The function of platelets in hemostasis is well known, but only recently their role in modulating the adaptive immune response has been increasingly recognized. Platelets are able to stimulate B lymphocytes, enhance T lymphocytes responses and to adhere to dendritic cells modulating their activation. In addition to their ability to release a wide variety of inflammatory mediators, activated platelets can directly interact with pathogens, releasing attractant mediators for immune cells and acting as antigen presenting cells themselves. Once activated, platelets express numerous immune or inflammatory receptors and ligands normally present in lymphocytes. These observations support the active participation of platelets in immunological processes and their direct interaction with immune responding cells. The active role of platelets in these events is further substantiated by their ability to process and translate specific mRNAs into functional proteins. During the performance of studies related with protein synthesis by platelets, we observed that pure human platelets expressed CD3-ε and γ chains mRNAs. CD3 complexes are integral components of the T cell receptor and their main function is to signal through their immunoreceptor tyrosin-based activation sequence (ITAM). Based on the role of platelets in immunity and given that platelet function depends on specific activation-induced signaling, the aims of this work was to demonstrate that platelets express CD3-ε chain mRNA, can be translated into de-novo protein and detected in platelet membranes by Western blot and by immunofluorescence. Platelets were isolated as referred (
) and purity was assessed by flow cytometry using anti-CD-14 and anti-CD-45 antibodies, by microscopic detection of nucleated cells using propidium iodide and by lack of amplification of CD-14 by RT-PCR. All our platelet preparations contained less than 1 leukocyte per 105 platelets and had no CD14 transcripts evidenced in silver-stained polyacrilamide gels. Nevertheless, all the platelet preparations analyzed showed abundant platelet-specific GPIbα and CD3-ε and γ chains mRNA′s that slightly enhanced when platelets were stimulated with 10μM TRAP for 30 min. Confocal analysis of immuno-stained platelets with either anti-CD3 FITC conjugated monoclonal antibody or a polyclonal antibody (ABCAM) recognizing the CD3ε chain, showed a barely detectable label in non stimulated platelets that was poorly enhanced after TRAP activation. However, analysis of peripheral blood mononuclear cells (PBMNC) revealed a strong lymphocyte CD3 label and a visible CD3 staining in the contaminating platelets which are always present in these suspensions. Platelet CD3 in these preparations was enhanced after PBMNC stimulation with 10 mg/mL LPS for 2h. We co-incubated platelets with dendritic cells as a non bearing CD3 cell and known to adhere to platelets. In parallel, aliquots of the same platelet preparation were incubated only with the mature dendritic media, containing 1000 U/ml TNF-α. Platelets in contact with dendritic cells were negative for CD3 immunostaining even in the presence of contaminating CD3 positive lymphocytes carried from the dendritic cell preparation. However, after 30 min incubation of isolated platelets with the media containing TNF-α, a prominent CD3 platelet staining was apparent (confocal microscopy). Metabolic labeling of isolated, non-stimulated platelet suspensions with 35S- methionine, followed by immune-precipitation with the polyclonal antibody, disclosed the expected ≈23kD radio-labeled band corresponding to monomeric CD3 and an additional band of Mr ≈49kD. Both bands were reduced by puromycin, but not by actinomycin D treatment. These findings demonstrate that human platelets have CD3-ε chain mRNA and that they are able to translate this mRNA into immune-detectable protein. Notably, we observed that immune-inflammatory stimuli rather than specific hemostatic platelet activators induced expression of CD3-ε protein. Together our results showed that expression of CD3-ε is not restricted to T lymphocytes and suggest that its presence in platelets play a role in inflammatory-immune responses.
Disclosures: No relevant conflicts of interest to declare.
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