A Mechanism for Exacerbation of Chronic ITP by Infection: Toll-Like Receptor 4 (TLR4) Activation Enhances Antibody-Mediated Platelet Phagocytosis by Human Macrophages.

Infection often triggers an exacerbation of chronic ITP. The precise mechanism for the decreased platelet count associated with infection is not completely understood. However, interference with megakaryopoiesis is often invoked, and increased production of autoantibodies is also possible. We hypothesized that stimulation of innate immunity could enhance phagocytosis in the reticuloendothelial system and thus contribute to an exacerbation of chronic ITP in the setting of infection. The innate immune response involves activation of Toll-like receptors (TLRs) that initiate intracellular signaling through MyD88 kinase as well as through other incompletely characterized MyD88-independent pathways. We have previously shown that Syk is a crucial signaling tyrosine kinase involved in Fcγ receptor-mediated platelet phagocytosis and that Syk-specific dsRNA inhibits antibody-mediated platelet phagocytosis. Furthermore, the ligand binding requirements for Syk are not stringently limited to classical tandem immunoreceptor tyrosine-based activation motifs. We speculated that TLR4 might employ Syk tyrosine kinase intracellular signaling in human macrophages as a mechanism to enhance immune platelet phagocytosis. The present study was undertaken to address the following questions: Does lipopolysaccharide (LPS) stimulation, an in vitro model for innate immunity triggered by gram-negative bacterial infection, augment antibody-dependent phagocytosis of platelets? Does TLR4 associate with Syk tyrosine kinase in macrophages? Does LPS stimulation induce phosphorylation of Syk?

Methods: Antibody-mediated phagocytosis of platelets by THP-1 (human macrophage) cells in culture was assayed by co-incubation of THP-1 cells with or without LPS stimulation at 37°C with platelets that were labeled with 5-(and 6-) carboxyfluorescein diacetate mixed isomers (CFDA) in the presence of human serum containing antihuman GPIIIa IgG antibody. Association of TLR4 with the tyrosine kinase, Syk, was studied in THP-1 cells by immunoprecipitation and immunoblotting after ligation with anti-TLR4 monoclonal antibody (mAb) or stimulation with LPS. Phosphorylation of TLR4-associated Syk after LPS stimulation was studied by immunoprecipitation and immunodetection with anti-TLR4, antiphosphotyrosine (PY99), and anti-Syk mAbs.

Results: Antibody-mediated platelet phagocytosis by THP-1 cells was enhanced by 50% after LPS exposure. In five experiments a mean of 21.8% ± 4.1% CFDA-labeled platelets were ingested by THP-1 cells upon antiplatelet antibody exposure in the absence of LPS, while 32.8% ± 6.3% were phagocytosed after LPS (p<0.001.) Syk protein was coimmunoprecipitated with TLR4 from THP-1 cell lysates by anti-TLR4 mAb. Furthermore, intracellular Syk became phosphorylated upon LPS stimulation of THP-1 cells.

Conclusions: These studies support the hypothesis that activation of TLR4 in the setting of infection contributes to enhanced antibody-mediated platelet phagocytosis by macrophages and thus exacerbates chronic ITP. Syk becomes phosphorylated upon TLR4 stimulation of macrophages and may play a role in signal transduction triggered by the innate immune system through TLR4. These novel findings may have an impact on the understanding and management of immune thrombocytopenia in the setting of infectious diseases and offer a new therapeutic target for chronic ITP.