Freezing injury of granulocytes during slow cooling: role of the granules.

The granulocyte is an interesting model for studying the behavior of fragile cells at low temperature. A previous study suggested that during slow freezing the plasma membrane was not the primary site of injury and, as a corollary to this, we examined the damage occurring in the cytoplasmic compartment. The present study was designed to investigate the role of granules during cryoinjury of the granulocytes. Granulocytes were prepared from fresh blood and a population of granules was extracted by nitrogen cavitation. A graded freeze-thaw protocol was used to follow the release of beta-glucuronidase (beta-Glu), one of the hydrolytic enzymes present in granules. Granulocytes and granules were subjected to treatments simulating slow freezing conditions, and the release of beta-Glu was evaluated after exposure to increased hydrolytic enzymes and hypertonic salt concentration. It was found that, after thawing, granulocytes generally expressed more release than granules during graded freezing. The postthaw incubation period had no effect on enzyme release. Increase in salt concentration reduces beta-Glu activity. Direct exposure to hydrolytic enzymes produced similar injury on both granules and granulocytes, and salt combined with enzymes did not increase granule disruption. It is concluded that the effect of injured granules may be more apparent during rewarming when isotonicity is reestablished. Enzymes released extracellularly induce no extra injury to the granulocyte because of dilution effect; however, their release in the cytosol can cause a defect resulting in the loss of cell viability but no membrane disruption. Overall, the release of enzymes is seen as a secondary factor contributing to whole cell or membrane damage during slow freezing.