Sequential expression of neutrophil chemoattractants in cerebrospinal fluid after subarachnoid hemorrhage

Background:Neutrophils induce inflammation through the exocytosis of cytotoxic granule proteins. Recently, neutrophils have been reported to be an independent parameter associated with unfavorable outcomes after subarachnoid hemorrhage (SAH). However, the mechanism by which neutrophils accumulate within the CSF after SAH remains undetermined.Findings:Concentrations of C5a, epithelial neutrophil activating peptide 78 (ENA-78), interleukin-8 (IL-8), growth-regulated oncogene-α (GRO-α), neutrophil gelatinase-associated lipocalin (NGAL) and myeloperoxidase (MPO) were measured serially until day 14 in the CSF of 10 patients with SAH. CSF samples obtained from patients suffering from unruptured aneurysms were used as controls. The concentrations of C5a and ENA-78 were significantly increased on day 1, while those of IL-8 and GRO-α significantly increased during days 3-7 compared with those of the control samples. The levels of NGAL and MPO, components of neutrophil granules, significantly increased during days 1~5 after SAH and gradually decreased thereafter. The correlations between ENA-78 and C5a on day 1, IL-8 and GRO-α on days 3~7, and NGAL and MPO on days 1~3 were significant.Conclusions:These neutrophil chemoattractants might be serially involved in the infiltration of neutrophils into the CSF after SAH. Migrated neutrophils play an important role in inflammatory reactions in the central nervous system after SAH.

Cytotoxic granule endocytosis depends on the Flower protein

Cytotoxic T lymphocytes (CTLs) kill target cells by the regulated release of cytotoxic substances from granules at the immunological synapse. To kill multiple target cells, CTLs use endocytosis of membrane components of cytotoxic granules. We studied the potential calcium dependence of endocytosis in mouse CTLs on Flower, which mediates the calcium dependence of synaptic vesicle endocytosis in Drosophila melanogaster. Flower is predominantly localized on intracellular vesicles that move to the synapse on target cell contact. Endocytosis is entirely blocked at an early stage in Flower-deficient CTLs and is rescued to wild-type level by reintroducing Flower or by raising extracellular calcium. A Flower mutant lacking binding sites for the endocytic adaptor AP-2 proteins fails to rescue endocytosis, indicating that Flower interacts with proteins of the endocytic machinery to mediate granule endocytosis. Thus, our data identify Flower as a key protein mediating granule endocytosis.

VAMP8-dependent fusion of recycling endosomes with the plasma membrane facilitates T lymphocyte cytotoxicity

Cytotoxic T lymphocytes (CTLs) eliminate infected and neoplastic cells through directed release of cytotoxic granule contents. Although multiple SNARE proteins have been implicated in cytotoxic granule exocytosis, the role of vesicular SNARE proteins, i.e., vesicle-associated membrane proteins (VAMPs), remains enigmatic. VAMP8 was posited to represent the cytotoxic granule vesicular SNARE protein mediating exocytosis in mice. In primary human CTLs, however, VAMP8 colocalized with Rab11a-positive recycling endosomes. Upon stimulation, these endosomes rapidly trafficked to and fused with the plasma membrane, preceding fusion of cytotoxic granules. Knockdown of VAMP8 blocked both recycling endosome and cytotoxic granule fusion at immune synapses, without affecting activating signaling. Mechanistically, VAMP8-dependent recycling endosomes deposited syntaxin-11 at immune synapses, facilitating assembly of plasma membrane SNARE complexes for cytotoxic granule fusion. Hence, cytotoxic granule exocytosis is a sequential, multivesicle fusion process requiring VAMP8-mediated recycling endosome fusion before cytotoxic granule fusion. Our findings imply that secretory granule exocytosis pathways in other cell types may also be more complex than previously appreciated.