Transient increase in cyclic AMP localized to macrophage phagosomes

Abstract

Cyclic AMP (cAMP) regulates many biological processes and cellular functions. The importance of spatially localized intracellular gradients of cAMP is increasingly appreciated. Previous work in macrophages has shown that cAMP is produced during phagocytosis and that elevated cAMP levels suppress host defense functions, including generation of proinflammatory mediators, phagocytosis and killing. However, the spatial and kinetic characteristics of cAMP generation in phagocytosing macrophages have yet to be examined. Using a Förster resonance energy transfer (FRET)-based cAMP biosensor, we measured the generation of cAMP in live macrophages. We detected no difference in bulk intracellular cAMP levels between resting cells and cells actively phagocytosing IgG-opsonized particles. However, analysis with the biosensor revealed a rapid decrease in FRET signal corresponding to a transient burst of cAMP production localized to the forming phagosome. cAMP levels returned to baseline after the particle was internalized. These studies indicate that localized increases in cAMP accompany phagosome formation and provide a framework for a more complete understanding of how cAMP regulates macrophage host defense functions.

Figure 1. Changes in cAMP measured by biochemical and FRET microscopic methods in macrophages.

A. RAW cells were plated overnight at 2×10⁶ cells per well and stimulated with 200 µM forskolin for 20 min or with EdTx for 3 h. During the final 20 min incubation, either PBS (blank bars) or opsonized sRBCs (open bars) were added to cells as indicated. Total cAMP was quantified as described in material and methods. Data represent the mean ± SEM. B. and C. Cells expressing C4 control (B) or the Epac-camps biosensor (C) were either analyzed directly or following treatment with the indicated compounds. The relative amount of FRET after each condition was determined and the results are graphed as mean ± SEM (n = 50–100 cells per condition). D. A representative phase-contrast (top) and corresponding E_A image (bottom) of an untreated or EdTx-treated macrophage. Color bar indicates scale of ratio and scale bar is 10 µm.

Figure 2. No change in total cAMP during phagocytosis.

RAW cells were transfected with plasmids for C4 control or the Epac-camps biosensor. Total cellular E_A was measured over time and plotted relative to the first measured value. A. Measurements of unfed macrophages showed small decreases in FRET, indicating selective photobleaching of mCit. B. Transfected cells were fed opsonized targets and phagocytosis was synchronized as described in material and methods. No significant changes in cAMP were detectable during phagocytosis. Results are shown as mean ± SEM of 4–7 cells.

Figure 3. Levels of intracellular cAMP are independent of the number of particles phagocytosed.

RAW cells were plated at 5×10⁵ cells per coverslip and transfected with plasmids encoding the C4 control (A) or the mCFP-mCit Epac-camps biosensor (B). Opsonized sRBCs were then added to the cultures and cells were permitted to phagocytose for 1 h at 37°C. Non-ingested sRBCs were washed away and the images were collected and analyzed. There was no significant correlation between E_A and the number of sRBCs ingested in the C4 control (A, p = 0.5509) or Epac-camps (B, p = 0.7879) cells, when analyzed by linear regression (n = 50–52 cells per condition).

Figure 4. Transient burst of cAMP at the developing phagosome.

RAW cells expressing C4 or the Epac-camps biosensor were fed opsonized targets and component images for phase-contrast and FRET were taken every 30 sec to capture the phagocytic process from initiation to closure of the phagocytic cup. A. and B. Left insert: A phase-contrast (top) and corresponding E_A image (bottom) of intact live macrophages transfected with C4 control (A) or Epac-camps (B), from the designated time intervals. Right inserts: One minute time course of a magnified portion of the cell transfected with the specified plasmid (beginning immediately after the sRBCs were added to the culture). The red circle denotes the location of the opsonized sRBC on the E_A image. Color bar indicates scale of ratio and scale bar is 10 µm in the left insert and 5 µm.in the right insert C. To normalize for non-specific cAMP-mediated effects during phagocytosis, the data are shown as the phagosome-specific difference in E_A between C4 control and Epac-camps (ec) biosensor-expressing cells. (ie., (E_A(C4-phago)/E_A(C4-cell)) − (E_A(ec-phago)/E_A(ec-cell)); n = 10 cells) D. To verify that the differences seen between cells transfected with the C4 control construct and the Epac-camps construct were not due to selective bleaching of one of the fluorescent proteins, the R_I values are plotted (ie., (R_I(C4-phago)/R_I(C4-cell)) − (R_I(ec-phago)/R_I(ec-cell))).