Experimental Biology 2010: Mechanisms Of Platelet Granzyme B Induced End-Organ Apoptosis In Sepsis

Purpose of Study: A frequent precursor to mortality from sepsis is Multiple Organ Dysfunction Syndrome (MODS). End-organ apoptosis is considered a diagnostic hallmark of progressive sepsis and MODS. Importantly, platelets have been shown to accumulate in many of the commonly affected end-organs (e.g. spleen and lung) in sepsis. We recently reported acute sepsis-induced alterations in the megakaryocyte-platelet transcriptional axis that resulted in a strongly lymphotoxic platelet phenotype via expression of the potent serine protease granzyme B (GzmB). In the ensuing experiments, our goal was to define the site(s) of and mechanism(s) by which platelet GzmB induces end-organ apoptosis in sepsis.

Methods Used: Platelets from septic mice 18 hours status post cecal ligation and puncture were co-incubated for 90 minutes ex vivo with splenocytes isolated from healthy control mice. Splenocyte apoptosis was measured using TUNEL-based assays. Organs were assayed for TUNEL staining by immunohistochemistry. Data were generated to explore potential mechanisms of GzmB delivery (i.e. perforin-dependent vs. independent, contact vs. non-contact mediated) and action (i.e. caspase vs. non-caspase).

Summary of Results: There was evident apoptosis in spleen, lung, and kidney sections from septic wild type (WT) mice (i.e. C57BL6). In contrast, there was a lack of TUNEL staining in spleens and lungs from septic GzmB-/- mice (i.e. B6.129S2-Gzmbtm1Ley). Because GzmB is typically delivered to target cells via co-released perforin, platelet GzmB-induced rates of splenocyte apoptosis were compared among platelets from healthy (4.8±2.9%), septic WT (25.1±1.4; p<0.001), and septic perforin null (-/-) (i.e. C57BL/6-Pfptm1Sdz) mice (24.0±5.2%; p<0.01). Additionally, incubation across a semi-permeable membrane reduced splenocyte apoptosis (10.3±3 vs. 5.6±2.6; p<0.01) to a rate indistinguishable from non-platelet treated controls (5.6±2.5%; p=NS). When direct contact was permitted, TUNEL+ splenocytes were >98% Caspase+.

Conclusions: In sepsis, platelet GzmB-mediated apoptosis occurs in spleen and lung tissue. This process appears to proceed in a perforin-independent, caspase-mediated, and contact-dependent manner. Therapeutics should be developed to target these parameters of platelet-mediated cytotoxicity in sepsis.