The involvement of phospholipase C in crystal induced human neutrophil activation.
OBJECTIVE
To investigate the involvement of phospholipase C (PLC) in the signal transduction pathway leading to neutrophil activation by inflammatory microcrystals.


METHODS
Neutrophil chemiluminescence, superoxide anion generation, and degranulation responses to opsonized crystals were measured in the presence of U73122, a specific inhibitor of PLC. Phospholipase Cgamma2 (PLCgamma2) activity was measured by Western blotting.


RESULTS
U73122 was shown to inhibit neutrophil activation induced by plasma opsonized crystals of calcium pyrophosphate dihydrate (CPPD) and monosodium urate monohydrate. The IC50 for inhibition of crystal induced respiratory burst (measured by chemiluminescence) was roughly 3 microM. No inhibition of crystal induced respiratory burst was observed using U73343, an inactive analog of U73122. These results show the pivotal role of PLC in neutrophil respiratory burst activation by both crystals. Superoxide anion generation and degranulation responses of neutrophils to CPPD crystals were also strongly inhibited by U73122 at a concentration of 10 microM, supporting the concept of direct involvement of PLC in signal transduction pathways leading to crystal induced oxidase activation and degranulation responses. CPPD crystals caused a sustained increase in the tyrosine phosphorylation levels of PLCgamma2 in neutrophils.


CONCLUSIONS
We conclude that PLC plays a central role in signal transduction pathways leading to respiratory burst and degranulation responses in neutrophils activated by inflammatory microcrystals, and that PLCgamma2 may be the isoform involved in these pathways.
Jackon
J K
JK
Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada.
Tudan
C
C
Burt
H M
HM
D002118:Calcium / Q000378:metabolism; D002131:Calcium Pyrophosphate / Q000737:chemistry / Q000494:pharmacology; D015550:Cell Degranulation / Q000187:drug effects; D019069:Cell Respiration / Q000187:drug effects / Q000502:physiology; D003460:Crystallization; D004075:Diglycerides / Q000494:pharmacology; D004347:Drug Interactions; D004963:Estrenes / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D008163:Luminescent Measurements; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D018375:Neutrophil Activation / Q000502:physiology*; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011760:Pyrrolidinones / Q000494:pharmacology; D015398:Signal Transduction; D013481:Superoxides / Q000378:metabolism; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D014527:Uric Acid / Q000737:chemistry / Q000494:pharmacology
D004075:Diglycerides; D004963:Estrenes; D007527:Isoenzymes; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; C044295:1,2-dioctanoylglycerol; D013481:Superoxides; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C074854:U 73343; D014527:Uric Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002118:Calcium; D002131:Calcium Pyrophosphate