The inhibition of spontaneous and tumor necrosis factor-alpha induced neutrophil apoptosis by crystals of calcium pyrophosphate dihydrate and monosodium urate monohydrate.

OBJECTIVE

Spontaneous neutrophil apoptosis may be inhibited by various proinflammatory stimuli. which may result in prolonged lifetimes and responses of these phagocytic cells with the potential for extended inflammation. We investigated the effect of short term incubation of opsonized crystals of monosodium urate monohydrate (MSUM) or calcium pyrophosphate dihydrate (CPPD) on both spontaneous and tumor necrosis factor-alpha (TNF-alpha) induced neutrophil apoptosis.

METHODS

Peripheral neutrophils were incubated with plasma opsonized crystals of CPPD or MSUM in the presence or absence of TNF-alpha for 4 h at 37 degrees C. Apoptosis was determined using 3 separate assays: (1) an agarose DNA fragmentation assay, (2) a cytoplasmic histone associated DNA fragmentation assay, and (3) a caspase 3 fluorometric assay.

RESULTS

All 3 assays showed similar results. Both MSUM and CPPD crystals inhibited spontaneous apoptosis in neutrophils. TNF-alpha induced high levels of apoptosis in neutrophils. However, co-incubation of the cells with TNF-alpha and crystals resulted in the inhibition of apoptosis to levels below those of control cells. Pretreatment of neutrophils with the protein synthesis inhibitor cycloheximide prevented the inhibition of apoptosis by crystals.

CONCLUSION

These data support the concept of crystal induced inhibition of neutrophil apoptosis as part of the pathophysiology of the diseases collectively known as crystal induced arthritis.