IgG autoantibodies against deposited C3 inhibit macrophage-mediated apoptotic cell engulfment in systemic autoimmunity - PubMed (original) (raw)

IgG autoantibodies against deposited C3 inhibit macrophage-mediated apoptotic cell engulfment in systemic autoimmunity

Karla D Kenyon et al. J Immunol. 2011.

Abstract

Defective clearance of apoptotic cells has been shown in systemic lupus erythematosus (SLE) and is postulated to enhance autoimmune responses by increasing access to intracellular autoantigens. Until now, research has emphasized inherited rather than acquired impairment of apoptotic cell engulfment in the pathogenesis of SLE. In this study, we confirm previous results that efficient removal of apoptotic cells (efferocytosis) is bolstered in the presence of wild-type mouse serum, through the C3 deposition on the apoptotic cell surface. In contrast, sera from three mouse models of SLE, Mer(KD), MRL(lpr), and New Zealand Black/WF1 did not support and in fact actively inhibited apoptotic cell uptake. IgG autoantibodies were responsible for the inhibition, through the blockade of C3 recognition by macrophages. Consistent with this, IgG removal reversed the inhibitory activity within autoimmune serum, and purified autoimmune IgG blocked both the detection of C3 on apoptotic cells and C3-dependent efferocytosis. Sera from SLE patients demonstrated elevated anti-C3b IgG that blocked detection of C3 on apoptotic cells, activity that was not found in healthy controls or patients with rheumatoid arthritis, nor in mice prior to the onset of autoimmunity. We propose that the suppression of apoptotic cell disposal by Abs against deposited C3 may contribute to increasing severity and/or exacerbations in SLE.

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Figures

Figure 1

Figure 1. C3 deposition was necessary but not sufficient for serum enhancement of apoptotic cell engulfment

(A) Comparison of apoptotic Jurkat T cell uptake by J774 macrophages in 10% heat-inactivated fetal bovine serum (FBS) or 10% fresh C57BL/6 mouse serum (BL/6). Arrows indicate engulfed apoptotic cells or cell fragments. (B) Quantification of engulfment assay described in (A) showed enhanced uptake using serum from BL/6, ICR or Balb/c mice compared to FBS, n=3. (C) Engulfment for apoptotic, but not viable, Jurkat T cells was seen using BL/6 serum, n=3. (D) C3 on the surface of apoptotic Jurkats was detected by FACS after treatment with BL/6 serum, but not after no serum or heat-inactivated BL/6 serum (Δ H BL/6), n=3. Compared to BL/6 serum, engulfment of apoptotic Jurkats was reduced in media containing (E) ΔH BL/6 and C3 knockout mouse serum (C3 ™/ ™), n=3. (F) Treatment of BL/6 serum with neutralizing goat anti-mouse C3 also reduced efferocytosis compared to non-treated (NT) or control antibody (IgG), n=3. (G) C3 could be detected on apoptotic Jurkats by FACS after treatment with BL/6 serum, even after a second incubation in serum free media (BL/6 Serum → No Serum), n=3. (H) Phagocytosis of Jurkats was observed when the assay was preformed in BL/6 serum but not without serum despite the presence of C3 on apoptotic Jurkats preincubated in BL/6 serum (grey bar) compared to no serum (black bar), n=3. The results in this figure are expressed as Phagocytic Index (PI), which is defined as the total number of apoptotic targets ingested divided by the total number of phagocytes counted multiplied by 100. PI is expressed as a percent of non-autoimmune serum control. Unfilled histograms are isotype labeling, filled histograms are anti-C3 labeling.

Figure 2

Figure 2. Serum from autoimmune mice did not support, and in fact inhibited, apoptotic cell engulfment

(A, B, C) Uptake was assessed after macrophages were incubated with apoptotic cells in media containing either C57BL/6 (black bars) or MerKD serum (grey bars). (A) Uptake of apoptotic targets, either Jurkat T cells (n=37), a mouse CD4+ T cell line (CD4+) (n=3) or mouse splenocytes (n=2), was reduced by MerKD serum compared to C57BL/6. (B) MerKD serum reduced apoptotic Jurkat engulfment by J774 macrophages (n=37), thioglycollate-elicited peritoneal macrophages (Elicited) (n=4), or bone marrow-derived macrophages (BMDM) (n=3). (C) Serum from 6 week-old Merkd mice, which have not yet developed autoimmunity, supported similar levels of efferocytosis as C57BL/6 serum, n=4. (D, E, F) J774 macrophages were co-cultured with apoptotic Jurkats in the presence of 5% non-autoimmune serum (black bar), 5% non-autoimmune serum mixed with 2.5% autoimmune serum (grey bar with black lines) or 5% autoimmune serum (grey bar). Serum from autoimmune (D) MRL_lpr_ (n=9), (E) NZBWF1 (n=4) and (F) MerKD (n=3) did not support uptake of apoptotic Jurkats by themselves and inhibited the uptake of apoptotic Jurkats promoted by non-autoimmune (MRL or BL/6) serum. PI is expressed as a percent of non-autoimmune serum control.

Figure 3

Figure 3. Sera from autoimmune mice inhibited C3 protein detection on the surface of apoptotic cells

C3 on apoptotic Jurkats incubated in media containing non-autoimmune serum (black), non-autoimmune serum mixed with autoimmune serum (dark grey) and autoimmune serum (light grey) was detected by FACS (A,C) or Western blot (B, D). Inclusion of serum from autoimmune MRL_lpr_ mice with pre-autoimmune MRL serum (A) blocked detection of C3 on apoptotic cells by FACS analysis (n=5) but not (B) total C3 fragment deposition determined by Western blotting of the apoptotic cell extracts (n=3). (C,D) Similar results were obtained with autoimmune MerKD serum mixed with non-autoimmune serum from C57BL/6 mice (n=3). Unfilled histograms are isotype labeling, filled histograms are anti-C3 labeling. The geometric mean fluorescence for C3 (C3 Protein MFI) is expressed as a percent of non-autoimmune serum control.

Figure 4

Figure 4. The inhibitory activity in autoimmune serum co-eluted with immunoglobulin

Serum from (A) MerKD mice or (B) control C57BL/6 was fractionated on a Superdex™200 size gel filtration column. For each fraction, the relative serum protein levels (grey histogram) and apoptotic Jurkat T cell engulfment by J774 macrophages after mixing with C57BL/6 serum (black bars) are depicted. For phagocytosis assay, whole serum controls (grey bars) were 5% C57BL/6 serum, 5% C57BL/6 serum + 2.5% MerKD serum (mixed serum) and 5% MerKD serum. Analysis of the inhibitory activity in serum fractions from each strain was performed separately, and data represent the mean of samples run in duplicate from a single experiment.*Fractions in which both duplicates demonstrated inhibitory activity.

Figure 5

Figure 5. IgG depletion with protein G-sepharose removed the inhibitory activity present in autoimmune serum

Analyses of MRL_lpr_ (A–C) and MerKD (D–E) sera treated with protein G-sepharose beads to remove serum IgG. (A, D) Engulfment assays using J774 macrophages and apoptotic Jurkat T cells. Single serum samples contained 5% serum each while “Mixed Serum” samples contained 5% untreated pre- or non-autoimmune serum + 2.5% autoimmune serum. Autoimmune serum was either untreated (black bars), control sepharose bead treated (dark grey bars) or protein G-sepharose bead treated (light grey bars). Removing IgG from (A) MRL_lpr_ serum and (C) Merkd serum using Protein G Sepharose did not restore the ability of these sera to promote uptake of apoptotic cells but did prevent these autoimmune sera from inhibiting the uptake of apoptotic cells when mixed with non-autoimmune sera, n=3. (B, E) C3 was detected by FACS on apoptotic Jurkats treated with either pre- or non-autoimmune serum (black), autoimmune serum (dark grey), pre- or non-autoimmune serum mixed with autoimmune serum (medium grey) or pre- or non-autoimmune serum mixed with protein G treated, IgG-depleted autoimmune serum (light grey). Protein G removal of IgG from (B) MRL_lpr_ serum, but not (E) Merkd serum allowed C3 to be detected with the autoimmune sera was mixed with pre- or non -autoimmune sera, n=3. Western Blot demonstrated that C3 protein and distribution of its fragments bound to the apoptotic cells was unchanged when exposed to (C) MRL, MRL_lpr_ or a mixture of both sera or (F) C57BL/6, Merkd or a mixture of both sera, n=3. PI is expressed as a percent of non-autoimmune serum control. Unfilled histograms are isotype labeling, filled histograms are anti-C3 labeling. The geometric mean fluorescence for C3 (C3 Protein MFI) is expressed as a percent of non-autoimmune serum control.

Figure 6

Figure 6. Apoptotic cell engulfment and C3 protein detection were inhibited by IgG purified from autoimmune sera

(A, C) Engulfment of apoptotic Jurkats by J774 macrophages in pre- or non-autoimmune sera alone or mixed with IgG purified from either pre- or non-autoimmune or autoimmune sera. IgG purified from (A) MRL_lpr_ and (C) MerKD sera inhibited the uptake of apoptotic Jurkats seen in the presence of pre- or non-autoimmune sera, n=4. (B, D) C3 was detected by FACS on apoptotic Jurkat T cells incubated with pre- or non-autoimmune serum alone (black), or pre- or non-autoimmune serum mixed with IgG purified from pre- or non-autoimmune (dark grey) or autoimmune (light grey) mouse serum. The detection of C3 was inhibited by IgG purified from (B) MRL_lpr_ and (D) MerKD, n=3. IgG purified from non-autoimmune sera had no effect on uptake or FACS detection of C3, n=3. PI is expressed as a percent of non-autoimmune serum control. Unfilled histograms are isotype labeling, filled histograms are anti-C3 labeling. The geometric mean fluorescence for C3 (C3 Protein MFI) is expressed as a percent of non-autoimmune serum control. (E) C3 deposition was unaltered as detected by Western blot analysis of total C3 protein and distribution of its fragments bound to apoptotic cells exposed to pre- or non-autoimmune serum mixed with IgG purified from either autoimmune serum or pre- or non-autoimmune controls. (F) IgG from autoimmune MRL_lpr_ and MerKD sera bound purified C3 and its degradation products by immunoblotting, n=3. (G) J774 macrophage phagocytosis of apoptotic Jurkats (Efferocytosis) but not anti-CD3 coated viable Jurkats (Fc-Mediated) was enhanced by C57BL/6 sera, n=3. (H–I) Sera from 23 MerKD mice were tested by ELISA for anti-C3b and markers of autoimmunity (anti-dsDNA or anti-chromatin). Each dot represents an individual animal, with the line representing the mean for the group and the whiskers representing the SEM. Elevated levels of anti-C3b were detected in the sera of MerKD mice with (H) anti-dsDNA (n=17) or (I) anti-chromatin (n=20) autoantibodies compared to the MerKD mice lacking these autoantibodies, n=6 and 3 respectively

Figure 7

Figure 7. SLE sera contained IgG against C3b that blocked opsonized C3 detection

(A) ELISA showed that the mean anti-C3b titers were higher in sera from SLE patients (n=53) compared to sera from healthy controls (HC) (n=35) or patients with rheumatoid arthritis (n =18). (B) FACS detection of C3 on apoptotic Jurkats pre-incubated with fresh human sera (Control) was reduced by mixing heat-inactivated SLE (n=16) but not heat-inactivated HC sera (n=16). (C) Removal of IgG with Protein G treatment restored the FACS detection of C3 from selected SLE sera (n=3) while the IgG purified from these SLE sera blocked C3 detection (n=3). Removal of IgG and purifed IgG from HC sera had no effect on the detection of C3 (n=3). (D) HMDM uptake of apoptotic Jurkats was enhanced by fresh, but not heat inactivated (ΔH) human sera (n=4). (E) Mixing sera from SLE patients (n=14) or healthy controls HC (n=16) with fresh human sera did not inhibit uptake of apoptotic Jurkats compared to fresh sera alone (Control). (F) Uptake of apoptotic cells with fresh human sera mixed with either SLE (n=6) or HC (n=7) was increased by protein G treatment of these sera while the purified IgG had no effect. (G) Western blots of lysates revealed no change in total C3 bound to apoptotic cells incubated in fresh sera and mixed with SLE (n=6) or HC (n=7). Dots represent each individual sera, horizonal lines are mean for the group and whiskers are the SEM. The geometric mean of C3 staining (MFI) on apoptotic Jurkats pre-incubated in 15% human sera alone was used as the control. Phagocytic index of apoptotic Jurkats by HMDM in 10% fresh human sera alone was used as the control.

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