Activated platelets mediate inflammatory signaling by regulated interleukin 1beta synthesis

Abstract

Platelets release preformed mediators and generate eicosanoids that regulate acute hemostasis and inflammation, but these anucleate cytoplasts are not thought to synthesize proteins or cytokines, or to influence inflammatory responses over time. Interrogation of an arrayed cDNA library demonstrated that quiescent platelets contain many messenger RNAs, one of which codes for interleukin 1beta precursor (pro-IL-1beta). Unexpectedly, the mRNA for IL-1beta and many other transcripts are constitutively present in polysomes, providing a mechanism for rapid synthesis. Platelet activation induces rapid and sustained synthesis of pro-IL-1beta protein, a response that is abolished by translational inhibitors. A portion of the IL-1beta is shed in its mature form in membrane microvesicles, and induces adhesiveness of human endothelial cells for neutrophils. Signal-dependent synthesis of an active cytokine over several hours indicates that platelets may have previously unrecognized roles in inflammation and vascular injury. Inhibition of beta3 integrin engagement markedly attenuated the synthesis of IL-1beta, identifying a new link between the coagulation and inflammatory cascades, and suggesting that antithrombotic therapies may also have novel antiinflammatory effects.

Figure 1.

mRNA for IL-1β is present in platelets and associated with polysomes. (A) Total RNA from quiescent platelets was isolated and analyzed using an arrayed cDNA library. The circle indicates duplicate hybridizations with the cDNA for pro–IL-1β. (B) Total RNA from resting platelets (pl) or lipopolysaccharide- stimulated monocytes (mo) was isolated and analyzed by reverse transcription PCR for COX-1, IL-8, GM-CSF, ENA-78, Bcl-3, or IL-1β. (C) The ribosomal profiles of resting platelets (left) or platelets activated with thrombin (0.1 U/ml; 1 h) (right) were determined (see Materials and methods). 28S and 18S rRNA and mRNA for IL-1β and GAPDH were assayed in each ribosomal fraction. Fractions 1–3 contain monosomes and low-order polysomes, and fractions 4–6 contain higher order polysomes. This figure is representative of three independent experiments.

Figure 2.

IL-1β is synthesized by platelets in fibrin clots. (A) Immunolocalization of IL-1β or P-selectin with actin in resting platelets (Baseline) or platelets activated with thrombin (0.1 U/ml) after pretreatment with puromycin (100 μM) or control buffer. Freshly isolated platelets were incubated as indicated for 8 h and examined by immunocytochemical analysis. Red immunofluorescence is staining for IL-1β (top right) or P-selectin (bottom right). Green immunofluorescence is staining for actin. In online supplemental Fig. S1, immunostaining for IL-1β was quenched when the antibodies were preincubated with purified IL-1β protein (http://www.jcb.org/cgi/content/full/154/3/485/DC1). Representative of five independent experiments. (B) Total IL-1β (mean ± SEM, n = 6) synthesized by resting platelets (Co) or platelets activated with thrombin (IIa) (8 h) in the presence or absence of puromycin (Puro; 100 μM) was determined by ELISA. (C) Platelets were pretreated with puromycin or DMSO, activated with thrombin, and incubated in the presence of [³⁵S]methionine for 8 h. The cells were then lysed and the proteins were immunoprecipitated with an antibody that preferentially recognizes pro–IL-1β. A single labeled protein with a molecular mass corresponding to pro–IL-1β was identified (arrow). A second experiment yielded a similar result. Bars, 10 μm.

Figure 3.

Platelet agonists trigger prolonged IL-1β accumulation in fibrin complexes. Platelets were activated with thrombin (0.1 U/ml) (A) or PAF (1 nM) (B) in the presence of fibrinogen for the designated time periods. Precursor and mature IL-1β were measured by ELISA. These data are the mean ± SEM for four independent experiments. (C) Control platelets or cells activated with thrombin (0.1 U/ml), ADP (20 μM), collagen (200 μg/ml), or epinephrine (10 μM) were incubated for 18 h and IL-1β was assayed (mean ± SEM; n = 4).

Figure 4.

Microvesicle-derived IL-1β induces endothelial adhesiveness for neutrophils (PMNs) and synthesis of this cytokine requires engagement of β₃ integrins. (A) Control platelets or platelets activated with thrombin (0.1 U/ml) or PAF (1 nM) were incubated for 18 h. IL-1β was then measured in the fibrin-rich cell lysates (C) or the supernatant fractions that contained soluble IL-1β (S) or IL-1β exported in microvesicles (MV). The same fractions were immunoblotted for P-selectin (P-Sel) and β₃ integrins (inset and unpublished data). (B) A portion of the microvesicle fraction assayed in A, recombinant pro–IL-1β, or recombinant mature IL-1β was incubated with endothelial cell monolayers in the presence or absence of a specific IL-1β receptor antagonist. After 4 h, the endothelial cells were washed, ¹¹¹In-labeled PMNs were added, and adhesion was measured after an additional 30-min incubation. The data in A and B are the mean ± SEM of three independent experiments. (C) Platelets were lysed immediately (Co; baseline) or incubated for 18 h alone, with thrombin (IIa), or with thrombin plus a control antibody (CoAb) or mAb c7E3 (100 μg/ml). The inset illustrates platelet– fibrin complex formation induced by thrombin in the presence of mAb c7E3 or the control antibody. The bar graph illustrates values for the measurements of total mature IL-1β levels by ELISA (mean ± SEM of four independent experiments), and the asterisks designate P < 0.05 compared with maximum IL-1β synthesis over 18 h (Max).