Critical role of macrophages in the marginal zone in the suppression of immune responses to apoptotic cell-associated antigens - PubMed (original) (raw)

Critical role of macrophages in the marginal zone in the suppression of immune responses to apoptotic cell-associated antigens

Yasunobu Miyake et al. J Clin Invest. 2007 Aug.

Abstract

Injection of apoptotic cells can induce suppression of immune responses to cell-associated antigens. Here, we show that intravenous injection of apoptotic cells expressing a fragment of myelin oligodendrocyte glycoprotein (MOG) reduced MOG-specific T cell response and prevented the development of EAE. Since injected apoptotic cells accumulated initially in the splenic marginal zone (MZ), the role of macrophages in the MZ in immune suppression was examined using transgenic mice in which these cells could be transiently deleted by diphtheria toxin (DT) injection. DT-treated mice became susceptible to EAE even though MOG-expressing apoptotic cells were preinjected. Deletion of the macrophages caused delayed clearance of injected dying cells in the MZ. In wild-type mice, injected apoptotic cells were selectively engulfed by CD8 alpha(+) DCs, which are responsible for suppression of immune responses to cell-associated antigens. In contrast, deletion of macrophages in the MZ caused aberrant phagocytosis of injected dying cells by CD8 alpha(-)CD11b(+) DCs. These results indicate that macrophages in the MZ regulate not only efficient clearance of apoptotic cells but also selective engulfment of dying cells by CD8 alpha(+) DCs and that functional failure of these unique macrophages impairs suppression of immune responses to cell-associated antigens.

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Figures

Figure 1

Figure 1. Expression of HB-EGF–MOG fusion proteins in W3/MOG cells.

(A) Schematic diagrams of the HB-EGF–MOG fusion protein constructs. The amino acids 32–97 (HB-EGF–MOG-S) or 1–120 (HB-EGF–MOG-L) of MOG are attached at the C terminus of the signal (SIG), extracellular (EC), and transmembrane (TM) domains of HB-EGF. (B) Expression of HB-EGF–MOG/FLAG on the cell surface of transformants. W3 cells and their transformants expressing HB-EGF–MOG-S (W3/MOG-S) or HB-EGF–MOG-L (W3/MOG-L) were stained with Alexa Fluor 488–conjugated anti–HB-EGF antibody and analyzed by flow cytometry. (C) Immunodetection of HB-EGF–MOG proteins in the transformants. W3 cells (lanes 1, 4, and 7) and W3 cells expressing MOG-S (lanes 2, 5, and 8) or MOG-L (lanes 3, 6, and 9) were lysed and analyzed by Western blotting (WB) with anti–HB-EGF antibody (lanes 1–3). In order to confirm the expression of full-length HB-EGF–MOG/FLAG fusion proteins, cell lysates were subjected to immunoprecipitation with an anti-FLAG antibody. Immunoprecipitates were analyzed by Western blotting with anti–HB-EGF (lanes 4–6) or anti-MOG antibodies (lanes 7–9). (D) Induction of apoptosis in W3 cells and their transformants. W3 cells and their transformants were treated with (solid line) or without (dashed line) recombinant Fas ligand for 4 hours. Cells were then stained with PE-conjugated annexin V and analyzed by flow cytometry. More than 95% of W3 and W3/MOG cells underwent apoptosis by treatment with Fas ligand.

Figure 2

Figure 2. Intravenous administration of apoptotic W3/MOG cells prevents the progression of EAE.

(A) We treated 2 × 107 W3, W3/MOG-S, and W3/MOG-L cells with Fas ligand to induce apoptosis. Then the treated cells were intravenously injected into mice 8 days prior to immunization. Mice were then immunized with MOG35–55 in CFA on day 0. (B) Dose-dependent effects of apoptotic cell injection on EAE. The indicated number of apoptotic W3/MOG-L cells was injected into mice on 4 days prior to immunization with MOG35–55 in CFA on day 0. (C) Pretreatment with apoptotic cells is required for suppression of EAE. Mice were treated with 2 × 107 apoptotic W3/MOG-L cells 8 or 3 days prior to immunization or 8 days afterwards. Immunization was on day 0. (D) Subcutaneous injection of apoptotic cells does not suppress EAE. We subcutaneously injected 2 × 107 apoptotic W3/MOG-L cells into the tail base 6 days before mice were immunized with MOG35–55 in CFA. (E) Intravenous injection of MOG35–55 peptide does not induce tolerance to EAE. We intravenously injected 1 μg of MOG35–55 peptide, which corresponds to more than 200 times the amount of MOG fragment expressed in 2 × 107 apoptotic W3/MOG-L cells in molar ratio, into mice. Four days later, the mice were immunized with MOG35–55 in CFA. (F) Secondary necrotic cells did not suppress EAE. W3/MOG-L cells were treated with Fas ligand either for 4 hours or 24 hours and were injected intravenously into mice 4 days prior to immunization with MOG35–55 in CFA on day 0. (A–F) The disease severity of each mouse was scored, and the mean clinical scores at the indicated times were plotted. These results are representative of 3 (A–D) or 2 (E and F) independent experiments.

Figure 3

Figure 3. Administration of apoptotic W3/MOG cells induced antigen-specific T cell unresponsiveness.

(A and B) We intravenously injected 2 × 107 apoptotic W3/MOG-L cells into mice. Three days later, 100 μg of MOG35–55 (A) or KLH (B) in CFA was injected subcutaneously into the bilateral foot pads. Popliteal and inguinal LNs were collected from the mice 5 days after immunization. In vitro, 5 × 105 LN cells were restimulated with MOG35–55 (A) or KLH (B) for 70 hours. T cell proliferation was quantified based on the [3H] thymidine uptake in the last 20 hours of the culture. Mean values are shown with SD. Numbers indicate mouse 1 and mouse 2. (C and D) Apoptotic W3/MOG-L cell injection reduces IFN-γ and IL-17 production in splenocytes. We intravenously injected 2 × 107 apoptotic W3 or W3/MOG-L cells into mice 4 days prior to immunization of MOG35–55 on day 0. Splenocytes obtained 10 days after immunization were restimulated in vitro with MOG35–55 for 72 hours. Production of IFN-γ (C) and IL-17 (D) was measured by ELISA. Mean values are shown with SEM. *P < 0.05. These results are representative of 3 independent experiments.

Figure 4

Figure 4. Generation of CD169-DTR mice.

(A) Schematic diagram of the CD169-DTR targeting construct. Exons of the CD169 gene are indicated by solid boxes. Human DTR (hDTR) cDNA, the loxP-franked neomycin resistant gene (Neo), and the thymidine kinase gene (TK) are indicated by open boxes. The probe used for Southern blot analysis is indicated as a solid line together with the predicted hybridizing fragments. (B) Genomic Southern blot analysis of wild-type (+/+) and CD169-DTR (+/CD169DTR) mice using a Bgl II digest in combination with the indicated probe. (C) Depletion of macrophages in the MZ by DT administration in CD169-DTR mice. The indicated amount of DT was intraperitoneally injected into wild-type (+/+) or CD169-DTR (+/CD169DTR) mice. Spleens were obtained from these mice 48 hours later. Spleen sections were immunostained with CD11b, ER-TR9, CD169, and CD11c for red pulp macrophages, MZM, MMM, and DCs, respectively. (D) We intraperitoneally injected 10 μg/kg of DT into wild-type mice (+/+) and CD169-DTR (+/CD169DTR) mice. Spleens were obtained from these mice on the indicated days after DT administration and immunostained with ER-TR9 and CD169. Original magnification, ×100 (C and D).

Figure 5

Figure 5. Failure of apoptotic cell–mediated tolerance induction after depletion of MZM.

We intraperitoneally injected 10 μg/kg of DT into wild-type (+/+) or CD169-DTR (+/CD169DTR) mice 8 days prior to immunization. We intravenously injected 2 × 107 apoptotic W3 or W3/MOG-L cells into mice 4 days prior to immunization with MOG35–55 in CFA on day 0. (A) Disease severity of each mouse was scored, and the mean clinical scores at the indicated times were plotted. (B) Spinal cords were obtained 18 days after immunization, fixed with paraformaldehyde, and embedded in paraffin. Paraffin sections were stained with H&E. Results are representative of 3 independent experiments. Original magnification, ×40.

Figure 6

Figure 6. Delay of apoptotic cell clearance in DT-treated CD169-DTR mice.

We intraperitoneally injected 10 μg/kg of DT into wild-type (+/+) or CD169-DTR (+/CD169DTR) mice. Four days after DT treatment, CMFDA-labeled (green) apoptotic W3/MOG-L cells (2 × 107 cells) were intravenously injected, and the spleen was obtained at the indicated times after injection. (A) Cryosections of the spleens were stained with F4/80 (red pulp macrophage), ER-TR9 (MZM), CD169 (MMM), CD11c (DC), or MAdCAM-1 (marginal sinus-lining cell) (red). (B) Fluorescence intensity in MZ on the slices was quantified at the indicated time points, and mean values are shown with SEM. These results are representative of 2 independent experiments. Original magnification, ×200.

Figure 7

Figure 7. DCs were not affected by DT treatment in CD169-DTR mice.

Wild-type (+/+) or CD169-DTR (+/CD169DTR) mice were injected with (+DT) or without (–DT) 10 μg/kg of DT. Spleens were obtained 4 days after DT treatment. (A) Cryosections of the spleen were stained with CD11c (DCs) and B220 (B cells). Original magnification, ×200. (B) Single-cell suspensions of splenocytes were stained with CD11c alone (total DCs) or in combination with CD8α (lymphoid DCs) or CD11b (myeloid DCs) and analyzed by flow cytometry.

Figure 8

Figure 8. Aberrant engulfment of apoptotic cells by CD11b+ DCs in MZM-depleted mice.

We intraperitoneally injected 10 μg/kg of DT into wild-type (+/+) or CD169-DTR (+/CD169DTR) mice. Four days after DT treatment, CMFDA-labeled apoptotic W3/MOG-L cells (2 × 107 cells) were intravenously injected, and the spleens were obtained at the indicated times after injection. Splenic DCs were enriched by cell sorting with anti-CD11c microbeads and stained with CD11c-PE, CD8α-PE, or CD11b-PE. PE and CMFDA double-positive cells were considered to have engulfed the injected apoptotic cells. (A) Kinetics of apoptotic cell engulfment by DC subsets. (B) Engulfment of apoptotic cells by DC subsets 1 hour after injection. These results are representative of 3 independent experiments, and mean values are shown with SD. *P < 0.01. (C) Antigen-presentation activity of DC subsets. We intravenously injected W3/OVA cells (2 × 107 cells) into wild-type (+/+) and DT-treated CD169-DTR (+/CD169DTR) mice, and spleen was obtained 3 hours after injection. CD11c+ DCs were further separated into CD8α+ DCs and CD11b+ DCs by the cell sorter. Indicated number of DC subsets was cocultured with OT-II cells (2 × 105 cells) for 60 hours. T cell proliferation was quantified based on [3H] thymidine uptake in the last 12 hours of the culture. These results are representative of 2 independent experiments, and mean values are shown with SD.

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