Granzyme-b is involved in mediating post-ischemic neuronal death during focal cerebral ischemia in rat model.
Although peripheral immune cells infiltrate ischemic infarct tissue and elicit immune injury, the role of Cytotoxic T Lymphocytes (CTLs) and the toxins they release in mediating neuronal death is not well understood. Granzyme-b (Gra-b), a serine protease found in the cytoplasmic granules of CTLs and natural killer cells, plays an important role in inducing target cell death by activating several caspases and by initiating caspase-independent pathways that contribute to target cell death. To determine if CTLs and Gra-b are involved in post-ischemic cerebral cell death; we investigated the role of CD8(+) CTLs and Gra-b in ischemic rat brain infarct after transient middle cerebral artery occlusion (tMCAO) and in sham-operated animals. We observed that CTLs infiltrate the ischemic infarct within 1 h of reperfusion. There was a significant increase in Gra-b levels in the ischemic region starting from 1 h until 3 day which correlated with increased levels of chemokines (IP-10/CXCL10, IL-2) and TNF-alpha. Co-immunoprecipitation experiments show that Gra-b interacts with Bid, PARP, and caspase-3 in ischemic samples. Immunofluorescence analysis of Gra-b and TUNEL showed that Gra-b is present both in apoptotic and necrotic cells. Triple immunostaining further confirmed that the Gra-b positive degenerating cells were neurons. CTLs in close spatial proximity to degenerating neurons, increased levels of Gra-b, localization in neurons positive for TUNEL, and interaction with other pro-apoptotic proteins indicate that Gra-b and CTLs play a significant role in neuronal death following cerebral ischemia in the rat brain after tMCAO. Based on the above findings we support our hypothesis that Gra-b secreted from activated CTLs might be involved in aggravating post-ischemic damage by mediating neuronal death.
Chaitanya
G V
GV
Departments of Biotechnology and Animal Sciences, School of Life Sciences, University of Hyderabad, India.
Schwaninger
M
M
Alexander
J S
JS
Babu
P Prakash
PP
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D001921:Brain / Q000201:enzymology / Q000503:physiopathology*; D002545:Brain Ischemia / Q000201:enzymology / Q000503:physiopathology; D016827:CD8 Antigens / Q000378:metabolism; D016923:Cell Death / Q000502:physiology; D004195:Disease Models, Animal; D053804:Granzymes / Q000378:metabolism*; D020244:Infarction, Middle Cerebral Artery / Q000201:enzymology / Q000503:physiopathology*; D008297:Male; D009336:Necrosis / Q000201:enzymology / Q000503:physiopathology; D009410:Nerve Degeneration / Q000201:enzymology / Q000503:physiopathology; D009474:Neurons / Q000201:enzymology / Q000502:physiology*; D016716:PC12 Cells; D011897:Random Allocation; D051381:Rats; D017208:Rats, Wistar; D013602:T-Lymphocytes, Cytotoxic / Q000201:enzymology / Q000502:physiology; D013997:Time Factors
D016827:CD8 Antigens; D053804:Granzymes; C509215:Gzmb protein, rat