In vivo identification of lymphocyte subsets exhibiting transcriptionally active NF-{kappa}B/Rel complexes

Jean Feuillard1, Sylvie Mémet, Bertrand Goudeau, Alain Lilienbaum, Ruth Schmidt-Ullrich2, Martine Raphaël1 and Alain Israël

Unité de Biologie Moléculaire de l'Expression Génique, URA 1773 CNRS, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France
1 Département d'Hématologie, Hôpital Avicenne, 93000 Bobigny, France
2 Present address: Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Strasse 10, 13122 Berlin-Buch, Germany

Correspondence to: S. Mémet. Email: symemet{at}pasteur.fr


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
To analyze the NF-{kappa}B/Rel activity pattern in a living organism, we previously generated transgenic mice carrying a {kappa}B-dependent lacZ gene. In situ analysis of both primary and secondary lymphoid organs revealed a strong NF-{kappa}B transcriptional activity in antigen-presenting cells, some endothelial cells and sinus lining cells of the lymph node capsula with very little activity in lymphocytes and thymocytes. Using fluorescein-di-ß-D-galactopyranoside (FDG) as a vital substrate for the ß-galactosidase, we re-examined by flow cytometry the NF-{kappa}B/Rel transcriptional activity in our mouse model. We report here that such constitutive NF-{kappa}B/Rel activity was significantly detected in thymocytes at the CD44+CD25 stage. This constitutive activity extended with CD25 expression to the majority of the CD44CD25+ thymocytes and was then restricted to a few mature T cells. In the spleen, constitutive NF-{kappa}B/Rel activity was found in most B cells, unlike T cells which were largely negative. Virgin IgD+ B cells expressed higher levels of NF-{kappa}B transcriptional activity than other B cell types. Altogether, these results suggest that NF-{kappa}B/Rel complexes are key players in the in vivo differentiation of IgD+ B lymphocytes and possibly CD25+ thymocytes.

Keywords: lymphocyte, NF-{kappa}B, Rel, spleen, thymus, transgenic mice


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The NF-{kappa}B/Rel family of transcription factors is thought to play pleiotropic roles during the immune response. Five members have been described in mammals, i.e. p50, p52, p65, c-Rel and RelB. These proteins share an N-terminal Rel homology domain and are functionally active as homo and/or hetero-dimers, with the exception of RelB which exists only as an heterodimer with p50 or p52 (for a review, see 1).

Cellular NF-{kappa}B/Rel complexes can be subdivided into two parts. The first corresponds to constitutively nuclear complexes found mainly in accessory cells of the immune system and in B lymphocytes, as well as in neurons of certain regions of the brain (see 2 for a review and 3). The second includes the cytoplasmic NF-{kappa}B/Rel complexes containing c-Rel or p65. These complexes are trapped in the cytoplasm by interaction with members of the I{kappa}B family. A broad range of signals, such as tumor necrosis factor (TNF), IL-1 or lipopolysaccharide, induces NF-{kappa}B activation, i.e. phosphorylation of I{kappa}B molecules, followed by their degradation through the proteasome pathway, thus allowing nuclear translocation of NF-{kappa}B/Rel complexes (for a review, see 4). These nuclear complexes will in turn bind to the {kappa}B sites located in the promoter region of target genes. NF-{kappa}B/Rel complexes have been reported to enhance the transcription of a wide variety of genes of the immune response such as genes encoding cytokines (IL-2, IL-6, IL-8, TNF-{alpha}, granulocyte macrophage colony stimulating factor, etc.), proteins of the inflammatory response, cytokine receptors, adhesion molecules, MHC class I molecules, etc. (for a review, see 5).

In situ analysis of NF-{kappa}B/Rel complexes, characterization of NF-{kappa}B binding activity in different lymphocyte populations, and generation of transgenic and/or knockout mice for NF-{kappa}B/Rel or I{kappa}B brought about some major breakthroughs in the understanding of the role of NF-{kappa}B/Rel proteins in the immune system. For example, in situ analysis and generation of knockout mice for RelB revealed a major role of RelB-containing complexes in the differentiation of dendritic cells (69). Knockout mice for p52 or Bcl3 underscored the role of these proteins in germinal center formation (1013). Knockout mice for p65 suggested a role for NF-{kappa}B complexes in protection against apoptosis (14). Genetic defects for c-Rel or p50 led to altered capacities of lymphocytes to be activated following antigen stimulation (15,16). Characterization of NF-{kappa}B/Rel complexes in B cells expressing surface IgM revealed that c-Rel/p50 heterodimers constitute the major species responsible for their constitutive NF-{kappa}B binding activity (17). Generation of mice deficient for NF-{kappa}B/Rel family members has clearly demonstrated the prominent role of these proteins in the maturation and activation of lymphocytes (for a review, see 18). However, because conclusions are drawn from phenotypes resulting from a missing protein, it is possible that defects in lymphocytes may result from an indirect effect, as a consequence of the absence of a particular cell type such as dendritic cells for instance (6,9,13). Furthermore, these experiments do not really indicate in which cell types the transcriptionally active NF-{kappa}B/Rel complexes are located.

NF-{kappa}B transcriptional activity consists of a constitutive and an activated fraction. This suggests that NF-{kappa}B/Rel complexes may play a role in both a well-defined cell differentiation program in some cells of the immune system and the response to some local activation events in other cells (e.g. by protecting them against apoptosis). Therefore, in vivo identification of cells exhibiting constitutive transcriptionally active NF-{kappa}B/Rel complexes would allow us to better define the cell types in which NF-{kappa}B/Rel complexes participate in cell differentiation.

To analyze the NF-{kappa}B/Rel activity pattern during mouse development and in adults, we previously generated transgenic mice carrying a {kappa}B-dependent lacZ gene (3). Results obtained with this model showed that it is possible to identify in situ cells harboring transcriptionally active NF-{kappa}B/Rel complexes. In situ analysis of both thymus and secondary lymphoid organs, using the X-gal reagent on frozen tissue sections, showed a strong NF-{kappa}B transcriptional activity in antigen-presenting cells, some endothelial cells and sinus lining cells of the lymph node capsula. However, by this in situ approach, we failed to reveal any significant transcriptional NF-{kappa}B/Rel activity in either thymocytes or lymphocytes.

Fluorescein-di-ß-D-galactopyranoside (FDG) was first used as a vital substrate of the ß-galactosidase enzyme in eukaryotic cells in order to characterize the NF-AT and NF-{kappa}B/Rel transcriptional activity at the single-cell level by flow cytometry (19). This method has been claimed to detect as few as five ß-galactosidase molecules per viable cell (20). Moreover, in conjunction with immunolabeling, FDG has been reported to be a valuable ß-galactosidase substrate to analyze simultaneously by flow cytometry the ß-galactosidase activity and surface antigen expression in viable hematopoietic cells isolated from lacZ transgenic mice (21). Therefore, we decided to reinvestigate the NF-{kappa}B/Rel transcriptional activity in our Ig{kappa}lacZ mouse model using FDG.

In this report, we present the characterization by flow cytometry of subsets of lymphocytes and thymocytes containing a constitutive NF-{kappa}B/Rel transcriptional activity, using double immunolabeling of cells plus fluorescent staining of the ß-galactosidase activity with the FDG vital substrate. Analysis of thymocyte subpopulations revealed a significant constitutive NF-{kappa}B/Rel activity in cells at the CD44+CD25 stage. This activity was detected in the majority of the T cells at a following differentiation stage, CD44CD25+, and then restricted to a few mature T cells. In the spleen, NF-{kappa}B transcriptional activity was found in most B cells, whereas most T cells were negative. Our results also showed that virgin IgD+ B cells had higher levels of NF-{kappa}B transcriptional activity than other B cell types. Altogether, these results suggest that NF-{kappa}B/Rel complexes are likely to play a significant role in the in vivo differentiation of IgD+ B lymphocytes and possibly CD25+ thymocytes.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
The transgenic mice ((Ig{kappa})3conalacZ line 197-6) used in the present study have been described previously (3). The transgene consists of the gene encoding for ß-galactosidase with a nuclear localization sequence appended to the N-terminus. Expression of the lacZ reporter gene is driven by an artificial promoter made up of three direct repeats of the NF-{kappa}B binding site from the Ig {kappa} light chain enhancer, fused upstream of the conalbumin (cona) minimal promoter. In these mice, the expression of lacZ reflects the presence of a nuclear NF-{kappa}B binding activity, whatever constitutive or induced. For this study, 2- to 3-month-old heterozygote animals from matings between a F1 hybrid C57Bl6xSJL wild-type mouse and a homozygote transgenic mouse on a mixed F1 hybrid C57Bl6xSJL background were used. Control non-transgenic mice were F1 hybrid C57Bl6xSJL individuals. Mice were kept in clean housing conditions. Histological control of spleens of the animals after sacrifice showed the absence of any visible secondary follicules, suggesting the absence of recent local antigenic immune response.

Antibodies
For flow cytometric analyses, the following mAb were used: antibodies against CD4 (clone CT-CD4), CD8 (clone CT-CD8a) and CD45R/B220 (clone RA3-6B2), conjugated to phycoerythrin (PE) were purchased from Caltag (Burlingame, CA) and used at a dilution of 1/20. Antibodies against CD3 (clone 145-2C11), CD25 (clone 3C7), CD44 (clone IM7) and CD45R/B220 (clone RA3-6B2) conjugated to CyChrome, and antibodies against CD25 (clone 3C7) and IgD (clone 217-170) conjugated to PE, were purchased from PharMingen (San Diego, CA) and used at a dilution of 1/50.

For electrophoretic mobility shift assay (EMSA), sera raised against murine p50 (no. 1263), p65 (no. 1226), p52 (no. 1267) and c-rel (no. 1051) were kind gifts of N. Rice (Frederick, MD), and serum against RelB was kindly provided by R. Bravo (Princeton, NJ).

EMSA
Nuclear extracts from whole organs were prepared and bandshift assays were performed as previously described (3), using the {kappa}B site derived from the promoter of the MHC class I H-2 Kb gene as a probe.

ß-Galactosidase flow cytometric analysis
The ß-galactosidase activity was assessed in lymphocytes of transgenic mice according to Fiering et al. (19), using the FDG vital substrate, purchased from Sigma (St Quentin Fallavier, France). Briefly, after dissection of thymus or spleen, cells were immediately resuspended in cold RPMI. Tissue debris was eliminated by sedimentation for 1 min at 1 g. After washing, lymphocytes were isolated by gradient centrifugation through Ficoll-Isopaque (Pharmacia, Uppsala, Sweden). Cells were washed and resuspended at 107 cells/ml in cold RPMI. Both cells and FDG (diluted in water at 3.1 mg/ml) were then prewarmed for 5 min at 37°C. One volume of FDG solution (usually 100 µl) was added to an identical volume of cells. The mixture was gently agitated at 37°C for exactly 90 s and the FDG loading was stopped by addition of 4 ml of cold RPMI. Cells were incubated for 2 h on ice and washed once before immediate flow cytometric analysis or additional immunolabeling of cells. Fluorescein background was determined by performing the ß-galactosidase assay on lymphocytes isolated in parallel from a non-transgenic C57Bl/6xSJL F1 hybrid mouse and the threshold was set at <3% of positive cells from the negative control mice.

Immunolabeling and flow cytometric analysis
After FDG staining of lymphocytes from both transgenic and non-transgenic mice, additional immunolabeling was performed. Five thousand cells were resuspended in 100 µl of cold PBS supplemented with 2% BSA and PE- and/or CyChrome-conjugated antibodies were added at the appropriate dilution. After 30 min incubation on ice in the dark, cells were washed twice with cold PBS and resuspended in 500 µl of PBS. Flow cytometric analysis was performed immediately on an Epics XL apparatus from Coulter (Margency, France). Lymphocytes were gated on the basis of their forward scatter and side scatter profiles, and fluorescein, PE and CyChrome emissions were measured at 525 (FL1 sensor), 575 (FL2 sensor) and 675 (FL4 sensor) nm respectively. For each combination of FDG plus antibody labeling, both background of fluorescein emission and non-specific labeling of cells with the antibodies used were evaluated on cells from non-transgenic mice labeled with both FDG and irrelevant antibodies. Furthermore, in order to correctly set the quadstat thresholds for the ß-galactosidase activity in the different lymphocyte subpopulations, each combination of FDG plus antibody labeling of lymphocytes from transgenic mice was run on the flow cytometer immediately after running the same combination of labeling from non-transgenic cells. Each experiment was repeated at least 3 times.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
lacZ expression in the spleen of Ig{kappa}–lacZ transgenic mice
To determine the NF-{kappa}B/Rel activity pattern in vivo, we have previously generated transgenic mice carrying a {kappa}Bdependent ß-galactosidase transgene (3). In these mice, expression of the ß-galactosidase reflects the presence of a nuclear {kappa}B-binding activity. This model allowed us to identify by in situ staining of the ß-galactosidase cells with a constitutive transcriptional {kappa}B activity in the thymus and in secondary lymphoid organs. However, very few thymocytes and lymphocytes exhibited a detectable transcriptional NF-{kappa}B activity. We thus decided to pursue our analysis using another, potentially more sensitive assay of the ß-galactosidase, the flow cytometry assay combined with the use of a vital fluorogenic substrate, FDG, to quantitate the ß-galactosidase activity in individual viable cells (22).

In a first step, we analyzed the ß-galactosidase activity in freshly isolated lymphocytes from the spleen of Ig{kappa}lacZ transgenic mice. CD4 and CD8 T lymphocytes ranged from 20 to 50% of total gated events according in four experiments. Very few T lymphocytes were found to be significantly stained with FDG compared to the negative control (Fig. 1A and BGo). No correlation was found with CD25 expression (data not shown). By contrast, a majority of B lymphocytes were strongly stained with FDG (Fig. 1CGo). Among them, most IgD+ B lymphocytes were FDG+ (Fig. 1DGo). In order to analyze the ß-galactosidase activity in B cells, we performed triple staining of cells with CyChrome-conjugated anti-B220 mAb plus PE-conjugated anti-IgD mAb together with FDG staining. This allowed us to specifically discriminate the ß-galactosidase activity from both naive IgD+ B cells and antigen-primed IgD B lymphocytes. Results shown in Fig. 2Go indicate that IgD+ B lymphocytes have higher levels of ß-galactosidase than IgD B cells.



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Fig. 1. ß-Galactosidase activity of splenic lymphocytes from Ig{kappa}lacZ transgenic mice. CD4 (A), CD8 (B), B220 (C) and IgD (D) immunolabeling against FDG staining. Percentages are indicated in each quadrant of the different dot-plots.

 


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Fig. 2. ß-Galactosidase activity of IgD+ versus IgD B lymphocytes isolated from the spleen of Ig{kappa}lacZ transgenic mice. Triple staining with CyChrome-conjugated mAb against the B220 marker, PE-conjugated mAb against IgD and FDG allowed us to define specific gates on the B220 versus IgD dot-plot (A) corresponding to B220+IgD cells (quadrant 1 of the quadstat thresholds), B220+IgD+ cells (quadrant 2 of the quadstat thresholds) and B220IgD cells (quadrant 3 of the quadstat thresholds). Analysis of FDG staining of these differently gated events is presented on (B)–(D). Percentages of FDG+ events are indicated in each graph. Each single histogram contains at least 2000 events. This figure is representative of three independent experiments.

 
lacZ expression in the spleen of Ig{kappa}–lacZ transgenic mice correlates with the presence of a nuclear NF-kB binding activity
We checked that the ß-galactosidase expression detected by FACS analysis in the spleen of our transgenic mice reflected the presence of a constitutive nuclear NF-{kappa}B binding activity. Splenic nuclear extracts were incubated with the {kappa}B site from the MHC class I gene H-2 Kb promoter and submitted to EMSA. This probe binds the same complexes in vitro as the {kappa}B site from the Ig {kappa} light chain enhancer. Figure 3Go(A and B, lane 1) shows that two complexes, I and II, could be detected. Antisera directed against each of the five known mammalian members of the NF-{kappa}B/Rel family were used to identify the components of these nuclear complexes. Serum against p50 (Fig. 3A and BGo, lane 2) considerably reduced the intensity of complex I, inhibited the formation of complex II and led to the formation of an intense supershifted complex. Serum against p52 (Fig. 3AGo, lane 3) had no effect. Alone, the anti-p65 serum (Fig. 3AGo, lane 4) or the anti-c-rel serum (Fig. 3AGo, lane 6 and B, lane 4) very slightly decreased the intensity of complex I, this diminution being more visible when both antisera were used (Fig. 3AGo, lane 7). Alone or together with anti-c-rel, anti-RelB serum strongly reduced the intensity of complex I (Fig. 3A and BGo, lanes 5, and A, lane 9). Further addition of anti-p50 serum (Fig. 3BGo, lane 6) totally abolished the faint remaining complex. A combination of serum directed against p65 (Fig. 3AGo, lane 8) or p65 and c-rel (Fig. 3AGo, lane 10) together with anti-RelB serum also inhibited the formation of complex I without significantly affecting formation of complex II. From these results, we conclude that complex I mainly consists of the heterodimeric species p50/RelB with a low contribution of p50/p65 and c-rel/c-rel and possibly p50/ c-rel, p65/c-rel heterodimers. Complex II is composed of p50/p50 homodimers only. We thus demonstrate that the expression of ß-galactosidase detected in the spleen of our Ig{kappa}lacZ transgenic mice by FACS analysis corresponds to a genuine constitutive nuclear {kappa}B-binding activity, which has been attributed to particular dimers by EMSA.



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Fig. 3. NF-{kappa}B binding activity of splenic nuclear extract. Nuclear extracts prepared from spleen from F1 B6xSJL hybrid mice were incubated with a double-stranded oligonucleotide corresponding to a canonical {kappa}B site located in the promoter of the MHC class I gene H-2 Kb. The addition of specific antisera against the different members of the NF-{kappa}B/Rel family is indicated at the top. Lane 1 corresponds to extracts incubated with preimmune serum. Arrowheads indicate the different NF-{kappa}B/Rel complexes discussed in the text. ns corresponds to a non-specific band.

 
Altogether, the results obtained from the analysis of both the ß-galactosidase activity by flow cytometry and the {kappa}B-binding activity from freshly isolated splenic cells allow us to conclude that, in the spleen, B lymphocytes contain constitutive transcriptionally active NF-{kappa}B/Rel complexes, in contrast to T lymphocytes, which do not exhibit such activity. Furthermore, our data suggest that levels of {kappa}B-transcriptional activity are higher in IgD+ virgin B cells than in IgD antigen-primed B cells.

lacZ expression in the thymus of Ig{kappa}–lacZ transgenic mice
In a next step, we analyzed the ß-galactosidase activity on freshly isolated thymocytes from Ig{kappa}lacZ transgenic mice. Early steps of thymocyte cortical maturation have been correlated with CD25 and CD44 expression (for a review, see 23). They include the CD44+CD25, CD44CD25+, CD44CD25+ and CD44CD25 stages followed by the CD3low CD4/CD8 double-positive (DP) stage. To assess the NF-{kappa}B/Rel activity at these different stages of T cell development, we performed triple staining of cells with various combinations of CyChrome-conjugated and PE-conjugated mAb specific for CD3, CD4, CD8, CD25 and CD44, together with FDG staining (Fig. 4Go).



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Fig. 4. ß-Galactosidase activity of the different thymocyte subsets from Ig{kappa}lacZ transgenic mice. Triple staining with CyChrome-conjugated mAb against CD3, PE-conjugated mAb against CD25 and FDG (A) or CyChrome-conjugated mAb against CD44 and a cocktail of PE-conjugated mAb against CD4 and CD8, and FDG (B) or CyChrome-conjugated mAb against CD44 and PE-conjugated mAb against CD25 and FDG (C) were performed. These combinations allowed us to define specific gates (D, E and F boxes in A; G, H and I boxes in B; and J, K and L boxes in C) corresponding to CD3+CD25, CD3CD25, CD25+, CD4+ and/or CD8+, CD44/DN, CD44high/DN, [CD44– or low]CD25, CD44highCD25 and [CD44– or low]CD25+ cells respectively. Analysis of FDG staining of these differently gated events is presented on the corresponding single fluorescence graphs (D–L). Percentages of FDG+ events are indicated in each graph. Panels (A–C) contain at least 100 000 events and each single graph (D–L) contains at least 1500 events. This figure is representative of three independent experiments.

 
The first triple combination used consisted of CyChrome-conjugated anti-CD3 mAb (FL4 sensor) plus PE-conjugated anti-CD25 mAb (FL2 sensor) and FDG (FL1 sensor). Analysis of the ß-galactosidase activity in the different cell subsets was done by drawing three boxes corresponding to CD3+CD25, CD3CD25 and CD25+ cells on the FL4/FL2 (CD3/CD25) dot-plot graph (Fig. 4AGo). The next combination used was composed of CyChrome-conjugated mAb against CD44 plus a cocktail of PE-conjugated mAb against CD4 and CD8, and FDG. Three boxes were drawn on the CD44/(CD4–CD8) dot-plot graph corresponding to CD4+ and/or CD8+, CD44 double-negative (DN) and CD44high/DN cells (Fig. 4BGo). Finally, we performed a triple staining with CyChrome-conjugated mAb against CD44 plus PE-conjugated mAb against CD25 in combination with FDG. The CD44/CD25 dot-plot graph was used to define boxes corresponding to CD44– or lowCD25+, CD44highCD25 and CD44– or lowCD25 cells (Fig. 4CGo). Specific gating on the boxes defined on the three FL4/FL2 dot-plot graphs then allowed us to assess the ß-galactosidase activity in each of the defined cell subsets using the FL1 sensor to set up the FITC emission fluorescence graph (Fig. 4D–LGo).

CD44high/DN and CD44highCD25 cells exhibited significant ß-galactosidase activity (Fig. 4H and KGo). This suggests that the acquisition of a constitutive NF-{kappa}B activity precedes the expression of CD25 at the very early steps of thymocyte ontogenesis. However, the highest levels of ß-galactosidase activity were correlated with CD25 expression, even in CD44 or low cells (Fig. 4F and LGo). A relationship between NF-{kappa}B activity and CD25 expression seems then to exist in thymocyte precursors. Gating on CD44/DN cells showed two peaks of FDG staining, one which corresponded to negative cells and the other to cells with ß-galactosidase activity (Fig. 4HGo). The CD44/DN subpopulation includes two thymocyte subsets which are CD44CD25 and CD44CD25+. Since CD25 expression was found to be correlated with the highest levels of FDG labeling (Fig. 4FGo) and [CD44– or low]CD25+ cells are uniformly positive for FDG (Fig. 4LGo), it seems likely that the peak of negative cells in the CD44/DN subset corresponds to the CD44CD25 cells, which form the latest stage of thymocyte precursor differentiation. CD4 and/or CD8 DP and CD3+CD25 thymocytes exhibited a weak ß-galactosidase activity (Fig. 4D and 4GGo) suggesting that the vast majority of mature T cells do not express significant constitutive NF-{kappa}B/Rel activity. However, CD25 expression was found in a few CD3+ thymocytes (Fig. 4AGo) and specific gating on the CD3+CD25+ subset showed levels of FDG staining similar to the ones observed for the CD3CD25+ cells (data not shown), suggesting that CD25 expression was still correlated to NF-{kappa}B activity at the cortical and/or medullary stages of thymocyte ontogenesis.

In summary, our results suggest that a constitutive NF-{kappa}B/Rel activity is present in T cells at the very early CD44highCD25 stage, then extends at both the CD44+CD25+ and CD44CD25+ stages and is lost at the CD44CD25 stage. Later, at the CD3low or high stages, the vast majority of cortical and medullary thymocytes do not harbor any detectable NF-{kappa}B activity.

lacZ expression in the thymus of Ig{kappa}–lacZ transgenic mice correlates with the presence of a nuclear NF-{kappa}B binding activity
We checked that the ß-galactosidase expression detected by FACS analysis in the thymus of our transgenic mice reflected the presence of a constitutive nuclear NF-{kappa}B binding activity. Thymic nuclear extracts were incubated with the {kappa}B site from the MHC class I gene H-2 Kb promoter and submitted to EMSA. Figure 5Go(A and B, lanes 1) shows that two complexes, I and II, could be detected. Antisera directed against each of the five known mammalian members of the NF-{kappa}B/Rel family were used to identify the components of these nuclear complexes. Serum against p50 (Fig. 5A and BGo, lanes 2) considerably reduced the intensity of complex I, inhibited the formation of complex II and led to the formation of an intense supershifted complex. Serum against p52 (Fig. 5AGo, lane 3) alone or in combination with anti-p50 antibodies (Fig. 5BGo, lane 4) had very little effect. Alone, the anti-p65 serum (Fig. 5AGo, lane 5) significantly decreased the intensity of complex I. Serum against c-rel alone (Fig. 5AGo, lane 6 and B, lane 5) or in combination with p65 (Fig. 5AGo, lane 8) had a very slight effect on complex I. Alone, anti-RelB serum (Fig. 5AGo, lane 7 and B, lane 6) strongly reduced the intensity of complex I. The remaining upper band disappeared when anti-RelB serum was used together with anti-p65 (Fig. 5AGo, lane 9), leaving a faint but visible complex I, which was not modified by further addition of anti-p50 serum (Fig. 5BGo, lane 9). This diffuse complex I was also revealed by a combination of sera against p50 and RelB (Fig. 5BGo, lane 8), and remained unchanged after further addition of anti-p65 serum (Fig. 5BGo, lane 9). It thus can be attributable to c-rel/c-rel homodimers. Anti-p50 and anti-c-rel sera plus serum against RelB (Fig. 5BGo, lane 7) or against p65 (Fig. 5BGo, lane 10) did not totally eliminate complex I, revealing the presence of p52/RelB heterodimers. The only combination which totally inhibited the formation of complex I without significantly affecting formation of complex II was a mixture of sera directed against p65, c-rel and RelB (Fig. 5AGo, lane 10).



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Fig. 5. NF-{kappa}B binding activity of thymic nuclear extract. Nuclear extracts prepared from the thymus of F1 B6xSJL hybrid mice were incubated with a double-stranded oligonucleotide corresponding to a canonical {kappa}B site located in the promoter of the MHC class I gene H-2 Kb. The addition of specific antisera against the different members of the NF-{kappa}B/Rel family is indicated at the top. Lane 1 corresponds to extracts incubated with preimmune serum. Arrowheads indicate the different NF-{kappa}B/Rel complexes discussed in the text. `ns' corresponds to a non-specific band.

 
From these results, we conclude that complex I mainly consists of the heterodimeric species p50/RelB with a low contribution of p50/p65 and p52/RelB heterodimers as well as some c-rel/c-rel homodimers. p50/p50 homodimers form complex II. We thus demonstrate that the expression of ß-galactosidase detected in the thymus of our Ig{kappa}lacZ transgenic mice by FACS analysis reflects a genuine constitutive nuclear {kappa}B-binding activity, which has been attributed to particular NF-{kappa}B dimers by EMSA.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
{kappa}B–lacZ transgenic mice have been generated as a tool to identify accurately cells with a constitutive or inducible NF-{kappa}B/Rel transcriptional activity. Initial characterization of these transgenic mice showed a positive correlation between in situ ß-galactosidase activity and NF-{kappa}B/Rel binding activity (3). However, we failed to detect any significant in situ NF-{kappa}B/Rel transcriptional activity in most thymocytes and lymphocytes from both lymph nodes and spleen. This result was somehow surprising since a constitutive NF-{kappa}B binding activity has been reported in both thymocytes and peripheral B cells (8,24,25). One explanation could be a question of sensitivity of the in situ ß-galactosidase assay using the X-gal reagent. Therefore, in order to address the issue of the constitutive NF-{kappa}B/Rel transcriptional activity in both lymphocytes and thymocytes, we performed simultaneous detection of surface antigen expression and ß-galactosidase activity in lymphocytes and thymocytes using the FDG vital substrate which allows the detection by flow cytometry of very low levels of ß-galactosidase expression at the single-cell level (19,22). In this report, we present the phenotypic characterization of thymocytes and peripheral lymphocytes displaying a constitutive NF-{kappa}B/Rel transcriptional activity.

Our results showed that it was possible with the FDG substrate to detect a constitutive ß-galactosidase activity in lymphocytes isolated from both spleen and thymus of our Ig{kappa}lacZ transgenic mice. Control in situ experiments performed in parallel on frozen tissue sections from the same organ failed to provide evidence for any substantial ß-galactosidase activity using the X-gal reagent as a substrate (data not shown), which points out the greater sensitivity of FDG versus X-gal. The absence of detectable ß-galactosidase activity in a many cell subtypes rules out the possibility that FDG loading and reaction protocol by themselves activate NF-{kappa}B, in accordance with previous findings of Nolan and Fiering (19,22). EMSA experiments revealed that the ß-galactosidase activity detected with the FDG substrate was correlated with a constitutive NF-{kappa}B binding activity in the nuclei of cells from both spleen and thymus. These data demonstrate that the FDG substrate allowed the detection of cells which possess a genuine nuclear NF-{kappa}B/Rel transcriptional activity.

High levels of NF-{kappa}B/Rel transcriptional activity were found in lymphocytes isolated from the spleen. Phenotypic analysis of FDG+ lymphocytes revealed that this NF-{kappa}B/Rel transcriptional activity was mainly due to B cells, whereas CD4 and CD8 cells presented only a few FDG+ cells. Furthermore, triple labeling of lymphocytes with the B220 marker, IgD mAb and FDG revealed that the ß-galactosidase activity was higher in IgD+ B lymphocytes than in IgD cells; the former subset corresponds to IgD+/IgM+ naive B cells of the mantle zone whereas the latter one corresponds to antigen-primed B cells (26). A recent report indicates that NF-{kappa}B/Rel constitutive activity in B cells is a feature of lymphocytes having rearranged their Ig genes (27). Analysis of NF-{kappa}B/Rel complexes during B cell differentiation showed that c-Rel containing complexes are associated with the so-called `mature' B lymphocytes (25,27,28), which correspond to B220+IgM+ non-antigen-primed (i.e. virgin) B lymphocytes. B220+IgM+ B cells from both c-Rel- and p65-deficient mice failed to proliferate in response to IgM and B220+IgM+ B cells failed to differentiate into switched IgA or IgG1 B-secreting lymphocytes in p65-deficient mice (15,29). B lymphocytes from mice deficient for the transactivation domain of c-Rel failed to switch in vitro to IgG1, IgG3 and IgE (30). Our results show that B lymphocytes are the only lymphocyte subset in which high levels of NF-{kappa}B/Rel transcriptional activity could be detected in the majority of cells, pointing out the role of NF-{kappa}B/Rel complexes in B cell maturation. Furthermore, we found that IgD+ B cells have significantly higher levels of ß-galactosidase activity than IgD cells, while no ß-galactosidase activity was detected in B220CD43+ cells (data not shown). This result, in conjunction with the analysis of deficient mice, suggests that NF-{kappa}B/Rel complexes are likely to be essential at the IgM+IgD+ B cell maturation stage by allowing virgin B cells to proliferate and differentiate in response to antigen.

Analysis of the NF-{kappa}B/Rel transcriptional activity in cells freshly isolated from the thymus revealed that only a minority of thymocytes were FDG+. This may be due to the fact that most of the thymocytes did not contain nuclear NF-{kappa}B/Rel complexes. It is noteworthy that a constitutive NF-{kappa}B/Rel binding activity has been reported to be a characteristic of freshly isolated thymocytes (8,24). Our bandshift assays confirm the existence of a constitutive NF-{kappa}B binding activity in thymic cells, made up of a mixture of p50/RelB, p50/p65 heterodimers as well as some c-rel/c-rel, c-rel/p65 and p52/RelB complexes. This could suggest that the NF-{kappa}B/Rel binding activity detected by EMSA in thymic nuclear extract is ascribable to only a few thymocytes with nuclear NF-{kappa}B/Rel complexes. On the other hand, the data presented in the initial publication of Fiering et al. (19), which describes the single-cell characterization of transcriptional activity of NF-AT and NF-{kappa}B/Rel complexes, suggested that the detection of ß-galactosidase activity with FDG is under the dependence of a threshold effect related to the amount of the transcriptional complex within the nucleus. Therefore, another hypothesis which could account for the low frequency of FDG+ thymocytes would be that, in the majority of thymocytes, levels of NF-{kappa}B/Rel complexes are not high enough to switch on the transcription of the {kappa}B-dependent lacZ reporter gene. One may also argue that the transgenic reporter construct which consists of three copies of the NF-{kappa}B site from the Ig{kappa} intronic enhancer placed upstream of a minimal promoter is not sensitive to all biologically relevant NF-{kappa}B complexes. We cannot exclude this possibility, but the fact that several independently generated transgenic lines containing distinct {kappa}B-responsive promoter, an artificial one (used in this study) or a natural one (p105), gave the same overall pattern of NF-{kappa}B activity in a whole organism (3), supports our present data.

A constitutive NF-{kappa}B/Rel activity has been detected in thymocytes at the very early CD44+CD25 stage. This transcriptional NF-{kappa}B/Rel activity increased to the majority of cells at the CD44CD25+ stage and was presumably lost at the CD44CD25 stage. Expression of CD25 and CD44 is associated with the maturation of the CD3CD4CD8 triple-negative (TN) mouse thymocytes (31). Rearrangement of both TCR ß and {gamma} loci, followed by the production of TCR ß transcripts, parallels the transition from the CD44+CD25+ TN to the CD44CD25+ TN stage (31). This latter stage, where the majority of our transgenic cells exhibit a transcriptional NF-{kappa}B/Rel activity, constitutes a control point of thymocyte development at which cells expressing a productively rearranged TCR ß chain are selected for further differentiation (32). In mice overexpressing a trans-dominant form of I{kappa}B{alpha} under the control of a proximal lck promoter (33,34); and in I{kappa}B{varepsilon}-nullizygous mice (35), a 50% reduction of this CD25+ subspecies was observed together with abnormal composition of thymocyte subsets, impaired proliferation and increased sensitivity to apoptosis after mitogenic stimulation in the former case. Moreover, fetal thymocytes infected with adenovirus expressing a mutated I{kappa}B{alpha} developed normally until the CD44CD25+ TN stage, whereas the transition to CD44CD25 TN thymocytes was impaired and the production of mature DP CD4+CD8+ and single-positive populations was strongly decreased (36). The developmental block observed in these infected fetal thymocytes, which was associated with increased apoptosis, occurs exactly at the transition from constitutive nuclear NF-{kappa}B activity to inactive cytoplasmic NF-{kappa}B/Rel complexes that we have observed in our transgenic mice. Altogether our data and results obtained with different mouse models suggest that NF-{kappa}B/Rel plays a crucial role in the differentiation and survival of thymocytes at early stages of development, and especially at the transition from the CD44CD25+ TN to the CD44CD25 TN stage. NF-{kappa}B/Rel complexes are involved in the positive regulation of CD25 gene, and a {kappa}B site has been described in its promoter region (37,38). Is the role of NF-{kappa}B on thymocyte differentiation correlated with CD25 gene expression? Further characterization of the regulatory sequences of the CD25 gene indicated that other regulatory adjacent sites are necessary for full promoter activity (39) and that cooperation with other transcription factors like SRF may be important (40). So far, CD25 gene expression has not been reported to be modified in homozygous deficient mice for any of the NF-{kappa}B/Rel family members (18) nor in I{kappa}B{alpha}- or I{kappa}B{varepsilon}-deficient mice (41,42,35). In addition, the proliferative defect of CD25+ cell subset in transgenic mice overexpressing a transdominant mutated form of I{kappa}B{alpha} is not due to an inhibition of CD25 expression (34). These data suggest that CD25 gene expression regulation in vivo does not depend on NF-{kappa}B/Rel solely and that other transcriptional factors are likely to play a role. No strict correlation could be established between CD25 expression and FDG staining, since some CD25 thymocytes (i.e. CD44+CD25) had levels of NF-{kappa}B/Rel transcriptional activity equivalent to that observed for CD25+ cells (Fig. 4Go). The correlation of NF-{kappa}B activity with CD25 expression appears coincidental and CD25 does not constitute a critical target of NF-{kappa}B at these early stages of development.

NF-{kappa}B/Rel proteins are ubiquitously distributed among most cell types of the immune system, at least at the cytoplasmic level. Results presented here and in our previous publications allow us to identify only four subsets of cells of the immune system in which the majority of cells harbor a constitutive NF-{kappa}B/Rel transcriptional activity. These are the following: CD25+ thymocytes, B220+ B lymphocytes (with a partial partition regarding the IgD+ versus IgD B lymphocytes), antigen-presenting cells and sinus lining cells from the capsula of lymph nodes. CD25 expression on thymocytes is associated with the TCR rearrangement, IgD+ B lymphocytes represent the emergent repertoire of newly produced B cells from the bone marrow, antigen-presenting cells are critical in antigen-dependent immune response and sinus lining cells are controlling the entrance of lymphocytes from lymphatic vessels to lymph nodes. These results suggest that, as constitutive nuclear complexes, NF-{kappa}B/Rel proteins have some very precise functions in a limited range of cell types of the immune system. This raises the obvious question of the in vivo identification of NF-{kappa}B/Rel target genes in the resting state or in conditions where the immune system is challenged to various stresses. In this respect, our {kappa}B-dependent mice will be of great help to unravel the cell types involved in vivo in the defence of the organism during viral or bacterial infections.


    Acknowledgments
 
We would like to thank Philippe Bissières, Martine Motty and Séverine Ferrand for technical assistance, as well as Clare Strong for critical reading of the manuscript. We also thank N. Rice and R. Bravo for providing us with sera. This work is supported by the grant 96000990 from the Fondation de France. A. I. is supported by grants from INSERM, ARC, Ligue National Franciaise contre le Cancer, ANRS and EEC.


    Abbreviations
 
DN double negative
DP double positive
EMSA electrophoretic mobility shift assay
FDG fluorescein-di-ß-D-galactopyranoside
PE phycoerythrin
TN triple negative
TNF tumor necrosis factor

    Notes
 
The first two authors contributed equally to this work

Transmitting editor: A. Fischer

Received 22 April 1999, accepted 13 January 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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