From the Department of Medicine II,
University of Mainz, D-55101 Mainz, Germany, the ¶ Institute of
Clinical Biochemistry and Pathobiochemistry, and the ** Institute of
Virology and Immunobiology, University Medical Clinic,
D-97080 Wuerzburg, Germany
Received for publication, October 26, 2000
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ABSTRACT |
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Several major functions of type I
cGMP-dependent protein kinase (cGK I) have been established
in smooth muscle cells, platelets, endothelial cells, and cardiac
myocytes. Here we demonstrate that cGK I Effective T cell activation can be elicited by a combination
of (i) engagement of the T cell antigen receptor
(TCR)1·CD3 complex
with foreign antigen presented on major histocompatibility complex molecules and (ii) ligation of the CD28 T cell antigen by B7 molecules (CD80 and CD86) on the surface of antigen-presenting cells, resulting in transcription factor activation, interleukin 2 (IL-2) production, and cell proliferation (1-4). Activation and
subsequent proliferation of resting T lymphocytes is an essential process in the T cell-mediated immune response. In vitro, T
cells respond to stimulation of the TCR·CD3 complex (without
costimulation) with IL-2-dependent induction of cell
proliferation (2, 5). A costimulatory signal from the CD28 pathway
synergizes with the signal from the TCR·CD3 complex to further
increase cell proliferation and IL-2 production, and T cell
proliferation becomes largely independent of IL-2 (6). Intracellular
signaling pathways, which may mediate T cell activation, include
phosphorylation cascades that stimulate MAP kinases including ERK (3,
7, 8) and p38 kinase (9, 10), as well as phosphatidylinositol 3-kinase (11, 12) and its target Akt/protein kinase B (13).
Reports of cyclic nucleotide effects on T cell activation
indicate that cAMP is inhibitory, whereas effects of cGMP have not been
conclusively elucidated. In fact, lowering of cAMP levels by induction
of phosphodiesterase-7 was reported to be required for T cell
activation (14). Nitric oxide, which activates soluble guanylate
cyclase and increases intracellular cGMP, has inhibitory effects on T
cell proliferation (15) and cytokine secretion (16), stimulates c-Jun
N-terminal kinase, ERK, and p38 activities in Jurkat T cells (17), and
decreases tyrosine phosphorylation of Jak3/STAT5 and proliferation in
cultured T cells (18). However, NO can elicit effects via both
cGMP-dependent and -independent pathways, and increasing
intracellular cGMP may affect not only cGMP-dependent
protein kinase but also cGMP-regulated phosphodiesterases or channels
and cAMP-dependent protein kinase (19, 20).
The present study focuses on the role of cGK in mediating cGMP
effects on human peripheral blood T cells. In general, there exist two
mammalian isoforms of cGMP-dependent protein kinase, cGK I
and cGK II (19-24), as well as two splice variants of cGK I (cGK I Materials--
Anti-CD3-, CD4-, or CD8-conjugated Dynabeads used
for affinity purification of T cells were purchased from Dynal
(Hamburg, Germany). Stimulatory anti-CD3 antibodies were either 12F6, a kind gift from Johnson T. Wang (Massachusetts General Hospital, Boston,
MA), or produced from the OKT3 hybridoma obtained from the American
Tissue Culture Collection. For flow cytometry, fluorochrome-conjugated antibodies against CD2, CD3, CD14, and CD19 were obtained from Immunotech (Hamburg, Germany), against CD8 from Pharmingen (Hamburg, Germany), against CD4 from Exalpha (Boston, MA), and against CD41a from
Southern Biotechnology Associates (Birmingham, AL).
Fluorochrome-conjugated isotype control antibodies were from Dako
(Hamburg, Germany).
SNP, 3-(5'-hydroxymethyl-2'furyl)-1-benzylindazol (YC-1), and forskolin
were from Calbiochem (Bad Soden, Germany). cGMP analogs PET-cGMP and
8-pCPT-cGMP, as well as the cAMP analog Sp-5,6-dichlorobenzimidazole riboside-3',5'-cyclic monophosphorothioate (cBIMPS) were from BioLog
(Bremen, Germany).
Jurkat-E6 cells were obtained from American Tissue Culture Collection
and A3.01 human T lymphoma cells from the Center for Disease Control
(Atlanta, GA), fetal calf serum and cell culture media were from Life
Technologies, Inc., nitrocellulose membranes were from Schleicher & Schuell, and polyvinylene difluoride membranes from Millipore
(Eschborn, Germany). Standard chemicals were obtained from Sigma.
Purified recombinant cGK I protein, used for preabsorbing anti-cGK I
antibody, has been described (28). Purified dephospho-VASP was kindly
provided by Christiane Bachmann (29). Polyclonal anti-cGK I antiserum
(25) and monoclonal anti-phospho(Ser239)-VASP 16C2 antibody
(30) were produced as described. Antibodies against dual
phosphorylated, activated MAP kinases, ERK1/2, and p38, as well as
antibodies for total ERK1/2 and p38 were obtained from New England
Biolabs (Schwalbach, Germany).
Isolation of Human Peripheral Blood Mononuclear Cells, Purified T
Lymphocytes, and Platelets--
PBMC were isolated from freshly drawn
heparinized human blood using Ficoll gradient centrifugation (31) and
then resuspended in RPMI 1640 medium. T cells were purified from PBMC
using anti-CD3-conjugated Dynabeads, or CD4+ and
CD8+ T cell subsets were obtained by incubating PBMC with
anti-CD4- or anti-CD8-conjugated Dynabeads at 4 °C for 1 h and
then following the supplier's protocol. Affinity purification of T
cells was performed using either fluorochrome-conjugated antibodies
against T cell antigens together with fluorescence-activated cell
sorting (see below) or using T cell immunoaffinity columns and a
protocol provided by Cedar Lane (Hornby, Canada) for negative-selection of T cells (removal of B cells and monocytes). Purified human platelets
were prepared as described elsewhere (32).
For experiments, PBMC or purified T cells were incubated at 37 °C in
RPMI 1640 medium containing 5% fetal calf serum, 100 units/ml
penicillin, 100 µg/ml streptomycin, or in the case of long term
culturing (less than 2 weeks), in the same buffer except with 10%
fetal calf serum plus 100 units/ml IL-2, and an initial addition of 100 ng/ml anti-CD3 antibody.
Fluorescence-activated Cell Sorting of Human T
Lymphocytes--
PBMC were resuspended in phosphate-buffered saline
containing 0.1% bovine serum albumin and then incubated with
fluorochrome-conjugated anti-CD3 and anti-CD41a antibodies at 4 °C
for 1 h. After washing to remove excess antibodies, cells were
sorted to obtain pure T cells devoid of platelets
(CD3+/CD41a Reverse Transcription-PCR--
Total RNA was isolated from
immunoaffinity column-purified T cells using Trizol (Life Technologies,
Inc.) reagent and reverse transcription was performed with random
hexamers using a first strand cDNA synthesis kit from MBI Fermentas
(St. Leon-Rot, Germany) according to the supplier's protocol. PCR
amplification of cGK was performed using oligonucleotide primer pairs
specific for each cGK I splice variant (cGK I Analysis of cGK Activation by Detection of VASP Phosphorylation
in Human PBMC, Purified T Cells, and Platelets Either in Lysates or
Intact Cells--
In vitro cGK I activity was determined by
analyzing VASP phosphorylation in cell lysates of affinity-purified T
lymphocytes as well as platelets, following a previously published
protocol (33). Briefly, purified T cells were lysed in 10 mM Hepes, pH 7.4, 5 mM MgCl2, 0.2 mM EDTA, and a protease inhibitor mix
(CompleteTM, Roche Molecular Biochemicals), and then
lysates were passed through 25 gauge needles to shear chromosomal DNA.
In vitro kinase reactions with cell lysates were carried out
in buffer (20 mM Tris-Cl, pH 7.4, 10 mM
MgCl2, 5 mM
In intact cell experiments, PBMC (preincubated at 37 °C for 3 h
to overcome potential preactivation arising from handling during
preparation) or platelets were resuspended in serum-free RPMI medium
and stimulated in the absence or presence of agents that elevate cGMP
or cAMP levels (see details under "Results" and in the figure
legends) and briefly centrifuged to obtain cell pellets which were
processed for SDS-PAGE and Western blot analysis of VASP
phosphorylation as described below.
Analysis of cGMP-dependent Activation of the MAP
Kinases ERK 1/2 and p38, in Human PBMC and Immunoaffinity
Column-Purified T Lymphocytes--
PBMC or purified T lymphocytes were
incubated in serum-free RPMI medium for 3 h at 37 °C and then
stimulated in the absence or presence of agents which elevate cGMP
levels and briefly centrifuged to obtain cell pellets that were
processed for SDS-PAGE and Western blot analysis of phosphorylation as
described below.
Western Blot Analysis of Protein Kinases (cGK I, ERK1/2, and p38)
and VASP Phosphorylation--
Intact cells or cell lysates were added
to SDS gel loading buffer and heated at 95 °C for 10 min, then
analyzed by SDS-PAGE (8% gels, for cGK I and VASP) and Western
blotting. Western blot nitrocellulose membranes were blocked with 1%
hemoglobin in phosphate-buffered saline and divided horizontally to
stain the high molecular weight range of proteins with polyclonal
anti-cGK I antiserum (diluted 1:3000) and the lower range with
monoclonal anti-phospho(Ser239)-VASP 16C2 antibodies (1 µg/ml), followed by either horseradish peroxidase-coupled goat
anti-rabbit or goat anti-mouse secondary antibodies (1:5000; Bio-Rad),
respectively. Signals were visualized using the ECL system (Amersham
Pharmacia Biotech).
For determining ERK and p38 activation, stimulated cells were analyzed
by SDS-PAGE (using 10% gels) and blotted to polyvinylene difluoride
membranes, which were then blocked with TBST (10 mM Tris-Cl, pH 7.5, 100 mM NaCl, 0.1% Tween 20) containing
3% nonfat dry milk (Bio-Rad). Signals were visualized with polyclonal
antibodies that recognize activated dual phosphorylated ERK1/2 or dual
phosphorylated p38, followed by horseradish peroxidase-coupled goat
anti-rabbit IgG (Bio-Rad). To determine total expression of MAP kinase
for verification of equal protein loading, membranes were stripped at
58 °C for 1 h in buffer containing 62.5 mM Tris-Cl,
pH 6.8, 100 mM Determination of T Cell Proliferation--
PBMC (2 × 105/well of 24-well plates) were incubated with or without
agents that elevate cAMP or cGMP levels, at 37 °C for 1 h under
serum-free conditions prior to adding anti-CD3 antibody (12F6 or OKT3,
diluted 1:2500), alone or together with anti-CD28 antibody (0.1 µg/ml), and incubating in 10% serum-containing medium for analysis
of proliferation after 5 days. Subsequently, PBMC were counted using a
Cell Counter (Coulter, Krefeld, Germany), and the inhibition of
anti-CD3 antibody-induced cell proliferation caused by cAMP or cGMP
analogs or SNP was determined.
Proliferation of cGK I Enzyme-linked Immunosorbent Assay of IL-2--
PBMC cultured in
triplicate at 3 × 106/ml (24-well plate) in RPMI
1640/10% fetal calf serum were stimulated with anti-CD3 (1:2500) in
the presence of various concentrations of 8-pCPT-cGMP. After 48 h
of stimulation, cell supernatants were collected, and IL-2 concentrations were determined using the OPTEIA IL-2 set according to
the supplier's protocol (BD/Pharmingen, Heidelberg, Germany).
Highly Purified Primary Human T Lymphocytes, but Neither Cultured T
Cells nor T Cell Lines, Display Endogenous cGK I Expression and
Activity--
A rich source of cGK I in human blood is platelets that
can easily contaminate crude lymphocyte preparations. Therefore, for examining the expression of cGK in T lymphocytes, it was crucial to use
highly purified cells (Fig.
1A). T cells were prepared from freshly isolated PBMC using anti-CD3-conjugated Dynabeads or were
isolated from PBMC by labeling with fluorochrome-conjugated anti-CD3
and anti-CD41a (platelet-specific marker) and using FACS to separate
pure T cells devoid of platelets (CD3+/CD41a
Anti-CD8+- and anti-CD4+-conjugated Dynabeads
used to purify T cell subsets (with less than 5% contaminating
platelets; data not shown) demonstrated endogenous cGK I expression in
both CD8+ and CD4+ T cells (Fig.
1B). However, transformed T cell lines (Jurkat-E6 or A3.01)
contained no detectable cGK I, and that observed in primary T cell
cultures was lost after 48 h of culture (Fig. 1, B and
C, respectively). None of the T cells investigated (freshly isolated, cultured, or transformed) expressed detectable cGK II by
either Western or PCR analysis (data not shown). The cGK I isoform
expressed in T cells was identified by reverse transcriptase-PCR amplification of RNA derived from immunoaffinity column-purified T
cells using cGK I
Evidence that the band identified by Western blot analysis as cGK I in
fresh T cells indeed demonstrated that cGK activity was obtained by
studying cGK I phosphorylation of the endogenous substrate VASP in
lysates of T cells (affinity-purified using anti-CD3-conjugated
Dynabeads) in response to 0.5 µM PET-cGMP, a hydrolysis
resistant, cGK-selective cGMP analog (Fig.
2). VASP phosphorylation was determined
by Western blot analysis using the
anti-phospho(Ser239)-VASP antibody 16C2, which detects a
46-kDa VASP phosphorylated on Ser239, as well as a 50-kDa
VASP phosphorylated on both Ser239 and Ser157
(30). The signal for VASP phosphorylation obtained from fresh T cell
extracts (Fig. 2A, fourth lane) was less
prominent in comparison with that from an equal amount of platelet
extract (Fig. 2C, fourth lane). This appeared to
be in part due to lower VASP levels in T cells (determined using a
phosphorylation-independent antibody which recognizes total VASP; data
not shown), and the signal in fresh T cells could be increased by
adding exogenous purified recombinant VASP (Fig. 2A, compare
fourth lane with sixth lane), which is isolated
primarily in the de-phospho form (Fig. 2A, second and fifth lanes). The exogenously added dephospho-VASP
ensured saturating amounts of substrate and increased the sensitivity of the in vitro kinase assay in T cells (Fig. 2A,
sixth lane). In contrast to freshly purified T cells, T
cells from the same donor cultured for 10 days in vitro no
longer demonstrated PET-cGMP-stimulated VASP phosphorylation activity
(Fig. 2B).
Distinct Patterns of cGK I Activation in Freshly Isolated Human
PBMC versus Platelets, and Loss of cGMP-dependent but Not
cAMP-dependent VASP Phosphorylation in Cultured
PBMC--
The stimulation of VASP phosphorylation by cGMP-elevating
agents was also demonstrated in intact PBMC (Western blots; Fig. 3). After 25 min of stimulation, VASP
phosphorylation in PBMC in response to 200 µM PET-cGMP, a
membrane-permeant, hydrolysis-resistant, cGK-selective cGMP analog, and
in response to 100 µM SNP, a NO donor, was markedly less
than VASP phosphorylation in platelets (Fig. 3, A and
B, respectively, showing analysis of equal amounts of
protein), as had also been observed in cell lysates above. Examination
of a detailed time course of SNP stimulation revealed that peak VASP
phosphorylation occurred transiently after 5 min SNP and then rapidly
declined (Fig. 3B), unlike the SNP stimulation in platelets
that persisted throughout 2 h. YC-1 (50 µM), a
nitric oxide-independent activator of guanylate cyclase and an
inhibitor of several phosphodiesterases, caused strong and long lasting stimulation of VASP phosphorylation in PBMC (Fig. 3). SNP plus YC-1
treatment produced somewhat more VASP phosphorylation than YC-1 alone.
cBIMPS, a selective activator of cAK, also strongly stimulated VASP
phosphorylation in PBMC and platelets. Whereas the cBIMPS stimulatory
effect was preserved in cultured PBMC, none of the effects of
cGMP-elevating agents were preserved (Fig. 3A), indicating
that their effects were lost along with cGK I and could not be mediated
by cAK.
SNP and cGMP Analog Activation of cGK Leads to
Dose-dependent Transient Activation of ERK and p38 in
Immunoaffinity Column-Purified T Cells--
Stimulation of purified T
cells with 200 µM PET-cGMP or 8-pCPT-cGMP or with 100 µM SNP caused dual phosphorylation/activation of both ERK
and p38 over a time period of 5-60 min (Western blot; Fig.
4A), with peak activation
between 10 and 15 min. Induction of both ERK- and p38-phosphorylation
by 8-pCPT-cGMP for 15 min was dose-dependent (Fig.
4B).
SNP and cGMP or cAMP Analog Activation of cGK I Inhibits
TCR·CD3-induced Proliferation in PBMC--
Proliferation of smooth
muscle cells and other cGK I positive cells is inhibited by cGMP
analogs (see "Discussion"). Because cAMP is a known inhibitor of T
cell proliferation, stimulators of cGMP/cGK were compared with the cAMP
analog cBIMPS with regard to their effects on proliferation of
TCR·CD3-activated PBMC (Fig. 5; note
that cell number increase is shown in A, percentage of control proliferation in the absence of inhibitor in B-D).
cBIMPS (200 µM) abolished CD3-activated T cell
proliferation, whereas 200 µM PET-cGMP (or 8-pCPT-cGMP)
and 100 µM SNP inhibited proliferation about 50, 40, and
70%, respectively, in freshly prepared PBMC (Fig. 5, A and
B) shown to contain cGK I (Figs. 1-3). In cultured PBMC
(cultured for 3 days prior to the proliferation assay) that have lost
cGK I, the inhibitory effect of cBIMPS was highly preserved; however,
no inhibition by 8-pCPT-cGMP was observed, and inhibition by SNP was
reduced from 70% in fresh PBMC to 50% (Fig. 5, B and C). Jurkat-E6 cells (Fig. 5D) containing no detectable cGK I
(Fig. 1B) also did not respond to 8-pCPT-cGMP, but Jurkat
proliferation was inhibited by cBIMPS (60%) and by SNP (40%). Not
only the proliferation of T cells stimulated with anti-CD3 antibody
alone (Fig. 5B) but also that of T cells costimulated with
anti-CD3 plus anti-CD28 (Fig. 5E) was inhibited by
8-pCPT-cGMP (40 and 30% inhibition, respectively).
cGMP/cGK Inhibition of T Cell Proliferation Partially Results from
Reduced IL-2 Expression--
IL-2 is an important regulator of T cell
proliferation and is released by activated T cells. Anti-CD3-stimulated
IL-2 release was reduced by 8-pCPT-cGMP to 42% of control (from 64 to
37 pg of IL-2/107 cells; Fig.
6). To test whether the observed
cGMP-dependent inhibition of T cell proliferation was due
to reduction of IL-2 levels, recombinant human IL-2 was added to
anti-CD3-stimulated PBMC cultures in the presence or absence of
8-pCPT-cGMP. IL-2 (500 units/ml) effectively diminished 8-pCPT-cGMP
inhibition of anti-CD3-stimulated proliferation (Fig.
7). Costimulation with anti-CD3 plus
anti-CD28 elicits IL-2 independent proliferation, which is also
inhibited by 8-pCPT-cGMP (Fig. 5E), but this was, as
expected, not reversed by addition of exogenous IL-2 (data not
shown).
Reconstitution of cGK Expression in Jurkat-E6 Cells Restores
cGK-mediated Inhibition of Cell Proliferation--
Jurkat-E6 cells
were found to be deficient in cGK I (Fig. 1B), and their
proliferation was resistant to inhibition by 8-pCPT-cGMP (Fig.
5D). To provide clear evidence that
cGMP-dependent inhibition of T cell proliferation was
indeed mediated by cGK, cytomegalovirus-promoter-driven cDNA
constructs encoding either cGK I Human T cells devoid of platelets were shown to express endogenous
cGK I, which could be stimulated by NO and cGMP analogs to inhibit T
cell proliferation. Both CD4+ and CD8+ T cells
expressed endogenous cGK I In our experiments, stimulation of cGK I activated ERK and p38 MAP
kinases, and in contrast to cAMP (37), cGMP did not inhibit anti-CD3-induced MAP kinase activation (data not shown). NO, cGMP, or
cGK I stimulation or inhibition of MAP kinases has been demonstrated in
other cell types including cGK I-transfected baby hamster kidney cells
(38), 293T fibroblasts (39), CHO cells (40), Jurkat T cells (17),
macrophages (41), mesangial cells (42), and also low passage rat aorta
vascular smooth muscle cells (43). Responses to NO, cGMP, or cGK not
only varied in different cell types with respect to activation or
inhibition of MAP kinases but also proliferation. In many cases, a
definitive relationship between cGK and effects on MAP kinases and
proliferation is not possible because certain cells may not contain cGK
(e.g. Jurkat cells), and this was not always investigated.
Also some criteria that have been used to define cGK effects in cells,
such as inhibition by agents such as KT5823, may be unreliable because
this agent has been shown to actually enhance cGMP effects in certain
cell types such as platelets and mesangial cells (44). Our own results in T cells indicate that short term cGK I activation of MAP kinases does not activate T cells, instead cGK I inhibits proliferation of
activated T cells. This is in distinct contrast to
cAMP-dependent inhibition of T cell proliferation reported
to be related to MAP kinase inhibition (37) and thus represents a major
difference in cAMP and cGMP effects on T cells.
Selective activation of cGK I by cGMP analogs, as well as by SNP,
inhibited proliferation of human T cells, although these were only
40-70% as effective as the cAMP analog cBIMPS, which caused complete
inhibition. In Jurkat T cells and PBMC cultured for 3 days prior to the
proliferation assay, i.e. cells that contain no detectable
cGK I, the inhibitory effect of 8-pCPT-cGMP was lost, whereas that of
cBIMPS was unchanged and that of SNP was only somewhat reduced. These
results indicated that cGMP effects are not mediated by cAK and that
SNP has additional inhibitory effects on T cell proliferation that are
clearly cGK-independent. cGMP inhibition of cell proliferation could be
reconstituted in Jurkat cells overexpressing wild type cGK I In our studies, the effect of cGMP analogs on proliferation appeared
specific because the effect is absent in cGK I-deficient cells (whereas
that of cAMP analogs is intact) but can be reconstituted by
transfection of cGK I. This agrees with other publications that also
show specific effects of cGMP on cGK. In contrast to the respective
normal cells containing endogenous cGK I, platelets of cGK I-deficient
mice lose 8-pCPT-cGMP-dependent inhibition of platelet
aggregation (50), cGK I-deficient platelets of chronic myelocytic
leukemia patients lose 8-pCPT-cGMP-dependent inhibition of
intracellular Ca+2 (51), and cGK-deficient endothelial
cells lose 8-pCPT-cGMP-dependent inhibition of
intracellular Ca+2 and endothelial permeability (52). In
contrast, the effect of a cAMP analog (50) or cAMP-elevating agent (52)
was intact.
Activation of T cells is associated with increased expression and
release of IL-2, which stimulates T cell proliferation (4). cGK
activation reduced antigen-receptor-stimulated IL-2 release and
proliferation, and inhibition of anti-CD3-stimulated cell proliferation
could be significantly reduced by addition of exogenous recombinant
IL-2. These results indicate that inhibition of IL-2 release represents
at least one mechanism by which cGK inhibits T cell proliferation.
Inhibition of T cell activation by the NO/cGMP/cGK signaling pathway
may be of potential significance for counteracting inflammatory or
lymphoproliferative processes. Studies have shown that NO (53, 54), as
well as cGMP (18), is involved in inhibition of T cell activation and
inflammation. NO has also been reported to inhibit the proliferation of
human leukemia cells (55); however, this requires study in greater
detail. Because there are also cGMP-independent effects of NO and cGMP
can affect other mediators than cGK, the role of cGMP and cGK in
inflammation and lymphoproliferative disorders needs to be directly
investigated in the future.
In summary, our data show that cGK I is endogenously expressed
in freshly purified human peripheral blood T lymphocytes and inhibits
their proliferation and interleukin 2 release. Incubation of
human T cells with the NO donor, sodium nitroprusside, or the
membrane-permeant cGMP analogs PET-cGMP and 8-pCPT-cGMP, activated cGK
I and produced (i) a distinct pattern of phosphorylation of
vasodilator-stimulated phosphoprotein, (ii) stimulation of the
mitogen-activated protein kinases ERK1/2 and p38 kinase, and, upon
anti-CD3 stimulation, (iii) inhibition of interleukin 2 release and
(iv) inhibition of cell proliferation. cGK I was lost during in
vitro culturing of primary T cells and was not detectable in
transformed T cell lines. The proliferation of these cGK I-deficient
cells was not inhibited by even high cGMP concentrations indicating
that cGK I, but not cGMP-regulated phosphodiesterases or channels,
cAMP-dependent protein kinase, or other potential cGMP
mediators, was responsible for inhibition of T cell proliferation.
Consistent with this, overexpression of cGK I
, but not an inactive
cGK I
mutant, restored cGMP-dependent inhibition of cell
proliferation of Jurkat cells. Thus, the NO/cGMP/cGK signaling system
is a negative regulator of T cell activation and proliferation and of
potential significance for counteracting inflammatory or
lymphoproliferative processes.
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DISCUSSION
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and I
) (21, 22) with differing N-terminal ends. cGK I is expressed
in platelets, vascular smooth muscle cells, fibroblasts, certain
endothelial cells, lung, cerebellum, and heart, whereas cGK II is
expressed in intestinal epithelium and many regions of brain and kidney
(19, 20, 23-26). In human T cells, effects of the NO donor sodium
nitroprusside (SNP) were compared with those of membrane-permeant,
selective activators of cGK such as PET-cGMP and 8-pCPT-cGMP (27). Our
results demonstrate that primary human T cells contain endogenous cGK
I
, which mediates inhibitory effects of cGMP on IL-2 production and
proliferation. In contrast to the bulk of published studies, only
freshly prepared cells were used for examining cGK I effects on T cells
because our studies demonstrated that cGMP effects were absent in cGK I-deficient cultured cells; however, cGMP effects could be
restored by transfection of these cells with cGK I.
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DISCUSSION
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) separate from a T cell fraction
with adherent platelets (CD3+/CD41a+). T cell
purity was determined by distinguishing T cells from other cells, using
fluorochrome-conjugated antibodies directed against cell-specific
antigens such as CD2, CD4, CD8 (T cells), CD19 (B cells), CD14
(monocytes), and CD41a (platelets), Briefly, affinity-purified cells
were incubated with a combination of up to three differently labeled
antibodies at 4 °C for 1 h and then washed with
phosphate-buffered saline containing 0.1% bovine serum albumin, and
analyzed using a FACScan cell sorter (Becton-Dickenson, Hamburg,
Germany) together with LysisII and Cellquest software. Purified T cells
contained at least 95% T lymphocytes, less than 2% monocytes or B
cells, and either no detectable platelets (FACS-sorted cells) or less
than 5% platelets (immunoaffinity column-purified cells). For FACS
calibration purposes, fluorescein isothiocyanate- and
phycoerythrin-labeled isotype-matched control antibodies were used.
-83 sense:
5'-GTCAGAGAAGGAGGAAGAAAT-3' and cGK I
-954 antisense:
5'-CAATCACAAGGCAGGTTACAG-3', or cGK I
-69 sense:
5'-ATGGGCACCTTGCGGGATTTA-3' and cGK I
-1269 antisense: 5'-TTTTGGATTCTTGACTTTTCA-3', resulting in PCR products of 872 nucleotides (cGK I
) and 1201 nucleotides (cGK I
), respectively. PCR amplification was performed using 40 cycles of 96 °C for 30 s, 62 °C for 30 s, and 72 °C for 1 min. Products were
analyzed by agarose gel electrophoresis.
-mercaptoethanol, 0.1% bovine serum albumin, and 50 µM ATP), in the absence or presence
of 0.5 µM PET-cGMP and 0.1 µg of purified recombinant
dephospho-VASP, at 30 °C for 30 min. Subsequently, samples were
processed for SDS-PAGE and Western blot analysis of VASP
phosphorylation as described below.
-mercaptoethanol, and 2% SDS and then
blocked in 5% nonfat dry milk (Bio-Rad) and incubated with anti-ERK1/2
or anti-p38 phosphorylation-independent antibodies, followed by ECL
detection (data not shown).
-expressing Jurkat Cells--
Jurkat
E6.1 cells (5 × 105/ml) were cotransfected with
pCMV-cGK I
(34) or with pCMV-cGK I
-K405A mutant (35), and
pCMV-EGFP (kindly provided by S. Schneider-Rasp) at a ratio of 3:2;
using Fugene-6 reagent (Roche Molecular Biochemicals). After 16 h
of transfection, cells were seeded at a density of 200,000 cells/well and subsequently incubated for 72 h in the presence of various concentrations of 8-pCPT-cGMP. Using flow cytometry, the impact of cGK
I expression and activation with 8-pCPT-cGMP on Jurkat cell
proliferation was determined. In the presence of 8-pCPT-cGMP, the ratio
of green fluorescent cells (e.g. containing cGK I and EGFP)
to nonfluorescent cells was calculated and divided by the same ratio
for cells exposed to medium only (non-8-pCPT-cGMP treated cells). This
gave a Jurkat proliferation ratio which was converted to percent.
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ABSTRACT
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DISCUSSION
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)
from T cells with adherent platelets
(CD3+/CD41a+). Western blot analysis using
polyclonal anti-cGK I antibody (Fig. 1A) detected cGK I in
unsorted as well as both of the FACS-sorted T cell fractions,
indicating that T cells devoid of platelets (CD41a
)
indeed contain endogenous cGK I. To estimate the abundance of cGK I
expression in T cells, signal intensities from FACS-sorted T cells were
compared with those observed in increasing amounts of platelet extracts
with a known content of cGK I (38.1 ± 3.9 pmol cGK
I/109 platelets, equivalent to 3 ng of cGK I
protein/106 cells (32). T cell-specific cGK I expression
was estimated at ~5 ng/106 T cells (Fig.
1A).
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Fig. 1.
Demonstration of endogenous cGK I expression
in freshly isolated, affinity-purified human T lymphocytes
(A and D) but not in T cell lines
(B) or cultured T lymphocytes
(C). A, T cell-specific endogenous cGK
I expression was estimated by comparison of T cell signals with
standards of increasing amounts of platelet extracts previously
demonstrated to contain 3 ng of cGK I/106 platelets (32). T
cells (unsorted) were purified from PBMC using anti-CD3-conjugated
Dynabeads. Pure T cells devoid of platelets
(CD3+/CD41 ), as well as a T cell fraction
with adherent platelets (CD3+/CD41+) were
separated using anti-CD3 and anti-CD41a antibodies and FACS sorting. T
cells (106 cells/lane) and purified platelets were added to
SDS gel loading buffer and examined for cGK I expression by Western
blot analysis (see "Experimental Procedures"). The data shown are
representative of three experiments. B, endogenous
expression of cGK I in CD8+ and CD4+ primary T
cells but not in transformed T cell lines Jurkat-E6 and A3.01
(106 cells/lane). T cell subtypes were purified using
anti-CD4 and anti-CD8-conjugated Dynabeads, and separation from
monocytes, B cells, and platelets was verified by FACS analysis (not
shown). cGK I antibody specificity was demonstrated by preabsorbing
(Preabs.) the antibody (Ab) with immobilized
recombinant cGK I protein prior to staining of a parallel Western blot
(lower panel in B). The data shown are
representative of four independent experiments. C, loss of
endogenous cGK I expression upon cultivation of purified T cells
in vitro. T cells, purified as described for A
above, were either freshly prepared (Fresh) or cultured
in vitro for 48 h (Cultured) for examination
(106 cells/lane) of endogenous cGK I expression. Shown is
an example of results obtained in three independent experiments. A
platelet-derived extract (Std) was used as a molecular
weight marker for identifying cGK I in B and C. D, reverse transcriptase-PCR demonstration of cGK I
but
not cGK I
mRNA in immunoaffinity column-purified T cells.
Reverse transcribed T cell RNA as well as positive control cDNA
standards (cGK I
and cGK I
) were PCR-amplified using cGK I
and
cGK I
specific primer pairs (see "Experimental Procedures"). A
control (ctrl) PCR reaction without template gave no
product.
and cGK I
specific primer pairs (see
"Experimental Procedures"). As shown in Fig. 1D, only
the cGK I
splice variant was detected in human T cells.
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Fig. 2.
Demonstration of cGK activity,
i.e. cGMP-dependent phosphorylation of
VASP, in lysates of freshly isolated, affinity- purified human T cells
(A) but not in cultured T lymphocytes
(B). Purified T cells either freshly isolated
(A) or cultured for 10 days in vitro
(B) were compared with purified platelets (C)
with respect to cGK I activity in in vitro assays. T cells
purified from PBMC using anti-CD3-conjugated Dynabeads or purified
platelets were lysed as described under "Experimental Procedures"
and then incubated in the absence (lanes 1-3 and
5) or presence (lanes 4 and 6) of 0.5 µM PET-cGMP for 30 min, and in some cases with 0.1 µg
exogenously added, purified recombinant dephospho-VASP (rVASP,
lanes 2, 5, and 6) to ensure
saturating concentrations of substrate. The phosphorylation of VASP, a
well established substrate of cGK, was subsequently determined using
monoclonal anti-phospho(Ser239)-VASP antibody (16C2) on
Western blots. Shown for T cell and platelet lysates (30 µg of
protein/lane) is the phosphorylation of VASP on Ser239,
either without (46-kDa VASP), or with (50-kDa VASP) concomitant
phosphorylation of Ser157. Phospho-VASP in samples was
identified using a standard (Std) of phosphorylated VASP
derived from extracts made from 8-pCPT-cGMP-stimulated platelets
(lane 1, A-C). The experiments shown were
performed five times with cell preparations from five different
donors.
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Fig. 3.
Analysis of VASP phosphorylation
demonstrating distinct characteristics of cGK I activation in freshly
isolated human PBMC versus platelets and loss of cGMP-
but not cAMP-dependent VASP phosphorylation in cultured
PBMC. PBMC either freshly isolated or cultured for 10 days
in vitro or isolated platelets were incubated in the absence
(control) or the presence of 100 µM SNP, 50 µM YC-1, SNP+YC-1, 200 µM PET-cGMP, or 500 µM cBIMPS, for 25 min (A) or the times
indicated (B). Cells immediately harvested in SDS gel
loading buffer were subsequently analyzed (30 µg of protein/lane) for
VASP phosphorylation using the monoclonal
anti-phospho(Ser239)-VASP antibody 16C2. Observed is the
phosphorylation of VASP on Ser239, either without (46-kDa
VASP) or with (50-kDa VASP) concomitant phosphorylation of
Ser157. Phospho-VASP was identified in lymphocyte samples
using a standard (Std) of phosphorylated VASP derived from
extracts made from 8-pCPT-cGMP-stimulated platelets. A, VASP
phosphorylation in response to elevation of cAMP was observed in both
fresh and cultured PBMC; however, in response to elevation of cGMP only
in fresh PBMC (cGK I is lost from cultured T cells, as shown in Fig.
1C). Note the absence of SNP-stimulated VASP phosphorylation
at the 25-min time point for fresh PBMC in A, a time at
which stimulation by SNP has already returned to base line in PBMC but
not platelets (see SNP time course in the description of B
below). Shown are data representative of those obtained with PBMC from
three different donors and platelets from two different donors.
B, time course of SNP- and YC-1-induced VASP phosphorylation
in freshly isolated PBMC and platelets. The data shown are
representative of three independent experiments with cells obtained
from different donors.
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Fig. 4.
cGMP-dependent activation (dual
phosphorylation) of ERK and p38 in immunoaffinity column-purified human
T lymphocytes. A, purified T cells
(106/lane) were incubated in serum-free medium in the
absence (ctrl) or presence of 200 µM PET-cGMP
or 8-pCPT-cGMP or 100 µM SNP for the times indicated and
then harvested immediately in SDS gel loading buffer for Western blot
analysis using polyclonal antibodies which recognize dual
phosphorylated, activated ERK, or p38 MAP kinases. The Western blots
shown are representative of three independent experiments.
B, purified T cells (106 cells/lane) were
incubated in serum-free medium in the absence or presence of the
indicated concentrations of 8-pCPT-cGMP for 15 min, followed by Western
blot analysis as described for A. Data are representative of
three independent determinations with cells from three different
donors.
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Fig. 5.
cAMP- and cGMP-dependent
inhibition of T cell receptor/CD3-induced proliferation of human
PBMC. A, the effect of preincubation of PBMC (2 × 105/well) in the absence ( ) or presence of 200 µM PET-cGMP (
) or 200 µM cBIMPS (
),
on 3-30 ng/ml anti-CD3 antibody (12F6)-stimulated PBMC proliferation
in medium containing 10% serum for 5 days (results shown as cell
number increase in A only). In B-D, the
inhibitory effect of PET-cGMP and cBIMPS on proliferation (expressed as
percentages of anti-CD3-stimulated proliferation) was compared with
those of SNP (
) and 8-pCPT-cGMP (
) on anti-CD3 (OKT3,
1:1000)-induced proliferation of freshly isolated PBMC (B),
PBMC cultured for 72 h prior to stimulation (C), and
Jurkat-E6 cells (D). In E, PBMC were costimulated
with anti-CD3 plus anti-CD28 (0.1 µg/ml) antibodies in the presence
of the indicated concentrations of 8-pCPT-cGMP. Data represent average
values ± S.E. of two (A), six (B), four
(C), three (D), and six (E)
determinations with cells obtained from different donors. In each
experiment, at least triplicate determinations of cell proliferation
were done for each condition.
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Fig. 6.
cGMP-dependent inhibition of IL-2
release in anti-CD3- stimulated human PBMC. PBMC (3 × 106/well) stimulated with anti-CD3 (1:2500) were incubated
with or without 8-pCPT-cGMP, and subsequently, IL-2 concentrations were
determined in supernatants using enzyme-linked immunosorbent assay. The
data represent the mean values ± S.E. of four independent
experiments with triplicate determinations performed on blood from
different donors. Data significantly differing from control values are
indicated with asterisks (p < 0.05, Student's paired t test).
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Fig. 7.
cGMP-dependent inhibition of
anti-CD3-stimulated proliferation of PBMC is diminished in the presence
of exogenously added IL-2. PBMC were incubated in the presence of
the indicated concentrations of 8-pCPT-cGMP, in the presence or absence
of 500 units/ml exogenously added recombinant human IL-2, and the
effect on anti-CD3-stimulated T cell proliferation was determined. The
data represent the mean values ± S.E. of five independent
experiments, each performed with triplicate samples.
Asterisks indicate statistically significant differences
between IL-2-treated and untreated samples at a given 8-pCPT-cGMP
concentration (p < 0.05, Student's paired
t test).
or an inactive cGK I
-mutant (cGK
I
K405A) were transfected into Jurkat cells, and, subsequently, the
number of cGK expressing cells were determined in the presence of
increasing concentrations of 8-pCPT-cGMP. As observed previously for
cGK I-deficient Jurkat cells in Fig. 5D, the proliferation of cGK I
-K405A-transfected, 8-pCPT-cGMP treated Jurkat cells was not
inhibited relative to that of similarly transfected,
non-8-pCPT-cGMP-treated cells (Fig. 8).
In contrast, the proliferation of cGK I
(wild type)-transfected,
8-pCPT-cGMP treated Jurkat cells was inhibited relative to that of the
corresponding non-8-pCPT-cGMP-treated cells, indicating that
cGMP-dependent inhibition of T cell proliferation can
indeed be reconstituted by cGK I
transfection.
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Fig. 8.
cGMP-dependent inhibition of T
cell proliferation is restored in Jurkat cells expressing cGK
I but not in those expressing the inactive
mutant cGK I
-K405A.
cGMP-dependent inhibition of cell proliferation was
determined in Jurkat-E6 cells transfected with either pCMV-cGK I
or
with inactive pCMV-cGK I
-K405A as described under "Experimental
Procedures." Data represent the average values of four independent
experiments each performed with triplicate samples ± S.E. The
proliferation of cells expressing wild type but not inactive cGK
I-K405A was significantly decreased by 100 and 200 µM
8-pCPT-cGMP (p < 0.05, Student's paired t
test).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
; however, neither cell lines such as
Jurkat and A3.01 nor cultured T cells retained cGK I. Thus, NO or cGMP
effects reported in such cells are unlikely to be mediated by cGK I. In
our experiments employing freshly isolated T cells, effects of NO and
cGMP analogs were clearly mediated by cGK I and not cAK because these
agents were unable to phosphorylate VASP in cultured PBMC that had lost
cGK I, but contained cAK that did phosphorylate VASP in response to the
cAMP analog cBIMPS. The pattern of VASP phosphorylation by cGK I in
response to SNP was particularly different in PBMC compared with
platelets. Whereas SNP-stimulated VASP phosphorylation persisted for
over 2 h in platelets, it lasted only about 5 min in lymphocytes,
suggesting the presence of a highly efficient cGMP phosphodiesterase in
lymphocytes. Experiments using other types of agonists gave results in
agreement with this. PET-cGMP (a hydrolysis-resistant analog), YC-1 (an activator of soluble guanylate cyclase but also an inhibitor of several
phosphodiesterases including ones that hydrolyze cGMP (36)), as well as
SNP plus YC-1, had much longer lasting effects than SNP alone.
but not
the inactive mutant cGK I
K405A, further demonstrating that the
observed inhibition of proliferation was indeed
cGK-dependent and not due to cross-activation of cAK.
cGK-mediated inhibition of cellular proliferation has also been
described for other cell types such as vascular smooth muscle cells
(45, 46) and cardiac fibroblasts (47). Furthermore, vector-mediated
overexpression of eNOS or cGK I has been reported to decrease neointima
proliferation in balloon catheter-damaged carotid arteries (48,
49).
is present only in freshly
purified T cells, that cGK I-dependent inhibition of T cell proliferation is clearly distinct from that which is cAMP mediated, that cGK I-dependent inhibition of T cell proliferation
results in part from inhibition of IL-2 release, and that not all NO
inhibitory effects on T cell proliferation are controlled by cGK I. cGK
I suppression of T cell proliferation may be an important independent adjunct to the inhibition mediated by cAMP and have implications for
diverse aspects of immune function.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank S. Rotzoll for performing the cell sorting experiments and I. Euler-König and U. Sauer for excellent technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by the Wilhelm Sander Stiftung and Deutsche Forschungsgemeinschaft Grant SFB 355/B4.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors equally contributed to this work.
To whom correspondence should be addressed: Institut für
Klinische Biochemie und Pathobiochemie, Medizinische
Universitaetsklinik, Josef-Schneider Str. 2, D-97080 Wuerzburg,
Germany. Tel.: 49-931-201-2782; Fax: 49-931-201-3137; E-mail:
palme@klin-biochem.uni-wuerzburg.de.
Published, JBC Papers in Press, November 9, 2000, DOI 10.1074/jbc.M009781200
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ABBREVIATIONS |
---|
The abbreviations used are:
TCR, T cell
receptor;
cAK, cAMP-dependent protein kinase;
cBIMPS, Sp-5,6-dichlorobenzimidazole riboside-3',5'-cyclic
monophosphorothioate;
8-pCPT-cGMP, 8-(4-chlorophenylthio) guanosine-
3',5'-cyclic monophosphate;
cGK, cGMP-dependent protein
kinase;
ERK, extracellular signal-related kinase;
FACS, fluorescence
activated cell sorting;
MAP, mitogen-activated protein;
PET-cGMP, -phenyl-1,N2-ethenoguanosine-3',5'-cyclic monophosphate;
PBMC, peripheral blood mononuclear cells;
VASP, vasodilator stimulated
phosphoprotein;
and YC-1, 3-(5'-hydroxymethyl-2'furyl)-1-benzylindazol;
IL, interleukin;
SNP, sodium nitroprusside;
PCR, polymerase chain
reaction.
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