TLR2-induced surface mobilization and release of CD14 in human platelets

Abstract

Abstract In addition to hemostasis, platelets are involved in immunological processes and express toll-like receptors (TLR) like TLR2 and TLR4. Since CD14 represents an essential co-receptor for TLR in immune cells, this study investigated the presence, the release and the distribution of CD14 in human platelets. Washed human platelets (WP) were used for analysis, either unstimulated or stimulated with the TLR2 agonist Pam3CSK4 or with the TLR4 agonist LPS (lipopolysaccharides from Escherichia coli). CD14 expression was detected by flow cytometry, soluble CD14 (sCD14) by an immunoassay. The platelet content of CD14 was measured with Western Blot analysis. Visualization of CD14 was performed with transmission electron microscopy or high-resolution fluorescence microscopy. CD14 expression was detectable after TLR2- or collagen-induced stimulation, but not in resting and TLR4-stimulated platelets. TLR2 activation induced the release of sCD14. In adherent non-permeabilized WP, CD14 expression was found locally condensed in the central region. In permeabilized platelets, an intracellular CD14-specific thread-like staining was visible predominantly along the membrane. CD14 showed a distribution pattern in co-localization with TLR2 and TLR4. In conclusion, platelets contain CD14 molecules co-localized with TLR2 and TLR4, which can be recruited to the surface after specific platelet stimulation and released as sCD14.