The Journal of Immunology
Lymphotoxin and TNF Produced by B Cells Are Dispensable for Maintenance of the Follicle-Associated Epithelium but Are Required for Development of Lymphoid Follicles in the Peyer’s Patches1,2 Alexei V. Tumanov,*† Dmitry V. Kuprash,† Julie A. Mach,* Sergei A. Nedospasov,†‡ and Alexander V. Chervonsky3* Organogenesis of Peyer‘s patches (PP), follicle-associated epithelium, and M cells is impaired in mice lacking B cells. At the same time, lymphotoxin (LT) and TNF are known to be critical for the development of PP. To directly address the function of LT and TNF expressed by B cells in the maintenance of PP structure, we studied the de novo formation of PP in B cell-deficient mice after the transfer of bone marrow from mice with targeted mutations in LT, TNF, or their combinations. We found that although the compartmentalization of T and B cell zones and development of follicular dendritic cells were affected by the lack of B cell-derived LT and TNF, the development of follicle-associated epithelium and M cells in PP was completely independent of LT/TNF production by B cells. The Journal of Immunology, 2004, 173: 86 –91.
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2 The contents of this publication do not necessarily reflect the view or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government. The publisher or recipient acknowledges the right of the U.S. government to retain a nonexclusive, royalty-free license in and to any copyright covering this article.
tecture of T and B cell zones in the spleen (6 – 8), and the full development of PP, including propagation of M cells (5). M cells are specialized epithelial cells in the follicle-associated epithelium (FAE) and play an important role in transporting foreign Ags to the underlying organized lymphoid tissue, where protective immune responses are initiated (for review, see Refs. 9 and 10). At the same time, many pathogens exploit M cells as a route of invasion, including bacterial pathogens (9, 11) and prions (12). The ability of B cells to promote the generation of FDC and follicle architecture has been attributed to three members of the TNF family of cytokines: surface lymphotoxin (LT), consisting of LT␣ and LT, and TNF (6 – 8). LT and TNF differ in their effects on the early development of PP. LT␣ knockout (KO) and LT KO mice completely lack PP because of defects in anlagen formation (13, 14), while the early stages of PP development do not appear to be impaired in TNF KO mice (15, 16). The effect of tnf-ri gene removal on PP development remains unclear, as conflicting results have been published (15–18). Although PP in TNF-RI KO mice are reduced in number and size and show abnormal microarchitecture (15), their FAE contains M cells (5). We have previously shown that mice lacking B cells have intact anlagen, but that their FAE and the number of M cells are significantly reduced. These defects can be restored by bone marrow (BM) transfer from normal mice (5). In vitro studies also provided evidence that mature lymphocytes can deliver signals converting epithelial cells into M cells (19). The exact nature of such signals provided by B cells remains unknown. Clearly, there are late postembryonic events that allow B lymphocytes to send signals to both FAE and stroma to induce full development of PP. Thus, we sought to determine whether TNF/LT family cytokines produced specifically by B cells are the mediators of the molecular conversation between B cells and epithelial cells that lead to development of FAE, M cells, and organized lymphoid follicles.
3 Address correspondence and reprint requests to Dr. Alexander Chervonsky, 600 Main Street, Bar Harbor, ME 04609. E-mail address:
[email protected]
Materials and Methods
econdary lymphoid organs are essential for efficient immune responses. Lymphocytes not only populate these organs, become activated, and exert their functions, but they also participate in the generation and maintenance of the architecture of lymphoid tissues. Organogenesis of Peyer‘s patches (PP),4 secondary lymphoid organs located in the small intestine, begins with the formation of primary anlagen (at ⬃16 days of gestation) that become populated with mature lymphocytes just before birth (1, 2). The lymphocyte-like cells that express CD4 and lack CD3 surface marker were shown to be involved in the early stages of the generation of lymph nodes and PP (1, 3, 4), while B lymphocytes are required for the later stages of lymph nodes (4) and PP organogenesis (2, 5). B cells were shown to regulate the generation of follicular dendritic cells (FDC), maintenance of the normal archi*The Jackson Laboratory, Bar Harbor, ME 04609; †Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia; and ‡Basic Research Program, SAIC Frederick, Inc. and Laboratory of Molecular Immunoregulation, Center for Cancer Research, National Cancer Institute, Frederick. MD 21702 Received for publication December 13, 2003. Accepted for publication April 22, 2004. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grant DK52210 (to A.V.C.) and with U.S. federal funds from the National Cancer Institute, National Institutes of Health, under Contract NO1-CO-12400. A.V.T. was supported by The Jackson Laboratory grant to A.V.C. and by a Grant 03-04-49097a from the Russian Foundation for Basic Research. D.V.K. was supported by Grant 02-04-49105a from the Russian Foundation for Basic Research and a Molecular and Cell Biology program grant from the Russian Academy of Sciences. S.A.N. is an International Research Scholar of the Howard Hughes Medical Institute.
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Abbreviations used in this paper used in this paper: PP, Peyer’s patches; FAE, follicle-associated epithelium; LT, lymphotoxin; FDC, follicular dendritic cell; BM, bone marrow; SEM, scanning electron microscopy; KO, knockout; WT, wild type; BLC, B lymphocyte chemoattractant; SLC, secondary lymphoid tissue chemokine. Copyright © 2004 by The American Association of Immunologists, Inc.
Mice C57BL/6J (B6) wild-type mice, B6.CB17-Prkdcscid/SzJ (B6 scid) mice, C57BL/6J-Igh-6 tm1Cgn (Igh6) mice, and C57BL/6J-Rag1tm1Mom (RAG1 0022-1767/04/$02.00
The Journal of Immunology KO) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The following mice with targeted mutations of the genes in the lt/tnf locus were bred and genotyped as previously described: LT␣ KO (13), LT KO (20), LT/TNF double-deficient mice (LT/TNF KO) (21), and LT␣/LT/ TNF triple-deficient mice (TNF/LT KO), in which all three genes were simultaneously deleted (22). B-TNF KO mice and T-TNF KO mice, generated similarly to B-LT KO and T-LT KO mice (23) will be described elsewhere (A. V. Tumanov, S. I. Grivennikov, M. Heikenwalder, A. Aguzzi, D. V. Kuprash, and S. Nedospasov, manuscript in preparation). All mice were housed under specific pathogen-free conditions.
Immunohistology Immunolabeling was performed as described (8) with Abs purchased from BD PharMingen (San Diego, CA) unless otherwise indicated. Briefly, PP were visualized by stereomicroscopy, embedded in tissue-freezing medium, and snap-frozen in 2-methylbutane (Sigma-Aldrich, St. Louis, MO) prechilled by liquid nitrogen. FDC were visualized using anti-CR1 (8C12) and anti-FDC-M1 Abs. We have not seen discordance between the two types of FDC staining in this study. For double CD3/B220 labeling, antiB220 and biotinylated anti-CD3 were used in combination with secondary HRP-conjugated mouse anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and alkaline phosphatase-conjugated streptavidin (Sigma-Aldrich); Vector Red or Vector Blue substrate kits were obtained from Vector Laboratories (Burlingame, CA). Sections were counterstained with Mayer’s hematoxylin, mounted with glycerol-gelatin, and documented using digital microscopy. For immunofluorescence, FITC-conjugated anti-B220 and biotin-conjugated anti-CR1 or FITC-conjugated anti-CD3 and biotin-conjugated antiB220 were used, followed by rhodamine-streptavidin (Jackson ImmunoResearch Laboratories). Fluorescence images were mounted with Vectashield (Vector Laboratories) and documented using a photomicroscope.
Transfer of BM cells BM cell suspensions were prepared from femurs and tibiae of donor mice. Igh6 mice were lethally irradiated (900 rad) and reconstituted with 4 ⫻ 106 donor BM cells on the same day. For mixed BM donor transfers, a 1:1 mixture (total 4 ⫻ 106) of BM cells was used and recipients were irradiated with 650 rad. The degree of chimerism was established by staining the recipient’s splenocytes for the markers of B cells. PP were collected under a stereomicroscope 8 wk later.
Scanning electron microscopy (SEM) SEM was done essentially as described previously (5). Briefly, intestinal fragments containing PP were cleaned from mucus and fixed in 2.5% glutaraldehyde in 0.1% cacodylate buffer overnight at ⫹4°C. Specimens were treated with alternating 1% osmium tetroxide and 1% thiocarbohydrazide (OTOTO method), dehydrated in a graded series of acetone, critical point dried, and sputter coated to produce a 15-nm gold coating. Samples were examined using a Hitachi S-3000N Variable Pressure scanning electron microscope (Hitachi, Tokyo, Japan) at an accelerating voltage of 20 kV.
In situ hybridization In situ hybridization on frozen sections was performed as described in Ref. 24. Frozen 6-m sections were fixed in 4% paraformaldehyde/PBS solution for 10 min at room temperature, washed three times with PBS, and acetylated in 0.25% acetic anhydride for 10 min at room temperature. After three washes in PBS, slides were prehybridized in mRNA hybridization buffer (DakoCytomation, Carpinteria, CA) for 1 h. Hybridization was performed with dioxigenin -labeled antisense RNA (200 – 400 ng/ml) at 72°C prepared and purified using a Boehringer Mannheim T7 kit (Indianapolis, IN) according to the recommended protocol. After overnight hybridization, slides were washed in 0.2⫻ SSC for 3– 4 h at 72°C. Anti-dioxigenin Abs conjugated with alkaline phosphatase were applied after a 1/2000 dilution in buffer B (0.1 M Tris-HCl (pH 7.6) and 0.15 M NaCl) containing 1% heat-inactivated sheep serum and incubated overnight at ⫹4°C. After extensive washing in buffer B (three to five times by 10 min) phosphatase reaction was performed in buffer B containing 50 mM MgCl2 supplemented with 0.34 mg/ml nitroblue tetrazolium, 0.23 mg/ml 5-bromo-4chloro-3-indolyl-phosphate, and 0.24 mg/ml levamisole (pH 9.5) for 3 h. All reagents for in situ hybridization, if not indicated otherwise, were purchased from Boehringer Mannheim.
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Results and Discussion Development of FAE and M cells does not require TNF and LT cytokines To address the issue of the role of TNF/LT subfamily cytokines in the development of FAE and specialized M cells within the FAE, we used a BM transfer approach. Mice from a strain lacking B lymphocytes (Igh6) and having bona fide anlagen, but only rudimentary PP (5, 25) were used as recipients of BM. This approach allows the separation of two important stages in PP development: formation of the anlagen and complete development into a functional organ with lymphoid follicles. BM was derived from normal C57BL/6J (wild type (WT)) donors or from LT␣ KO, LT KO, double LTTNF KO, or triple-deficient LT/TNF KO mice. To ensure that only B cells developing in chimeras would lack the genetically deleted cytokines, BM from cytokine-deficient donors was mixed with BM from TNF/LT-sufficient Igh6 mice so that all other cytokine-sufficient cell types were provided by Igh6 BM. Clearly, the lack of TNF/LT cytokines did not have much effect on the ability of B cells to stimulate advanced development of PP (Table I). When the numbers of PP that have developed in B celldeficient recipients after transfer of BM from normal and cytokinedeficient mice were compared, it became obvious that they did not differ: very similar numbers of PP were found in all recipients (Table I). Thus, expression and/or secretion of TNF/LT by B cells were not required for the appearance of the developed PP. The method of choice to study FAE is SEM. The advantages of this method are that it can be used on mouse strains that lack Ulex europaeus lectin (UEA-1) binding by M cells (which is true for all strains used in this study), and that it is not prone to artifacts such as nonspecific staining by reagents revealing M cells (e.g., alkaline phosphatase substrates). This is because M cells have a very characteristic morphology of the cell membrane facing the intestinal lumen: shortened microvilli and lack of mucus, two features that distinguish them from enterocytes and goblet cells, respectively. Upon examination of FAE with SEM, we found that there was no difference in the formation of the domes and generation of M cells between mice that received the mixtures of BM from Igh6 and from WT mice and those that lacked major TNF family members (Fig. 1A). Few (3.8 ⫾ 0.5% in B63 Igh6 domes and 2.9 ⫾ 0.6% in triple LT/TNF KO3 Igh6 domes) goblet cells were found in the FAE of all chimeric mice (Fig. 1A), another feature of normal FAE. In addition to conducting BM transfer experiments, we have also examined FAE of mice with genetic cell-specific deletion of LT from B cells (B-LT KO mice) (8). SEM revealed normal distribution and numbers of M cells in the FAE of B-LT KO mice (Fig. 1B). Taken together, these results clearly demonstrate that none of the three major members of the TNF family cytokines (or their Table I. De novo formation of PP in Igh6 mice does not require major TNF family membersa Donor (n)b
B6 (10) Igh6 KO (5) LT␣ KO (6) LT KO (6) LT/TNF KO (3) (LT␣/LT/TNF) KO (9)
PP Numbers in Igh6 Recipient Micec
5.3 ⫾ 0.9 1.0 ⫾ 0.9 6.3 ⫾ 1.5 7.3 ⫾ 0.5 6.3 ⫾ 1.2 5.4 ⫾ 1.6
a 1:1 mixtures of BM cells from Igh6 and mutant mice were injected into irradiated (650 rad) Igh6 mice. b Numbers in parentheses indicate the number of mice analyzed. c Mean ⫾ SD of PP numbers in recipient mice.
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FIGURE 1. Development of FAE and M cells does not require LT/TNF cytokines. A, Igh6 mice were reconstituted with a mixture of BM from Igh6 and WT or BM from indicated KO mice. SEM shows that M cells (arrows) are clearly present in all chimeras. Magnification, ⫻100 (left panels) and ⫻3000 (right panels). Examples of size bars (10 M) are also shown for higher magnification. Histogram of anti-Ig-stained splenocytes shows equal reconstitution with donor B cells in Igh6 chimeras receiving a mixture of BM from Igh6 and WT (solid line) or BM from TNF/LT KO mice (dashed line). Arrows indicate M cells (A and B). G, Goblet cell. B, SEM of PP of B-LT KO mice shows normal M cells in the FAE. Magnification, ⫻3000.
combination) is required for the B lymphocyte-dependent development of FAE and M cells in PP. It is an open question whether B lymphocytes are necessary to stimulate a differentiation step that leads to M cell generation from a common epithelial precursor of other intestinal epithelial cell types, or whether the predominant role of B cells is in the maintenance of M cell phenotype. The latter option seems to be more plausible since the presence of reduced M cell numbers in B celldeficient mice (5) and in Rag1 KO mice (26) has been documented. Interestingly, in mice completely lacking T and B lymphocytes (Rag1 KO), residual M cell numbers were further (although very moderately) reduced by treatment with LTR-Ig fusion protein (26). In that case, LT could not be produced by T or B lymphocytes and the source of this cytokine needs to be identified. However, generation of M cells can be also induced by microbial infection (27), suggesting intermediation by the innate immune system or direct influence of microbial flora on the epithelium. Thus, it is possible that LTR signaling may play some role in lymphocyte-independent induction of M cells. TNF and LT expressed by B cells are required for maintenance of lymphoid follicle architecture in PP The role for B cell-derived TNF and LT in the maintenance of the splenic structure has been well documented (6 – 8, 28). Remarkably, other secondary lymphoid organs were affected differently by such deficiencies: lack of LT production by B cells had only a moderate effect on the structure of PP and had no effect on the
structure of lymph nodes (8). Mice with targeted deletions of TNF and TNF-RI have rudimentary PP that show abnormal microarchitechture with mixed T and B cell zones and the absence of FDC (15, 16). To determine the role of TNF and LT expressed by B cells in the maintenance of lymphoid follicle structure in the PP of adult mice, we again used a BM transfer approach. We addressed the issue of whether cooperation between TNF and LT produced by BM-derived cells is necessary at all. Igh6 mice were reconstituted with BM from LT KO, or triple LT/TNF KO mice, after which PP from BM chimeras were examined using immunohistochemical staining of cryostat sections (Fig. 2). Transfer of BM from LT KO mice lacking LT in all cell types partially restored FDC development in Igh6 mice (Fig. 2, middle panel), suggesting that LTR signaling is not completely dispensable. In contrast, transfer of BM from triple LT/TNF KO mice did not result in any FDC development in the PP of recipient mice (Fig. 2, right panel). Thus, we concluded that TNF and LT may cooperate in the maintenance of FDC in the PP of adult mice. However, cells other than B lymphocytes could have been a source of TNF required for FDC development, as all donor cells were LT and TNF deficient in these experiments. To elaborate specifically the role of B cells in the LT- and TNFdependent maintenance of PP structure in adult mice, we used other approaches. We transferred mixed BM grafts from triple LT/ TNF KO and Igh6 donors into Igh6 recipient mice (Fig. 3A).
FIGURE 2. LT and TNF are required for generation of FDC in PP of adult mice. Igh6 mice were reconstituted with BM from WT, LT KO, or LT/TNF KO mice. Frozen sections of PP were stained with anti-CR1 (red) and anti-B220 (blue). Original magnification, ⫻100. Note remaining FDC clusters in PP of LT KO3 Igh6 chimera shown by arrows.
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FIGURE 3. LT and TNF expressed by B cells are required for the maintenance of lymphoid follicles in PP of adult mice. A, Igh6 mice were reconstituted with a mixture of BM from WT ⫹ Igh6 (left column) or LT/TNF KO and Igh6 mice (right column). Serial frozen sections of PP were stained with anti-CD3 (blue), anti-B220 (yellow; upper row), anti-CR1 (red), and anti-B220 (blue; lower row). Magnification, ⫻100. B, Serial frozen sections of PP from mice with B cell-specific deletion of TNF (B-TNF KO) and T cell-specific deletion of TNF (T-TNF KO) mice were stained with Abs: anti-B220 (red)/anti-CD3 (blue), antiCR1 (red), and anti-FDC-M2 (red). PP from TNF-sufficient mice and mice with ubiquitous deletion of TNF (TNF KO) were used as controls. Magnification, ⫻100.
Transfer of WT BM resulted in the formation of normal PP structure in Igh6 recipient mice, with segregated B and T cell zones and prominent FDC clusters (Fig. 3A, left column). In contrast, the
structure of PP in mice receiving the mixture of BM from LT/TNF KO and Igh6 mice was markedly disturbed: B cells did not form distinct B cell follicles but were scattered along the domes of the
FIGURE 4. LT and TNF cooperate to control BLC expression essential for correct positioning of T and B cells in the PP. Igh6 mice were reconstituted with a mixture of BM from WT, LT KO, TNF KO, and LT/TNF KO mice. Serial frozen sections of PP were stained with anti-CD3 (blue), anti-B220 (yellow, upper row), and antiCR1 (red, second row) and were hybridized with BLC and SLC antisense RNA (two lower rows). Arrow shows remaining FDC clusters in PP of LT KO3 Igh6 chimeric mice. Magnification, ⫻100.
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PP, while no FDC clusters were found (Fig. 3A, right column). These results suggest that LT and TNF both expressed exclusively by B cells are required for the generation of FDC and compartmentalization of T and B cell zones in PP. LT and TNF from other cell types (originating from Igh6 donor) were not sufficient for the maintenance of refined PP structure in the absence of LT and TNF from B cells in these mixed BM transfer experiments. Further confirmation of the role of TNF expressed by B cells in the maintenance of lymphoid follicles was obtained by analysis of the PP structure in mice with inactivation of tnf gene, specifically in B or T cells (Fig. 3B). B-TNF KO mice lacked organized B cell follicles and FDC in PP while T-TNF KO mice showed normal PP structure (Fig. 3B), suggesting the primary role of TNF produced by B cells, but not by T cells, in the maintenance of PP microarchitecture. Thus, LT/TNF cytokines not only participate in the early embryonic organogenesis of PP, but their production by B lymphocytes contributes to the maintenance of normal PP structure in adult mice. To address the issue of how B cell-derived cytokines influence follicular microarchitecture, we sought to evaluate the influence of LT and TNF deficiencies on the expression of relevant chemokines in the PP. Migration of T and B cells to the secondary lymphoid organs and formation of T and B cell zones in lymphoid follicles is regulated by specific chemokines. In the spleen, expression of these chemokines, B lymphocyte chemoattractant (BLC; CXCL13) and secondary lymphoid tissue chemokine (SLC; CCL21), is strongly dependent on LT and TNF signaling (29, 30). Moreover, it has been suggested that TNF and LT cooperate for the regulation of BLC expression in the spleen (22). Recent studies identified BLC as one of the principal chemokines required for the homing of B cells to the lymphoid follicles in PP (31, 32). To examine whether abnormalities in follicle architecture in the PP of triple LT/TNF3 Igh6 reconstituted mice can be attributed to impaired chemokine expression, we analyzed expression of BLC and SLC chemokines in the PP of mice reconstituted with BM from mice deficient in LT and TNF (Fig. 4). SLC expression was clearly detected in the PP of all reconstituted mice (Fig. 4), suggesting that, in contrast to the spleen, LT and TNF do not play a major role in the regulation of SLC expression in PP. PP of LT KO3 Igh6 chimeras displayed reduced expression of BLC (Fig. 4), although compartmentalization of T and B cell areas was not affected, suggesting that the remaining level of BLC expression was capable of supporting proper B cell migration. BLC expression was also not significantly reduced in PP of TNF KO3 Igh6 chimeras, and their T and B cell areas were relatively intact, although lacking FDC (Fig. 4). Interestingly, this result clearly shows that BLC can be produced by cells other than FDC. Therefore, elimination of LT or TNF production by BM-derived cells did not affect BLC production enough to influence positioning of T and B cell zones in PP. In contrast, B and T cells were mixed and scattered within the PP of triple LT/TNF KO3 Igh6 chimeras. Importantly, BLC expression was clearly down-regulated in the PP of these mice. Our data suggest that similar to the spleen, LT and TNF cooperate in BLC expression essential for correct positioning of T and B cells in the PP. Biochemical inhibition of LTR and TNFR signaling has been shown to be a promising therapeutic approach in mouse models of inflammatory bowel disease and experimental colitis (33, 34). It remains to be determined whether such effects of LTR or TNFR blockers are linked to inhibition of BLC function in the areas of local inflammation in the gut where ectopic expression of BLC has been shown (35, 36). This study reveals the distinct contribution of LT/TNF cytokines produced by B lymphocytes to the development of PP and FAE.
Although LT/TNF cytokines are critical for FDC generation and maintenance of T and B cell zones, they are dispensable for the formation of FAE and M cells. These results are clearly important for the understanding of lympho-epithelial interactions and for the design and application of mucosal vaccines (37).
Acknowledgments We thank Lesly Bechtold for excellent technical help.
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