Activation of protein kinase C beta II by the stereo-specific phosphatidylserine receptor is required for phagocytosis of apoptotic thymocytes by resident murine tissue macrophages

Abstract

We showed previously that protein kinase C (PKC) is required for phagocytosis of apoptotic leukocytes by murine alveolar (AMø) and peritoneal macrophages (PMø) and that such phagocytosis is markedly lower in AMø compared with PMø. In this study, we examined the roles of individual PKC isoforms in phagocytosis of apoptotic thymocytes by these two Mø populations. By immunoblotting, AMø expressed equivalent PKC eta but lower amounts of other isoforms (alpha, betaI, betaII, delta, epsilon, mu, and zeta), with the greatest difference in betaII expression. A requirement for PKC betaII for phagocytosis was demonstrated collectively by phorbol 12-myristate 13-acetate-induced depletion of PKC betaII, by dose-response to PKC inhibitor Ro-32-0432, and by use of PKC betaII myristoylated peptide as a blocker. Exposure of PMø to phosphatidylserine (PS) liposomes specifically induced translocation of PKC betaII and other isoforms to membranes and cytoskeleton. Both AMø and PMø expressed functional PS receptor, blockade of which inhibited PKC betaII translocation. Our results indicate that murine tissue Mø require PKC betaII for phagocytosis of apoptotic cells, which differs from the PKC isoform requirement previously described in Mø phagocytosis of other particles, and imply that a crucial action of the PS receptor in this process is PKC betaII activation.

Figure 1. Resident murine AMø and PMø differ in expression of multiple PKC isoforms

Whole cell lysates of AMø and PMø of normal C57BL/6 mice cultured as adherent monolayers for 2 hours were analyzed for expression of PKC isoforms by Western blotting using isoform-specific antibodies.

Figure 2. Overnight PMA treatment decreases expression of cPKC and nPKC isoforms by AMø and PMø

Resident AMø and PMø were incubated overnight at 37° C and 5% CO₂ with either PMA (8.1 μM final concentration) or the appropriate amount of ethanol as the control, and then analyzed for expression of PKC isoforms by Western blotting using isoform-specific antibodies.

Figure 3. Depletion of cPKCs and nPKCs by overnight PMA treatment reduces Mø phagocytosis of apoptotic thymocytes

Phagocytosis of apoptotic thymocytes by resident AMø and PMø incubated overnight in medium containing a final concentration of 8.1 μM PMA (black bars) or the appropriate amount of ethanol as a control (gray bars) was determined by examining H & E stained slides under oil immersion. (A) percentage of phagocytic Mø; (B) phagocytic index. Data are mean ± SEM of 4 replicates in two experiments. *, p<0.05, unpaired Student t-test.

Figure 4. Rottlerin inhibits Mø phagocytosis of apoptotic thymocytes in a dose-dependent fashion

Phagocytosis of apoptotic thymocytes by resident AMø and PMø was determined after 30 minutes incubation under the following conditions: control medium (light gray bars), 10 μM rottlerin (black bars), 40 μM rottlerin (white bars), and 100 μM rottlerin (dark gray bars). (A) percentage of phagocytic Mø; (B) phagocytic index. Data are mean ± SEM of 3 replicates and are representative of results of two experiments. *, p<0.05, ANOVA with post-hoc Dunnett's testing.

Figure 5. Ro-32-0432 inhibits Mø phagocytosis of apoptotic thymocytes in a dose-dependent fashion

Phagocytosis of apoptotic thymocytes by resident AMø and PMø was determined after 30 minutes incubation under the following conditions: control medium (gray bars), 9 μM Ro-32-0432 (black bars), 28 μM Ro-32-043 (white bars). (A) percentage of phagocytic Mø; (B) phagocytic index. Control, gray bars;, black bars; 2, white bars. Data are mean ± SEM of 4 replicates in two experiments. *, p<0.05, ANOVA with post-hoc Dunnett's testing.

Figure 6. Myristylated PKC βII blocking peptide specifically inhibits Mø phagocytosis of apoptotic thymocytes

Phagocytosis of apoptotic thymocytes by resident AMø and PMø was determined after 30 minutes incubation in control medium (light gray bars) or in medium containing a final concentration of 100 μM in water of one of the following myristylated blocking peptides: PKC α peptide (black bars), PKC βI (white bars), PKC βII peptide (dark gray bars). (A) percentage of phagocytic Mø; (B) phagocytic index. Data are mean ± SEM of three replicates and are representative of two experiments.

Figure 7. PS liposomes induce translocation of multiple PKC isoforms into the membrane and cytoskeletal fractions

Resident murine PMø were incubated for various times with liposomes consisting of L-α-PS (left-hand column) or control L-α-PI (right-hand column), and then expression of PKC isoforms in the cytosolic fraction and in the membrane & cytoskeletal fraction was analyzed by Western blotting. C, control (no liposomes).

Figure 8. Resident murine AMø & PMø express PS-R'

A. RT-PCR analysis of mRNA. Equally loaded PCR reactions were normalized to achieve the same expression of the housekeeping gene GAPDH (right side of the figure), by adjusting the volume of the cDNA product used in each reaction. The black and white image was inverted after scanning. Neg is a negative control where no RT reaction was performed and aliquots of RNAs from both resident Mø were mixed. The reference ladder is 1 kb Plus. Results are representative of two experiments performed with identical outcomes. B, C. Flow cytometric analysis of surface protein expression. AMø & PMø from normal C57BL/6 mice were stained in suspension with mAb 217 culture-supernatant or with control IgM from a murine myeloma, with biotinylated goat antimouse IgM F(ab'₂) and with streptavidin-phycoerythrin, and then analyzed by flow cytometry, counting 10,000 cells per condition. B, AMø; C, PMø. The narrow line depicts staining with isotype control and the dark line depicts staining with mAb 217. Representative histograms are shown; similar results were obtained in four independent experiments.

Figure 9. mAb against PS-R' blocks Mø phagocytosis but not adhesion of apoptotic thymocytes

Resident AMø and PMø from normal C57BL/6 mice were pre-treated with saturating amounts of control antibody (gray bars) or anti-PS-R' mAb 217 (black bars) and then phagocytosis or adhesion of apoptotic thymocytes was determined by examining H-E stained slides under oil immersion. A, B. Phagocytosis. Data are mean ± SEM of 3-5 replicates per condition in a single experiment; *, p<0.05, unpaired t-test. C, D. Adhesion. Data are mean ± SEM of 3-5 replicates per condition in a single experiment. Similar results were found in an additional experiment.

Figure 10. Antibody against PS-R' inhibits PKC βII translocation induced by PS liposomes

Resident PMø were incubated with saturating amounts of control rabbit IgM (top row) or anti-PS-R' mAb 217 (bottom row), exposed to PS-liposomes to a final concentration of 0.11 mM for up to 15 min at 37° C and 5% CO₂ , and then PKC βII expression in cytosolic versus membrane and cytoskeletal fractions was assayed by Western analysis. C, control (no liposomes).