Developmental expression of the mouse c-rel ... - Semantic Scholar

Development 120, 2991-3004 (1994) Printed in Great Britain © The Company of Biologists Limited 1994

2991

Developmental expression of the mouse c-rel proto-oncogene in hematopoietic organs Daniel Carrasco, Falk Weih and Rodrigo Bravo Department of Molecular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 4000, Princeton, New Jersey 08543-4000, USA

SUMMARY We have studied the expression of the c-rel proto-oncogene during mouse embryonic development and adult animals using in situ hybridization and immunocytochemical analysis. c-rel transcripts were detected late in development with an expression pattern that parallels the emergence and diversification of hematopoietic cells. In the embryo, crel is expressed first in the mesoderm-derived hematopoietic cells of the liver and later also in other hematopoietic tissues such as thymus and spleen. This correlation between c-rel expression and places of hematopoietic infiltration is conserved in the postnatal period, with expression of c-rel mRNA in the medullary region of the thymus and in splenic B cell areas, including the marginal zone and the outer region of the periarterial sheath. High levels of c-rel transcripts were also detected in the splenic germinal centers,

lymph nodes and Peyer’s patches. Using double immunofluorescence and cell preparations from different embryonic and adult hematopoietic organs, we have defined the pattern and cell types of c-rel expression in different hematopoietic cell lineages and in the stromal cell content of the thymus. By using electrophoretic mobility shift assays, we have also correlated c-Rel expression in spleen with κB-binding activity in the form of c-Rel/p50 and c-Rel/p52 heterodimers. The timing and pattern of expression of the c-rel proto-oncogene in the different cell lineages suggest that temporally regulated changes in c-Rel expression may be required for vertebrate hematopoiesis.

INTRODUCTION

1993). In addition to the RHD, the proteins of this family also exhibit similar biochemical properties. They exist as homo- and heterodimers and their activities are modulated by interaction with the IκB molecules. Activation of the cellular NF-κB activity by mitogens and cytokines involves the dissociation of the Rel/NF-κB dimers from IκB, allowing their translocation to the nucleus. Similarly, the IκB protein homolog in Drosophila, cactus (Geisler et al., 1992; Kidd, 1992), regulates the activity of dorsal by controlling its translocation to the nucleus (Steward, 1989; Whalen and Steward, 1993). The mammalian IκB proteins, IκBα, IκBγ and Bcl-3, not only regulate the movement of the different Rel/NF-κB complexes to the nucleus but also can affect their DNA-binding and transcriptional activities. Furthermore, the different IκBs interact differentially with the various Rel/NF-κB dimers (for review see Blank et al., 1992; Nolan and Baltimore, 1992; Beg and Baldwin, 1993; Gilmore and Morin, 1993; Liou and Baltimore, 1993). The NF-κB activity plays an important role in the inducible expression of genes involved in immune response and/or acute phase reactions, such as the regulated expression of cytokines like interleukin-2, interleukin-6 and tumor necrosis factor α, as well as cell adhesion molecules, the interleukin-2 receptor α chain, major histocompatibility complex (MHC) classes I and II, and the immunoglobulin kappa light chain. In addition, some viral enhancers, including human immunodeficiency virus (HIV) and cytomegalovirus (CMV), contain κB sequences

The c-rel proto-oncogene is the cellular homolog of v-rel, the viral oncogene of reticuloendotheliosis virus strain T, an acutely oncogenic retrovirus that induces a rapidly fatal lymphoma in young birds (Theilen et al., 1966; Stephens et al., 1983). c-rel cDNAs from chicken (Capobianco et al., 1990), mouse (Bull et al., 1989; Grumont and Gerondakis, 1989) and human (Brownell et al., 1986) cells have been isolated and characterized. Comparison of c-rel and v-rel sequences show that the vrel oncogene encodes a truncated and mutated form of c-rel protein (c-Rel) (Capobianco et al., 1990; Stephens et al., 1983; Brownell et al., 1986). c-Rel and v-Rel are members of the Rel/NF-κB family of transcriptional regulators which includes NF-κB1 (p105/p50), NFκB2 (p100/p52), RelA (p65) and RelB (for review see Grilli et al., 1993; Liou and Baltimore, 1993). The family also includes the Drosophila proteins dorsal, which is involved in establishing the dorsal-ventral polarity in the developing embryo (Steward, 1987), and Dif, which mediates an immune response in the larval fat body (Ip et al., 1993). Members of the Rel/NFκB family are defined by a conserved region of approximately 300 amino acids, termed the Rel homology domain (RHD), which includes the DNA-binding and dimerization domains (for review see Blank et al., 1992; Bose, 1992; Gilmore, 1992; Nolan and Baltimore, 1992; Grilli et al., 1993; Grimm and Baeuerle,

Key words: c-rel expression, mouse development, B cells, protooncogene, hemopoiesis

2992 D. Carrasco, F. Weih and R. Bravo (reviewed in Grilli et al., 1993). Recent work has demonstrated that the various Rel/NF-κB homo- and heterodimers distinctly bind to different κB sites and that the combination of these subunits determines the specificity of the transcriptional activation, providing strong support for the selective involvement of Rel/NF-κB complexes in the expression of genes (Schmid et al., 1991; Fujita et al., 1992; Hansen et al., 1992; Kunsch et al., 1992; Nakayama et al., 1992; Perkins et al., 1992; Tan et al., 1992; Sica et al., 1992; Shu et al., 1993). Therefore, cell-type and tissue-specific expression of certain NF-κB responsive genes could be achieved by the differential expression of Rel/NF-κB subunits. RelB presents a differential expression during mouse development, which is highly restricted in lymphoid tissues to interdigitating dendritic cells (Carrasco et al., 1993), thus supporting this notion. We have extended our analysis of Rel/NF-κB family members to the expression of the c-rel proto-oncogene in mouse embryos and adult animals, focusing on hematolymphoid organ development. The results show that c-rel is expressed in tissues engaged in hematopoietic activity. Expression of c-rel is primarily restricted to erythroid precursors as well as B and T cell lineages, matching the process of cell migration and colonization of hematopoietic organs characteristic of the various stages of erythroid and lymphoid cell development. This pattern of expression clearly differs from that described previously for relB (Carrasco et al., 1993). Our results demonstrate that members of the rel family of genes are differentially expressed, suggesting distinct roles during hematopoietic diversification and immune regulation. MATERIALS AND METHODS PCR analysis Total RNA preparation from different tissues and polymerase chain reaction (PCR) were performed as previously described (Carrasco et al., 1993). The two oligonucleotide primers used in all PCR experiments were 5′-TGGCTGACTGACTCACTGACTGACTGACTCGTGCCTTGC-3′ and 5′-CCAACTAAATCATGAGGATGAGGCTTATATGGATCATTC-3′, complementary to nucleotides 309 to 348 and 603 to 642 of c-rel cDNA (Bull et al., 1989), respectively. Tissue preparation Embryos were obtained from natural matings of B6C3F1 mice. The time of pregnancy was established by the presence of the vaginal plug and was regarded as day 0.5 of gestation. After dissection the tissues were prepared for in situ hybridization and immunocytochemistry as previously described (Carrasco et al., 1993). In situ hybridization A murine cDNA fragment of c-rel, positions 1957 to 2188 (Bull et al., 1989) was cloned into Bluescript KS+ (Stratagene). Preparation of the [α-35S]UTP-labeled sense and antisense probes and in situ hybridization were performed essentially as described (Carrasco et al., 1993; Carrasco and Bravo, 1994). Antibody generation and immunopurification c-rel cDNA (coding for amino acids 1 to 183) was cloned into the bacterial expression vector pEx34 (Strebel et al., 1986). The antibody was generated and immunopurified according to Kovary and Bravo (1991). Immunohistochemistry Immunohistochemistry was performed as previously described

(Carrasco et al., 1993). Tissue sections were incubated with immunoaffinity-purified primary antibodies or an IgG fraction of preimmune rabbit serum at a concentration of 5 µg/ml (diluted in 10% non-immune goat serum) overnight at 4°C. The anti-IgM and antiCD45R (clone RA3-6B2) monoclonal antibodies were obtained from Pharmingen and visualized with biotinylated goat anti-rat antibody coupled to streptoavidin-peroxidase complex (Zymed Laboratories Inc). Isolation of T and B cell subpopulations Spleen single cell suspensions were obtained from 4- to 5-week-old BALB/c male mice (Taconic) and red blood cells were removed by hypotonic lysis. T cell enrichment was achieved by cytotoxic elimination of B cells and accessory cells using anti-mouse I-Ad monoclonal antibody (Pharmingen) and rabbit complement (Cedarlane). B cells were prepared by T cell cytotoxic depletion after incubation of the splenic cell suspension with a mixture of anti-Thy-1.2 (Cedarlane), anti-CD4 and anti-CD8 (both Pharmingen) monoclonal antibodies. Immunofluorescent staining Cytospin samples of cell suspensions from thymus or spleen were prepared in medium containing 10% FCS using a cytocentrifuge (Shandon, Astmoor, UK). Cytospins were fixed at RT for 2 minutes in acetone:methanol (1:1), air-dried and stained. For double immunofluorescence, cytospins were incubated at room temperature for 1 hour with anti-c-Rel antibodies together with one of the following antibodies: anti-CD45R (clone RA3-6B2), anti-CD25 (both from Immuno Select), anti-CD4, anti-CD8, anti-IgM (all Pharmingen), anti-Mac-1 (Boehringer Mannhein), anti-thymic medullary epithelium and antineutrophils (Serotec). Αnti-c-Rel antibodies were visualized with a donkey anti-rabbit immunoglobulin conjugated with Texas red (TR, Amersham). Anti-CD4, anti-CD8, anti-IgM, anti-Mac-1, anti-CD25, anti-neutrophils and anti-CD45R antibodies were visualized with a goat anti-rat IgG conjugated with FITC (Boehringer). The anti-thymic medullary epithelium antibody was visualized with anti-rat IgM conjugated with FITC (Serotec). For the identification of erythroid precursors in fetal liver, incubation and detection were done in the following order: First, incubation with rabbit anti-mouse c-Rel and then detection with anti-rabbit immunoglobulin TR-labelled (Amersham). Second, incubation with rabbit-anti-mouse hemoglobin (Cappel) and detection with anti-rabbit immunoglobulin FITC-labeled (Amersham). Apoptotic cells were detected with anti-digoxigenin using the Apop Tag kit from Oncor. Electrophoretic mobility shift assays (EMSA) The κB-binding site used, labeling of the oligonucleotide and EMSA were described previously (Dobrzanski et al., 1993). Whole-cell extracts from spleen (mouse strain C57BL/6) were prepared as described (Lernbecher et al., 1993). In reactions where specific antisera were included, extracts were preincubated with 1-3 µl of rabbit serum for 10 minutes on ice before the addition of the κB-containing oligodeoxynucleotide. Extracts were activated with 0.6% sodium deoxycholate (DOC) for 10 minutes on ice followed by the addition of NP-40 to a final concentration of 1%. DNA-binding reaction mixtures were incubated for 30 minutes at room temperature, loaded onto a 6% acrylamide-bisacrylamide (40:1) gel and run in 0.25× TBE buffer (22 mM Tris base, 22 mM boric acid, 0.5 mM EDTA) at 4°C.

RESULTS Expression of the c-rel proto-oncogene is high in hematopoietic tissues of the adult animal and is temporally regulated during embryogenesis To investigate c-rel gene expression, PCR analyses of adult mouse tissues and embryos at different stages of development

c-rel expression in lymphoid tissues 2993

Fig. 1. Expression of c-rel in various mouse tissues (A) and in mouse lymphoid cells (B). Analysis of c-rel gene expression was performed by PCR-mediated cDNA amplification. B and T splenic cells were isolated by cytotoxicity. Control, no cDNA was added to the PCR reaction. NIH 3T3, quiescent cells stimulated with 20% FCS for 4 hours in the presence of cycloheximide (5 µg/ml).

were performed. In adult tissues, c-rel transcripts were detected at highest levels in spleen, thymus, intestine and lung. Lower levels of c-rel transcripts were detected in heart, testis and kidney, and no c-rel mRNA was detected in liver (Fig. 1A). These results prompted us to analyze c-rel expression in the two major cellular subpopulations that reside in the spleen. When T- or B-enriched splenic cell preparations were analyzed for c-rel expression, the highest levels of c-rel mRNA were detected in the B-cell-enriched preparation (Fig. 1B). Since the bone marrow is the primary hematopoietic organ after birth and the source of B cell precursors in mammals, the detection of low levels of c-rel transcripts in bone marrow compared to splenic B cells would indicate that c-rel expression increases in later stages of lymphocyte maturation. PCR analysis of mouse embryos from E8.5 to E17.5, revealed the presence of c-rel transcripts during later development, but at much lower levels than those observed in adult tissues (not shown). c-rel expression was first observed with RNA preparations isolated from E11.5 embryos and increased thereafter until birth. These results indicate that c-rel transcripts are primarily distributed in tissues of hematopoietic origin in the adult animal and that c-rel is expressed during embryonic development in a temporally regulated manner.

Fig. 2. c-rel expression during mouse fetal liver development. (A,B) Sagittal sections obtained from a 13.5 and (C,D) coronal sections from a 14.5 day embryo were hybridized with specific antisense riboprobes for c-rel and photographed under (A,C) brightfield and (B,D) dark-field illumination. (E) A partial view of a coronal section through a 14.5 fetal liver stained with anti-c-Rel antibody. Li, liver; Th, thymus. Arrows indicate clusters of c-Relbearing cells. Arrowheads indicate isolated megakaryocytes. (*) indicates peripheral erythrocytes. Microphotographs were taken at 9× (A, B), 16× (C, D) and 50× (E) magnification.

Fetal expression of c-rel is restricted to hematopoietic tissues and correlates with the process of hematopoietic diversification In agreement with our PCR results, c-rel expression was not detected during early embryonic development. No c-rel

specific labelling was observed in the embryo itself nor in embryonic derived tissues when sections of a 7.5 and 8.5 days p. c. embryos embedded in the decidua were hybridized with a c-rel anti-sense probe (data not shown). However, as soon as

2994 D. Carrasco, F. Weih and R. Bravo the hematopoietic activity appears in the fetal liver, c-rel (Fig. 1A and data not shown). These results indicate that c-rel expression could be found in this organ. As shown in Fig. 2A expression in liver is restricted to a subpopulation of the and B, c-rel expression was detected as a weak signal heterohematopoietic precursors that colonize this organ during fetal geneously distributed throughout the liver in a E13.5 embryo. life, particularly of erythroid characteristics. After day 13.5, the levels of c-rel mRNA in liver increased c-rel expression in embryonic and adult thymus is rapidly reaching a maximun around E14.5-E15.5 (Fig. 2C,D restricted to medullary thymic epithelial cells and data not shown), coinciding with the time of maximal hematopoietic activity of this organ. Fig. 2C,D also illustrates After E12.5, the thymus undergoes a series of rapid changes, that other surrounding embryonic structures such as the neural becoming later the main source of T cell differentiation. c-rel tube, vertebrae, heart, muscle and bones were devoid of c-rel mRNA was first detected, in the embryonic thymus, around expression. Additional analysis of c-rel expression by immunohistochemistry in an E14.5 embryo showed that the cells expressing c-Rel constitute a subpopulation of the total cells in the liver (Fig. 2E). This subset of c-rel expressing cells (arrows) was distributed in numerous islands of cells filling the sinusoid of the liver. Circulating mature erythrocytes did not display c-rel expression (Fig. 2E *). Megakaryocytes, which are scattered throughout the fetal liver and easily identified as giant cells containing a polylobulated nucleus and large cytoplasm, were not labeled by anti-c-Rel antibodies (arrowheads). To identify further the cells expressing c-Rel in fetal liver, cytospin preparations were analyzed by double immunofluorescence using a variety of hematopoietic cell markers (Fig. 5A). A large percentage of the cells bearing cRel protein were identified as the different forms of erythroid precursors recognized by the presence of a nucleus and cytoplasmic hemoglobin (Fig. 5Aa, b). Although the fetal liver produces predominantly cells of the erythroid lineage, it also generates some myeloid and lymphoid cells. In addition to the erythroid precursor, B-cell precursors as recognized by the CD45R marker were also positive for c-Rel (not shown); however, no significant expression of c-Rel was detected in other cell lineages such us macrophages (Fig. 5Ac, d) and neutrophils (Fig. 5Ae, f). The identity of an additional cell type, which displayed c-Rel expression but was not recognized by the markers used in this study, remains to be determined. After day 16.5 of embryonic development, c-rel expression in the liver appeared to be down-regulated and was almost undetectable in newborn mice (not shown), paralleling the tran- Fig. 3. c-rel expression during mouse thymic development. Sections obtained from (A,B) a 13.5 sition of the liver from a hematopoietic and (C,D) 17.5 day embryo, (E,F) newborn mouse and (G,H) 6-day-old mouse thymus were to a nonhematopoietic organ. In situ hybridized with specific antisense riboprobes for c-rel and photographed under (A,C,E,G) hybridization and PCR analysis bright-field and (B,D,F,H) dark-field illumination. In A-F, only the thymic region from the performed in adult liver failed to reveal original section is shown. Co, cortex; Me, medulla; Th, thymus. Microphotographs were taken any significant levels of c-rel mRNA at 40× (A,B), 31× (C,D), 26× (E,F) and 12× (G,H) magnification.

c-rel expression in lymphoid tissues 2995

α-c-Rel

Control

Fig. 4. Immunohistochemical localization of c-Rel protein in the thymic medulla. Immunoperoxidase staining of thymus sections from a 6week-old mouse incubated with affinity purified anti-c-Rel antibody (α-c-Rel) or with preimmune rabbit IgG (Control). Co, cortex; Me, medulla. Microphotographs were taken at 25× (A,B), 100× (C,D) and 250× (E,F) magnification.

E13.5 as an homogeneous signal distributed throughout the organ (Figs 2A,B; 3A,B). Around day 17.5 of fetal development the thymus shows cortical and medullar differentiation. Changes in the c-rel expression pattern can be correlated with this histological differentiation of the thymus (Fig. 3C-H). As shown in Fig. 3C,D, by day 17.5 of fetal development, c-rel expression was strongest in the medulla with a much weaker signal in the cortex of the thymus. This differential pattern of expression continued into the postnatal period; the total levels of c-rel transcripts in newborn animals were comparable to those observed in the thymus of a E17.5 day embryo but the differences in the level of expression between medulla and cortex become more evident (compare Fig. 3C,D with E,F). c-rel expression rapidly increased postnatally reaching

maximum levels around 1 week after birth and remaining constant throughout adulthood (Fig. 3G,H and data not shown). To confirm further that the expression of c-rel is mainly in the medulla and not in the cortex of the thymus, immunohistochemical staining was performed using immunoaffinitypurified rabbit anti-c-Rel antibodies. As shown in Fig. 4A-D, c-Rel staining was mainly confined to the medullary region. Close examination of the thymic medulla demonstrated that the c-Rel-bearing cells are large, present a lighter nuclear hematoxylin staining and constitute less than 40% of the total cells found in this region (Fig. 4E-F). To identify the cells expressing c-rel in the thymus, cytospin preparations of single cell suspensions were analyzed by double immunofluorescence. Consistent with the results

2996 D. Carrasco, F. Weih and R. Bravo

A

B

Fig. 5. Double immunofluoresence analysis of c-Rel expression in fetal liver and thymus. (A) Fetal liver cell suspensions prepared according to standard procedures, were spun onto slides and incubated with a mixture of anti-hemoglobin and anti-c-Rel (a,b) or anti-Mac-1 and anti-c-Rel antibodies (c,d) or anti-Neutrophils and anti-c-Rel antibodies (e,f). The antibodies were visualized as described in Material and Methods. (b,d,f) c-Rel; (a) hemoglobin; (c) Mac-1; (e) neutrophils. (B) Thymic cell suspensions were spun onto slides and incubated with a mixture of (a,b) anti-CD4 and antic-Rel antibodies or (c,d) anti-CD8 and anti-c-Rel antibodies or (e,f) anti-CD45R and anti-c-Rel antibodies or (g,h) anti-thymic medullary epithelial (TME) cell and anti-c-Rel antibodies. The antibodies were visualized as described in Material and Methods. (b,d,f,h) c-Rel; (a) CD4; (c) CD8; (e) CD45R; (g) TME. Microphotographs were taken at 250× magnification.

obtained by in situ hybridization and immunohistochemistry, c-Rel expression was found to be differentially distributed among the thymic cells (Fig. 5B). The cells displaying c-Rel staining represented a fraction of the total thymic population and most of them were large oval to round shaped, and characterized by the absence of cellular processes (Fig. 5Bb, d, f and h). Some cells presented a strong and homogeneous cytoplasmic staining with a weaker and patchy nuclear staining (Fig. 5Bb, d and h), while others showed an homogeneous labeling of both the cytoplasm and the nucleus (Fig. 5Bf). The analysis of several cytospin preparations demonstrated no or very low expression of c-Rel in thymocytes which stained positive for the CD4 (Fig. 5Ba, b) or CD8 (Fig. 5Bc, d) T cell markers. In addition, immunofluorescence with M342, a monoclonal antibody specific for dendritic cells (Agger et al., 1992), did not stain c-Rel-bearing cells (not shown). However, high levels of c-Rel protein were detected in B cells (Fig. 5Be, f) and in medullary epithelial cells (Fig. 5Bg, h). Because of the few B cells that colonize the thymic medulla and the intensity of the signal observed by in situ hybridization, our results indicate that the signal is originated primarily by the medullary epithelial cells.

c-rel expression in spleen correlates with the appearence of lymphoid cells The spleen displays a complex pattern of hematopoietic activity in which myelopoiesis occurs during late embryogenesis, whereas lymphopoiesis takes place after birth (Medvinsky, 1993). In situ hybridization showed that c-rel transcripts were almost undetectable in the spleen of newborn animals, indicating that c-rel is not expressed in myeloid cells (Fig. 6A,B). Upon the onset of splenic lymphopoiesis, c-rel mRNA could be detected in specific areas of this tissue. Significant c-rel expression was first detected in the emerging white pulp around the central and follicular arteries in the spleen of 2-day- (not shown) and 4-day-old animals (Fig. 6C,D). In older animals, c-rel transcripts increased in the lymphatic follicles (Fig. 6E,F; 1-week old) and was localized preferentially in the outer region of these structures (Fig. 6G,H; 6-week old). Detailed analysis of several spleen sections from 4- to 6-week-old mice showed that the highest levels of c-rel mRNA were confined to the B cell areas, the outer region of the periarterial lymphatic sheath and the marginal zone (Figs 6G,H, 8A,B). c-rel transcripts were also detected in the germinal center, a structure formed by the accumulation of

c-rel expression in lymphoid tissues 2997 actively dividing B cell blasts, which were present in some lymphatic follicles (Fig. 8A,B). Much lower levels of c-rel transcripts were detected in the T cell area, the inner region of the periarterial lymphatic sheath. In contrast, c-rel transcripts were not detected in the red pulp that derives its name by the high content in mature erythrocytes. To study whether B cells express high levels of the c-rel proto-oncogene, we analyzed spleen sections of lipopolysaccharide (LPS)-treated animals by in situ hybridization. The treatment of mice with this polyclonal specific B cell activator has been associated with the development of plasma blasts in the white pulp and memory B cells in the marginal zone of the spleen (Zhang et al., 1988). As shown in Fig. 6I,J, LPStreated animals showed a strong increase in number and size of lymphatic follicles in the spleen, and an increase in the intensity of the c-rel signal. The hybridization signal was mainly detected in the outer region of the periarterial sheath and in the marginal zone. These results indicate that c-rel gene expression is induced in splenic B cells after polyclonal activation. The expression of c-rel was further investigated by immunohistochemistry using affinity-purified rabbit antibodies against c-Rel. As shown in Fig. 7A,B, c-Rel-positive cells were mainly located in the marginal zone, in agreement with the in situ hybridization results. Much lower levels were detected in the inner region of the periarterial sheath and red pulp. Some staining was also detected in cells in the outer region of the periarterial sheath and in germinal centers (not shown). A similar pattern of staining was observed when spleen sections were incubated with antiIgM monoclonal antibody (Fig. 7G). A few c-Rel-positive cells, resembling plasma blasts, were observed scattered throughout the red pulp (not shown). Analysis at higher magnification revealed that the c-Rel-expressing cells were excluded from the inner region of the periarterial sheath (Fig. 7C-F).

Fig. 6. In situ hybridization analysis of c-rel expression in mouse spleen. Serial spleen sections of (A,B) newborn, (C,D) 4 day, (E,F) 1 week, (G,H) 6 week and (I, J) 6 week LPS-injected animals hybridized with c-rel antisense riboprobes and photographed under (A,C,E,G,I) bright-field or (B,D,F,H,J) dark-field illumination. RP, red pulp; WP, white pulp. Microphotographs were taken at 32× (A,B), 25× (C,D), 20× (E,F,G,H) and 12.5× (I,J) magnification.

2998 D. Carrasco, F. Weih and R. Bravo For comparison, the staining of a spleen section with a CD45R monoclonal antibody that specifically recognizes B cells is shown in Fig. 7H. Anti-CD45R staining was detected in the marginal zone and in the outer region but excluded from the inner region of the periarterial sheath. A comparative analysis of the staining patterns between c-Rel and anti-IgM as well as c-Rel and anti-CD45R indicated that c-Rel is expressed primarily in B cell lineages and most probably in B cells of more mature phenotype that have already encountered an antigen. The expression of the c-rel proto-oncogene in the spleen was further analyzed by double immunofluorescence. Cytospin preparations of splenic single cell suspensions were incubated in the presence of anti-c-Rel antibodies and a variety of B and T cell markers (Fig. 9A). When splenic B cells were double labeled with anti-CD45R and anti-c-Rel antibodies, a complex pattern of c-Rel expression was observed. Most but not all the CD45R-positive cells (Fig. 9Aa, c) were positive for c-Rel staining (Fig. 9Ab, d). This finding indicates that not every splenic B cell expresses c-Rel, in agreement with the results presented in Figs 7A and 8B, where not all the cells in the B cell compartment were positives for c-rel expression. Fig. 9A also demonstrates that c-Rel protein is also present in splenic T cells (Fig. 9Ae, f). c-rel expression in lymph nodes and Peyer’s patches In contrast to the spleen, the vast majority of B and T cells in secondary lymphoid organs, such as the lymph nodes and Peyer’s patches are activated (Burger and Vitetta, 1991). In lymph nodes, B cells are located in the cortical region forming lymphatic follicles, while T cells are largely confined to the paracortical area. As shown in Fig. 8C,D, the strongest hybridization signal was detected in the lymphatic follicles but significant levels of c-rel transcripts were also observed in the paracortical areas. Considering the intensity of the signal and the cellular density observed after hematoxylin/eosin staining, this result indicates that the levels of c-rel expression are also increased in T cells of more mature phenotype. To prove further that c-rel is expressed in T cells of mature phenotype, we performed double immunofluorescence with lymph node cytospin preparations using the interleukin-2 receptor (IL2R) as a T cell marker for activated T cells. Fig. 9B demonstrates that helper (CD4; Fig. 9Ba, b), cytotoxic (CD8; Fig. 9Bc, d) and activated (IL-2R; Fig. 9Be, f) T cells expressed high levels of c-Rel protein. This data, together with the results shown in Fig. 5B, clearly demonstrate that c-Rel expression is triggered after migration of T cells from the thymus to the spleen and lymph nodes, suggesting that c-Rel plays a role in the process of homing or activation that takes place in secondary lymphoid organs. Peyer’s patches are clusters of confluent

lymphatic nodules situated in the walls of the small intestine, particularly in the ileum. In situ hybridization experiments indicated that c-rel expression was homogeneously distributed in the lymphoid component of the patches (Fig. 8E,F). No signal was associated with the overlying villi, and the underlying muscularis mucosa and submucosa. Analysis of c-Rel DNA-binding complexes in spleen extracts It has been shown that c-Rel is capable of forming homodimers

α-c-Rel

Control

α-IgM

α-CD45R

Fig. 7. Immunohistochemical localization of c-Rel in the B cell areas of the spleen. Cross sections through the spleen of a six week old mouse incubated with affinity purified anti-c-Rel antibodies (α-c-Rel) or with preimmune rabbit IgG (Control). Staining is seen in cells localized in the marginal zone, outer region of the periarterial lymphatic sheath and germinal centers. MZ, marginal zone; PS, periarterial lymphatic sheath; RP, red pulp; WP, white pulp. Sections stained with anti-IgM (G) or anti-CD45R (H) monoclonal antibodies are included for comparison. Microphotographs were taken at 25× (A,B,G,H), 100× (C,D) and 250× (E,F).

c-rel expression in lymphoid tissues 2999 as well as heterodimers with p50, p52 and p65 (Hansen et al., 1992; Rice et al., 1992; Tewari et al., 1992; Mercurio et al., 1993). We performed electrophoretic mobility shift assays (EMSA) to correlate c-Rel expression in spleen with κBbinding activity and to determine which c-Rel complexes are active in this organ. Two major complexes could be observed when spleen extracts were incubated with a labeled oligodeoxynucleotide containing the κB site from the immunoglobulin kappa light chain enhancer (Fig. 10). The upper band consisted mainly of RelB and p65 complexes, since the addition of specific anti-RelB and anti-p65 antiserum almost completely eliminated this activity (compare lanes 1 and 2). A faint complex that did not migrate as a sharp band remained but was eliminated by anti-c-Rel antiserum after the addition of anti-RelB and anti-p65 antiserum (compare lanes 2 and 3), demonstrating the presence of c-Rel in this complex. Anti-p50 antiserum not only destroyed the faster migrating band but also strongly reduced the c-Rel-specific complex (compare lanes 2 and 4). When anti-p52 antiserum was included, in addition to antisera directed against RelB, p65 and p50, the DNA-binding activity was completely destroyed. An identical result was obtained when only anti-p50 and anti-p52 antisera were added to the binding reaction (lane 6), suggesting that the c-Rel-specific complex consists of p50/c-Rel and p52/c-Rel heterodimers. Identical results were obtained with extracts prepared from splenocytes and with different κB-binding sites (data not shown). In addition, we tested the effect of deoxycholate (DOC) on Rel/NF-κB complexes in spleen extracts. DOC has been reported to dissociate IκB molecules from Rel/NF-κB heterodimers (Baeuerle and Baltimore, 1988), resulting in increased DNA-binding activity in vitro. As shown in Fig. 10 (lanes 7-12), DOC treatment of spleen extracts resulted in a strong increase of the upper complex, whereas p50 homodimers were slightly affected. The majority of the induced, slower migrating κB-binding activity (lane 7) consisted of p50/p65 heterodimers (not shown). However, c-Rel-specific complexes, consisting of p50/c-Rel and p52/c-Rel heterodimers (compare lane 8 with 10-12), were also significantly increased. No other c-Rel-containing complexes could be observed. A very similar result was obtained after DOC treatment of a cytoplasmic fraction prepared from splenocytes (not shown). This result suggests that a significant part of the c-Rel κB-binding activity in splenocytes is inhibited by IκB or an IκB-like activity. Analysis of c-rel expression in apoptotic cells and B cell lineages To correlate c-rel expression with some physiological processes in lymphoid cells, we analyzed its expression in splenic apoptotic cells and its sequential expression during B cell differentiation. Fig. 11A shows two representative examples from the analysis of several splenic cytospin preparations stained with anti-c-Rel antibodies and anti-digoxigenin-labeled DNA (corresponding Hoechst stainings are also shown). Nuclear chromatin fragmentation is the hallmark of cells undergoing programmed cell death (Krammer et al., 1994) and has been used as a

biochemical marker in our studies. In general, a large percentage of splenic cells were stained with anti-c-Rel antibody (Fig. 11Aa, d) and only a very small percentage (less than 1-2%) of the total cells showed anti-digoxigenin nuclear staining (Fig. 11Ab, c). Of the anti-digoxigenin-labeled splenocytes, some were negative for c-Rel expression (Fig. 11Aa, b) while others presented some degree of anti-c-Rel staining (Fig. 11Ad, e). These results indicate that c-Rel expression does not correlate with apoptosis in splenocytes. The production of immunoglobulin is one of the earliest markers for cells of B lineages, and is considered their most reliable and unambiguous identifying feature (Rolink and Melchers, 1991). Using anti-IgM monoclonal antibodies, we have analyzed c-Rel expression in the three general stages of differentiation representing pre-B, B and plasma cells. As shown in Fig. 11Ba, b, bone marrow-derived pre-B cells expressed low levels of c-Rel protein and expression was restricted to the cytoplasm. Splenic B cells recognized by surface anti-IgM expressed high levels of c-Rel protein in both, cytoplasm and nucleus (Fig. 11Bc, d; see also Fig. 9Aa-d). Compared with most of the splenic B cells, the majority of the plasma cells expressed low levels of c-Rel protein. In a minor population of plasma cells, c-Rel was detected at significant levels and the protein was primarily localized in the nucleus

Fig. 8. In situ hybridization analysis of the c-rel gene expression in secondary lymphoid tissues. Frozen sections from the (A,B) spleen, (C,D) lymph nodes and (E,F) ileum of a 6-week-old mouse were hybridized with c-rel antisense riboprobes and photographed under (A,C,E) bright-field and (B,D,F) dark-field illumination. GC, germinal center; LF, lymphatic follicle; LM, lymphatic mantle; MZ, marginal zone; PA, paracortical area; PS, periarterial lymphatic sheath; Se, serose; Vi, villi. Microphotographs were taken at 60× (A,B), 38× (C,D) and 25× (E,F) magnification.

3000 D. Carrasco, F. Weih and R. Bravo (Fig. 11Be, f). Our observations indicate that c-Rel is expressed during all stages of B cell differentiation and maturation. Furthermore, increased levels of c-Rel expression, as observed during the transition from pre-B to plasma cell, correlate with its activation and translocation to the nucleus. DISCUSSION c-rel expression during hematopoiesis Previous studies have indicated that the c-rel proto-oncogene is expressed in a variety of hematopoietic cell lines (Brownell et al., 1987, 1988; Grumont and Gerondakis, 1989; Rice and Ernst, 1993). In this report, we have utilized in situ hybridization and immunocytochemical techniques to determine the pattern of c-rel gene expression during mouse embryogenesis and the postnatal period, as well as in certain adult tissues and isolated (or purified) cell types. These techniques allowed the identification of the cells expressing c-rel in the microenvironment in which the development of the hematopoietic system is taking place. c-rel transcripts have been identified in most of the hematopoietic A related tissues, including fetal but not adult liver, embryonic and adult thymus, spleen, blood, lymph nodes, Peyer’s patches and bone marrow. In all cases, c-rel expression matched the timing of hematopoietic activity in these organs. c-rel expression during erythroid development During embryonic life, the earliest place of c-rel expression was detected in the fetal liver. Within this tissue, c-rel expression is heterogeneously distributed, with high expression in the mesoderm-derived bloodforming islands, but not in megakaryocytes, granulocytes and endodermal prehepatocytes. c-Rel protein was found in several erythropoietic precursors but not in nonnucleated circulating erythrocytes. The yolk sac is the original site of erythropoiesis in the developing embryo (reviewed in Medvinsky, 1993). At day 10 of fetal development, the yolk sac is an active hematopoietic center. It is devoted strictly to erythropoiesis and produces a relatively large nucleated erythrocyte carrying the

embryonic complement of hemoglobin. By day 12.5, however, the major site of hematopoiesis shifts from the yolk sac to the liver, where adult-type erythrocytes, which are slighly smaller and lacking a nucleus are produced in the peripheral blood. Fetal liver erythropoiesis and yolk sac erythropoiesis are distinct in several ways (Tavassoli and Yoffey, 1983). Our observation that c-rel is not expressed in the blood islands of the yolk sac but is detected in the fetal liver erythrocyte precursors, suggests a potential role in these functional differences. In addition, our observation raises the possibility that the c-rel proto-oncogene may not be needed for the earliest stages of hematopoietic embryonic development. However, it is not clear whether the yolk sac-derived cells are the progenitors of these hematopoietic cells that colonize the fetal liver. It has been recently demonstrated that the splachnopleura and the aorta, gonads and mesonephros (AGM) region may be an alternative embryonic source of early hematopoietic precursors in mouse (Godin et al., 1993; Medvinsky et al., 1993), structures not analyzed in our study. Erythropoiesis has provided a long established model for

B

Fig. 9. c-Rel expression in peripheral B and T cells. (A) Splenic cell suspensions prepared according to standard procedures were spun onto slides and incubated with a mixture of (a-d) anti-CD45R and anti-cRel or (e,f) anti-CD4 and anti-c-Rel antibodies. The antibodies were visualized as described in Material and Methods. (b,d,f) c-Rel; (a,c) CD45R; (e) CD4. (B) Lymph node single cell suspensions were spun onto slides and incubated with a mixture of (a,b) anti-CD4 and anti-c-Rel antibodies or (c,d) CD8 and anti-c-Rel antibodies or (e,f) anti-IL-2R and anti-c-Rel antibodies. The antibodies were visualized as described in Material and Methods. (b,d,f) c-Rel; (a) CD4; (c) CD8; (e) IL-2R. Microphotographs were taken at 250× magnification.

c-rel expression in lymphoid tissues 3001 studying the developmental regulation of gene expression and the identification of transcriptional regulators such as GATA1, c-myb and c-fos among others (Orkin, 1990; Ellis and Grosveld, 1993). Until now, the expression of Rel/NF-κB proteins has not been described during erythroid development. Our observation constitutes the first indication of Rel/NF-κB activity in erythropoiesis.

precursors in fetal liver as early as E14.5 by using a anti-CD45R monoclonal antibody as a marker. However, the highest levels of c-rel expression in B cell lineages were detected after birth in the spleen following lymphocyte infiltration. At this stage, crel expression was mainly found in the B cell compartments, including the outer region of the periarterial sheath in the white pulp and the marginal zone. This expression pattern prevailed into adulthood. Further analysis of c-rel expression in splenocytes by double immunofluorescence confirmed its presence in B cell lineages. The presence of c-rel mRNA in the lymph node follicles, as well as in the Peyer’s patches, corroborated the tight correspondence between this proto-oncogene and the development of B cell lineages. Analysis of the lymphatic follicles revealed heterogeneity in the level of expression of c-rel during B cell development. For example, germinal centers that contain B cells in active proliferation, and the marginal zone that is enriched in plasma and memory B cells (Liu et al., 1988; Zhang et al., 1988) presented the highest levels of c-rel expression. In contrast, in the lymphatic mantle that contains mainly resting B cells, low levels of c-rel expression were detected. This heterogeneity in the level of c-rel expression was also observed in isolated splenocytes and in B cell lineages at various stages of differentiation. These in vivo observations indicate that c-rel is expressed throughout the process of B cell development, especially in the pool of mature, antigen-sensitive B cells. These cells are characterized by their ability to respond to T cell-independent (LPS) and T cell-dependent antigens, which occurs in the germinal centers of secondary lymphoid organs (Szakal et al., 1989; Kroese et al., 1990). These data confirm and extend previous studies performed in established B cell lines showing c-rel expression in more mature B cells (Grumont and Gerondakis, 1989; Rice and Ernst, 1993). Whether c-Rel plays a func-

c-rel expression during T cell development As hematopoiesis is established in the thymus, c-rel gene expression is concomitantly turned on in this tissue. Contrary to what was suspected, c-rel expression was undetectable in either immature double positive (CD4+CD8+) cortical thymocytes or in single positive (CD4+CD8− or CD4−CD8+) medullary thymocytes, indicating that the c-rel proto-oncogene is not required for the early stages of T cell development. This result is in contrast to the findings of Brownell et al. (1987) in which they concluded that medullary T cells were the major source of c-rel expression. Brownell et al. (1987) performed northern blot analysis with thymocyte preparations enriched on the basis of the differential susceptibility of cortical and medullary thymocytes to agglutination by peanut lectin, PNA. The fraction of medullary (PNA−) thymocytes may have been contaminated with the medullary cells detected in our study. Most medullary thymocytes display markers typically observed in functionally mature T cells and are therefore believed to represent a relatively mature thymic population from which peripheral T cells are derived (Spits, 1994). The fact that we detected c-rel expression in splenic and lymph node-derived T cells is in aggrement with previous observations (Brownell et al., 1987; Grumont and Gerondakis, 1989) and indicates that c-rel may have a role in mature T cells, particularly during T cell activation that takes place in secondary lymphoid organs. Our observation that c-rel is expressed in medullary epithelial cells is particularly interesting since it suggests an indirect role of c-Rel in early T cell development. c-Rel might be necessary in the expression of target genes required for the functional interaction between T cells and medullary epithelial cells. Several cytokines produced by cells comprising the thymic epithelium are thought to be important in the homing of T cell progenitors to the thymus (Champion et al., 1986) and in supporting some stages of T cell maturation (Okazaki et al., 1989; Sakata et al., 1989). Medullary epithelial cells express MHC class II complex and a ligand for CTLA4 important for T cell/epithelial cell interaction (Mizuochi et al., 1992; Nelson et al., 1993). In addition, medullary epithelial cells can process and present antigen to phenotypically mature T cells within the medullary environment (Mizouchi et al., 1992; Farr et al., 1990). Fig. 10. Analysis of c-Rel DNA-binding complexes. Spleen extracts were incubated with the c-rel expression during B cell development We have detected c-rel expression in B cell

murine Ig κ gene κB-binding site in the absence (lanes 1-6) or in the presence (lanes 7-12) of 0.6% DOC. The addition of specific antisera directed against different members of the Rel/NF-κB family is indicated at the top. p.i., preimmune serum. The arrow indicates heterodimeric Rel/NF-κB complexes whereas the arrowhead indicates p50 homodimers.

3002 D. Carrasco, F. Weih and R. Bravo tional role during the early stages of B cell development remains to be determined. Its cytoplasmic localization in pre-B cells indicates that it may be in an inactive form. Our findings also suggest that the genetic program of the developing thymocyte depends on the hematopoietic microenvironment provided by the primary and secondary lymphoid organs in which this development takes place. c-rel expression does not correlate with apoptosis in lymphoid cells Apoptosis plays an important role in development and maturation of both normal T and B lymphocytes and in lymphoid malignancies (Schwartz and Osborne, 1993; Krammer et al., 1994). In the normal lymphoid system, apoptosis occurs in primary lymphoid organs such as the bone marrow, liver and thymus and is used to eliminate useless precursor cells with non-rearranged or aberrantly rearranged, non-functional receptors. The cortex of the thymus is mainly composed of double positive (CD4+CD8+) cells which are being negatively selected (von Boehmer, 1992). Approximately 90 to 95% of these thymocytes are eliminated by apoptosis. Although a recent report correlates c-rel expression in several non- A hematopoietic tissues with programmed cell death (Abbadie et al., 1993), we could not detect c-rel expression in the cortical zone of the thymus. In peripheral lymphoid organs, such as lymph nodes and spleen, a similar apoptotic deletion mechanism is operative in T and B cells. Using splenic cytospin preparations, we did not find a correlation between c-Rel expression and programmed cell death. Our observation, together with B Abbadie et al. results (1993), indicate that c-Rel might be involved in different cellular programs depending on the cell type. c-Rel DNA-binding activity in spleen extracts We performed EMSA with spleen extracts to correlate crel expression with κBbinding activity. We have shown that c-Rel specific complexes consisted of p50/c-Rel and p52/c-Rel heterodimers. Neither c-Rel homodimers nor p65/c-Rel heterodimers could be detected in our analysis since anti-p50 together with antip52 antisera completely

abolished c-Rel DNA-binding activity. In situ hybridization studies demonstrated that in spleen, both nfkb1 (coding for p105/p50) and nfkb2 (coding for p100/p52) were coexpressed with c-rel (Weih et al., 1994). The major κB-binding activity in spleen extracts, however, consists of p50/RelB, p52/RelB and p50/p65 heterodimers (see Fig. 10, lanes 1, 2 and 7, 8; F. W., unpublished; Lernbecher et al., 1993). Our immmunofluorescence studies, performed with splenocytes, have demonstrated c-Rel staining in both the nucleus and the cytoplasm of most of the c-Rel positive cells. The increase in c-Rel-specific κB-binding activity detected in EMSA after DOC treatment most probably reflects the release of cytoplasmic c-Rel from its inhibitor(s). Furthermore, our observations indicate that from the several potential dimeric combinations between the different Rel/NF-κB family members observed in vitro (Hansen et al., 1992; Rice et al., 1992; Tewari et al., 1992; Mercurio et al., 1993), only a subset might exist in vivo within a particular cell, allowing the differential expression of Rel/NF-κB target genes in a cell typespecific manner.

Fig. 11. (A) Apoptosis does not correlate with c-Rel expression in splenocytes. Two examples of splenic cell suspensions, prepared according to standard procedures, spun onto slides and triple stained (as described in Material and Methods) for the detection of c-Rel (a,d), nuclei in apoptotic cells (b,e). In c,d, the Hoechst nuclear staining is shown. (B) c-Rel expression during B cell development. (a,b) Bone marrow or (c-f) splenic cytospin preparations were labeled with a mixture of anti-IgM and anti-c-Rel antibodies. The double immunofluorescence of a (a,b) pre-B cell, (c,d) B cell and (e,f) plasma cell is shown. (a,c,e) IgM and (b,d,f) c-Rel staining was performed as described in Materials and Methods. Microphotographs were taken at 250× magnification.

c-rel expression in lymphoid tissues 3003 Differential expression of Rel/NF-κB family members during lymphoid and erythroid development We have previously characterized the pattern of expression of another Rel/NF-κB family member, RelB (Ryseck et al., 1992; Carrasco et al., 1993). In contrast to the expression pattern observed for c-Rel, RelB was detected in dendritic antigen-presenting cells and in all places where dendritic cells are mainly distributed, such as the medulla of the thymus and the T cell areas of the secondary lymphoid organs, the inner region of the periarterial sheath in the spleen and the paracortical area in the lymph nodes. These differences clearly demonstrate that members of the Rel/NF-κB family of transcription factors are differentially expressed in lymphoid organs and that they play distinct functions during the process of hematopoietic diversification. We have also observed that other members of the Rel/NF-κB family are differentially expressed in lymphoid tissues. In contrast to c-rel and relB, the highest levels of nfkb1 and relA (coding for p65) mRNAs were detected in the cortex of the thymus (Weih et al., 1994), indicating a potential role for these two genes during the earlier steps of T cell development. Until now, no other member of the Rel/NF-κB family of transcription factors has been described to be involved in the development of erythroid cell lineages. The precise role of c-Rel and other Rel/NF-κB family members in regulating specific critical stages of lymphoid and erythroid development remains to be defined. In particular, it will be important to identify the target genes involved in c-Rel transcriptional activation. Geneknockout experiments currently underway in several laboratories using homologous recombination in embryonic stem cells should help to determine the importance of individual Rel/NFκB family members in the development of different hematopoietic cell lineages. The responsiveness of the Rel/NF-κB family to several signal transduction pathways makes the c-Rel protein an ideal candidate for the transcriptional regulation of target genes resulting from cell-cell and cytokine-cell communication during hematopoietic cell development. We would like to thank Rolf-Peter Ryseck for plasmid constructs and helpful suggestions, Karla Kovary for affinity-purified c-Rel antibodies and Anne Lewin for technical help. We would also like to thank Xose Bustelo, Maryann Gruda and Heather Macdonald-Bravo for valuable comments on this manuscript.

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