SIMULTANEOUS CALCIUM-DEPENDENT DELIVERY OF NEUTROPHIL LACTOFERRIN ANDREACTIVE OXYGEN METABOLITES TO ERYTHROCYTE TARGETS - EVIDENCE SUPPORTING GRANULE-DEPENDENT TRIGGERING OF SUPEROXIDE DEPOSITION

Optical microscopic techniques have been utilized to study the deposit ion of lactoferrin, a specific granule marker, and superoxide anions i nto target erythrocytes during antibody-dependent phagocytosis. Previo us studies from this laboratory have shown that the entry of superoxid e anions into erythrocytes can be sensitively monitored with Soret ban d transmitted light microscopy. When neutrophils were incubated with B APTA/AM, an intracellular Ca2+ chelator, they phagocytosed IgG-opsoniz ed sheep red blood cells (SRBC) but did not affect the microscopically detected absorption of their Soret band. When these same erythrocytes were observed after the infusion of 20 muM ionomycin, a Ca2+ ionophor e, 58% of the cell-bound SRBC targets were destroyed immediately. Howe ver, neutrophils from chronic granulomatous disease (CGD) patients wer e unable to affect the Soret absorption of erythrocyte targets under a ny conditions. These results suggest that a Ca2+ signal can participat e in triggering superoxide deposition in targets. Since Ca2+ signals a re known to participate in the exocytic release of granules, we tested the hypothesis that specific lactoferrin-bearing granules are deliver ed to targets in parallel with superoxide anions. Lactoferrin delivery to phagosomes was monitored using resonance energy transfer (r.e.t.) microscopy. SRBCs were opsonized with both unconjugated and rhodamine B isothiocyanate (RBITC)-conjugated rabbit anti-SRBC IgG. After incuba tion with adherent neutrophils, the samples were washed, fixed with 3. 7% paraformaldehyde, then labeled with fluorescein isothiocyanate (FIT C)-conjugated antilactoferrin IgG. Energy transfer between FITC and RB ITC was imaged microscopically and quantitated by photon counting. Sig nificant levels of r.e.t. between antilactoferrin and anti-SRBC labels were observed after phagocytosis, but not in the absence of acceptor fluorochromes. To control for r.e.t. specificity, neutrophil membranes were labeled with FITC-conjugated, anti-HLA IgG after internalization of rhodamine B-tagged SRBCs (RSRBCs). Although r.e.t. between lactofe rrin and RSRBCs labels was observed, no r.e.t. between HLA and RSRBC l abels could be found. Further studies showed that treatment of neutrop hils with BAPTA inhibited r.e.t. between anti-lactoferrin and RSRBCs. However, addition of ionomycin relieved this inhibition of energy tran sfer. These experiments show that both lactoferrin and superoxide deli very to targets are regulated in parallel by a Ca2+-dependent pathway. Furthermore, by combining Soret microscopy with r.e.t. microscopy, we have shown that superoxide anions and lactoferrin are delivered to th e same phagosomes. We speculate that the NADPH oxidase, which produces superoxide anions, is assembled on specific granule membranes, thus a ccounting for their parallel Ca2+-dependence, activation, and delivery . (C) 1993 Wiley-Liss, Inc.