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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: lymphocyte, NF-B, Rel, spleen, thymus, transgenic mice
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cellular NF-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-
B/Rel complexes containing c-Rel or p65. These complexes are trapped in the cytoplasm by interaction with members of the I
B family. A broad range of signals, such as tumor necrosis factor (TNF), IL-1 or lipopolysaccharide, induces NF-
B activation, i.e. phosphorylation of I
B molecules, followed by their degradation through the proteasome pathway, thus allowing nuclear translocation of NF-
B/Rel complexes (for a review, see 4). These nuclear complexes will in turn bind to the
B sites located in the promoter region of target genes. NF-
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-
, 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-B/Rel complexes, characterization of NF-
B binding activity in different lymphocyte populations, and generation of transgenic and/or knockout mice for NF-
B/Rel or I
B brought about some major breakthroughs in the understanding of the role of NF-
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-
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-
B/Rel complexes in B cells expressing surface IgM revealed that c-Rel/p50 heterodimers constitute the major species responsible for their constitutive NF-
B binding activity (17). Generation of mice deficient for NF-
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-
B/Rel complexes are located.
NF-B transcriptional activity consists of a constitutive and an activated fraction. This suggests that NF-
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-
B/Rel complexes would allow us to better define the cell types in which NF-
B/Rel complexes participate in cell differentiation.
To analyze the NF-B/Rel activity pattern during mouse development and in adults, we previously generated transgenic mice carrying a
B-dependent lacZ gene (3). Results obtained with this model showed that it is possible to identify in situ cells harboring transcriptionally active NF-
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-
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-
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-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-
B/Rel transcriptional activity in our Ig
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-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-
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-
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-
B transcriptional activity than other B cell types. Altogether, these results suggest that NF-
B/Rel complexes are likely to play a significant role in the in vivo differentiation of IgD+ B lymphocytes and possibly CD25+ thymocytes.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In a first step, we analyzed the ß-galactosidase activity in freshly isolated lymphocytes from the spleen of IglacZ 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 B
). No correlation was found with CD25 expression (data not shown). By contrast, a majority of B lymphocytes were strongly stained with FDG (Fig. 1C
). Among them, most IgD+ B lymphocytes were FDG+ (Fig. 1D
). 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. 2
indicate that IgD+ B lymphocytes have higher levels of ß-galactosidase than IgD B cells.
|
|
|
lacZ expression in the thymus of IglacZ transgenic mice
In a next step, we analyzed the ß-galactosidase activity on freshly isolated thymocytes from IglacZ 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-
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. 4
).
|
CD44high/DN and CD44highCD25 cells exhibited significant ß-galactosidase activity (Fig. 4H and K). This suggests that the acquisition of a constitutive NF-
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 L
). A relationship between NF-
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. 4H
). 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. 4F
) and [CD44 or low]CD25+ cells are uniformly positive for FDG (Fig. 4L
), 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 4G
) suggesting that the vast majority of mature T cells do not express significant constitutive NF-
B/Rel activity. However, CD25 expression was found in a few CD3+ thymocytes (Fig. 4A
) 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-
B activity at the cortical and/or medullary stages of thymocyte ontogenesis.
In summary, our results suggest that a constitutive NF-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-
B activity.
lacZ expression in the thymus of IglacZ transgenic mice correlates with the presence of a nuclear NF-
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-B binding activity. Thymic nuclear extracts were incubated with the
B site from the MHC class I gene H-2 Kb promoter and submitted to EMSA. Figure 5
(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-
B/Rel family were used to identify the components of these nuclear complexes. Serum against p50 (Fig. 5A and B
, 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. 5A
, lane 3) alone or in combination with anti-p50 antibodies (Fig. 5B
, lane 4) had very little effect. Alone, the anti-p65 serum (Fig. 5A
, lane 5) significantly decreased the intensity of complex I. Serum against c-rel alone (Fig. 5A
, lane 6 and B, lane 5) or in combination with p65 (Fig. 5A
, lane 8) had a very slight effect on complex I. Alone, anti-RelB serum (Fig. 5A
, 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. 5A
, lane 9), leaving a faint but visible complex I, which was not modified by further addition of anti-p50 serum (Fig. 5B
, lane 9). This diffuse complex I was also revealed by a combination of sera against p50 and RelB (Fig. 5B
, lane 8), and remained unchanged after further addition of anti-p65 serum (Fig. 5B
, 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. 5B
, lane 7) or against p65 (Fig. 5B
, 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. 5A
, lane 10).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 IglacZ 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-
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-
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-
B/Rel transcriptional activity.
High levels of NF-B/Rel transcriptional activity were found in lymphocytes isolated from the spleen. Phenotypic analysis of FDG+ lymphocytes revealed that this NF-
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-
B/Rel constitutive activity in B cells is a feature of lymphocytes having rearranged their Ig genes (27). Analysis of NF-
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-
B/Rel transcriptional activity could be detected in the majority of cells, pointing out the role of NF-
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-
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-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-
B/Rel complexes. It is noteworthy that a constitutive NF-
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-
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-
B/Rel binding activity detected by EMSA in thymic nuclear extract is ascribable to only a few thymocytes with nuclear NF-
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-
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-
B/Rel complexes are not high enough to switch on the transcription of the
B-dependent lacZ reporter gene. One may also argue that the transgenic reporter construct which consists of three copies of the NF-
B site from the Ig
intronic enhancer placed upstream of a minimal promoter is not sensitive to all biologically relevant NF-
B complexes. We cannot exclude this possibility, but the fact that several independently generated transgenic lines containing distinct
B-responsive promoter, an artificial one (used in this study) or a natural one (p105), gave the same overall pattern of NF-
B activity in a whole organism (3), supports our present data.
A constitutive NF-B/Rel activity has been detected in thymocytes at the very early CD44+CD25 stage. This transcriptional NF-
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
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-
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
B
under the control of a proximal lck promoter (33,34); and in I
B
-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
B
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-
B activity to inactive cytoplasmic NF-
B/Rel complexes that we have observed in our transgenic mice. Altogether our data and results obtained with different mouse models suggest that NF-
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-
B/Rel complexes are involved in the positive regulation of CD25 gene, and a
B site has been described in its promoter region (37,38). Is the role of NF-
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-
B/Rel family members (18) nor in I
B
- or I
B
-deficient mice (41,42,35). In addition, the proliferative defect of CD25+ cell subset in transgenic mice overexpressing a transdominant mutated form of I
B
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-
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-
B/Rel transcriptional activity equivalent to that observed for CD25+ cells (Fig. 4
). The correlation of NF-
B activity with CD25 expression appears coincidental and CD25 does not constitute a critical target of NF-
B at these early stages of development.
NF-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-
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-
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-
B/Rel target genes in the resting state or in conditions where the immune system is challenged to various stresses. In this respect, our
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 |
---|
![]() |
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 |
---|
Transmitting editor: A. Fischer
Received 22 April 1999, accepted 13 January 2000.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|