NaCl (150 mM) was added to the lysate. Mitochondria were removed by further centrifugation (5 min, 14,000 g) and the resulting supernatant was placed on a 4-ml sucrose gradient (0.52 M) and centrifuged at 4°C for 2 h (43,700 rpm) using an SW55 Ti swinging bucket rotor (Beckman Coulter).
Sucrose gradients were fractionated using an ISCO UA-6 254-nm continuous flow chamber into 760-µl aliquots. Total RNA in each fraction was isolated for analysis of ribosomal RNA and mRNA for IL-1ß and glutaraldehyde 3-phosphate dehydrogenase (GAPDH) as described previously (Weyrich et al., 1998). The total mRNA pool in platelets was also determined by interrogation of cDNA libraries according to the manufacturer's instructions (array 7742-1; CLONTECH Laboratories, Inc.). In selected experiments, sucrose cushions were used to isolate mRNAs associated with polysomes of resting and thrombin-activated platelets as described previously (Davies and Abe, 1995). The sucrose cushion was based on ribosomal profiles from complete sucrose gradients that were fractionated with the ISCO continuous flow chamber (see above).
ELISA
ELISAs for proIL-1ß, mature IL-1ß, and M-CSF were performed according to the manufacturer (R&D Systems; Bender Med Systems). The pro and mature IL-1ß ELISAs did not cross react with one another.
Immunoprecipitation of [35S]methioninelabeled proteins
Immunoprecipitation of metabolically labeled IL-1ß was conducted as described previously (Weyrich et al., 1998) using an antibody that preferentially recognizes proIL-1ß (Research Diagnostics, Inc.).
Immunolocalization of platelet-derived IL-1ß
Platelets were left quiescent or stimulated with 0.01 U/ml thrombin for 8 h as described above. Immunocytochemistry for IL-1ß, P-selectin, and polymerized actin was conducted as described previously (Weyrich et al., 1996, 1998).
Isolation of platelet microvesicles
Platelets were activated with PAF or thrombin as indicated above. After 8 h, the platelet suspensions were centrifuged for 4 min at 15,500 g. The pellet was lysed in RIPA buffer and the supernatant was collected and recentrifuged at 100,000 g in a Ti80 rotor (Beckman Coulter) for 90 min at 4°C to collect platelet microvesicles (George et al., 1982). The resulting pellet from high-speed centrifugation of the supernatant pool, which contained microvesicles, was lysed in RIPA buffer. Mature IL-1ß was assayed in the pellet lysates, the microvesicle fraction, and the soluble supernatant by ELISA. The same samples were also examined by Western analysis using mAb S12 to detect P-selectin in the microvesicle fraction.
PMN adhesion to activated endothelial cells
Human umbilical endothelial cells were incubated with platelet supernatants or microvesicle fractions for 4 h in the presence or absence of a recombinant IL-1ß receptor antagonist (R&D Systems). In parallel, human umbilical endothelial cells were also stimulated with tumor necrosis factor (100 U/ml), recombinant active IL-1ß, or recombinant proIL-1ß. After 4 h, the human umbilical endothelial cells were thoroughly washed and PMN adhesion to the cell surface was quantitated as described previously in detail (Lorant et al., 1991; Lindemann et al., 2000).
Online supplemental material
Fig. S1(available at http://www.jcb.org/cgi/content/full/154/3/485/DC1) demonstrates that immunostaining of platelet-derived IL-1ß is quenched when the antibodies are preincubated with purified IL-1ß protein. Platelets were prepared for immunocytochemical detection of IL-1ß protein as described in Fig. 2 A, the exception being that the antiIL-1ß antibody was quenched with excess IL-1ß protein. The left panel depicts staining for IL-1ß with quenched antibody. The middle panel shows the same cells stained for actin. The right panel is an overlay of the two panels. Note that staining for IL-1ß is absent in the left and right panels.
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Footnotes |
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* Abbreviations used in this paper: COX, cyclooxygenase; GAPDH, glutaraldehyde 3-phosphate dehydrogenase; IL, interleukin; M-CSF, macrophage colony stimulating factor; PAF, platelet-activating factor; PMN, polymorphonuclear leukocyte; proIL-1ß, IL-1ß precursor.
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health (HL56713 to A.S. Weyrich, HL44525 to G.A. Zimmerman), the Atorvastatin Research Awards (A.S. Weyrich), the Western State Affiliate of the American Heart Association (S. Lindemann, AHA Grant 0020030Y), the American Heart Association (D.A. Dixon, AHA Grant 9930102N), and a National Institutes of Health Special Center of Research in Adult Respiratory Distress Syndrome (P50 HL50153).
Submitted: 10 May 2001
Revised: 22 June 2001
Accepted: 25 June 2001
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