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. 2004 Jul;142(5):890-8.
doi: 10.1038/sj.bjp.0705858. Epub 2004 Jun 14.

Stimulus-specific defect in the phagocytic pathways of annexin 1 null macrophages

Simon Yona  1 , Julia C Buckingham, Mauro Perretti, Roderick J Flower
Affiliations

Stimulus-specific defect in the phagocytic pathways of annexin 1 null macrophages

Simon Yona et al. Br J Pharmacol. 2004 Jul.

Abstract

The role of the glucocorticoid-regulated protein annexin 1 during the process of phagocytosis has been studied using annexin 1 null peritoneal macrophages. Wild type and annexin 1 null macrophages were incubated with several distinct phagocytic targets. No differences were observed in rate or the maximal response with respect to IgG complexes or opsonised zymosan phagocytosis, as assessed by monitoring the production of reactive oxygen species. When annexin 1 null macrophages were incubated with non-opsonised zymosan particles, they exhibited impaired generation of reactive oxygen species, which was linked to a defect in binding of cells to the particles, as determined with fluorescent zymosan. This phenomenon was further confirmed by electron microscopy analysis, where annexin 1 null macrophages internalised fewer non-opsonised zymosan particles. Specific alterations in macrophage plasma membrane markers were observed in the annexin 1 null cells. Whereas no differences in dectin-1 and FcgammaR II/III expression were measured between the two genotypes, decreased membrane CD11b and F4/80 levels were measured selectively in macrophages lacking annexin 1. These cells also responded with an enhanced release of PGE(2) and COX-2 protein expression following addition of the soluble stimulants, LPS and heat-activated IgG. In conclusion, these results suggest that participation of endogenous annexin 1 during zymosan phagocytosis is critical and that this protein plays a tonic inhibitory role during macrophage activation.

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Figures

Figure 1
Figure 1
Annexin 1 null macrophages display a stimulus-dependent defect in phagocytosis. Peritoneal MØ from WT and annexin-1 null mice were tested for their ability to phagocytose-specific particles. (a) Peritoneal cells (2.5 × 105 per sample) were incubated with IgG complexes and monitored by real-time flow cytometry for phagocytosis-related oxidation and associated fluorescence over a 204-s time frame. (b,c) As above, except that cells were loaded with DHR 123 and stimulated by addition of either (b) non-opsonised zymosan or (c) opsonised zymosan. (d) Analysis of non-opsonised FITC-zymosan association, cytochalasin D control for WT and annexin-1 null Mø shown by formula image and ▴, respectively. For all panels, data are mean ± s.e.m. of n=4 experiments performed with n=3 mice each. *P<0.05 vs respective WT value.
Figure 2
Figure 2
Electron micrographs of peritoneal MØ before and after non-opsonised zymosan incubation. Peritoneal MØ from WT and annexin-1 null mice were incubated with 200 μg ml−1 non-opsonised zymosan for 3 h, prior to fixation and processing as described in the Methods section. (a) Resting WT MØ. (b) Resting annexin-1 null MØ. (c) WT MØ engulfed with several zymosan particles (asterisks highlight some of these phagosomes). (d) Annexin-1 null MØ with fewer zymosan particles. Arrowheads indicate particles associated with the plasma membrane rather than fully internalised. Inset: lower magnification image showing an example of frustrated phagocytosis with several zymosan particles in close vicinity, but not internalised, of the cell. Images are representative of 14 distinct animals. Panels (a–d), bar 1 μm; inset, bar 2 μm.
Figure 3
Figure 3
Quantification of zymosan particle association with WT and annexin-1 null MØs. A ‘blind' analysis of fully internalised or loosely associated (clearly in contact with the plasma membrane) particles was performed on nine distinct electron micrographs prepared as in Figure 2. Data are mean±s.e.m. of n=9 from 14 distinct animals of a genotype. *P<0.05 vs WT value.
Figure 4
Figure 4
Comparison of membrane marker expression between WT and annexin 1 null peritoneal MØs. Peritoneal MØ from WT and annexin-1 null mice (2.5 × 105 per sample) were stained with specific mAbs to quantify membrane expression of dectin-1 (a), FcRg-II/III (b), F4/80 (c) and CD11b (d). Panel (d) also shows CD11b immunoreactivity in permeabilised MØ to estimate total antigen expression. Inset: immunofluorescent image of total CD11b staining in WT and annexin 1 null MØ. Data are the mean ± s.e.m. of n=4–5 experiments performed with five mice each. *P<0.05 vs respective WT values.
Figure 5
Figure 5
WT and annexin 1 null MØ activation as measured by PGE2 generation and COX-2 or iNOS expression. Peritoneal MØ from WT and annexin-1 null mice (1 × 106 per sample) were seeded in a 24-well plate and activated with zymosan (200 μg ml−1), heat aggregated IgG (HA IgG; 200 μg ml−1) or LPS (10 ng ml−1) for a 0.5–20-h period. (a) Time-course of PGE2 release in response to zymosan. Data are mean±s.e.m. of three experiments performed in triplicate with cells pooled from 7–10 mice. (b) COX-2 and iNOS immunoreactivity in cell extracts as measured following 20-h cell incubation. Gel representative of two experiments.
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