Increased Protein Kinase C Activity in Low Density Eosinophils

Eosinophil heterogeneity is expressed in cell density, membrane receptors and function. It has been observed that increases in some functional activities correlate with decreased sedimentation density in human eosinophils. However, the cellular mechanisms to explain the up-regulation of eosinophil function have not been fully explored. Protein kinase C (PKC) is an important family of enzymes mediating signal transduction for a wide variety of functions in many different cell types. Changes in the activity of PKC could explain some of the observed differences in function. In these experiments, PKC activity of human granulocyte lysate supernatants was measured as the phosohatidyl serine-dependent transfer of ³³P from [γ-³²P]ATP to a protein substrate under conditions of maximal stimulation; a measure of activatable PKC concentration. We observed that the activity present in eosinophils (87.2 ± 8.4 pmol PO₄ incorporated into histone per minute per 10⁶ cells, n = 30) was not significantly different from that of neutrophils assayed under the same conditions (91.5 ± 5.6 U, n = 31) but the percent of total activity that was phosphatidyl serine dependent was greater in eosinophils (97 ± 1% vs 81 ± 1 % for neutrophils, p = 0.001). Blood eosinophils isolated from low density Percoll fractions had a higher activity (120 ± 16 U) than that found in the higher density cells from the same subjects (81 ± 19 U, n = 9, p = 0.011). When eosinophils recovered from bronchoalveolar lavage (BAL) fluid after segmental Ag challenges were assayed, the PKC activity of BAL eosinophils was similar to that of blood-derived eosinophils of equal density and low density BAL eosinophil PKC tended to be equal to or greater than higher density cells. The β isozyme of PKC but not the α or γ was detected in eosinophils by Western blotting with isozyme-specific mAb. These data indicate that eosinophil PKC activity is primarily caused by the β isozyme, is related to cell density in blood-derived cells, and may have a relationship to cell function.