IgM promotes the clearance of small particles and apoptotic microparticles by macrophages

Abstract

Background: Antibodies are often involved in enhancing particle clearance by macrophages. Although the mechanisms of antibody-dependent phagocytosis have been studied for IgG in greater detail, very little is known about IgM-mediated clearance. It has been generally considered that IgM does not support phagocytosis. Recent studies indicate that natural IgM is important to clear microbes and other bioparticles, and that shape is critical to particle uptake by macrophages; however, the relevance of IgM and particle size in their clearance remains unclear. Here we show that IgM has a size-dependent effect on clearance.

Methodology/principal findings: We used antibody-opsonized sheep red blood cells, different size beads and apoptotic cells to determine the effect of human and mouse IgM on phagocytosis by mouse alveolar macrophages. Our microscopy (light, epifluorescence, confocal) and flow cytometry data show that IgM greatly enhances the clearance of small particles (about 1-2 micron) by these macrophages. There is an inverse relationship between IgM-mediated clearance by macrophages and the particle size; however, macrophages bind and internalize many different size particles coated with IgG. We also show that IgM avidly binds to small size late apoptotic cells or bodies (2-5 micron) and apoptotic microparticles (<2 µm) released from dying cells. IgM also promotes the binding and uptake of microparticle-coated beads.

Conclusions/significance: Therefore, while the shape of the particles is important for non-opsonized particle uptake, the particle size matters for antibody-mediated clearance by macrophages. IgM particularly promotes the clearance of small size particles. This finding may have wider implications in IgM-mediated clearing of antigens, microbial pathogens and dying cells by the host.

Figure 1. IgG, but not IgM, drastically enhances the uptake of sRBCs by macrophages.

Light microscopy analyses show that macrophages effectively phagocytose IgG-coated sRBCs (∼7–10 µm) compared to control sRBC with no coating antibody (p<0.05). IgM-coated sRBCs are not effectively taken up by the macrophages. Although IgM coating conditions shows a small dose-dependence at low concentrations of the antibody, this effect is less likely to represent a biologically significant effect. Phagocytic index represents the number of sRBCs internalized by 100 macrophages. Antibody dilutions used for coating sRBCs are shown within parenthesis. Data are representative of 3 experiments performed on different days with similar results. Error bars are ± SEM from 3 technical replicates.

Figure 2. Human IgG, but not IgM, drastically enhances uptake of large beads by macrophages.

(A) Macrophages effectively bind and internalize IgG-coated, but not IgM-, BSA-, or PBS-coated, large beads (∼8.31 µm; p<0.05). (B) Counting numbers of “beads/cell” shows that IgG-coated beads are taken up in greater quantities. Only a small number of cells binds or internalize IgM-coated beads. Bead uptake saturates at 100–500 µg/ml IgG-coating concentrations. Percentage values for each IgG and IgM condition will add up to the corresponding values shown in panel A. (C) Differential interference contrast (DIC) images show examples of IgG-coated 8.31 µm large beads that associate with or are engulfed by macrophages. A similar effect was not seen with IgM-, or BSA- or PBS-coated beads. Scale bar is 10 µm.

Figure 3. Human IgM preferentially enhances the binding and uptake of smaller-sized particles.

Macrophages were incubated with different size beads that are coated with IgM, IgG or PBS. (A) The percentage of macrophages involved in bead binding and uptake compared to bead size and bead coating condition. Phagocytic macrophages are the cells that contained at least one bead. Non-linear regression analyses between phagocytic microphage (%) and bead diameter: IgG (y = 60.1 x^0.1354; r² = 0.49) and IgM (y = 51.6 x^−0.815; r² = 0.94). These two regression lines are different from zero and to each other (p<0.05). PBS condition does not fit a similar mathematical equation. (B–E) Phagocytic macrophages shown in (A) were examined and counted for the number of beads bound and internalized under varying protein coating conditions and the bead diameter. The percentages of phagocytic macrophages were plotted separately for beads with (B) 1.58 µm, (C) 3.87 µm, (D) 5.19 µm and (E) 8.31 µm in mean diameter. X-axis is the same for charts B–E: 1–2, one to two; 3–4, three to four; 5+, 5 or more beads/cell. Note: Only the phagocytic macrophages are shown in the bar charts; hence, the values for each condition shown in (B–E) will add up to the corresponding values shown in (A); *, p<0.05 (Tukey's test).

Figure 4. Epifluorescence microscopy reveals that both human and mouse IgM enhance the binding and uptake of small particles by macrophages.

(A) Mouse macrophages incubated with human IgM-, human IgG-, BSA-, or DPBS-coated 1 µm beads show distinct fluorescence bead binding or internalization patterns. Left panel shows macrophages and their binding and uptake of beads coated with PBS, BSA (100 µg/ml), IgG (100 µg/ml) or dissociated IgG (100 µg/ml). Right panel shows the uptake of IgM (0–400 µg/ml) or dissociated IgM (100 µg/ml)-coated beads by representative macrophages. Coating of beads with increasing concentrations of IgM shows increasing fluorescence in the macrophages. Dissociating both IgG and IgM eliminates antibody-dependent uptake of these beads by macrophages. Numerical values denote the concentrations of IgM (µg/ml) used for coating the beads. (B) Macrophages phagocytose mouse IgG (100 µg/ml) and mouse IgM (100 µg/ml)-coated small beads (1 µm). Both human (A) and mouse (B) antibodies show similar effects. Scale bar is 10 µm.

Figure 5. Confocal microscopy images show that macrophages internalize large numbers of human IgG- and IgM-coated beads.

Confocal images show that macrophages internalize only a few BSA- or PBS-coated beads (1 µm); these beads are primarily bind to macrophages. In contrast, macrophages effectively internalize large numbers of IgG- and IgM-coated beads. Most of these beads are found as 3-D masses within the macrophages. Top and left strips in each panel show different views (X, Y, Z) of the same cells at a confocal plane. Scale bar is 10 µm.

Figure 6. Flow cytometry reveals that IgM enhances the binding and uptake of small particles by macrophages in a concentration-dependent manner.

(A) Overlay comparison (% of Max) of IgM-coated (10, 100, 200, 400 µg/ml), IgG-coated (100 µg/ml), BSA-coated (100 µg/ml) or DPBS-suspended beads. IgM conditions are represented with varying hues of green lines, IgG in a blue line, BSA in dark red, PBS in bright red, and macrophages alone with a black line. Gates A and B partition cells containing beads to resolve the population of cells containing few beads from those with many beads. (B) Gate A represents the population of cells (% of total events) with few beads as indicated by lower fluorescent values. (C) Gate B represents the population of cells (% of total events) with many beads as indicated by higher fluorescent values.

Figure 7. Human IgM preferentially binds to a subpopulation of late apoptotic cells and vesicle-like small apoptotic particles (v-SAPs) or apoptotic “microparticles”.

(A) Differential interference contrast (DIC) microscopy image. (B) Epifluorescence microscopy image showing the preferential interaction between IgM (green) and small apoptotic cells (arrow) and microparticles (arrowheads). (C) Fluorescence image showing that these cells still contain DNA stained blue with DAPI. (D) Overlay of (B) and (C) shows the locations of IgM binding in relation to the location of the nuclei. (E) Overlay of (A) and (C) shows the locations of nuclei in these cells. (F) Overlay of (A) and (B) shows IgM also binds to larger cells as small faint punctate dots. Overall, these images show that IgM avidly binds to a subpopulation of relatively smaller cells with smooth regular surfaces (arrows) and v-SAPs or microparticles (arrowheads). Scale bar is 10 µm. (G–H) Flow cytometry showing that IgM binds small size late apoptotic cells and microparticles or v-SAPs. (G) Flow cytometry shows that large amounts of IgM can bind to smaller apoptotic cells using a voltage setting that is sensitive to events measured at 2 µm or greater. (H) Flow cytometry confirming that IgM can bind to v-SAPs using a voltage setting that is sensitive to events measured at 0.5 µm or greater.

Figure 8. Flow cytometry shows that human IgM increases the uptake of fluorescent beads coated with v-SAPs by alveolar macrophages.

Fluorescent beads coated with v-SAPs were incubated with (A) PBS or IgG (100 µg/ml) or (B) with increasing concentrations of IgM (10, 100, 200, 400 µg/ml). Macrophage negative control is represented by the red line in both (A) and (B). In (A), the blue line represents PBS and the orange line is IgG. In (B), increasing concentrations of IgM are represented by the increasing darkness of the green lines.