Simultaneous calcium‐dependent delivery of neutrophil lactoferrin and reactive oxygen metabolites to erythrocyte targets: Evidence supporting granule‐dependent triggering of superoxide deposition

Abstract

Optical microscopic techniques have been utilized to study the deposition of lactoferrin, a specific granule marker, and superoxide anions into target erythrocytes during antibody-dependent phagocytosis. Previous studies from this laboratory have shown that the entry of superoxide anions into erythrocytes can be sensitively monitored with Soret band transmitted light microscopy. When neutrophils were incubated with BAPTA/AM, an intracellular Ca²⁺ chelator, they phagocytosed IgG-opsonized 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 μM ionomycin, a Ca²⁺ ionophore, 58% of the cell-bound SRBC targets were destroyed immediately. However, neutrophils from chronic granulomatous disease (CGD) patients were unable to affect the Soret absorption of erythrocyte targets under any conditions. These results suggest that a Ca²⁺ signal can participate in triggering superoxide deposition in targets. Since Ca²⁺ signals are known to participate in the exocytic release of granules, we tested the hypothesis that specific lactoferrin-bearing granules are delivered 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 incubation with adherent neutrophils, the samples were washed, fixed with 3.7% paraformaldehyde, then labeled with fluorescein isothiocyanate (FITC)-conjugated antilactoferrin IgG. Energy transfer between FITC and RBITC was imaged microscopically and quantitated by photon counting. Significant 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). Althougth r.e.t. between lactroferrin and RSRBCs labels was observed, no r.e.t. between HLA and RSRBC labels could be found. Further studies showed that treatment of neutrophils with BAPTA inhibited r.e.t. between anti lactoferrin and RSRBCs. However, additon of ionomycin relieved this inhibition of energy transfer. These experiments show that both lactoferrin and superoxide delivery to targets are regulated in parallel by a Ca²⁺ -dependent pathway. Furthermore, by combining Soret microscoy with r.e.t. microscopy, we have shown that superoxide anions and lactoferrin are delivered to the same phagosomes. We speculate that the NADPH oxidase, which produces superoxide anions, is assembled on specific granule membranes, thus accounting for their parallel Ca²⁺ -dependence, activation, and delivery.