- Email: [email protected]
Role of follicular dendritic cells in the apoptosis of germinal center B cells
Immunology Letters 72 (2000) 107 – 111
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Role of follicular dendritic cells in the apoptosis of germinal center B cells Dae Y. Hur b, Dae J. Kim a, Seonghan Kim a, Young I. Kim a, Daeho Cho a, Dong S. Lee a, Young-il Hwang a, Ki-Won Bae b, Ka Y. Chang a, Wang J. Lee a,* a
Department of Anatomy, Seoul National Uni6ersity College of Medicine and Institute of Allergy and Clinical Immunology, Seoul National Uni6ersity, Medical Research Center, Seoul 110 -799 South Korea b Department of Anatomy, Inje Uni6ersity College of Medicine, Pusan 614 -735 South Korea Accepted 24 January 2000
Abstract Follicular dendritic cells (FDCs) provide the most obvious source of antigens, which are essential for the differentiation of GC B cells. It has been reported that most proliferating B cells in germinal centers undergo apoptosis. Quantitative histology shows macrophages with apoptotic debris throughout the germinal center, the highest frequency of these cells being found in the dense FDC network. Based on these findings, we hypothesized that FDC may be involved in an apoptotic pathway of the germinal center B cells. To prove this hypothesis, we performed double immunohistochemical analysis using anti-FDC mAb and peanut agglutinin (PNA), with their respective TUNEL kits. Collated data showed that a great proportion of the apoptotic cells, most of which were positive for PNA, were in close contact with FDC, which indicated an interaction between FDC and B cells in the apoptotic pathway. Further studies using double immunohistochemical staining and FACS analyses demonstrated the expression of Fas–ligand (FasL) in a subset of the FDC. These results suggest that FDC may play a role in the apoptosis of germinal center B cells via Fas–FasL interaction. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Follicular dendritic cells; Apoptosis; Germinal center B cells
1. Introduction Follicular dendritic cells (FDCs) are specialized antigen trapping cells which have only been detected in the lymphoid tissue, located within B cell follicles [1,2]. These cells have cytoplasmic extensions (dendrites), which form a dense network in the germinal center and contact with germinal center B cells [3]. FDC also express the complement receptors (CR1, CR2, CR3) and the Fc receptor, which are involved in holding antigen–antibody complexes and complement-associated antigen for extended periods (from months to years) [4–7]. It is known that this ability to hold intact antigen on their surfaces is essential to the survival of germinal center B cells, germinal center formation, and
* Corresponding author. Tel.: +82-2-7408208; fax: 7459528. E-mail address: [email protected] (W.J. Lee)
+82-2-
memory B cell generation [8–11], and it is believed that the positive selection of B cells is simply the result of the absence of a positive survival signal, which is dependent upon affinity for the original antigen [12,13]. Most studies on the role of FDC have focused on the rescue mechanisms of the germinal center B cells. However, it has been documented that most of GC B cells undergo apoptosis during B cell differentiation and that these apoptotic cells are to be found in the dense FDC network [14]. In addition, all germinal center B cells express Fas molecule highly on their surfaces, which suggests that there may be a signal for apoptosis. Recently expressions of FasL mRNA were reported in the germinal centers of the human tonsil [15–17]. In this study, we investigated the possible role of FDC in the apoptosis of germinal center B cells by visualizing the contact between apoptotic germinal center B cells and FDC, and the expression of FasL on FDC.
0165-2478/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 7 8 ( 0 0 ) 0 0 1 6 6 - 8
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D.Y. Hur et al. / Immunology Letters 72 (2000) 107–111
2. Materials and methods
2.1. Animals and tissues BALB/C mice were immunized with washed sRBC (5 × 108 cells/per mouse) intraperitoneally. Four weeks after the primary immunization, the same number of sRBC were intraperitoneally injected into the mice. Spleens were dissected from the animals on the fifth and seventh day after the secondary immunization. After extracting the spleens, they were immersed in a 25% sucrose solution, prior to being submerged in OCT embedding compound in liquid nitrogen cooled isopentane, 8 mm sections were prepared using a cryostat (Leica CM 3050).
2.2. (Immuno)histochemistry (IHC) 2.2.1. PNA (peanut agglutinin) staining for GC B cells and TUNEL staining Cryosections were dried in air and fixed in cold acetone at 4°C. These specimens were then incubated with biotinylated PNA(Vector) overnight at 4°C, and then incubated with alkaline phosphatase (AP)-conjugated streptavidin (Vector Co.) for 30 min at 37°C in a wet chamber. The labeled cells were visualized by the substrate solution (fast red, naphthol, levamisole, Sigma Chemical, St Louis, MO). Specimens were then stained with TUNEL (Terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP-biotin nick-end labeling) kit for apoptotic cells. Briefly, specimens were air dried and fixed in 1% paraformaldehyde for 10 min, and then postfixed in ethanol:acetic acid (2:1) for 5 min. After quenching the endogenous peroxidase in 3% hydrogen peroxide (Fluka Chemie AG, Switzerland) for 5 min, the sections were reacted with Apoptag kit (Oncor, Gaithersburg, MD). Sections were washed and then were visualized using diaminobenzidine tetrahydrochloride (DAB, Sigma Chemical, St Louis, MO), NiCl2 (0.4% w/v, Fluka Chemie AG, Switzerland) and 0.03% H2O2 in PBS (0.05 M, pH 7.4). 2.3. IHC staining for FDCs ( follicular dendritic cells) and TUNEL staining Cryosectioned spleen tissues were incubated with rat anti-mouse FDC (FDC-M2, a gift from Dr M.H Kosco-Vilbois) or biotinylated PNA (Vector), and then incubated with biotinylated anti-rat F(ab)%2 fragment (Boehringer Mannheim) for 30 min at 37°C in a wet chamber. Subsequently, they were incubated with AP-conjugated streptavidin (Vector). The labeled cells were visualized using fast red, naphthol, levamisole
substrate solution, and stained with the TUNEL kit for apoptotic cells as described above.
2.4. Double staining for FDCs and Fas-ligand (Fas-L) Cryosectioned spleen tissues were incubated with rat anti-mouse FDCs (FDC-M2) for 1 h at 37°C in a wet chamber, and then incubated with AP-conjugated anti-rat mAb (Sigma Chemical, St Louis, MO). The labeled cells were visualized using the substrate solution (described above). Tissues were incubated with biotinylated anti-Fas ligand (Pharmingen) for 1 h at 37°C in wet chamber, which was followed by an incubation with peroxidase-conjugated extravidin (Sigma Chemical, St Louis, MO). Labeled cells were visualized with diaminobenzidine tetrahydrochloride (DAB, Sigma Chemical, St Louis, MO), NiCl2 (0.4% w/v) and 0.03% H2O2 in PBS (0.05 M, pH 7.4).
2.5. Enrichment of FDC using a discontinuous Percoll gradient Discontinuous density gradients were prepared in 15 ml centrifuge tubes by layering 10% Percoll solution (1.030 g/ml) and 30% Percoll solution (1.052 g/ ml). The cell suspension (mice splenic cells) were layered on top of the gradient solution and centrifuged at 8500× g for 40 min. The cells in the interphase between 1.030 and 1.052 were collected, washed with PBS (0.01 M, pH 7.4) and prepared for flowcytometric analysis [18].
2.6. A flow-cytometric analysis The FDC-enriched cells were incubated with normal goat serum (Vector) for 1 h, and then with antiFDC mAb (FDC-M2) and biotinylated anti-FasL mAb (Pharmingen) for 30 min at 4°C. After washing with PBS, the samples were double-stained with antirat-FITC (Sigma Chemical, St Louis, MO) Ultravidin-PE (Leinco Technologies, St Louis, MO) for 30 min at 4°C. Using a FACStar (Bect and on Dickinson), samples were then analyzed.
3. Results
3.1. TUNEL positi6e cells in follicle were positi6e for PNA To characterize the TUNEL+ GC cells, we examined the possibility of in situ double staining using the TUNEL kit and PNA, which is usually used for the visualization of the germinal center forming cells [19,20]. The results of the double staining demonstrated
D.Y. Hur et al. / Immunology Letters 72 (2000) 107–111
109
3.2. In follicles, many TUNEL positi6e cells were obser6ed to be in close contact with FDC, appearing to interact mutually. To investigate correlation between FDC and TUNEL+ cells, the frozen sections were doublestained with anti-FDC mAb (FDC-M2) and the TUNEL kit. Some TUNEL+ cells were observed to be in close contact with FDC (Fig. 2), which suggested that TUNEL+ cells and FDC may interact mutually.
3.3. A subset of FDC express FasL We studied whether FDC would express FasL using in situ double staining and flowcytometric analysis. Double staining results showed that a few FDCs were positive for FasL, which suggested that a subset of FDC expressing FasL may exist in GC (Fig. 3). Dualcolor flow cytometic analysis revealed that a small percentage (20–30%) of FDC expressed FasL (Fig. 4).
4. Discussion Within the GC, the B cells closely interact with FDC, and provide them with antigenic stimuli which promotes their differentiation into memory cells or plasma cells. The integrin adhesion molecules LFA-1 and VLA-4 on the B cells through interaction with their counter receptor. ICAM-1 and VCAM-1 on the FDC, which have been shown to prevent GC B cells from entering into apoptosis. Triggering of Fas (CD95), on the other hand, has been shown to induce apoptosis of the GC B cells in the presence of anti-Ig or adhesionmediated rescue signals [13]. Our experimental results show that the TUNEL+ GC cells were also PNA-positive, which suggests that the apoptotic cells in the GC should be B cells. Moreover, in this experiment we have found that some GC B
that almost all TUNEL+ cells in GC were positive for PNA (Fig. 1), and that the majority of TUNEL+ cells were located in the dark and basal light zones. For comparison, tissue samples were also double stained using anti-CD3 mAb and the TUNEL kit. TUNEL+ GC cells were totally negative for CD3 (data not shown).
Fig. 1. Double staining using peanut agglutinin (PNA) and the TUNEL kit of frozen mouse spleen sections on the seventh day after secondary immunization with sRBC. The PNA + cells (red) delineate the germinal center (GC) over which The TUNEL+ cells (black) are scattered. Most of TUNEL + cells are also PNA-positive, which indicates that the apoptotic cells in the GC should be GC B cells. Fig. 2. Double staining using rat anti-FDC mAb (FDC-M2) and the TUNEL kit of frozen mouse spleen sections on the fifth day after secondary immunization with sRBC. In follicles, many TUNEL+ cells (dark brown) were observed to be in close contact with FDC (red), appearing to interact mutually. Fig. 3. Double immunohistochemical staining using anti-FDC mAb (FDC-M2) and anti-FasL mAb in frozen mouse spleen sections on the seventh day after secondary immunization with sRBC. Some FDCs (red color) were also positive for FasL (dark brown).
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Fig. 4. Two color flowcytometric analysis using anti-FDC mAb and anti-FasL mAb in mouse spleen cells on the seventh day after secondary immunization with sRBC. After enrichment of FDCs using a discontinuous Percoll gradient, single flowcytometric analysis for FDC showed that 20–30% of the mononuclear cells were FDC-positive (A). The same sample of FDC was analysed by two color flowcytometry using anti FDC mAb and anti-FasL mAb (B), demonstrated that some portion of FDC were FasL-positive.
cells undergoing apoptosis make close contact with FDC. It is known that apoptotic cells were found both outside and within the FDC-associated clusters [12]. Our results and this report suggest that FDC may participate in the apoptosis of GC B cells. It is documented that GC B cells express a high level of CD95 [15,16], which has been implicated in apoptosis induction [21 – 23]. Furthermore, our study shows by immunohistochemistry and flow cytometry that some FDC express FasL. The mode of participation of the Fas-FasL system in the clonal deletion of B lymphocytes still remains unclear. It is reported that not only anti-Fas mAb rapidly induces the apoptosis of germinal center cells, but that signalling through CD40 also up-regulates Fas expression, rendering the cells highly sensitive to Fas-mediated apoptosis. Although the precise mechanisms involved in this signalling have not been clarified, these reports suggest, therefore, that the Fas – FasL system may have role in the apoptosis of germinal center cells [24 – 26]. Although FasL has been reported predominantly in activated T cells, some germinal center B cells were also found to be capable of expressing functional FasL[17]. However, whether or not FDC express FasL remains unclear. Moreover, it has recently been reported that a subclass of dendritic cells expressing FasL have a role in the regulation of the response of primary peripheral T cells [27]. These findings suggest that FDC might express FasL. In our study, a population of FDC, which expressed FasL was clearly demonstrated by flow cytometry and immunohistochemistry. It would, therefore, appear in the light of the information available to date, that FDC may participate in the abolition process of germinal center B cells through Fas-mediated apoptosis.
Acknowledgements We would like to thank Dr M.H. Kosco-Vilbois for a gift of FDC-M2 mAb.
References [1] J.H. Humphrey, D. Grennan, V. Sundaram, Eur. J. Immunol. 14 (1984) 859 – 864. [2] D. Radoux, E. Heinen, C. Kinet-Denoel, E. Tihange, L. Simar, Cell. Tissue Res. 235 (1984) 267 – 274. [3] I.C. MacLennan, Annu. Rev. Immunol. 12 (1994) 117 –139. [4] Y. Fang, C. Xu, Y.X. Fu, V.M. Holers, H. Molina, J. Immunol. 160 (1998) 5273 – 5279. [5] K. Sellheyer, R. Schwarting, H. Stein, Clin. Exp. Immunol. 78 (1989) 431 – 436. [6] F. Schriever, A.S. Freedman, G. Freeman, E. Messner, G. Lee, J. Daley, L.M. Nadler, J. Exp. Med. 169 (1989) 2043 –2058. [7] Y.J. Liu, J. Xu, O. de Bouteiller, C.L. Parham, G. Grouard, O. Djossou, B. de Saint-Vis, S. Lebecque, J. Banchereau, K.W. Moore, J. Exp. Med. 185 (1997) 165 – 170. [8] N. Cormann, F. Lesage, E. Heinen, N. Schaaf-Lafontaine, C. Kinet-Denoel, L.J. Simar, Immunol. Lett. 14 (1986) 29–35. [9] A.K. Szakal, J.G. Tew, Cancer. Res. 52 (1992) 5554s –5556s. [10] J.G. Tew, J. Wu, D. Qin, S. Helm, G.F. Burton, A.K. Szakal, Immunol. Rev. 156 (1997) 39 – 52. [11] G. Grouard, I. Durand, L. Filgueira, J. Banchereau, Y.J. Liu, Nature 384 (1996) 364 – 367. [12] G. Koopman, R.M. Keehnen, E. Lindhout, D.F. Zhou, C. de Groot, S.T. Pals, Eur. J. Immunol. 27 (1997) 1 – 7. [13] P. Brandtzaeg, Acta. Otolaryngol. Suppl. (Stockh) 523 (1996) 55 – 59. [14] H. Orui, M. Yamakawa, Y. Imai, Immunology 90 (1997) 489– 495. [15] H. Martinez-Valdez, C. Guret, O. de Bouteiller, I. Fugier, J. Banchereau, Y.J. Liu, J. Exp. Med. 183 (1996) 971 – 977. [16] E. Schattner, S.M. Friedman, Immunol. Res. 15 (1996) 246–257. [17] E. Kondo, T. Yoshino, R. Nishiuchi, I. Sakuma, K. Nishizaki, N. Kayagaki, H. Yagita, T. Akagi, J. Pathol. 183 (1997) 75–79. [18] J. Schmitz, S. Petrasch, J. van Lunzen, P. Racz, H.D. Kleine, F. Hufert, P. Kern, H. Schmitz, K. Tenner-Racz, J. Immunol. Methods 159 (1993) 189 – 196.
D.Y. Hur et al. / Immunology Letters 72 (2000) 107–111 [19] M.L. Rose, M.S.C. Birbeck, V.J. Wallis, Nature 284 (1980) 364 – 366. [20] S.M. Hsu, H.J. Ree, J. Histochem. Cytochem. 31 (1983) 538 – 546. [21] P. Moller, C. Henne, F. Leithauser, A. Eichelmann, A. Schmidt, S. Bruderlein, J. Dhein, P.H. Krammer, Blood 81 (1993) 2067 – 2075. [22] C. Lagresle, C. Bella, P.T. Daniel, P.H. Krammer, T. Defrance, J. Immunol. 154 (1995) 5746–5756.
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[23] A.M. Cleary, S.M. Fortune, M.J. Yellin, L. Chess, S. Lederman, J. Immunol. 155 (1995) 3329 – 3337. [24] K. Nakanishi, K. Matsui, S. Kashiwamura, et al., Int. Immunol. 8 (1996) 791 – 798. [25] Y.J. Liu, C. Barthelemy, O. de Bouteiller, C. Arpin, I. Durand, J. Banchereau, Immunity 2 (1995) 239 – 248. [26] J. Choe, H.S. Kim, X. Zhang, R.J. Armitage, Y.S. Choi, J. Immunol. 157 (1996) 1006 – 1016. [27] G. Suss, K. Shortman, J. Exp. Med. 183 (1996) 1789 –1796.
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Role of follicular dendritic cells in the apoptosis of germinal center B cells Dae Y. Hur b, Dae J. Kim a, Seonghan Kim a, Young I. Kim a, Daeho Cho a, Dong S. Lee a, Young-il Hwang a, Ki-Won Bae b, Ka Y. Chang a, Wang J. Lee a,* a
Department of Anatomy, Seoul National Uni6ersity College of Medicine and Institute of Allergy and Clinical Immunology, Seoul National Uni6ersity, Medical Research Center, Seoul 110 -799 South Korea b Department of Anatomy, Inje Uni6ersity College of Medicine, Pusan 614 -735 South Korea Accepted 24 January 2000
Abstract Follicular dendritic cells (FDCs) provide the most obvious source of antigens, which are essential for the differentiation of GC B cells. It has been reported that most proliferating B cells in germinal centers undergo apoptosis. Quantitative histology shows macrophages with apoptotic debris throughout the germinal center, the highest frequency of these cells being found in the dense FDC network. Based on these findings, we hypothesized that FDC may be involved in an apoptotic pathway of the germinal center B cells. To prove this hypothesis, we performed double immunohistochemical analysis using anti-FDC mAb and peanut agglutinin (PNA), with their respective TUNEL kits. Collated data showed that a great proportion of the apoptotic cells, most of which were positive for PNA, were in close contact with FDC, which indicated an interaction between FDC and B cells in the apoptotic pathway. Further studies using double immunohistochemical staining and FACS analyses demonstrated the expression of Fas–ligand (FasL) in a subset of the FDC. These results suggest that FDC may play a role in the apoptosis of germinal center B cells via Fas–FasL interaction. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Follicular dendritic cells; Apoptosis; Germinal center B cells
1. Introduction Follicular dendritic cells (FDCs) are specialized antigen trapping cells which have only been detected in the lymphoid tissue, located within B cell follicles [1,2]. These cells have cytoplasmic extensions (dendrites), which form a dense network in the germinal center and contact with germinal center B cells [3]. FDC also express the complement receptors (CR1, CR2, CR3) and the Fc receptor, which are involved in holding antigen–antibody complexes and complement-associated antigen for extended periods (from months to years) [4–7]. It is known that this ability to hold intact antigen on their surfaces is essential to the survival of germinal center B cells, germinal center formation, and
* Corresponding author. Tel.: +82-2-7408208; fax: 7459528. E-mail address: [email protected] (W.J. Lee)
+82-2-
memory B cell generation [8–11], and it is believed that the positive selection of B cells is simply the result of the absence of a positive survival signal, which is dependent upon affinity for the original antigen [12,13]. Most studies on the role of FDC have focused on the rescue mechanisms of the germinal center B cells. However, it has been documented that most of GC B cells undergo apoptosis during B cell differentiation and that these apoptotic cells are to be found in the dense FDC network [14]. In addition, all germinal center B cells express Fas molecule highly on their surfaces, which suggests that there may be a signal for apoptosis. Recently expressions of FasL mRNA were reported in the germinal centers of the human tonsil [15–17]. In this study, we investigated the possible role of FDC in the apoptosis of germinal center B cells by visualizing the contact between apoptotic germinal center B cells and FDC, and the expression of FasL on FDC.
0165-2478/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 7 8 ( 0 0 ) 0 0 1 6 6 - 8
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D.Y. Hur et al. / Immunology Letters 72 (2000) 107–111
2. Materials and methods
2.1. Animals and tissues BALB/C mice were immunized with washed sRBC (5 × 108 cells/per mouse) intraperitoneally. Four weeks after the primary immunization, the same number of sRBC were intraperitoneally injected into the mice. Spleens were dissected from the animals on the fifth and seventh day after the secondary immunization. After extracting the spleens, they were immersed in a 25% sucrose solution, prior to being submerged in OCT embedding compound in liquid nitrogen cooled isopentane, 8 mm sections were prepared using a cryostat (Leica CM 3050).
2.2. (Immuno)histochemistry (IHC) 2.2.1. PNA (peanut agglutinin) staining for GC B cells and TUNEL staining Cryosections were dried in air and fixed in cold acetone at 4°C. These specimens were then incubated with biotinylated PNA(Vector) overnight at 4°C, and then incubated with alkaline phosphatase (AP)-conjugated streptavidin (Vector Co.) for 30 min at 37°C in a wet chamber. The labeled cells were visualized by the substrate solution (fast red, naphthol, levamisole, Sigma Chemical, St Louis, MO). Specimens were then stained with TUNEL (Terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP-biotin nick-end labeling) kit for apoptotic cells. Briefly, specimens were air dried and fixed in 1% paraformaldehyde for 10 min, and then postfixed in ethanol:acetic acid (2:1) for 5 min. After quenching the endogenous peroxidase in 3% hydrogen peroxide (Fluka Chemie AG, Switzerland) for 5 min, the sections were reacted with Apoptag kit (Oncor, Gaithersburg, MD). Sections were washed and then were visualized using diaminobenzidine tetrahydrochloride (DAB, Sigma Chemical, St Louis, MO), NiCl2 (0.4% w/v, Fluka Chemie AG, Switzerland) and 0.03% H2O2 in PBS (0.05 M, pH 7.4). 2.3. IHC staining for FDCs ( follicular dendritic cells) and TUNEL staining Cryosectioned spleen tissues were incubated with rat anti-mouse FDC (FDC-M2, a gift from Dr M.H Kosco-Vilbois) or biotinylated PNA (Vector), and then incubated with biotinylated anti-rat F(ab)%2 fragment (Boehringer Mannheim) for 30 min at 37°C in a wet chamber. Subsequently, they were incubated with AP-conjugated streptavidin (Vector). The labeled cells were visualized using fast red, naphthol, levamisole
substrate solution, and stained with the TUNEL kit for apoptotic cells as described above.
2.4. Double staining for FDCs and Fas-ligand (Fas-L) Cryosectioned spleen tissues were incubated with rat anti-mouse FDCs (FDC-M2) for 1 h at 37°C in a wet chamber, and then incubated with AP-conjugated anti-rat mAb (Sigma Chemical, St Louis, MO). The labeled cells were visualized using the substrate solution (described above). Tissues were incubated with biotinylated anti-Fas ligand (Pharmingen) for 1 h at 37°C in wet chamber, which was followed by an incubation with peroxidase-conjugated extravidin (Sigma Chemical, St Louis, MO). Labeled cells were visualized with diaminobenzidine tetrahydrochloride (DAB, Sigma Chemical, St Louis, MO), NiCl2 (0.4% w/v) and 0.03% H2O2 in PBS (0.05 M, pH 7.4).
2.5. Enrichment of FDC using a discontinuous Percoll gradient Discontinuous density gradients were prepared in 15 ml centrifuge tubes by layering 10% Percoll solution (1.030 g/ml) and 30% Percoll solution (1.052 g/ ml). The cell suspension (mice splenic cells) were layered on top of the gradient solution and centrifuged at 8500× g for 40 min. The cells in the interphase between 1.030 and 1.052 were collected, washed with PBS (0.01 M, pH 7.4) and prepared for flowcytometric analysis [18].
2.6. A flow-cytometric analysis The FDC-enriched cells were incubated with normal goat serum (Vector) for 1 h, and then with antiFDC mAb (FDC-M2) and biotinylated anti-FasL mAb (Pharmingen) for 30 min at 4°C. After washing with PBS, the samples were double-stained with antirat-FITC (Sigma Chemical, St Louis, MO) Ultravidin-PE (Leinco Technologies, St Louis, MO) for 30 min at 4°C. Using a FACStar (Bect and on Dickinson), samples were then analyzed.
3. Results
3.1. TUNEL positi6e cells in follicle were positi6e for PNA To characterize the TUNEL+ GC cells, we examined the possibility of in situ double staining using the TUNEL kit and PNA, which is usually used for the visualization of the germinal center forming cells [19,20]. The results of the double staining demonstrated
D.Y. Hur et al. / Immunology Letters 72 (2000) 107–111
109
3.2. In follicles, many TUNEL positi6e cells were obser6ed to be in close contact with FDC, appearing to interact mutually. To investigate correlation between FDC and TUNEL+ cells, the frozen sections were doublestained with anti-FDC mAb (FDC-M2) and the TUNEL kit. Some TUNEL+ cells were observed to be in close contact with FDC (Fig. 2), which suggested that TUNEL+ cells and FDC may interact mutually.
3.3. A subset of FDC express FasL We studied whether FDC would express FasL using in situ double staining and flowcytometric analysis. Double staining results showed that a few FDCs were positive for FasL, which suggested that a subset of FDC expressing FasL may exist in GC (Fig. 3). Dualcolor flow cytometic analysis revealed that a small percentage (20–30%) of FDC expressed FasL (Fig. 4).
4. Discussion Within the GC, the B cells closely interact with FDC, and provide them with antigenic stimuli which promotes their differentiation into memory cells or plasma cells. The integrin adhesion molecules LFA-1 and VLA-4 on the B cells through interaction with their counter receptor. ICAM-1 and VCAM-1 on the FDC, which have been shown to prevent GC B cells from entering into apoptosis. Triggering of Fas (CD95), on the other hand, has been shown to induce apoptosis of the GC B cells in the presence of anti-Ig or adhesionmediated rescue signals [13]. Our experimental results show that the TUNEL+ GC cells were also PNA-positive, which suggests that the apoptotic cells in the GC should be B cells. Moreover, in this experiment we have found that some GC B
that almost all TUNEL+ cells in GC were positive for PNA (Fig. 1), and that the majority of TUNEL+ cells were located in the dark and basal light zones. For comparison, tissue samples were also double stained using anti-CD3 mAb and the TUNEL kit. TUNEL+ GC cells were totally negative for CD3 (data not shown).
Fig. 1. Double staining using peanut agglutinin (PNA) and the TUNEL kit of frozen mouse spleen sections on the seventh day after secondary immunization with sRBC. The PNA + cells (red) delineate the germinal center (GC) over which The TUNEL+ cells (black) are scattered. Most of TUNEL + cells are also PNA-positive, which indicates that the apoptotic cells in the GC should be GC B cells. Fig. 2. Double staining using rat anti-FDC mAb (FDC-M2) and the TUNEL kit of frozen mouse spleen sections on the fifth day after secondary immunization with sRBC. In follicles, many TUNEL+ cells (dark brown) were observed to be in close contact with FDC (red), appearing to interact mutually. Fig. 3. Double immunohistochemical staining using anti-FDC mAb (FDC-M2) and anti-FasL mAb in frozen mouse spleen sections on the seventh day after secondary immunization with sRBC. Some FDCs (red color) were also positive for FasL (dark brown).
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Fig. 4. Two color flowcytometric analysis using anti-FDC mAb and anti-FasL mAb in mouse spleen cells on the seventh day after secondary immunization with sRBC. After enrichment of FDCs using a discontinuous Percoll gradient, single flowcytometric analysis for FDC showed that 20–30% of the mononuclear cells were FDC-positive (A). The same sample of FDC was analysed by two color flowcytometry using anti FDC mAb and anti-FasL mAb (B), demonstrated that some portion of FDC were FasL-positive.
cells undergoing apoptosis make close contact with FDC. It is known that apoptotic cells were found both outside and within the FDC-associated clusters [12]. Our results and this report suggest that FDC may participate in the apoptosis of GC B cells. It is documented that GC B cells express a high level of CD95 [15,16], which has been implicated in apoptosis induction [21 – 23]. Furthermore, our study shows by immunohistochemistry and flow cytometry that some FDC express FasL. The mode of participation of the Fas-FasL system in the clonal deletion of B lymphocytes still remains unclear. It is reported that not only anti-Fas mAb rapidly induces the apoptosis of germinal center cells, but that signalling through CD40 also up-regulates Fas expression, rendering the cells highly sensitive to Fas-mediated apoptosis. Although the precise mechanisms involved in this signalling have not been clarified, these reports suggest, therefore, that the Fas – FasL system may have role in the apoptosis of germinal center cells [24 – 26]. Although FasL has been reported predominantly in activated T cells, some germinal center B cells were also found to be capable of expressing functional FasL[17]. However, whether or not FDC express FasL remains unclear. Moreover, it has recently been reported that a subclass of dendritic cells expressing FasL have a role in the regulation of the response of primary peripheral T cells [27]. These findings suggest that FDC might express FasL. In our study, a population of FDC, which expressed FasL was clearly demonstrated by flow cytometry and immunohistochemistry. It would, therefore, appear in the light of the information available to date, that FDC may participate in the abolition process of germinal center B cells through Fas-mediated apoptosis.
Acknowledgements We would like to thank Dr M.H. Kosco-Vilbois for a gift of FDC-M2 mAb.
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