Cutting Edge: Identification of Marginal Reticular Cells as Phagocytes of Apoptotic B Cells in Germinal Centers

Kazuki Sato, Shin-ichiro Honda, Akira Shibuya and Kazuko Shibuya
J Immunol June 1, 2018, 200 (11) 3691-3696; DOI: https://doi.org/10.4049/jimmunol.1701293

Abstract

Germinal centers (GCs) in secondary lymphoid organs generate large numbers of apoptotic B cells that must be eliminated by phagocytes to prevent the development of autoimmune diseases. Although tingible body macrophages engulf apoptotic GC B cells, whether stromal cells are also involved in this process is unclear. In this study, we identified marginal reticular cells (MRCs) as novel nonprofessional phagocytes for the clearance of apoptotic GC B cells in the spleen. We used CD19eGFP (CD19creZ/EG) mice, which express enhanced GFP (eGFP) under the control of CD19cre expression, to track B cells in the GCs after immunization with NP-chicken γ globulin plus aluminum salt. We demonstrated that the MRC population, as determined by expression of podoplanin or Rankl, specifically showed an eGFP signal in the cytoplasm after immunization. These results suggest that MRCs contribute to the clearance of apoptotic B cells in GCs.

Introduction

High-affinity Abs are indispensable for host defense against pathogens. These Abs are produced by plasma cells that differentiate from B cells in the germinal centers (GCs) of secondary lymphoid tissues (1, 2). Within GCs, B cells undergo somatic hypermutation of the gene encoding the BCR to augment the affinity of BCR toward their cognate Ags (3). However, somatic hypermutation generates large numbers of B cells with BCR that are unable to recognize a cognate Ag; these B cells lack BCR-mediated survival signals and undergo programmed cell death (apoptosis) (4). Apoptotic GC B cells need to be eliminated to maintain optimal humoral immune responses and to prevent accumulation of autoantigens and production of autoantibodies (5–7). Tingible body macrophages localized in the GC play an important role in the clearance of apoptotic GC B cells by phagocytosis (8), and their dysfunction causes various autoimmune diseases in mouse models (6, 7, 9). However, exactly how apoptotic GC B cells are eliminated from the GC remains incompletely understood.

Removal of dead cells is mainly mediated by professional phagocytes, such as dendritic cells and macrophages, and by nonprofessional “neighboring” phagocytes that represent certain types of stromal cells, such as epithelial cells, endothelial cells, and fibroblasts (10). Although the phagocytic activity of nonprofessional phagocytes is lower than that of professional phagocytes, they are able to efficiently clear apoptotic debris from places that professional phagocytes find difficult to reach (11). In addition, nonprofessional phagocytes compensate for professional phagocytes when the function of professional phagocytes is impaired, such as in the clearance of apoptotic cells in interdigital spaces during embryo development (12). Therefore, whether nonprofessional phagocytes as well as professional phagocytes are involved in the clearance of apoptotic GC B cells warrants investigation.

The spleen is the lymphoid organ that initiates the GC reaction to blood-borne Ags, and it also works as a filter to eliminate debris and senescent RBCs from the circulation (13). The spleen contains several types of stromal cells: follicular dendritic cells (FDCs), fibroblastic reticular cells (FRCs), marginal reticular cells (MRCs), and blood endothelial cells (BECs). Stromal cells contribute to immune homeostasis and the efficient initiation of adaptive immune responses (14). FRCs and FDCs, respectively, regulate the migration of T cells and B cells by the production of chemokines (15, 16). To initiate an immune response, FDCs present Ags to B cells, and FRCs transport small Ags (<70 kDa) to T cell and B cell zones within lymph nodes via a conduit system (17, 18). Conversely, MRCs maintain their niche in the marginal zone in a steady state (19). However, the phagocytic activity of MRCs with regard to apoptotic cells remains totally unknown.

In this study, we aimed to determine whether splenic stromal cells act as nonprofessional phagocytes and engulf apoptotic B cells during GC reactions. We showed that MRCs are novel nonprofessional phagocytes of apoptotic B cells in the GCs.

Materials and Methods

Mice

C57BL/6J mice were purchased from Clea Japan (Tokyo, Japan) and maintained under specific pathogen-free conditions. Cd19-Cre mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Z/EG mice are Cre reporter mice that express the enhanced GFP (eGFP) by Cre excision (20). Z/EG mice on an ICR genetic background were kindly supplied by Prof. S. Takahashi of the University of Tsukuba, Japan. Z/EG mice were backcrossed onto a C57BL/6J background for four generations and then crossed with Cd19-Cre mice to obtain Cd19-Cre·Z/EG mice (CD19eGFP mice).

For immunization, 8- to 12-wk-old mice were i.p. administered 100 μg of 4-hydroxy-3-nitrophenyl acetyl–chicken γ globulin (NP-CGG; Santa Cruz Biotechnology, Dallas, TX) plus 100 μl of 2% Alhydrogel (alum; InvivoGen, San Diego, CA). Control littermates were left untreated (naive mice). Mice were killed 14 d after immunization and cells were isolated from spleen. All experiments were performed according to the guidelines of the Animal Ethics Committee of the University of Tsukuba Animal Research Center.

Abs and reagents

FITC-conjugated anti-CD31 Abs (clone 390), PE-conjugated anti–ICAM-1 Abs (clone 3E2), PE-conjugated anti-CD45R/B220 Abs (clone RA3-6B2), Alexa Fluor 647–conjugated anti-CD31 Abs (clone 390), Alexa Fluor 647 or biotin-conjugated GL7, V450-conjugated anti-CD45.2 Abs (clone 104), biotin-conjugated anti-CD45 Abs (clone 30-F11), and purified anti-CD107a (LAMP-1) Abs were purchased from BD Biosciences (San Jose, CA). Purified anti-CD16/32 Abs (clone 2.4G2) were purchased from Tonbo Biosciences (San Diego, CA). Alexa Fluor 488–conjugated anti–MAdCAM-1 Abs (clone MECA-367), PECy7-conjugated anti-CD21/35 Abs (clone 7G6), and biotin-conjugated anti-CD45R/B220 Abs (clone RA3-6B2) were purchased from BioLegend (San Diego, CA). eFluor 660–conjugated anti-podoplanin (PDPN) Abs (clone eBio8.1.1) were purchased from eBioscience (San Diego, CA). Biotin-conjugated peanut agglutinin (PNA) was purchased from Vector Laboratories (Burlingame, CA).

Isolation of stromal cells

Stromal cells were isolated by using a previously reported method with modifications (21). Briefly, the spleen was treated with an enzyme mixture of 1 mg/ml collagenase D (Roche Diagnostics, Indianapolis, IN), 100 μg/ml DNase I (Sigma-Aldrich, St. Louis, MO), and 0.6 U/ml dispase (Roche Diagnostics) in complete DMEM containing 2% FBS. After homogenization of splenocytes, stromal cells were enriched by incubating with anti-CD45 Abs followed by BD IMag streptavidin particles (BD Biosciences Pharmingen, San Diego, CA). The remaining stromal cell–enriched fraction was stained with each of the anti–stromal cell surface marker Abs. Flow cytometric analyses and cell sorting were performed by using a FACSAria special order research product (BD Biosciences). FlowJo software (Tree Star, Ashland, OR) was used for data analyses. Dead cells were stained and excluded by using propidium iodide solution (Sigma-Aldrich) or the Zombie Violet Fixable Viability Kit (BioLegend).

Phagocytosis assay using pHrodo-labeled apoptotic thymocytes

Apoptotic thymocytes were generated and labeled as described elsewhere (22). Briefly, thymocytes from C57BL/6J mice were incubated with 1 μM dexamethasone (Sigma-Aldrich) in complete RPMI 1640 medium for 16 h. Apoptosis was confirmed by the staining of the phosphatidylserine exposed on the plasma membrane with allophycocyanin-conjugated annexin V (BD Biosciences). Apoptotic and live thymocytes were labeled with 20 ng/ml pHrodo succinimidyl ester (Life Technologies, Carlsbad, CA) at room temperature for 30 min and were then injected i.v. at 2 × 107 cells per mouse. Mice were killed 30 min after thymocytes were injected, and cells were isolated from spleen.

TUNEL assays

For flow cytometry analysis, the stromal cell–enriched fraction was fixed with 4% paraformaldehyde and treated with 0.1% Triton X-100 (Sigma-Aldrich). TUNEL staining was then performed by ApopTag Fluorescein In Situ Apoptosis Detection Kit (Millipore, Temecula, CA) in accordance with the manufacturer’s instructions.

For immunohistochemistry, spleen sections (7 μm) were incubated for 10 min in cold acetone and stained with PNA and MAdCAM-1 by using the Opal 7-plex staining system (PerkinElmer, Waltham, MA) in accordance with the manufacturer’s instructions. After staining with TUNEL, sections were mounted and visualized with a fluorescent microscope (BZ-X710; Keyence, Osaka, Japan).

For immunocytochemistry, isolated cells were first immobilized in eight-well chamber slides (Thermo Fisher Scientific, Waltham, MA) and were then fixed with 4% paraformaldehyde and treated with 0.1% Triton X-100. B220+ B cells were isolated from spleen of wild-type (WT) mice on day 14 after immunization. CD11b+ macrophages were isolated from thymus of naive WT mice. Cells were then stained with TUNEL and/or anti-CD107a Abs, mounted with medium containing the nuclear counterstain DAPI (VECTASHIELD; Vector Laboratories), and analyzed by laser-scanning confocal microscopy (FV10i FLUOVIEW; Olympus, Tokyo, Japan).

Quantitative PCR

Total RNA was isolated from sorted stromal cells by using Isogen reagent in accordance with the manufacturer’s protocol (Nippon Gene, Tokyo, Japan). For reverse transcription, we used a High-Capacity cDNA Reverse-Transcription Kit (Applied Biosystems, Carlsbad, CA). Quantitative PCR analysis of Ccl19 and Rankl was performed by using an ABI 7500 sequence detector (Applied Biosystems), Power SYBR Green PCR master mix (Applied Biosystems), and the appropriate primers. The Actb expression level was measured as an internal control to normalize the data. The primer sequences for the target genes were as follows: Rankl, forward, 5′-ATA CAT GTG TAA GAC TAC TAA GAG AC-3′; reverse, 5′-AAT CTA ACA TCA CCT ATG GAC TTT AC-3′; Ccl19, forward, 5′-TTC ACG CCA CAG GAG GAC ATC T-3′; reverse, 5′-CCA CAC TCA CAT CGA CTC TCT AGG C-3′; and Actb, forward, 5′-ACT GTC GAG TCG CGT CCA-3′; reverse, 5′-GCA GCG ATA TCG TCA TCC AT-3′.

Statistical analysis

Statistical analysis was performed by using an unpaired, two-tailed Student t test or ANOVA followed by the post hoc Tukey–Kramer test in Prism 7 software (GraphPad Software, San Diego, CA). The p values <0.05 were considered statistically significant.

Results and Discussion

BECs and MRCs have phagocytic ability in the spleen

To address whether splenic stromal cells are capable of engulfing apoptotic cells, thymocytes were labeled with pHrodo before or after treatment with dexamethasone to induce apoptosis and were injected i.v. into WT C57BL/6J mice. Stromal cells were isolated from the spleens of mice 30 min after injection of pHrodo-labeled live or apoptotic thymocytes and were analyzed by flow cytometry. Stromal cells can be distinguished by the expression of their surface markers: FDCs by CD21/35 expression, FRCs/MRCs by PDPN expression, and BECs by CD31 expression (21) (Fig. 1A). In the spleen, mice injected with apoptotic thymocytes showed higher pHrodo signals in the BEC and FRC/MRC fractions, but not in the FDC fraction, than did mice injected with live thymocytes (Fig. 1B, 1C). To determine whether FRCs or MRCs phagocytosed apoptotic cells, we compared the gene expressions of MRC and FRC markers in the pHrodo+ population with those in the pHrodo− population. The pHrodo+ population showed higher expression of MRC marker (Rankl) and lower expression of FRC marker (Ccl19) than did the pHrodo− population, indicating that MRCs were the predominant phagocytes for apoptotic cells in the FRC/MRC fraction (Fig. 1D). Together, our results suggest that BECs and MRCs have the function of engulfing apoptotic cells.

FIGURE 1.
FIGURE 1.

BECs and MRCs have phagocytic ability against apoptotic cells. (A) Sorting strategy for FDCs (CD45−ICAM-1+CD21/35+), FRCs/MRCs (CD45−ICAM-1+PDPN+), and BECs (CD45−ICAM-1+CD31+) from the spleen. (B) Representative plots of the stromal cell subsets isolated from the spleens of WT C57BL/6J mice 30 min after being i.v. administered 2 × 107 pHrodo-labeled apoptotic thymocytes (Apo). Freshly prepared pHrodo-labeled thymocytes (Live) were administered as a control. The numbers indicate the percentage of the pHrodo+ population. (C) Percentage of pHrodo+ population in each stromal cell subset from mice administrated live thymocytes (Live, n = 4) and from mice administered apoptotic thymocytes (Apo, n = 9). (D) Quantitative PCR analyses of MRC and FRC marker expression in the pHrodo+ and pHrodo− FRC/MRC populations. Data were pooled from three (B and C) or two (D) experiments. Error bars show SEM. *p < 0.05, **p < 0.01, ****p < 0.0001.

To clarify the ability of splenic stromal cells to phagocytose endogenous dead cells, splenic stromal cells were isolated from WT C57BL/6J mice on day 14 after immunization by i.p. injection with NP-CGG plus aluminum salt (alum) adjuvant, which induces tissue damage and cell death (23), and then stained with TUNEL. Flow cytometry analysis showed the presence of TUNEL+ cells in the BEC and FRC/MRC populations, but not in the FDC populations, in the spleens of mice that had been immunized (Fig. 2A, 2B). In contrast, TUNEL+ cells were scarcely detected in those cell populations in the spleens of naive mice (Fig. 2A, 2B). Immunocytochemical analysis can distinguish apoptotic cells from phagocytes by the TUNEL staining pattern. B cells isolated from immunized mice showed colocalization of the TUNEL signal and the nucleus (DAPI signal), indicating that B cells underwent apoptosis (Supplemental Fig. 1). In contrast, in macrophages from the thymus, the TUNEL signal was localized in the cytosol, indicating that macrophage nuclei were intact and that the TUNEL signal showed engulfed dead cells (Supplemental Fig. 1). When the BEC and FRC/MRC fractions isolated from immunized mice were observed, the TUNEL signal was localized in the cytosol as it was with the macrophages (Fig. 2C). Furthermore, the TUNEL signal was colocalized with that of a lysosome marker (LAMP-1) (Fig. 2D). Interestingly, the sizes of TUNEL signals in the BEC and FRC/MRC were smaller than those in macrophages (Fig. 2C), which is consistent with the features of phagocytosis by nonprofessional phagocytes (11). These results indicate that the BEC and FRC/MRC themselves are not dead cells, but rather engulfed dead cells after immunization.

FIGURE 2.
FIGURE 2.

BECs and MRCs engulf dead cells induced by alum-adjuvant immunization. (A) Representative TUNEL staining plots of the stromal cell subsets isolated from spleens of naive or immunized mice on day 14. The numbers indicate the percentage of the TUNEL+ population. (B) Percentage of TUNEL+ population in each stromal cell subset isolated from naive mice (n = 5) and immunized mice (n = 3). (C and D) Cells were isolated from immunized mice on day 14 and stained with TUNEL and DAPI (C) or a lysosome marker (LAMP-1) (D). Arrows indicate the TUNEL signal localized in the nucleus. Scale bars, 10 μm. Data are representative of three (A and B) or two (C and D) independent experiments. Error bars show SEM. *p < 0.05, ****p < 0.0001.

MRCs contribute to clearance of apoptotic B cells during GC reactions

Alum-adjuvanted immunization not only mediates cytotoxicity in the tissue but also induces apoptosis of GC B cells in the follicle (24). Indeed, TUNEL signals were detected only in a subpopulation of GC B cells (PNA+GL7+), but not in any non-GC B cells (GL7−), in the spleen on day 14 after immunization with alum adjuvant (Fig. 3A). To investigate whether BECs and MRCs are involved in the clearance of apoptotic GC B cells in the spleen, we established CD19eGFP (CD19creZ/EG) mice, which express eGFP under the control of CD19cre expression. Because CD19 expression is restricted in B cells, CD19eGFP mice allow for tracking of apoptotic B cells, even after phagocytosis, by acquisition of eGFP. We did not observe an eGFP+ population in the any of the stromal cell subsets in naive CD19eGFP mice (Fig. 3B). However, the FRC/MRC population, but not the BEC population, showed an eGFP+ fraction 14 d after immunization with NP-CGG plus alum (Fig. 3B, 3C). Confocal microscopic analysis showed the eGFP signal was localized in the cytosol (Fig. 3D), suggesting that FRCs and/or MRCs engulfed B cells after immunization. To verify which of either FRCs or MRCs mediated phagocytosis of apoptotic GC B cells, we compared the gene expressions of MRC and FRC markers in the eGFP+ fraction with those in the eGFP− fraction sorted from the spleens of mice immunized with NP-CGG plus alum. The eGFP+ fraction showed higher expression of the MRC marker Rankl and lower expression of the FRC marker Ccl19 compared with the eGFP− population, suggesting that MRCs rather than FRCs engulfed apoptotic GC B cells (Fig. 3E). Finally, we analyzed immunohistochemistry of spleen after immunization by staining with an anti–MAdCAM-1 Ab, TUNEL, and the markers of the GC B cell (PNA and GL7). We found that TUNEL+ particles, merged with PNA and GL7, were localized in cells positive for MAdCAM-1, a marker for MRCs (25) (Fig. 3F, 3G). These results indicated that MRCs engulfed immunization-induced apoptotic GC B cells.

FIGURE 3.
FIGURE 3.

MRCs contribute to clearance of apoptotic B cells during GC reactions. (A) Representative TUNEL staining plots of GC B cells (PNA+GL7+) and non-GC B cells (GL7−) in the spleen of WT C57BL/6J mice on day 14 after immunization with alum adjuvant. (B) Representative plots of the stromal cell subsets isolated from spleens of naive CD19eGFP mice or immunized CD19eGFP mice on day 14. Numbers indicate the percentage of eGFP+ population. (C) Percentage of eGFP+ population in each stromal cell subset isolated from naive CD19eGFP mice (n = 3) and immunized CD19eGFP mice (n = 3). (D) The eGFP+ FRC/MRC population was isolated from immunized CD19eGFP mice on day 14. Cells were stained with DAPI. Scale bar, 10 μm. (E) Quantitative PCR analyses of MRC and FRC marker expression in the eGFP+ and eGFP− FRC/MRC populations isolated from immunized CD19eGFP mice on day 14. (F and G) Representative fluorescence microscopy image of a tissue section taken from the spleen of naive and immunized mice on day 14, stained with MAdCAM-1 Abs (blue), TUNEL (green) and PNA (red) (F) or GL7 (red) (G). Arrowheads indicate TUNEL-stained apoptotic cell particles taken up by MRCs; arrows indicate the TUNEL particles merged with PNA in MRCs. Scale bars, 25 μm. Data are representative of two independent experiments. Error bars show SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001.

MRCs have been identified as reticular cells localized in the marginal zone and expressed in MAdCAM-1 and RANKL (25). MRCs maintain the marginal zone niche for innate lymphoid cells and support the survival of marginal-zone B cells via production of BAFF (19, 26). Our results revealed a new function of MRCs as nonprofessional phagocytes of apoptotic GC B cells in the spleen. BECs, in contrast, although capable of phagocytizing apoptotic cells, did not engulf apoptotic GC B cells. Because MRCs are localized in the marginal zone, they seem to encounter apoptotic B cells more frequently than do BECs.

In nonlymphoid tissue, such as the lungs and intestine, nonprofessional phagocytes contribute to maintaining tissue homeostasis by clearing apoptotic cells (27). Moreover, nonprofessional phagocytes produce anti-inflammatory cytokines to suppress excessive inflammation after phagocytosis (28). The current study demonstrates that MRCs, one of the nonprofessional phagocytes in lymphoid tissue, also engulfed apoptotic cells, suggesting that MRCs might produce anti-inflammatory cytokines and contribute to the immune tolerance. Moreover, MRCs augment the production and class switching of Abs from marginal zone B cells (19). Therefore, our results, together with the previous reports, suggest a hypothesis that MRCs that have engulfed apoptotic B cells coordinate the optimal humoral immune responses. In addition, recent reports demonstrate that the cross-communication between professional and nonprofessional phagocytes plays an important role in the regulation of tissue inflammation (29). The marginal zone is a unique niche for heterogeneous cell populations in the spleen, including macrophages, dendritic cells, B cells, and innate lymphoid cells. Thus, MRCs are also presumed to be able to cross-communicate with other immune cells for optimal immune response. Further studies are needed to investigate the physiological role of MRC-mediated phagocytosis for optimal GC reaction and prevention of production of autoantibodies.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank S. Tochihara, Y. Nomura, and T. Abe for their secretarial assistance. We express our great appreciation to Prof. S. Takahashi for kindly suppling the Z/EG mice.

Footnotes

  • This work was supported by grants from the Japan Society for the Promotion of Science (KAKENHI) (16H06387 to A.S., 16H05169 to K. Shibuya, and 17H04362 to S.-i.H.) and by a grant-in-aid from the Japan Society for the Promotion of Science Fellows (14J00713 to K. Sato).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    BEC
    blood endothelial cell
    eGFP
    enhanced GFP
    FDC
    follicular dendritic cell
    FRC
    fibroblastic reticular cell
    GC
    germinal center
    MRC
    marginal reticular cell
    NP-CGG
    4-hydroxy-3-nitrophenyl acetyl–chicken γ globulin
    PDPN
    podoplanin
    PNA
    peanut agglutinin
    WT
    wild-type.

  • Received September 11, 2017.
  • Accepted April 5, 2018.

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