The effect of storage on degranulation by human neutrophils

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Abstract
The possibility that the functional impairment in neutrophil (PMN) chemotaxis which occurs during granulocyte concentrate storage might be due to autotoxicity from the release of neutrophil granule contents during storage was studied. Preliminary experiments confirmed that the exposure of fresh PMN to the intracellular contents of disrupted PMN, decreased the subsequent chemotaxis of the fresh PMN by 63 .+-. 5% compared to control PMN (P < 0.1). Freshly harvested neutrophils were stored at low (2 .times. 107 PMN/ml) or high cell concentration (8 .times. 107 PMN/ml) with or without 15 mM sodium bicarbonate (in order to maintain pH). Prior to storage, and 24 and 48 h after storage at 22.degree.-24.degree. C, the cell and unit plasma content of lactate dehydrogenase (LDH), .beta.-glucuronidase and lysozyme was measured. These enzymes served as markers for cell lysis, and primary and specific neutrophil granule contents, respectively. The effect on neutrophil chemotaxis of adding a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), to the storage medium was also measured along with the ability of PMN to degranulate in response to an inflammatory stimulus before and after storage. The cell content of granule markers was largely unchanged during storage, except in the case of the units at a concentration of 8 .times. 107 PMN per ml stored without bicarbonate. In these units, lysozyme activity decreased by 15 .+-. 7% after 48 h of storage (P < 0.02 vs. fresh PMN). Likewise, the plasma content of LDH, .beta.-glucuronidase and lysozyme increased significantly during storage, especially in units of high cell concentration stored without bicarbonate. The majority of the increase in neutrophil granule markers was due to cell lysis, not spontaneous degranulation. In units at a concentration of 8 .times. 107 PMN per ml stored without bicarbonate, however, degranulation accounted for a > 10% decrease in specific granule contents after 48 h of storage. The chemotaxis of units at a concentration of 8 .times. 107 PMN per ml stored without bicarbonate was severely impaired after 24 h of storage (58 .+-. 4% compared to fresh PMN, P < 0.01) and this was partially prevented by the addition of PMSF (74 .+-. 5% chemotaxis compared to fresh PMN, P < 0.01 vs. PMN stored at 8 .times. 107 PMN/ml without PMSF). This indicates a limited role for proteolysis as a possible mechanism for chemotactic impairment in PMN stored at high cell concentration. PMSF did not prevent chemotactic impairment after 48 h of storage. Finally, after 48 h of storage, the degranulation of .beta.-glucuronidase (in response to F-Met-Leu-Phe) was decreased by 54-75% compared to fresh PMN (P < 0.02 in all cases) and the degranulation of lysozyme was decreased by 53-55% (P < 0.02 in all cases). No differences were observed among units stored at 2 vs. 8 .times. 107 PMN per ml. Autotoxicity due to release of granule contents during granulocyte concentrate storage is likely to be of only minor importance to units stored at usual cell concentrations (2 .times. 107 PMN/ml) for 24 h but may impair the chemotaxis of PMN stored at high cell concentration (8 .times. 107 PMN/ml). Finally, PMN stored at 48 h, regardless of cell concentration, have defective capacity to degranulate in response to inflammatory stimuli.