Poster Board I-377
We have identified a 29 kDa protein from human neutrophils which binds to the NADPH oxidase component p67phox and enhances superoxide anion (O2−) production in a cell-free, reconstituted, NADPH oxidase system. The protein was identified as peroxiredoxin VI by sequence and the recombinant molecule was found to have both peroxiredoxin activity and calcium-independent PLA2 activity that is optimum at low pH (aiPLA2). Although p29 Prdx VI is found in many tissues, its role in myeloid cells is not well established. To explore other roles of p29, in addition to its effect on the respiratory burst, a PLB-985 cell line with shRNA mediated knockdown of p29 Prdx VI was established. Chemotaxis, as well as ingestion and killing S. aureus were determined in knockdown and control cells.
PLB-985 cells were transfected with a plasmid encoding a p29 Prdx VI targeting shRNA or a negative control plasmid and stable transfectants were selected in puromycin containing media. Knockdown of p29 Prdx VI was confirmed by Western blot with no changes in actin or other oxidase components. After maturation of the knockdown and control cells by DMSO for 4 days, each was combined with serum opsonized Staph. aureus in a 2 to1 human cell to bacterial cell ratio, bacterial cells remaining at various times were measured by plating aliquots of the cell mixtures and counting bacterial colonies which grew overnight. To evaluate ingestion, aliquots of the cell mixtures were transferred to slides by cytospin, stained, and examined under a light microscope to determine what proportion of PLB-985 cells had internalized bacteria. To evaluate chemotaxis, distances of migration toward chemo-attractant (opsonized zymosan) in a Boyden chamber were measured for differentiated p29 Prdx VI knockdown and control cells.
Using stable expression of shRNA p29 protein was reduced to 31+/-18% (SD) of that in non-knockdown control cells. In two separate assays of bactericidal activity, cells without knockdown of p29 Prdx VI had 17 and 13% of initial bacteria surviving at 30 min; cells with p29 Prdx VI knockdown had 30 and 56% of bacteria surviving. This defect in bactericidal activity since ingestion was no different between the two types of cells at 0, 5, 10, and 15 min after addition of the bacteria. In response to zymosan activated serum, stimulated directed migration (distance of leading front in response to zymosan activated serum minus distance of leading front in response to buffer) was greater in cells without knockdown (23.9 ± 3.0 microns, mean ± SEM, n = 4 separate experiments) than movement by cells with knockdown of p29 Prdx VI (18.3 ± 5.3 microns). The difference was significant, p<0.05 by paired t test.
Optimal O2− production during the respiratory burst in intact myeloid cells is dependent on p29. Deficient bactericidal activity was demonstrated at 30 min; this decrease could not be associated with a difference in ingestion. In addition, directed cell migration was also decreased in cells with decreased amounts of p29 Prdx VI. These results indicate that in addition to its effect on the respiratory burst, p29 Prdx supports multiple functions in neutrophils.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.