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TVW Telethon Institute for Child Health Research, Divisions of Cell Biology and Children’s Leukaemia and Cancer Research, West Perth, Australia; and
Royal Perth Hospital, U.W.A., Department of Biochemistry, Laboratory of Cancer Medicine, Perth, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The association between hyperexpression of iNOS and immunoinflammatory diseases appears particularly marked at mucosal tissue sites, especially in the respiratory tract in which NO has been invoked as a major pathogenic factor in a variety of diseases including viral pneumonitis (10), pertussis (11), bronchiectasis (12), and atopic asthma (13).
However, despite its potential for tissue damage, NO also appears to
contribute to the maintenance of immunologic homeostasis, by virtue of
its role in the well known phenomenon of T cell suppression by
"activated" macrophages. The archetypal example is the alveolar
macrophage (AM), which plays a key role in the down-regulation of T
cell activation in the lung (14, 15). The expression of the T cell
inhibitory activity by AM is not constitutive, but requires an initial
inductive signal from T lymphoblasts (14). The latter has been
identified as IFN-
(16), which, particularly in combination with
IL-2 and/or TNF-
(17, 18), triggers NO production by AM, thus
shutting off further T cell activation via a classical feedback
inhibitory loop. There is also indirect in vitro (19) and in vivo (20, 21) evidence that Th1 cells may be more susceptible than Th2 cells to
the inhibitory effects of NO, suggesting that this regulatory mechanism
may in some circumstances modulate host responses via immune
deviation.
Recent studies have revealed further complexities in this system. Notably, analysis of the surface and functional phenotype of T cells during exposure to specific Ag or mitogen in the presence of AM indicates that activation initially proceeds normally, up to and including IL-2 secretion and expression of IL-2R (22). However, these T cells are unable to respond to IL-2 and remain locked in the G0/G1 phase of the cell cycle until the source of NO is removed, after which they proliferate normally (23).
The present study sought to elucidate the molecular mechanisms
underlying suppression of T cell proliferation by macrophage-derived
NO, focusing on activation events downstream of IL-2R expression. One
of the known signaling pathways connecting the IL-2R and the
transcription machinery of T cells involves the IL-2R -chain, the
Janus kinase 3 (Jak3), and the signal transducer and activator of
transcription 5 (STAT5) (reviewed in 24 . STAT5 activation in T
cells requires the action of tyrosine kinases such as Jak3 and possibly
a yet unknown serine/threonine kinase (25). The activated STAT5 then
forms homodimers, in which the phosphorylated tyrosine residue of each
STAT5 interacts with the src homology (SH2) domain of its partner and
they comigrate to the nucleus, thereafter functioning as transcription
factors (26).
To determine whether NO secreted by macrophages can interfere with this signaling pathway, we analyzed the tyrosine-phosphorylation of Jak3 and STAT5 in mitogen-stimulated rodent T cells in coculture with AM. Our results indicate that the signaling pathway via Jak3/STAT5 is reversibly down-regulated through inhibition of tyrosine phosphorylation in the presence of NO.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inbred, specific pathogen-free female PVG rats aged 8 to 12 wk, maintained under barrier conditions, were employed in the series of experiments described below.
Cell preparation and culture
Methods for preparation of single cell suspensions from lymph nodes (LN) and spleens, collection of AM by bronchoalveolar lavage, and collection of peritoneal macrophages (PM) were as previously described (23). In experiments involving in vitro mitogenic T cell activation, splenic or mesenteric LN T cells were cultured at 106/700 µl in Costar Transwells (No. 3413 from Costar, Cambridge, MA). Cells were cultured in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with L-glutamine, 5% FCS (Trace Biosciences, Chapelhill, NSW, Australia) and stimulated with 5 µg/ml Con A. AM or PM were present in the bottom part of the Transwell or were added directly to the T cells at the indicated concentrations. For studies involving immunoprecipitation, the T cells were cultured in Transwells No. 3412 (5 x 106/3.5 ml) in the presence or absence of AM or PM in the bottom chamber (1.6 x 107/3.5 ml). For proliferation studies, DNA synthesis was measured at 72 h (unless otherwise indicated) via incorporation of [3H]thymidine (Amersham; Little Chalfont, England, sp. act. 5 Ci/mmol) over a 12-h pulse-labeling period; the results were initially expressed as mean disintegrations per minute per culture, and then normalized against control cultures containing T cells plus Con A alone.
In experiments examining the reversibility of AM-induced anergy, the Transwell containing the T cells was transferred into a new well containing fresh medium in the absence of Con A and AM.
Immunoprecipitation and immunoblotting
T cells were cultured in the presence or absence of AM or PM as described above. At the time points indicated, the T cells were washed in PBS and pelleted. Ice-cold lysis buffer was added at 100 µl/107 cells (containing 1 mM sodium orthovanadate, 2 mM EDTA, 10 mM iodoacetamide, 25 µg/ml p-nitrophenyl-guanidinobenzoate, 10 µg/ml leupeptin, 10 µg/ml aprotinin (Sigma, St. Louis, MO), and 0.5% Triton X-100 (Pharmacia, Uppsala, Sweden). Cells were vortexed and left on ice for 15 min. The lysates were cleared by centrifugation at 14,000 x g at 4°C for 15 min. The total protein content was measured using the protein assay from Bio-Rad (Hercules, CA). For phosphotyrosine immunoblots, an equal amount of protein lysate (50 µg) was used for each sample. Proteins (1 mg per lysate) were immunoprecipitated with 0.5 µg of anti-Jak3 (sc-1079, Santa Cruz Biotechnology, Santa Cruz, CA) or anti-STAT5b (sc-835, Santa Cruz Biotechnology) Ab and 10 µl of agarose-conjugated protein G (sc-2002, Santa Cruz Biotechnology). After incubation on a rocker at 4°C overnight, the immunoprecipitates were collected by centrifugation, washed three times with lysis buffer, and boiled in 3.3 x sample buffer (0.067 M Tris-HCl, 13% glycerol, 50 mM DTT, 1.3% w/v SDS, 0.0025% bromphenol blue, and 2% 2-ME; Sigma).
Samples were electrophoresed on a 10% SDS-polyacrylamide (Bio-Rad) gel and electroblotted to a Hybond-C Super membrane (Amersham). After blocking in 2% BSA at 4°C overnight, the membranes were washed in Tris-buffered saline (20 mM Tris, 150 mM NaCl) with 0.05% Tween and 0.1 mM Na3VO4 and incubated with 1 µg/ml anti-phosphotyrosine (4G10, 05-321, Upstate Biotechnology, Lake Placid, NY) or 1 µg/ml anti-Jak3 or anti-STAT5b Ab for 1 h at room temperature. Horseradish peroxidase (HRP)-conjugated second Abs (anti-mouse IgG-HRP, sc-2005; anti-rabbit IgG-HRP, sc-2004; and anti-goat IgG-HRP, sc-2020) were used for 1 h at a concentration suggested by Santa Cruz Biotechnology. After final extensive washing, the membranes were developed by the enhanced chemiluminescence method (ECL, Amersham) according to the manufacturer’s directions.
The amount of tyrosine phosphorylation was quantitated using a Kodak
digital camera in conjunction with Adobe Photoshop and NIH Image. For
Figure 3
, levels were adjusted for the amount of Jak3 or STAT5
immunoprecipitated.
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The decomposition of NO in solution produced stable anionic nitrite and nitrate ions in a molar ratio of 3:2, and this was measured using a colorimetric assay based on the Griess reaction. Samples (50 µl) were added to a microtiter plate containing 50 µl of 1% sulfanilamide and 0.1% naphthylethylenediamine in 2.5% H3PO4. After 10 min, the OD was measured at 550 nm and compared with a standard curve of NaNO3.
Other reagents
NG-methyl-L-arginine (NMMA) and S-nitroso-N-acetylpenicillamine (SNAP) were obtained from Molecular Probes (Eugene, OR). 8-Bromoguanosine-3'-5'-cyclic monophosphate (bromo-cyclic GMP) and methylene blue were purchased from Sigma. LY-83583 (440205-Q) was obtained from Calbiochem (San Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Macrophages from different sites have been shown to suppress T
cell proliferation. When cultured in direct contact with T cells, only
half the number of AM compared with PM were required to suppress T cell
proliferation by 50% (Fig. 1A). Cell viabilities at the
end of the incubation period were 
95% in both control and
macrophage-containing cultures, indicating that cytotoxic mechanisms
are not central to the suppression observed. The most effective
suppression of T cell proliferation was achieved by direct contact of
the two cell types. However, suppression could also be achieved across
a Transwell membrane, but a 222-fold higher AM:T cell ratio was
required. A ratio of > 1:1 was needed for a 50% suppression
across the Transwell membrane, while the same suppression was obtained
by an AM/T cell ratio of <1:100 if the two cell types were in direct
contact (Fig. 1B). This suppressive effect could be
inhibited by the NO-synthase inhibitor NMMA whether the cells were in
direct contact or not (Fig. 1B), indicating that the main
mechanism for the suppression of T cell proliferation by macrophages
was NO. Dose-dependent suppression of T cell proliferation was also
obtained using the NO-releasing compound SNAP (50–1000 µM), the 50%
suppression being at 300 µM SNAP (Fig. 1C).
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Because a common signaling pathway used by growth factor receptors
is the rapid induction of intracellular tyrosine kinase activity, we
studied the pattern of protein tyrosine phosphorylation in
mitogen-activated T cells in the presence or absence of AM. Figure 2A shows an immunoblot of
total cell extracts stained with anti-phosphotyrosine Ab
demonstrating increasing tyrosine phosphorylation of several proteins
16 h after mitogenic stimulation of T cells, and remaining
unchanged at 20 and 24 h. In the presence of AM (Fig. 2B) the same increase in tyrosine phosphorylation at 16
h was observed. However, at 20 and 24 h of coculture,
dephosphorylation of tyrosine-phosphorylated proteins in
mitogen-activated T cells was observed (Fig. 2B,
top). Protein tyrosine dephosphorylation due to the action
of AM most clearly occurred in proteins with molecular weights of 123,
96, 59, 56, and 42 kDa, respectively (* in Fig. 2B,
top). In follow-up time course experiments involving Con A
activation of T cells in the presence of 1 mm SNAP (not shown), protein
tyrosine dephosphorylation was observed with a delay of 6 to 10 h
after culture initiation.
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As shown in the bottom part of Figure 2
A, no
substantial nitrite (end product of NO) was produced by Con
A-stimulated T cells. There was a delay of 16 h until AM produced
nitrite at concentrations above 25 µM (Fig. 2B). This may
be due to the time necessary for IFN- production by the
mitogen-activated T cells in the coculture, which is the key signal for
iNOS activation in macrophages. The observed NO production inversely
correlates with the level of protein tyrosine phosphorylation detected
by immunoblotting shown in Figure 2B.
Activation status of Jak3 and STAT5 after mitogenic stimulation with and without the influence of NO
Because of our previous findings that T cells under control of AM
retain normal IL-2 secretion and IL-2R expression (23) and the observed
time lag of 20 h between stimulation and tyrosine
dephosphorylation in the T cells under control of AM (Fig. 2B, top), we hypothesized that the block in the
proliferative signal was to be found in the IL-2R signaling. We chose
to examine the functional status of Jak3 and STAT5 for two reasons.
First, the IL-2R is known to interact with the Jak3/STAT5-signaling
pathway (24). Second, the molecular weights of Jak3 (123 kDa) and STAT5
(96 kDa) match two of the tyrosine-containing proteins dephosphorylated
in the presence of AM (Fig. 2B, top).
Con A-activated T cells showed a clear proliferative response (Fig. 3, top) and increased tyrosine
phosphorylation of Jak3 (Fig. 3A) and STAT5 (Fig. 3B) after 24 h of culture. The effects of AM and SNAP
as donors of NO were examined using this experimental system. AM were
used at 3.2 times the T cell number and SNAP at 1 mM, both yielding a
95% suppression of the T cell proliferation (Fig. 1). AM and SNAP
again resulted in a 95% suppression of the T cell proliferation
compared with Con A stimulation (Fig. 3, top). This effect
was paralleled by decreased tyrosine phosphorylation of Jak3 and STAT5.
The addition of the NO synthase inhibitor NMMA to AM-containing
cultures completely restored the T cell proliferation, as well as the
tyrosine phosphorylation of Jak3 and STAT5. We conclude that the
inhibition of T cell proliferation is due to NO, regardless of its
origin, and that NO can reduce tyrosine phosphorylation of Jak3 and
STAT5, thereby inactivating this signaling pathway.
Mechanism of NO-induced disruption of signaling
To find a mechanism for the NO-induced inactivation of the Jak3/STAT5 signaling pathway, we tested two pathways. First, tyrosine nitrosylation has been described to prevent tyrosine phosphorylation in vitro (27), but we were unable to detect any difference in tyrosine nitrosylation of Jak3 or STAT5 in the presence or absence of AM using a monoclonal anti-nitrotyrosine Ab (data not shown).
Second, activation of guanylate cyclase by NO has been described in
smooth muscle cells, and many of the effects of NO in this cell type
appear to be mediated by cGMP (28). We therefore tested the effect of
bromo-cGMP, a cell-permeable analogue of cGMP on our T cells. It
clearly induced a dose-dependent suppression of T cell proliferation
(Fig. 4A), demonstrating that
T cells are susceptible to enhanced levels of cGMP. Most importantly,
the guanylate cyclase inhibitors (MB and LY) were able to reverse the
inhibition of T cell proliferation by SNAP, indicating that the
activation of guanylate cyclase by NO is a key step in its inhibitory
action on T cell proliferation (Fig. 4B).
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Previous work has demonstrated that after removal of AM from "suppressed" T cell cultures, the T cells regain their ability to proliferate normally (23). Additionally, in situ depletion of AM from rats markedly increased the capacity of lung-derived T cells to respond in vitro to polyclonal T cell mitogens (29). These results suggest that the anergy induced by AM is reversible.
Based on the findings of the present study, we accordingly hypothesized
that the tyrosine phosphorylation of the crucial signaling molecules
would be restored after the removal of the anergy-inducing AM. Figure 5A shows the suppressed T cell
proliferation rate at 24 h and the corresponding dephosphorylation
of most protein tyrosines at that time point. One day later, parallel T
cell cultures, which were removed from the suppressive AM after 24
h, (Fig. 5B) demonstrate a recovery of >70% of the
proliferation rate compared with the control Con A stimulation.
Equally, the level of the protein tyrosine phosphorylation was
restored, in particular with respect to proteins of
Mr 123 kDa and 96 kDa (indicated by * in the
bottom section of Fig. 5B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results show a clear relationship between the reduced
phosphorylation of tyrosine residues in Jak3/STAT5 and the suppression
of proliferation observed (Fig. 3). Both effects were readily reversed
by removal of NO from the system, either by blocking the NO-synthase
(Fig. 3) or by removing the NO-producing AM from the coculture (Fig. 5). We therefore concluded that NO is able to induce a reversible type
of T cell anergy by reducing phosphorylation of tyrosine residues on
Jak3 and STAT5.
The mechanism of the NO-induced signal transduction block appears to be
via activation of guanylate cyclase (Fig. 4), leading to higher levels
of cGMP, which in turn may influence a range of mechanisms such as the
activation of cGMP-dependent protein kinases (PKG), phosphodiesterases,
ion channels (33), or even a "cross-over" activation of
cAMP-dependent kinases (PKA) due to inordinately high concentrations of
NO and, consequently, cGMP (34). Other mechanisms (discussed below) may
also contribute to the NO-induced T cell anergy but the reversal of the
NO-induced T cell suppression by the guanylate-cyclase inhibitors MB
and LY at high (250 µM) SNAP concentrations (Fig. 4B)
clearly demonstrates the involvement of cGMP. NO may react with other
macrophage products, such as superoxide, to form highly toxic radicals
such as peroxynitrite (35), leading to tyrosine nitrosylation, thus
preventing tyrosine phosphorylation and signal transduction (27). This
mechanism appears highly unlikely in our system, since we could not
detect any difference in the tyrosine nitrosylation of Jak3 or STAT5
with or without AM in the coculture. Another possible mechanism is the
S-nitrosylation of proteins by nitrosonium (NO+) which
reacts with protein SH groups. This mechanism is responsible for the
inhibition of glutamate uptake and proton pumping in synaptic
vesicles (36).
The precise temporal relationship between the onset of NO production
and the subsequent effects on PTKs are not clear from these
experiments. However, our finding that PTK dephosphorylation in Con
A-activated T cells was delayed at least 6 to 10 h after the
addition of the rapid NO generator SNAP indicates that factors other
than NO concentration per se are rate limiting in the process. Indeed,
the interference of macrophage-derived NO with PTKs, which are normally
activated after mitogenic stimulation, does not seem to occur until
after IL-2 has been transcribed and IL-2Rs have bound the secreted
IL-2. This conclusion is based on two independent lines of evidence.
First, previous findings in our laboratory demonstrate that IL-2
secretion is not diminished to a significant extent by the presence of
AM (15, 23) and the binding of rIL-2 to IL-2R on T cells was comparable
in the presence and absence of AM (23). Second, from the present study,
the time course experiments (Fig. 3A) demonstrate comparable
levels of tyrosine phosphorylation of several proteins in T cells at
16 h in culture, regardless of the presence of AM. Decreasing
tyrosine phosphorylation occurred only after 20 to 24 h of
coculture with AM, allowing the secretion of IL-2 and expression of
IL-2Rs. At this time, however, DNA synthesis is not yet detectable in
Con A-stimulated T cells (23). These findings extend our previous
results (37), demonstrating that T cells under the influence of NO
remain locked in G0/G1 phase. In this respect,
the actions of NO show similarities with the actions of rapamycin, as
inhibition of cell proliferation by rapamycin and NO cannot be overcome
by addition of exogenous IL-2 (15) and the proliferation block occurs
before G1/S transition (37, 38) in both systems.
Our findings raise the question of whether NO specifically inactivates
the Jak3/STAT5 pathway in Con A-activated T cells. The phosphotyrosine
immunoblots (Fig. 2) show that, in addition to proteins of
Mr 123 and 96 kDa (presumably Jak3 and STAT5),
proteins of 59, 56, and 42 kDa lost their tyrosine phosphorylation
after prolonged (over 16 h) action of NO, indicating that other
known pathways in IL-2R signaling, such as the MAP kinases
(Mr 42 kDa), may well be affected in a similar
fashion: p56lck (39) and p59fyn (40) are physically associated with the
IL-2R ß-chain during activation, and lck in association with MAP
kinase is suggested to be directly involved in the control of
IL-2-mediated DNA synthesis of T cells (41). However, additional
experiments are necessary to examine the involvement of NO in the
inactivation of the MAP kinase pathway, and the possibility that the
reduction in tyrosine phosphorylation observed is due to the action of
cGMP-dependent phosphatase.
Our results on the reversible inhibition of tyrosine phosphorylation of
Jak3/STAT5 in T cells by AM-derived or chemically generated NO have
potentially important implications in relation to our understanding of
the role of NO in the pathogenesis of inflammatory diseases. The
current literature emphasizes the role of NO as a mediator of tissue
damage at inflammatory foci. However, the present study, taken together
with recent reports on the functional phenotype of T cells undergoing
activation in the presence of NO (29), suggests further possible
functions. Thus, while T cells activated in the presence of NO are
unable to proceed through the cell cycle, they nevertheless secrete
essentially normal amounts of cytokines, and retain their capacity for
clonal expansion if the NO source is removed (23) (Fig. 5). This
process provides a subtle mechanism to limit the contribution of
individual T cells to inflammatory responses at sites of NO generation
to a "single hit," while preserving their capacity to contribute to
T memory generation after subsequent emigration to regional lymph
nodes.
It is noteworthy in this context that NO is produced by a variety
of cell types other than macrophages, including neutrophils (42), some
T cells (43), and epithelial cells at sites of inflammation (13).
Moreover, NO production, particularly by macrophages, is subject to
down-regulation by a variety of cytokines including IL-4 (44), TGF-ß
(45), and the combination of granulocyte-macrophage-CSF and TNF-
(46), providing further opportunities for fine tuning of T cell
responses at sites of Ag challenge. Thus, analogous to a growing list
of secreted cell products that influence immunoinflammatory responses,
the net effects of NO are likely to be variable and dependent upon such
factors as microenvironmental concentration and stage of the
inflammatory process at the time of release.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Patrick G. Holt, TVW Telethon Institute for Child Health Research, PO Box 855, West Perth, Western Australia 6872.
3 Abbreviations used in this paper: NO, nitric oxide; iNOS, inducible form of the enzyme NO synthase; AM, alveolar macrophage; Jak3, Janus kinase 3; PM, peritoneal macrophages; HRP, horseradish peroxidase; NMMA, NG-methyl-L-arginine; SNAP, S-nitroso-N-acetylpenicillamine; PTK, protein tyrosine kinase; MAP, mitogen-activated protein; cGMP, cyclic guanylic acid; LN, lymph nodes.
Received for publication June 30, 1997. Accepted for publication February 9, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in the induction of the nitric oxide-synthesizing pathway. J. Immunol. 147:144.[Abstract]
-dependent production of reactive nitrogen intermediates mediates IFN- plus IL-2-induced murine macrophage tumoricidal activity. J. Immunol. 149:3290.[Abstract]
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D. Hongo, J. S. Bryson, A. M. Kaplan, and D. A. Cohen Endogenous Nitric Oxide Protects against T Cell-Dependent Lethality during Graft-versus-Host Disease and Idiopathic Pneumonia Syndrome J. Immunol., August 1, 2004; 173(3): 1744 - 1756. [Abstract] [Full Text] [PDF]
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 H.-O. Pae, B.-M. Choi, G.-S. Oh, M.-S. Lee, D.-G. Ryu, H.-Y. Rhew, Y.-M. Kim, and H.-T. Chung Roles of Heme Oxygenase-1 in the Antiproliferative and Antiapoptotic Effects of Nitric Oxide on Jurkat T Cells Mol. Pharmacol., July 1, 2004; 66(1): 122 - 128. [Abstract] [Full Text] [PDF]
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 C. Hucke, C. R. MacKenzie, K. D. Z. Adjogble, O. Takikawa, and W. Daubener Nitric Oxide-Mediated Regulation of Gamma Interferon-Induced Bacteriostasis: Inhibition and Degradation of Human Indoleamine 2,3-Dioxygenase Infect. Immun., May 1, 2004; 72(5): 2723 - 2730. [Abstract] [Full Text] [PDF]
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S. E. Macphail, C. A. Gibney, B. M. Brooks, C. G. Booth, B. F. Flanagan, and J. W. Coleman Nitric Oxide Regulation of Human Peripheral Blood Mononuclear Cells: Critical Time Dependence and Selectivity for Cytokine versus Chemokine Expression J. Immunol., November 1, 2003; 171(9): 4809 - 4815. [Abstract] [Full Text] [PDF]
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R. S. Mahidhara, R. A. Hoffman, S. Huang, A. Wolf-Johnston, Y. Vodovotz, R. L. Simmons, and T. R. Billiar Nitric oxide-mediated inhibition of caspase-dependent T lymphocyte proliferation J. Leukoc. Biol., September 1, 2003; 74(3): 403 - 411. [Abstract] [Full Text] [PDF]
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M. Dupuis, M. de Jesus Ibarra-Sanchez, M. L. Tremblay, and P. Duplay Gr-1+ Myeloid Cells Lacking T Cell Protein Tyrosine Phosphatase Inhibit Lymphocyte Proliferation by an IFN-{gamma}- and Nitric Oxide-Dependent Mechanism J. Immunol., July 15, 2003; 171(2): 726 - 732. [Abstract] [Full Text] [PDF]
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C. Guillot, S. Menoret, C. Guillonneau, C. Braudeau, M. G. Castro, P. Lowenstein, and I. Anegon Active suppression of allogeneic proliferative responses by dendritic cells after induction of long-term allograft survival by CTLA4Ig Blood, April 15, 2003; 101(8): 3325 - 3333. [Abstract] [Full Text] [PDF]
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C. Paolucci, S. E. Burastero, P. Rovere-Querini, C. De Palma, S. Falcone, C. Perrotta, A. Capobianco, A. A. Manfredi, and E. Clementi Synergism of nitric oxide and maturation signals on human dendritic cells occurs through a cyclic GMP-dependent pathway J. Leukoc. Biol., February 1, 2003; 73(2): 253 - 262. [Abstract] [Full Text] [PDF]
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V. Bronte, P. Serafini, C. De Santo, I. Marigo, V. Tosello, A. Mazzoni, D. M. Segal, C. Staib, M. Lowel, G. Sutter, et al. IL-4-Induced Arginase 1 Suppresses Alloreactive T Cells in Tumor-Bearing Mice J. Immunol., January 1, 2003; 170(1): 270 - 278. [Abstract] [Full Text] [PDF]
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 S. Blesson, J. Thiery, C. Gaudin, R. Stancou, J.-P. Kolb, J.-L. Moreau, J. Theze, F. Mami-Chouaib, and S. Chouaib Analysis of the mechanisms of human cytotoxic T lymphocyte response inhibition by NO Int. Immunol., October 1, 2002; 14(10): 1169 - 1178. [Abstract] [Full Text] [PDF]
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A. Rachlis, J. L. Watson, J. Lu, and D. M. McKay Nitric oxide reduces bacterial superantigen-immune cell activation and consequent epithelial abnormalities J. Leukoc. Biol., August 1, 2002; 72(2): 339 - 346. [Abstract] [Full Text] [PDF]
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 A. Moraska, J. Campisi, K. T. Nguyen, S. F. Maier, L. R. Watkins, and M. Fleshner Elevated IL-1beta contributes to antibody suppression produced by stress J Appl Physiol, July 1, 2002; 93(1): 207 - 215. [Abstract] [Full Text] [PDF]
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K.-J. Malmberg, R. Lenkei, M. Petersson, T. Ohlum, F. Ichihara, B. Glimelius, J.-E. Frodin, G. Masucci, and R. Kiessling A Short-Term Dietary Supplementation of High Doses of Vitamin E Increases T Helper 1 Cytokine Production in Patients with Advanced Colorectal Cancer Clin. Cancer Res., June 1, 2002; 8(6): 1772 - 1778. [Abstract] [Full Text] [PDF]
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 N. L. Harris, V. Watt, F. Ronchese, and G. Le Gros Differential T Cell Function and Fate in Lymph Node and Nonlymphoid Tissues J. Exp. Med., February 4, 2002; 195(3): 317 - 326. [Abstract] [Full Text] [PDF]
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A. Mazzoni, V. Bronte, A. Visintin, J. H. Spitzer, E. Apolloni, P. Serafini, P. Zanovello, and D. M. Segal Myeloid Suppressor Lines Inhibit T Cell Responses by an NO-Dependent Mechanism J. Immunol., January 15, 2002; 168(2): 689 - 695. [Abstract] [Full Text] [PDF]
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E. A. Patton, A. C. La Flamme, J. A. Pedras-Vasoncelos, and E. J. Pearce Central Role for Interleukin-4 in Regulating Nitric Oxide-Mediated Inhibition of T-Cell Proliferation and Gamma Interferon Production in Schistosomiasis Infect. Immun., January 1, 2002; 70(1): 177 - 184. [Abstract] [Full Text] [PDF]
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J. Wang, R. Charboneau, S. Balasubramanian, R. A. Barke, H. H. Loh, and S. Roy Morphine modulates lymph node-derived T lymphocyte function: role of caspase-3, -8, and nitric oxide J. Leukoc. Biol., October 1, 2001; 70(4): 527 - 536. [Abstract] [Full Text] [PDF]
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K.-J. Malmberg, V. Arulampalam, F. Ichihara, M. Petersson, K. Seki, T. Andersson, R. Lenkei, G. Masucci, S. Pettersson, and R. Kiessling Inhibition of Activated/Memory (CD45RO+) T Cells by Oxidative Stress Associated with Block of NF-{kappa}B Activation J. Immunol., September 1, 2001; 167(5): 2595 - 2601. [Abstract] [Full Text] [PDF]
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J. L. Jiménez, J. González-Nicolás, S. Alvarez, M. Fresno, and M. A. Muñoz-Fernández Regulation of Human Immunodeficiency Virus Type 1 Replication in Human T Lymphocytes by Nitric Oxide J. Virol., May 15, 2001; 75(10): 4655 - 4663. [Abstract] [Full Text]
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 A. Sydbom, A. Blomberg, S. Parnia, N. Stenfors, T. Sandstrom, and S-E. Dahlen Health effects of diesel exhaust emissions Eur. Respir. J., April 1, 2001; 17(4): 733 - 746. [Abstract] [Full Text] [PDF]
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M. Fleshner, T. Deak, K. T. Nguyen, L. R. Watkins, and S. F. Maier Endogenous Glucocorticoids Play a Positive Regulatory Role in the Anti-Keyhole Limpet Hemocyanin In Vivo Antibody Response J. Immunol., March 15, 2001; 166(6): 3813 - 3819. [Abstract] [Full Text] [PDF]
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C. Tang, M. D. Inman, N. van Rooijen, P. Yang, H. Shen, K. Matsumoto, and P. M. O'Byrne Th Type 1-Stimulating Activity of Lung Macrophages Inhibits Th2-Mediated Allergic Airway Inflammation by an IFN-{{gamma}}-Dependent Mechanism J. Immunol., February 1, 2001; 166(3): 1471 - 1481. [Abstract] [Full Text] [PDF]
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 U. Eriksson, M. O. Kurrer, R. Bingisser, H. P. Eugster, P. Saremaslani, F. Follath, S. Marsch, and U. Widmer Lethal Autoimmune Myocarditis in Interferon-{{gamma}} Receptor-Deficient Mice : Enhanced Disease Severity by Impaired Inducible Nitric Oxide Synthase Induction Circulation, January 2, 2001; 103(1): 18 - 21. [Abstract] [Full Text] [PDF]
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 P. G. HOLT Antigen Presentation in the Lung Am. J. Respir. Crit. Care Med., October 1, 2000; 162(4): S151 - 156. [Abstract] [Full Text] [PDF]
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C. A. JONES and P. G. HOLT Immunopathology of Allergy and Asthma in Childhood Am. J. Respir. Crit. Care Med., August 1, 2000; 162(2): S36 - 39. [Full Text] [PDF]
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M. M. Gherardi, J. C. Ramírez, and M. Esteban Interleukin-12 (IL-12) Enhancement of the Cellular Immune Response against Human Immunodeficiency Virus Type 1 Env Antigen in a DNA Prime/Vaccinia Virus Boost Vaccine Regimen Is Time and Dose Dependent: Suppressive Effects of IL-12 Boost Are Mediated by Nitric Oxide J. Virol., July 15, 2000; 74(14): 6278 - 6286. [Abstract] [Full Text]
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M. G. Espey, K. M. Miranda, R. M. Pluta, and D. A. Wink Nitrosative Capacity of Macrophages Is Dependent on Nitric-oxide Synthase Induction Signals J. Biol. Chem., April 6, 2000; 275(15): 11341 - 11347. [Abstract] [Full Text] [PDF]
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P. G. HOLT Key Factors in the Development of Asthma: Atopy Am. J. Respir. Crit. Care Med., March 1, 2000; 161(3): S172 - 175. [Full Text] [PDF]
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I. Angulo, F. G. de las Heras, J. F. Garcia-Bustos, D. Gargallo, M. A. Munoz-Fernandez, and M. Fresno Nitric oxide-producing CD11b+Ly-6G(Gr-1)+CD31(ER-MP12)+ cells in the spleen of cyclophosphamide-treated mice: implications for T-cell responses in immunosuppressed mice Blood, January 1, 2000; 95(1): 212 - 220. [Abstract] [Full Text] [PDF]
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L. R. Brunet, M. Beall, D. W. Dunne, and E. J. Pearce Nitric Oxide and the Th2 Response Combine to Prevent Severe Hepatic Damage During Schistosoma mansoni Infection J. Immunol., November 1, 1999; 163(9): 4976 - 4984. [Abstract] [Full Text] [PDF]
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S.-C. Lee, Z. H. Jaffar, K.-S. Wan, S. T. Holgate, and K. Roberts Regulation of Pulmonary T Cell Responses to Inhaled Antigen: Role in Th1- and Th2-Mediated Inflammation J. Immunol., June 1, 1999; 162(11): 6867 - 6879. [Abstract] [Full Text] [PDF]
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 A. Diefenbach, H. Schindler, M. Röllinghoff, W. M. Yokoyama, and C. Bogdan Requirement for Type 2 NO Synthase for IL-12 Signaling in Innate Immunity Science, May 7, 1999; 284(5416): 951 - 955. [Abstract] [Full Text]
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H. K. Koblish, C. A. Hunter, M. Wysocka, G. Trinchieri, and W. M.F. Lee Immune Suppression by Recombinant Interleukin (rIL)-12 Involves Interferon gamma Induction of Nitric Oxide Synthase 2 (iNOS) Activity: Inhibitors of NO Generation Reveal the Extent of rIL-12 Vaccine Adjuvant Effect J. Exp. Med., November 2, 1998; 188(9): 1603 - 1610. [Abstract] [Full Text] [PDF]
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T. A. Fischer, A. Palmetshofer, S. Gambaryan, E. Butt, C. Jassoy, U. Walter, S. Sopper, and S. M. Lohmann Activation of cGMP-dependent Protein Kinase Ibeta Inhibits Interleukin 2 Release and Proliferation of T Cell Receptor-stimulated Human Peripheral T Cells J. Biol. Chem., February 16, 2001; 276(8): 5967 - 5974. [Abstract] [Full Text] [PDF]
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