Myeloid cells have abundant levels of the glucocorticoid-regulated 37-kDa protein annexin A1 (ANX-A1), and this is particularly the case for serosal macrophages (MØ). Using conventional gene targeting technology we have recently generated a mouse strain lacking the ANX-A1 gene (Hannon et al., 2003). Here we have used this tool to investigate the role of ANX-A1 during MØ phagocytosis.

ANX-A1 null and littermate wild type (WT) control mice (6-8 weeks old) were killed and bone marrow extracted, washed and cultured for one week in RPMI-1640 supplemented with 10% bovine serum and 15% L929-cell conditioned medium to obtain bone marrow derived MØ (BMDM ) (Peiser et al., 2000).

Assays of MØ association: BMDM were incubated for 2 h at 37°C with FITC labelled zymosan and Neisseria. Control Cells were co-incubated with cytochalasin D (2 µM) throughout the experiment. Cells were washed and harvested with PBS containing 10 mM EDTA supplemented with 4 mg/ml lidocaine-HCl prior to fixation with 4% formaldehyde. Fluorescence was analyzed by confocal microscopy and on a FACScan (Becton Dickinson, Mountain View, US) in the FL-1 channel. The median fluorescence intensity (MFI) of unloaded control cells was subtracted from the mean fluorescence of each assay condition, and the average determined. In order to determine a better understanding of events occurring during phagocytosis, BMDM were incubated for 1 h at 37°C with FITC labelled zymosan, the phagocytic process was recorded by real time microscopy. Microscopy Assays of MØ phagocytosis and cytokine production : BMDM were incubated for LPS (10 ng/ml) zymosan or Neisseria (MOI 1:50) for 2, 4, 6, 12, 24 h at 37°C and cytokine release was measured by ELISA. In certain experiments ANX-A1 localisation was recorded by confocal microscopy.

ANX-A1 null BMDM exhibited a reduced particle association, with all particles tested. For instance, cell association after 30 min with non-opsonised FITC zymosan in WT cells yielded 1944 ±54 MFI relative to 1450±48 MFI in the ANX-A1 null cells (n=3 in duplicate) this phenomenon was confirmed by real time microscopy .

Incubation with Zymosan or Neisseria (MOI 1:50) for 6 h provoked a substantial increase from basal in TNF- production 45±6 pg/ml (WT) 188±5 pg/ml (KO) and 35±2 pg/ml (WT) 42±3 pg/ml (KO) respectively. By 24 h Zymosan or Neisseria provoked a substantial increase by in IL-6 production 252±4 pg/ml (WT) 396±28 pg/ml (KO) and 38620±2895 pg/ml (WT) 43427±3317 pg/ml (KO) respectively. LPS produced an increase of IL-6 at 24 h 7363±183 pg/ml (WT) 314131±1867 pg/ml (KO) (n= 3 in duplicate). This data suggests the absence of ANX-A1 augments the inflammatory response.

In conclusion, this data demonstrates that the lack ANX-A1 is functionally associated with a reduced ability of phagocytosis and supports the existence of a tonic inhibitory role for endogenous ANX-A1 in controlling MØ activation.

Hannon R et al., (2003) Faseb J 17, 253-255
Peiser L et al., (2000) Infect. Immum 68: 1953-1963

This work was supported by a PhD. studentship of the Nuffield Foundation (Oliver Bird Fund)