Murine hypodense eosinophils induce tumour cell apoptosis by a granzyme B-dependent mechanism.
OBJECTIVE
Eosinophils have been shown to potentiate anti-tumour cytotoxicity in both clinical and animal studies. The mechanism by which eosinophils induce tumour cell damage, however, has largely been speculative. The purpose of this study was to identify the mechanisms involved in eosinophil-induced tumour cell cytotoxicity.


METHODS
To investigate eosinophil cytotoxicity, eosinophils were isolated from the peritoneal cavity of Mesocestoides corti-infected BALB/c mice, and were separated into normodense (ND) and hypodense (HD) populations using discontinuous Percoll density gradient centrifugation. The tumoricidal activity of ND and HD eosinophils was assessed using the [51Cr]-release cytotoxicity assay (a measure of cytolytic activity) and the JAM assay (a measure of apoptotic activity). Investigation of apoptosis-inducing molecules in HD eosinophils was undertaken by RT-PCR. The calcium chelator EGTA, serine protease inhibitor aprotinin and a competitive substrate for granzyme B were used to assess the role of perforin and granzyme B in HD eosinophil killing.


RESULTS
Cytotoxic activity induced by HD eosinophils was significantly greater than that of ND eosinophils, and apoptosis was the principal killing mechanism. RT-PCR analysis revealed that HD eosinophils express mRNA for perforin, granzyme B and Fas ligand. Furthermore, HD eosinophil killing was markedly inhibited by EGTA, intracellular aprotinin and the granzyme B competitive substrate.


CONCLUSIONS
These data are consistent with a hypothesis that murine HD eosinophils elicit tumoricidal activity via a granzyme B-dependent mechanism.
Costain
D J
DJ
Faculty of Medicine, Department of Pathology, Dalhousie University, Halifax, Nova Scotia, Canada.
Guha
A K
AK
Liwski
R S
RS
Lee
T D
TD
D000818:Animals; D017209:Apoptosis / Q000276:immunology*; D002452:Cell Count; D002590:Cestode Infections / Q000276:immunology / Q000473:pathology; D004802:Eosinophilia / Q000276:immunology / Q000469:parasitology; D004804:Eosinophils / Q000276:immunology*; D053222:Fas Ligand Protein; D005260:Female; D053804:Granzymes; D016393:Lymphoma, B-Cell / Q000276:immunology* / Q000473:pathology; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000502:physiology; D008645:Mesocestoides; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000502:physiology
D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D014409:Tumor Necrosis Factor-alpha; D054353:Perforin; D053804:Granzymes; C509214:Gzmb protein, mouse; D012697:Serine Endopeptidases