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Prepublished online as a Blood First Edition Paper on September 5, 2002; DOI 10.1182/blood-2002-03-0744.
IMMUNOBIOLOGY
From the Institute of Molecular Biology, Academia
Sinica, Taipei, Taiwan, Republic of China; Graduate
Institute of Immunology, National Taiwan University School of Medicine,
Taipei, Taiwan, Republic of China; Department of
Immunology, Scripps Research Institute, La Jolla, CA; and Graduate
Institute of Microbiology and Immunology, National Yang-Ming
University, Taipei, Taiwan, Republic of China.
Positive selection of thymocytes during T-cell development is
mediated by T-cell receptor (TCR)-activated signals. For different mitogen-activated protein kinases (MAPKs) activated by TCR complex, a
selective involvement of extracellular signal-regulated kinase, but
not p38 MAPK, in positive selection has been suggested. Using transgenic mice with dominant-negative mutation of both MAP kinase kinase 3 (MMK3) and MKK6, we obtained mice with different extents of
inhibition of p38 MAPK activation. Partial inhibition of p38 MAPK
impaired CD4 p38 Mitogen-activated protein (MAP) kinase,
originally identified for its response to stress stimuli, is activated
by ligation of T-cell receptor (TCR) and costimulatory
molecules.1-7 p38 MAP kinase (MAPK) is specifically
activated by MAP kinase kinase 3 (MKK3) and MKK6.8-13 Of
the 4 isoforms of p38 MAPK, p38 Successful T-cell development is dependent on signals activated by
pre-TCR and TCR complexes. Assembly of pre-TCR after rearrangement of
the TCR p38 MAPK has been shown to participate in early thymocyte
development,25,26 yet conflicting results were obtained as
to the role of p38 MAPK in positive selection. By using fetal thymic organ culture and SB 203580, the pharmacologic inhibitor of p38 MAPK,
one study indicated that p38 is not required for positive selection,27 whereas another study illustrated that SB
203580 prevents positive selection.28 SB 203580, however,
inhibits both thymic T cells and thymic stroma cells, so the
controversy about whether TCR-activated p38 MAPK is involved in
positive selection cannot be directly addressed by using thymic organ
culture. Transgenic expression of the dominant-negative p38 Reagents and cell lines
Plasmids
Immunoblot Cell extracts (10-30 µg) were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) for 4 hours at 20 V. Membranes were washed in rinse buffer (phosphate-buffered saline with 2% Tween 20) at room temperature for 15 minutes and incubated in blocking buffer (5% nonfat milk in rinse buffer) for 1.5 hours. The membrane was then incubated with anti-p38 antibody C-20 (Santa Cruz Biotechnology, Santa Cruz, CA),
antiphosphorylated (T180/Y182) p38 MAPK antibody (Cell Signaling,
Beverly, MA), anti-MKK3, or anti-MKK6 antibody34 for 2 hours at room temperature and afterward washed 3 times with rinse
buffer. The membrane was then incubated with 1:1000 diluted horseradish
peroxidase-conjugated antirabbit immunoglobulin antibody (Santa
Cruz Biotechnology), followed by development with enhanced
chemiluminescence reagents (Amersham, Buckinghamshire, United Kingdom).
Protein kinase assay Thymocytes were treated with anti-CD3/anti-CD28 or hydrogen peroxide. Cell lysates were prepared 20 minutes after activation, and 100 µg lysate was precipitated with 1 µg anti-p38 or anti-p38 antibodies,38 followed by 20 µL protein A-Sepharose
(Pharmacia). The kinase activity of the immune complexes was determined
using GST-ATF-2 (1-109) as substrate. The reaction mixtures
were resolved on SDS-PAGE, subjected to autoradiography, and
quantitated by PhosphorImager (Molecular Dynamics).
Generation of lck-MKK3b(A)/MKK6b(A) transgenic mice We constructed transgenic mice with the dominant-negative mutant of p38, p38(AF). p38 activation was suppressed by 30% to 50%
in lck-p38(AF) (data not shown), an effect similar to that
previously reported from experiments using transgenic mice with a
dominant-negative p38.25 We were unable, however, to inhibit more than 50% of p38 activity by p38(AF) in several transgenic attempts. Consequently, because p38 is activated by both
MKK3 and MKK6, we used transgenic expression of MKK3b(A) and MKK6b(A),
the dominant-negative forms of MKK3b and MKK6b, for more effective
inhibition of p38 activation. Both MKK3b(A) and MKK6b(A) were subcloned
into p1017 vector with lck proximal promoter (Figure
1A). We coinjected lck-MKK3b(A) or
lck-MKK6b(A) with CD2 locus control region.37 The
generated transgenic founders were then bred to produce mice with both
MKK3b(A) and MKK6b(A). We also produced MKK3b(A)/MKK6b(A) double
transgenic mice by direct microinjection of lck-MKK3b(A), lck-MKK6b(A),
and CD2 locus control region. Simultaneous expression of MKK3b and
MKK6b in the double transgenic mice produced by either method was
confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR)
(Figure 1B). Total MKK3 and MKK6 protein expression was elevated in
transgenic mice (Figure 1C). Variation in the expression levels was
found between different transgenic lines.
As in previous reports,14,15 we found that p38 Inhibition of p38 MAPK reduces thymocyte number, suppresses thymocyte proliferation and interleukin-2 (IL-2) expression, and interferes with early thymocyte development Thymi from all lck-MKK3(A)/MKK6(A) transgenic mice were smaller than those of their normal littermate control (NLC) mice (Figure 2A). The reduction in total thymocyte number was in the range of 30% to 50%. The extent of the decrease in thymocytes is similar to that reported in mice with dominant-negative p38.25 Consistent with the notion of the requirement
for p38 in TCR signaling, activation-induced thymocyte proliferation
was profoundly reduced in all lck-MKK3(A)/MKK6(A) transgenic mice upon
activation with anti-CD3/anti-CD28, Con A, or TPA/A23187 (Figure 2B).
IL-2 production was similarly suppressed in transgenic (Tg) mice when
equal numbers of total thymocytes were analyzed (Figure 2C, NLC-[T]
and Tg-[T]). Because there was an apparent decrease of SP thymocytes,
the major IL-2-producing thymocytes, in MKK3(A)/MKK6(A) transgenic
mice (Figure 3A), IL-2 expression was
also determined in transgenic thymocytes with equal numbers of
SP thymocytes as in NLC thymocytes (Figure 2C, Tg-[SP]). IL-2
production was defective in transgenic thymocytes after such adjustment. Therefore, inhibition of p38 MAPK by MKK3(A)/MKK6(A) or by
p38(AF) expression led to reduced T-cell proliferation and decreased
IL-2 expression in all transgenic mice.
We also elucidated CD4 To summarize, decreased thymocyte number, reduced thymocyte
proliferation, diminished IL-2 production, and impaired early thymocyte
development were observed in transgenic mice with partial or with
profound inhibition of p38 MAPK. We observed the same phenotype in the
p38 Impaired positive selection in transgenic mice with low p38 activity Fluorescence-activated cell sorter (FACS) analysis of CD4 and CD8 expression on thymocytes from transgenic mice revealed 2 types of patterns. For those mice with moderate p38 activity, represented by line 9 of MKK3(A)/MKK6(A) (Figure 3A) and p38(AF) transgenic mice
(data not shown), thymocyte profiles were almost indistinguishable from
those of the NLC mice. This was in agreement with previous reports that
T-cell development is normal in MKK3 / or p38(AF)
transgenic mice.25,29,30 In contrast, thymocyte CD4/CD8
profiles were altered in transgenic mice with profound inhibition of
p38 activity, represented by line 2 (Figure 3A). There was a moderate
increase of CD4+CD8+ thymocytes, with a
proportional decrease in the CD4 SP and CD8 SP populations. The
disturbance of thymocyte development was more prominent when the
absolute cell numbers of individual thymic subpopulations from each
group of 6 mice were examined (Figure 3B). Consistent with a decrease
of total thymocyte number (Figure 2A), the cell numbers of
CD4+CD8+, CD4+CD8,
and CD4CD8+ thymocytes were reduced by at
least 30% in transgenic mice with moderate p38 inhibition (W-MKK3/6(A)
in Figure 3B). The reduction in the absolute cell numbers was more
dramatic in CD4+CD8+,
CD4+CD8, and
CD4CD8+ thymocytes from transgenic mice with
extensive p38 inhibition (S-MKK3/6(A) in Figure 3B). When comparing
transgenic mice with different extents of p38 inhibition, we found that
a 2.7% to 1.3% difference in CD8 SP thymocytes between line-9 and
line-2 transgenic mice (Figure 3A) was converted into a 3.6-million to
1.4-million difference in cell numbers (Figure 3B). In contrast, the
absolute number of CD4CD8 thymocytes was
not proportionally reduced in different MKK3(A)/MKK6(A) transgenic
mice. The partial arrest at the DN thymocyte stage, including
the accumulation of the CD25CD44
subpopulation (Figure 2D), is likely due to an impaired DN to DP
transition in the absence of sufficient p38
activity.26
The reduction in the transition from CD4+CD8+ to CD4 SP and CD8 SP thymocytes was correlated with the phenotype observed in peripheral lymphocytes. The percentage of lymph node CD4+ T cells in line-2 transgenic mice was reduced from 48.4% to 29.6% compared with NLC mice, concurrent with a decrease in CD8+ T cells from 23.2% to 8.9% (Figure 3A). The decrease in the absolute cell number was more conspicuous in peripheral T cells from MKK3(A)/MKK6(A) transgenic mice (Figure 3B). A nearly 30% decrease of CD4+ and CD8+ para-aortic lymph node cell number was found in transgenic mice with moderate p38 inhibition. For transgenic mice with extensive p38 inhibition, there was at least another 50% decrease in cell number (Figure 3B). Splenic T cells were also reduced by a similar degree compared with NLC mice (data not shown). We further examined molecules associated with positive selection in
transgenic mice with profound p38 inhibition. Thymocyte positive
selection is correlated with increased expression of CD5 and
CD69.41-43 The fraction of CD69+ thymocytes
was found to be lower in lck-MKK3(A)/MKK6(A) transgenic mice than in
NLC mice (Figure 4A). The reduction of
CD69+ expression was even more prominent in the absolute
number of CD69+ thymocytes, with a difference of 19.2 million to 6 million cells between NLC and MKK3(A)/MKK6(A) transgenic
mice. TCR expression on CD4+ SP and CD8+
SP thymocytes was also lower in lck-MKK3(A)/MKK6(A) transgenic mice (shadowed curve in Figure 4B) than in the NLC mice (open curve in
Figure 4B). Similar reductions in TCR expression were detected in
mature peripheral CD4+ and CD8+ T cells (not
shown). The expression of CD5 and CD69 in CD4+ SP and
CD8+ SP thymocytes was also lower in lck-MKK3(A)/MKK6(A)
transgenic mice than in NLC mice (Figure 4B). The expression of
costimulatory molecules, such as CD28, that play essential roles in p38
signaling was similar in NLC and transgenic mice.
p38 is required for selection of class II-restricted TCR transgene To further study the perturbation of thymocyte development in lck-MKK3(A)/MKK6(A) transgenic mice, we examined the selection of class II-restricted TCR transgene in mice in which p38 activity was profoundly suppressed. Line 2 and line 18 of MKK3(A)/MKK6(A) transgenic mice (in C57BL/6 background) were introduced into AND TCR transgenic mice44 in B10.A background through breeding. The expression of I-Ek and V 3 (from AND TCR) was confirmed
in the mice examined. The presence of the AND TCR transgene resulted in
an increase of CD4 SP thymocytes compared with normal thymocytes. The
introduction of MKK3(A)/MKK6(A) into AND TCR transgenic mice
significantly reduced CD4+ SP thymocytes from 70% to 48%
of the total population, with a more profound decrease in the absolute
number of CD4+ SP thymocytes, from 42.5 million to 19.3 million (Figure 5A). A similar effect was
found in peripheral T cells. Fractions of splenic CD4+ T
cells, representing mostly mature AND T cells, were reduced from 29%
to 10% of the total splenocytes in MKK3(A)/6(A) transgenic mice
(Figure 5B). The number of splenic CD4+ T cells was
decreased from 16.5 million in AND mice to 4.6 million in
MKK3(A)/MKK6(A)-bearing mice. When lymph node cells were gated by transgenic TCR expression (V3), the reduction in the AND T-cell population and absolute number was also conspicuous in
lck-MKK3(A)/MKK6(A) transgenic mice (Figure 5C). Positively selected
thymocytes, marked by the expression of either CD69 (Figure 5D) or CD5
(not shown), were also reduced in MKK3(A)/MKK6(A)-bearing mice. The
perturbation of T-cell development by MKK3(A)/MKK6(A) led to a profound
reduction of AND CD4+ T cells in thymus, spleen, and
lymph nodes.
p38 is required for the selection of class I-restricted TCR transgene We also examined the role of MKK3(A)/MKK6(A) in the selection of transgenic TCR restricted by class I MHC. H-Y TCR transgenic mice45 were crossed with line 2 and line 18 of lck-MKK3(A)/MKK6(A) transgenic mice, and the expression of Db, V8, and MKK3/6 was confirmed in the F1 generation.
The CD8 SP thymocytes in H-Y female transgenic mice were reduced from 24% to 13% of the total thymocytes by the presence of the
MKK3(A)/MKK6(A) transgene (Figure 6A). A larger decrease in the
absolute cell number of CD8 SP thymocytes was found in transgenic mice
carrying the MKK3(A)/MKK6(A) transgene. The
CD8+V8+ thymocyte population, representing
mostly H-Y TCR+ T cells, also decreased in mice with the
MKK3(A)/MKK6(A) transgene (Figure 6B).
The fraction and the cell number of CD69+ thymocytes were
reduced by the MKK3(A)/MKK6(A) transgene (Figure 6C). Among the
CD69+ population, the fraction of CD8 SP thymocytes was
also reduced in MKK3(A)/MKK6(A) transgenic mice (Figure 6D).
It may be noted that inhibition of p38 led to a prominent
accumulation of DN thymocytes in H-Y transgenic mice (Figure 6A), in
contrast to an arrest at DP stage in AND transgenic mice (Figure 5A).
Such interference was likely due to inhibition in the development from
CD4
In the present study, we illustrate the multiple roles of p38 in
the different stages of T-cell development. We confirm previous reports
that p38 is required for maintenance of normal thymocyte number,
proliferation of thymocytes, IL-2 production, and normal development of
the CD4 The differential sensitivity of T-cell developmental and activation
events to p38 Positive selection is mediated by TCR signals upon interaction with self-peptide/MHC complex. p38 is activated immediately upon TCR/CD28 engagement.5-7 Notably, ERK, another major MAPK critical for T-cell signaling, is activated by TCR alone22 and is independent of costimulation.7 The ERK pathway has long been known for its critical role in positive selection. Dominant-negative forms of Ras, Raf-1, or MEK-1 have been shown to inhibit positive selection.19,20 Moreover, positive selection is impaired in ERK1-deficient thymocytes.21 In one of the finest studies supporting a dominant role for ERK in positive selection,22 phosphorylated p38 MAPK, but not phosphorylated ERK, was detected in thymocytes when positive selection was blocked, suggesting that p38 MAPK is not required for positive selection. In the present study, we identified a role of p38 essential for the transition of CD4+CD8+ to mature CD4+ SP and CD8+ SP thymocytes (Figures 3-6). Our results suggest that ERK is not the sole MAPK that is involved in thymocyte-positive selection. Interestingly, re-examination of the previous studies (see Figures 4 and 5 in Werlen et al22) indicates that p38 activation is partially reduced in thymocytes not undergoing positive selection, which supports a significant contribution of p38 MAPK for positive selection. In addition, ERK and p38 MAPK have different activation modes. We have recently demonstrated that p38 MAPK, but not ERK, can be activated by CD28 alone.7 Conceivably, p38 may be stimulated by coactivators, such as CD28, during T-cell development in the absence of TCR engagement. This possibility is confirmed by the observation that a constitutively active p38 is detected in freshly isolated thymocytes.23,39 Therefore, the activity of p38 detected in unselected T cells does not necessarily exclude a role of p38 in positive selection. It may be noted that our study does not suggest an equal contribution
of ERK and p38 to T-cell-positive selection. Inhibition of
thymocyte-positive selection was detected only with extensive blockage
of p38 activation; little interference with positive selection could be
observed when p38 MAPK was partially suppressed (Figure 2).
Researchers25,29 including us have been unable to detect
any defects in thymic-positive selection in dominant-negative p38
We thank Dr Roger Perlmutter for p1017 vector, Dr Dimitris Kioussis for the CD2 enhancer, Dr John Kung for AND and H-Y transgenic mice, and Dr Bruce Boothby for helpful discussions.
Submitted March 11, 2002; accepted August 27, 2002.
Prepublished online as Blood First Edition Paper, September 5, 2002; DOI 10.1182/blood-2002-03-0744.
Supported by grants NSC 89-2320-B001-050 and 90-2320-B001-070 from the National Science Council, and a grant from Academia Sinica, Taiwan, Republic of China.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Ming-Zong Lai, Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan, Republic of China; e-mail: mblai{at}ccvax.sinica.edu.tw.
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  B. Liu, J.-f. Wu, Y.-y. Zhan, H.-z. Chen, X.-y. Zhang, and Q. Wu Regulation of the Orphan Receptor TR3 Nuclear Functions by c-Jun N Terminal Kinase Phosphorylation Endocrinology, January 1, 2007; 148(1): 34 - 44. [Abstract] [Full Text] [PDF]
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 A. L. Miller, M. S. Webb, A. J. Copik, Y. Wang, B. H. Johnson, R. Kumar, and E. B. Thompson p38 Mitogen-Activated Protein Kinase (MAPK) Is a Key Mediator in Glucocorticoid-Induced Apoptosis of Lymphoid Cells: Correlation between p38 MAPK Activation and Site-Specific Phosphorylation of the Human Glucocorticoid Receptor at Serine 211 Mol. Endocrinol., June 1, 2005; 19(6): 1569 - 1583. [Abstract] [Full Text] [PDF]
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 C.-C. Wu, S.-C. Hsu, H.-m. Shih, and M.-Z. Lai Nuclear Factor of Activated T Cells c Is a Target of p38 Mitogen-Activated Protein Kinase in T Cells Mol. Cell. Biol., September 15, 2003; 23(18): 6442 - 6454. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
was not detectable in
thymocytes. Because TCR engagement activated p38
B.
J Exp Med.
1997;185:1897-1907