| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
University Medicine, Southampton General Hospital, Southampton, United Kingdom
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. Such
immune regulation was tissue-specific, because T cell responses in the
lymph nodes and spleens were normal. This dramatic aerosol-induced
attenuation of parenchymal T cell proliferation was also observed in
BALB/c mice immunized with OVA and in BALB/c mice following adoptive
transfer of DO11.10 T cells bearing either a Th1 or Th2 phenotype. In
mice that had received Th2 cells, the reduced proliferative responses
were associated with a decrease in IL-2 expression but augmented IL-4
and IL-5 production. Invariably, the inhibition of proliferation was a
consequence of the action of F4/80+ interstitial
macrophages and did not involve alveolar macrophages or their products.
These observations demonstrate that clonal expansion of T cells in the
lung compartment is prevented following the onset of either Th1- or
Th2-mediated inflammation. This form of immune regulation, which
appears as a selective defect in IL-2-driven proliferation, may serve
to prevent the development of chronic pulmonary lymphoproliferative
responses. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, appears to result from the induction of
CD8+ T cells expressing IFN- (11, 12), which by
dampening Th2 responses may contribute to this phenomena. However, Ag
inhalation has recently been shown to result in reduced IgE responses
when mice are subsequently immunized by a mechanism that does not
require IFN-, CD8+ T cells, or 
T cells (13). The
inhalation of antigenic peptides has been shown to result in the
transient activation of T cells before the development of a systemic
tolerogenic state (14, 15). The mechanism by which tolerance is induced
by inhaled peptides is unclear. Immunosurveillance of a large
epithelial surface is likely to be an important characteristic of lung
mucosal immune responses. However, this has to be achieved without
detriment to the integrity of the epithelium. It is possible that the
development of tolerance prevents the onset of deleterious inflammatory
responses taking place in the lung mucosa to innocuous airborne Ags or
autoantigens.
We have analyzed the immunoregulatory events that follow repeated
aerosol challenge using a TCR transgenic mouse. DO11.10 transgenic mice
express an OVA-specific 
ß TCR (16). T cells from DO11.10
transgenic mice are specific for the OVA323–339 peptide
presented by the I-Ad molecule. The TCR transgenes are
expressed in BALB/c mice (i.e., H-2d haplotype), which
facilitates experiments in which transgenic animals can be exposed to
aerosolized Ag (17). These mice were used in this study to examine the
effect of inhaled Ag on the properties of parenchymal T cells during
pulmonary inflammation. We have demonstrated that following Ag
inhalation, the proliferative responses of lung parenchymal T cells and
their production of IL-2, but not IFN-, IL-4, or IL-5, were severely
attenuated. Such prevention of lymphproliferative responses was
tissue-specific and a consequence of the action of F4/80+
interstitial macrophages. These data suggest that the onset of clonal
expansion of T cells in the lung compartment is prevented at the site
of inflammation, but does not in itself obviate Th1- or Th2-driven
pulmonary inflammation.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Culture media RPMI was used throughout this study and comprised of RPMI 1640 (Life Technologies, Paisley, U.K.) supplemented with penicillin (50 U/ml), streptomycin (50 µg/ml), 5 mM HEPES, 2 mM glutamine, 2 ME (5 µM), and 5% FCS.
Animals
The DO11.10 animals used in this report were provided by Dr. Ethan Shevach (National Institutes of Health, Bethesda, MD). Animals were bred under aseptic conditions in a barrier facility. Mice were originally developed by Dr. D. Y. Loh (Howard Hughes Medical Institute, St. Louis, MO) and bred to normal BALB/c mice in the University of Texas Medical Branch facility (Texas). The expression of TCR transgenes was screened using the anti-clonotypic Ab KJ1-26. BALB/c mice were obtained from Harlan (Loughborough, U.K.), and in some instances they were immunized three times with OVA (100 µg/mouse) using an alum adjuvant as described previously (6).
Animal sensitization and adoptive transfer of DO11.10 T cells
Mice were intranasally challenged by exposure to aerosolized
solutions of either PBS or OVA (Grade V; Sigma, Poole, U.K.) for 20 min
a day over 8 consecutive days using a Wright’s nebulizer. Mice were
sacrificed on day 9, and the lungs, peripheral lymph nodes
(PLN),4 and spleen were
harvested for analysis. In some experiments, DO11.10 T cells were
adoptively transferred into BALB/c mice before exposure to OVA
aerosols. To drive T cell differentiation into a Th1 or Th2 effector
phenotype, PLN cells were incubated (5 x 105/ml) in
the presence of 1 µg/ml OVA323–329 peptide and either
400 ng/ml mouse IFN- (R&D Systems, Abingdon, U.K.) plus 20 µg/ml
anti-IL-4 Ab (11B11; American Type Culture Collection, Manassas,
VA) or 2 ng/ml murine IL-4 (R&D Systems) plus 10 µg/ml of the
anti-IFN- Ab (R4-6A2; American Type Culture Collection; used as
a hybridoma supernatant at 10% final concentration), respectively.
After 4 days of culture, cells were then injected i.v. into BALB/c mice
(107/mouse), and the mice were exposed to aerosolized OVA.
Isolation of lung inflammatory cells
Lung tissue was cut into small fragments and then washed extensively in culture medium RPMI. The tissue was then incubated for 1 h in culture medium RPMI containing 0.1% collagenase (Type IV; Sigma), 0.01% hyaluronidase (Sigma), and 0.002% DNase (Sigma). After digestion, cells were washed in media, and viable mononuclear cells were isolated over a Percoll density gradient (67.6%, 800 x g, 30 min). Bronchoalveolar lavage (BAL) was performed by cannulating the trachea and washing the lungs with 0.5 ml of PBS. In some experiments, lung mononuclear cells (LMCs) were fractionated using magnetic bead separation. This was achieved by pretreating LMC preparations with F4/80 and M1/70 (anti-CD11b) hybridoma supernatants (both from American Type Culture Collection). Cells were washed and mixed with sheep anti-rat IgG Dynabeads (Dynal, Oslo, Norway) before removal by magnetic separation.
Monoclonal Abs
The mAbs used in this study were F4/80, M1/70 (American Type
Culture Collection), MOMA-1, MOMA-2 (Serotec, Oxford, U.K.), DX5
(anti-NK cell, PharMingen), anti-class II (M5/114, rat IgG2b;
American Type Culture Collection), anti-CD4 (GK1.5, IgG2b; American
Type Culture Collection), anti-CD8 (5367, IgG2a; American Type
Culture Collection), anti-FcRII (2.4G2, IgG2b; American Type
Culture Collection), anti-CD3
(2C11, hamster IgG; American Type
Culture Collection), NLDC-145 (Serotec), and anti-CD103 (M290, a
gift from Dr P. J. Kilshaw; Babraham, Cambridge, U.K.). These Abs
were employed to deplete or to stain cells by indirect
immunofluorescence (using appropriate control Abs) for flow cytometric
analysis using a FACScan (Becton Dickinson, San Jose, CA).
In some experiments, blocking Abs to TNF- (R&D Systems), TGF-ß
(R&D Systems), GM-CSF (Sigma), and IL-12 (rabbit anti-mouse IgG;
Hoffmann-La Roche, Nutley, NJ) were used. Other blocking Abs were
obtained from PharMingen (Oxford, U.K.), and these include
anti-IFN- (R4-6A2, rat IgG1), anti-IL-4 (11B11, rat IgG1),
anti-IL-10 (SXC-1, rat IgM), anti-CTLA-4 (UC10, hamster IgG),
anti-CD40L (MR1, hamster IgG), anti-CD80 (1G10, rat IgG2a),
anti-CD86 (GL1, rat IgG2a), anti-CD28 (37.51, hamster IgG),
anti-Fas ligand (K10, mouse IgG2b), anti-VCAM-1 (429, rat
IgG2a), anti-CD31 (MEC 13.3, rat IgG2a), anti-CD44 (KM114, rat
IgG1), and ECCD-1 (R&D Systems). Briefly, cells were stimulated with
whole OVA (1 mg/ml), OVA peptide (1 µg/ml), or anti-CD3 (10
µg/ml) in the presence of the appropriate blocking Ab (20 µg/ml).
In some experiments, after 3 days 50 µl of supernatant was removed to
analyze for IL-2 and IFN- (described below). In all experiments,
[3H]thymidine was added to the wells, and the plates were
harvested after 16 h for ß counting to determine the level of
proliferation.
RT-PCR analysis of lymphokine expression
Lung tissue was homogenized and RNA extracted using TRIzol (Life
Technologies). Then, 1 µg total RNA was reverse transcribed by AMV
reverse transcriptase (RT Systems; Promega, Southampton, U.K.) at
42°C for 1 h using poly d(T)15 as a primer. The cDNA was then
amplified by PCR in the presence of PCR buffer, MgCl2 (1
mM), dNTPs (0.2 mM; Pharmacia), 1 U Taq DNA polymerase
(Promega), and cytokine-specific primer pair. The PCR was conducted for
35–40 cycles under the following conditions: denaturation at 95°C
for 60 s, annealing at 54°C for 30 s, and extension at
72°C for 60 s. Final extension was at 72°C for 10 min.
PCR-amplified products (10 µl) were electrophoresed through 2%
agarose gel containing ethidium bromide and visualized by UV
illumination. The expression of ß-actin (house-keeping gene), IL-2,
IL-4, IL-5, IFN-, and TNF- was determined in this way. Primers
used were ß-actin (5'-TGG AAT CCT GTC GCA TCC AT and 3'-TAA AAC GCA
GCT CAG TAA CA), IL-2 (5'-ACT TCA AGC TCC ACT TCA AGC and 3'-GCT TTG
AGA AAG TAT CCA), IL-4 (5'-GAA TGT ACC AGC AGC CAT ATC and 3'-CTC AGT
ACT ACG AGT AAT CCA), IL-5 (5'-CGC TCA CCG AGC TCT GTT GA and 3'-CAG
GTT TTG GAA TAG CAT TT), IFN- (5'-AAC GCT ACA CAC TGC ATC TTG G and
3'-GAC TTC AAA GAG TCT GAG G), and TNF- (5'-GCC CTT GCT GTT CTT CTC
TGT and 3'-GGC AAT CAG TTC CAG GTC AGT).
Eosinophil peroxidase (EPO) activity
The EPO activity in BAL cells was determined with a colorimetric assay. Five times 104 cells/well were added to 96-well U-bottom microtiter plates. The plates were centrifuged at 200 x g at 4°C for 10 min. The supernatants were aspirated, and 100 µl of substrate solution was added consisting of 0.1 mM orthophenylene diamine dihydrochloride in 50 mM Tris-HCl containing 0.1% Triton X-100 and 1 mM hydrogen peroxide. The plates were incubated at room temperature for 30 min, then 50 µl of 4 M sulfuric acid was added to stop the reaction. Absorbance was determined with an ELISA reader at 495 nm.
Cell cycle analysis
Cells were washed with PBS for 3 times, resuspended in 1 ml of hypotonic fluorochrome solution (50 µg/ml propidium iodide (PI) in 0.1% sodium citrate plus 0.1% Triton X-100), and then incubated for 1 h at room temperature. Cell cycle was measured using a FACScan flow cytometer (Becton Dickinson).
Analysis of cells isolated from lung tissue
Cytocentrifuge preparations of LMCs were stained using
Diff-Quick (Baxter, Abingdon, U.K.) and analyzed by microscopy.
Alternatively, cells were stained by indirect immunofluorescence, and
flow cytometric analysis was performed using a FACScan (Becton
Dickinson). To analyze T cell function, cells were plated onto
microtiter plates (2 x 105 per well) and in some
experiments stimulated with either whole OVA protein (1 mg/ml), OVA
peptide (1 µg/ml), or anti-CD3 Ab (10 µg/ml). After 3 days, 50
µl of supernatant was removed to analyze for IL-2 or IFN- as
described below. [3H]Thymidine was added to the wells,
and the plates were harvested after 16 h to monitor for
proliferation by ß counting.
In some experiments to determine whether physical contact between adherent and nonadherent cells was required for T cell responses, transwells (Costar, Cambridge, MA) were used in which we compared the proliferation of nonadherent LMCs from aerosol-challenged mice cultured alone or with adherent cells but physically separated from them by a 0.4-µm millipore filter.
Cytokine assay
Supernatants were assayed for IL-2 using a CTLL proliferation
assay. Briefly, 5 x 103 CTLL cells were added to
wells of a 96-well microtiter plate. Culture supernatants were then
titrated and the proliferation of the cells was determined in
triplicate by the addition of 1 µCi of [3H]thymidine
(Amersham, Little Chalfont, U.K.) to each well. After 18 h, cells
were harvested using a Dynatech harvester and tritium incorporation
measured by ß counting. IFN- was measured by ELISA using clone
R4.6A2 (PharMingen) as capture Ab and clone XMG1.2 (PharMingen) as the
detecting Ab. IL-4 was measured using a commercially available ELISA
kit (Biosource, Lifescreen, Watford, U.K.) according to the
manufacturer’s instructions. IL-5 was measured by ELISA using clone
TRFK-5 as capture Ab and TRFK-4 (PharMingen) as detection Ab.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cytokines mRNA expressed in lung tissue following aerosol exposure
To monitor the T cell response taking place in the lung following
Ag inhalation, cytokine mRNA expression in the lung tissue was
determined after exposure of the mice to aerosolized OVA. DO11.10 mice
were intranasally challenged with OVA for 2, 4, and 8 consecutive days.
After challenge, the lung tissue was removed, homogenized, and cytokine
mRNA transcripts screened by RT-PCR. We observed mRNA for IL-4,
IFN-, and TNF- in the lung tissue of animals that had been
intranasally challenged for 2–8 days (Fig. 1
A). In contrast, no cytokine
expression was observed in the lung tissue from naive mice not exposed
to aerosols of OVA. A similar cytokine profile was also observed when T
cells isolated from the PLN or LMCs of naive DO11.10 animals were
stimulated for 48 h with either whole OVA or immobilized
anti-CD3 (Fig. 1B). The production of these cytokines
has been previously demonstrated by other groups on stimulating DO11.10
spleen cells with OVA323–339 peptide (18). IL-2 mRNA was
also tested, but was undetectable in both naive and challenged tissue,
possibly a consequence of the rapid and transient kinetics of the
expression of IL-2 mRNA compared with the other cytokines.
|
The inflammatory response taking place in the lung tissue
following Ag inhalation was evaluated by monitoring the magnitude and
nature of any cellular infiltrate. This was determined by analyzing the
cells present in the BAL and in enzymatically dispersed lung tissue.
Aerosol exposure resulted in the development of a mild pulmonary
eosinophilia that was clearly evident after 2–4 days of challenge by
an increase in EPO activity and the number of eosinophils in the BAL
(Table I). The development of a pulmonary
eosinophilia was dependent on the concentration of OVA used in the
aerosol (Table I), with 0.5% OVA inducing the largest increase in
eosinophils in both the tissue and the BAL. Although eosinophils were
recruited to the lungs, the total cell yields from the lungs of
OVA-challenged animals did not increase significantly when compared
with naive or PBS-challenged animals. The number of OVA-specific T
cells present in the BAL, lung, spleen, and PLN of challenged mice was
determined by flow cytometry using the KJ1-26 mAb. Interestingly, the
frequency and total numbers of OVA-specific T cells in enzymatically
dispersed lungs of challenged animals increased only marginally
following inhalation of 0.5% OVA (Table II). The number of T cells present in the
lungs was not significantly affected by varying the concentration of
OVA used in the aerosol (Table II).
|
|
During the course of this study, we were particularly interested
in monitoring the effect of OVA aerosol inhalation on the properties of
T cells present in the lung tissue. Preparation of LMCs by dispersion
of the lung tissue in collagenase provided an opportunity to probe
events that were idiosyncratic to the lung environment. Stimulation of
LMCs isolated from naive DO11.10 mice with OVA protein and
OVA323–339 peptide resulted in strong proliferative
responses (Fig. 2). LMCs isolated from
mice challenged with the Ag for up to 8 days showed a reduced
proliferative response on restimulation in vitro with either
whole OVA protein or OVA323–339 peptide (Fig. 2A). The reduction was progressive over consecutive
challenges, eventually reaching a maximal attenuation after 8 days of
aerosol challenge (Fig. 2A). The proliferative response to
immobilized anti-CD3 was also diminished, implying that the
reduction of responses was not simply a consequence of a failure to
present Ag (Fig. 2A). This effect was not observed in the
PLN or spleen cells, where the T cell proliferative response to whole
OVA and OVA peptide stimulation remained high (Fig. 2A).
Moreover, the number of T cells present in the posterior mediastinal,
hilar, and tracheal lymph nodes increased 10- to 20-fold following
aerosol challenge, and restimulation with the Ag in vitro resulted in
normal proliferative responses (data not shown). The attenuated
responses in the lung tissue were dependent on the concentration of OVA
used in the aerosol with concentrations of >0.5% resulting in a
reduction of 96% (Fig. 2B). The addition of exogenous IL-2
did not restore LMC responses (2741 cpm vs 3070 cpm in response to
OVA323–339 peptide in the absence or presence of 2 U/ml
IL-2, respectively) implying that the failure to proliferate was not
simply due to an inability to produce IL-2. Presentation of
OVA323–339 peptide in the LMC cultures was likely to be by
B cells and interstitial macrophages because both cell types were
present in LMCs from aerosol-challenged animals. The dendritic cell
marker NLDC-145 was detected on <1% of LMCs (data not shown).
|
C). These data suggest that the attenuation of the
proliferative responses may be a downstream consequence of activation
of lung T cells (i.e., parenchymal T cells).
We next evaluated whether the attenuation of lung parenchymal T cell
responses was associated with either a Th1 or Th2 response. To resolve
this issue, DO11.10 T cells were driven into a Th1 or Th2 phenotype in
vitro by culture with OVA323–339 peptide and exogenous
IFN- or IL-4, respectively (Fig. 3A). T cells were injected
i.v. into BALB/c recipient mice that were then exposed to aerosolized
OVA. After six exposures, the lung tissue was removed and LMCs were
prepared. LMCs were restimulated with anti-CD3, and the
proliferation and production of IL-2 was then determined. Flow
cytometric analysis of LMCs revealed that the proportion of
CD4+ T cells that stained with the anti-clonotypic
KJ1-26 Ab increased after OVA inhalation from 36.6% to 54.1% for Th1
cells and 26.1% to 44.0% for Th2 cells. Following OVA inhalation, it
was found that the proliferation and production of IL-2 by LMCs in
response to anti-CD3 were severely attenuated in BALB/c mice that
had received DO11.10 T cells of either Th1 or Th2 phenotype (Fig. 3B). These data demonstrate that, OVA inhalation results in
the growth arrest of Ag-specific DO11.10 and host lung parenchymal T
cells. LMCs from control mice that had received DO11.10 T cells of
either Th1 or Th2 phenotype and not inhaled OVA responded normally
(Fig. 3B). In mice that received DO11.10 Th2 cells, an
increase in the peptide-induced production of IL-4 by LMCs in vitro was
observed following OVA inhalation, demonstrating that the Th2 phenotype
was retained in vivo (Fig. 3C). Neutralization of IL-4 or
IL-10 had no effect on the proliferative responses of lung parenchymal
T cells (data not shown). Collectively, these results show that lung
parenchymal T cell responses are dramatically attenuated following OVA
inhalation in BALB/c mice that received Ag-specific DO11.10 T cells of
either Th1 or Th2 phenotype.
|
). PLN or spleen cells from
aerosol-challenged DO11.10 animals produced high levels of IL-2 (data
not shown). Although the production of IL-2 by LMCs from challenged
mice was severely reduced, the expression of IFN- was high on
stimulation in vitro with 1000 and 100 ng/ml of OVA peptide. A total of
10 ng/ml of OVA323–339 peptide elicited the production of
IFN-, which was not observed by LMCs from naive animals under the
same conditions (Table III).
|
Coincident with the reduction in proliferative responses to
OVA323–339 peptide and anti-CD3 was a 2- to 3-fold
increase in the number of F4/80+ cells present in the lung
tissue digest of DO11.10 mice after OVA inhalation (Fig. 4A). The F4/80 Ab is specific
for a highly glycosylated protein expressed by splenic and tissue
macrophages, Langerhans cells, and dendritic cells. Isolation of
plastic adherent LMCs revealed that they were predominantly
F4/80+ cells (94%), which coexpressed CD80, CD86, CD54,
high levels of class II (Ia) (Fig. 4B), and were morphologically
indistinguishable from macrophages. The remaining cells comprised
mainly of stromal cells. Following aerosol challenge, the level of
expression of Ia, ICAM-1, and CD86 by F4/80+ cells was
increased.
|
Because the loss of T cell proliferative responses to Ag was
coincident with an increase in the number of interstitial
F4/80+ macrophages, we evaluated whether adherent cells
mediated the attenuation of T cell responses. Depleting adherent cells
from LMC preparations of aerosol-challenged DO11.10 animals resulted in
the restoration of the OVA323–339 peptide and anti-CD3
responses (Fig. 5A). Removal
of adherent cells from LMCs of naive animals did not affect
OVA323–339 peptide or anti-CD3 responses. Cell cycle
analysis by flow cytometry revealed that unfractionated (whole) LMCs
from aerosol-challenged mice remained predominantly in G0/G1 phase
following stimulation with OVA323–339 peptide, and only
10.4% were found to be in G2-M phase (Fig. 5B).
In contrast, removal of adherent cells restored the proliferative
response to OVA323–339 peptide, with 31.8% of cells found
to be in G2-M phase. Following stimulation with
OVA323–339 peptide, 36.2% and 34.4% of apoptotic cells
were observed in whole and adherent-depleted LMCs, respectively.
Alveolar macrophage-mediated inhibition of T cell responses has
previously been shown to be nitric oxide-dependent and not requiring
physical contact between the cells (19). To determine whether physical
contact between adherent and nonadherent cells was required for
attenuation of the T cell response, transwells were used in which we
compared the proliferation of nonadherent LMCs from aerosol-challenged
mice cultured alone or with adherent cells but physically separated
from them by a 0.4-µm millipore filter (Fig. 6). The proliferation of the nonadherent
LMCs was unaffected by adherent cells when they were physically
separated from them by the 0.4-µm filter, implying that cell-cell
contact was required for attenuation of parenchymal T cell responses.
However, when adherent cells were added to nonadherent LMCs, the
proliferative response was severely attenuated (Fig. 6).
|
|
Because the adherent cells were heterogeneous, it was important to
determine the specific contribution of interstitial macrophages to the
attenuation of the responses. Using the macrophage-specific mAbs F4/80,
M1/70, MOMA-1, or MOMA-2 with anti-rat Ig Dynabeads, it was
possible to specifically deplete interstitial macrophages. Removal of
tissue macrophages using the F4/80 Ab resulted in a 77% restoration of
the T cell response when compared with preparations depleted of
adherent cells (Fig. 7). The removal of
CD11b+ cells (using M1/70) completely restored responses.
In contrast, the depletion of NK cells using the DX5 Ab or
subpopulations of macrophages using the MOMA-1 and MOMA-2 Abs had no
effect. These data demonstrate that mature interstitial macrophages are
principally responsible for the immune modulation observed.
Interstitial macrophages expressed CD80, CD86, and class II, and thus
it is likely that they are effective at Ag-presentation. However,
adherent LMCs isolated from OVA-challenged animals were poor at driving
proliferative responses by both lung parenchymal and lymph node T cells
(data not shown).
|
A). In contrast to the
control mice, OVA inhalation by animals that had received DO11.10 Th2
cells resulted in a dramatic reduction in lung parenchymal T cell
proliferative responses and in IL-2 production (Fig. 8, A
and B). OVA inhalation by mice that had received the Th2
cells resulted in a 5-fold and 22-fold increase in the level of IL-4
and IL-5 production, respectively (Fig. 8B). These mice also
developed a pronounced airway eosinophilia (data not shown).
Importantly, growth arrest and attenuation of IL-2 production was
reversed by the removal of F4/80+ interstitial macrophages.
However, their depletion had no effect on the production of IL-4 or
IL-5 (Fig. 8B).
|
). Inhalation of OVA aerosol resulted
in the reduction of proliferative responses of LMCs to OVA protein, but
responsiveness was restored by the removal of adherent cells (Table IV). In contrast, the responses of spleen cells to OVA protein was not
influenced by Ag inhalation. LMCs from primed animals produced high
levels of IL-2; however, following aerosol challenge the production of
this cytokine was diminished. Interestingly, stimulation of LMCs from
challenged mice with the Ag induced the production of IL-5. These data
suggest that similar mechanisms of regulation of parenchymal T cell
responses are evident in nontransgenic mice.
|
, TGF-ß, GM-CSF, IFN-, IL-4, IL-10, or IL-12 did not
restore responses (data not shown), and coculture experiments failed to
implicate the involvement of soluble factors. CTLA-4 has been shown to
down-regulate T cell responses; however, parenchymal T cells did not
express CTLA-4 before or following activation. Similarly, blocking Abs
to CD40, CD40 ligand, CD80, CD86, CD28, Fas ligand, VCAM-1, CD31, and
CD44 failed to restore responses (data not shown). In other systems,
the immune modulatory effects of macrophages have been attributed to
nitric oxide. Inhibiting the production of nitric oxide with monomethyl
arginine or PG synthase activity with indomethacin restored responses
by 15% and 14%, respectively (when compared with adherent-depleted
LMCs). The action of these mediators was additive because blocking the
production of both resulted in a 22% restoration of responses (Table V). These data demonstrate that although
the action of such mediators contribute marginally to the attenuation,
the growth arrest is mediated via another mechanism. Interestingly, an
Ab to E-cadherin that blocks homeotypic adhesion (ECCD-1) has also been
shown to partially restore proliferative responses. In the experiment
depicted in Table V, the addition of an E-cadherin-specific Ab ECCD-1
(20 µg/ml) resulted in 10.8% restoration of OVA323–339
peptide responses. E-cadherin was expressed by a small population of
epithelial cells present in LMC preparations (data not shown), and its
role in modulating parenchymal T cell responses is unclear.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, and TNF- in the lung tissue
following 2–8 days of challenge. Coincident with the induction of
cytokine expression was the generation of a mild pulmonary
eosinophilia. In this context, TNF-, IL-4, and IL-5 have been
implicated in the recruitment of eosinophils in other mouse models
(20).
During the course of this study we were particularly interested in
monitoring the effect of OVA aerosol inhalation on the properties of T
cells present in the lung tissue. Conceivably, responses occurring in
the lung mucosa display characteristics which are idiosyncratic to the
lung environment. T cell responses in the pulmonary tissue are likely
to be secondary rather than primary, because the majority of the T
cells are of a memory phenotype (3). Memory T cells have been described
previously in the DO11.10 mouse, most of which express endogenous TCR
-chains (21). In the present study, the proportion of T cells in the
lungs of animals that stained with the anti-clonotypic Ab KJ1-26
was found to be 81.7% ± 9.8%.
Following repeated OVA aerosol challenge of DO11.10 mice, the number of lung parenchymal T cells, labeled with the anti-clonotypic Ab KJ1-26, did not change. More protracted Ag challenge did not elicit severe inflammatory responses as defined by evidence of T cell activation (such as expression of IL-2R or CD69). It is difficult to comment on the degree of T cell recruitment to the tissue because the dynamics of migration to, and from, the lungs is unknown. However, it appeared that severe pulmonary T cell responses were prevented as a consequence of the modulation of T cell function following Ag inhalation. The most clear demonstration of this phenomena was that parenchymal T cells isolated from lungs of mice exposed to OVA aerosols, when restimulated in vitro with OVA323–339 peptide or immobilized anti-CD3, displayed severely attenuated proliferative responses. This effect was dependent on the dose of aerosol administered and could not be explained by changes in the number of OVA-specific T cells in the tissue. Such immune regulation was tissue-specific because T cell responses in the lymph nodes and spleens were normal. Aerosol-induced growth arrest of lung parenchymal T cell responses was also observed in BALB/c mice that had received DO11.10 T cells bearing a Th1 or Th2 phenotype. During aerosol inhalation, the number of DO11.10 T cells in the lungs increased 2-fold. However, proliferative responses and the ability of parenchymal T cells to produce IL-2 was lost upon restimulation in vitro. The failure to proliferate was not only in response to OVA323–339 peptide but also following stimulation with a polyclonal T cell activator such as immobilized anti-CD3. A similar phenomena was also evident in OVA-primed BALB/c mice, demonstrating that the down-regulation of pulmonary T cell proliferative responses following OVA inhalation is not restricted to the DO11.10 mice used in this study.
On monitoring the kinetics of proliferation, stimulation of parenchymal
T cells in vitro with OVA323–339 peptide revealed that the
loss of response was progressive, with maximal inhibition occurring
after 24 h of stimulation. This possibly reflects a requirement
for activation of the T cells before the onset of inhibition. The
attenuated proliferative responses of LMCs from OVA-challenged DO11.10
mice, and from BALB/c mice that had received DO11.10 T cells, was
associated with a failure of the parenchymal T cells to produce IL-2 in
response to Ag stimulation in vitro. However, the production of IFN-
by LMCs from aerosol-challenged DO11.10 mice was not diminished on
restimulation with OVA323–339 peptide, demonstrating that
not all effector functions of the parenchymal T cells were lost.
Similarly, in BALB/c mice that had received DO11.10 Th2 cells and
inhaled OVA, stimulation of lung parenchymal T cells with either OVA
peptide or anti-CD3 cross-linking induced growth arrest and reduced
IL-2 production. Interestingly, this onset of growth arrest did not
prevent the production of IL-4 or IL-5. Moreover, these responses were
typically associated with the development of a pronounced pulmonary
eosinophilia. To our knowledge, this is the first demonstration of this
phenomena. Collectively, these observations imply that the onset of
growth arrest only serves to prevent T cell clonal expansion at the
site of inflammation, but in itself does not obviate Th1- or Th2-driven
pulmonary inflammation.
The failure of the T cells to proliferate was due to active inhibition of T cell effector function by adherent cells, comprising predominantly of tissue interstitial macrophages with 5% contamination by F4/80-negative stromal cells. The attenuation of proliferative responses required live cells because fixed macrophages did not attenuate proliferative responses but were effective APCs. Consequently, the attenuation of the proliferative responses was temporally separable from the process of Ag presentation. Because the tissue macrophages express both CD80 and CD86, the immunomodulatory function of these cells could not be explained by the presentation of Ag in the absence of costimulation. It is possible that an event that overrides CD80/CD86 costimulation serves to regulate the expansion of T cell responses in the lung mucosa. The onset of growth arrest is likely to serve to limit expansion of T cells during the course of inflammatory responses in the lung compartment. The prevention of clonal expansion in the lung implies that the majority of Ag-specific T cells present in the tissue have been recruited to this site. Consistent with this hypothesis is the recent report that following instillation of Ag into the trachea of mice the T cell expansion was restricted to the draining lymph nodes (22). The fate of T cells that enter a state of growth arrest but are being stimulated with Ag is unclear. Th1 cells may become anergic or enter into apoptosis, while Th2 cells may be less susceptible to these processes (23).
In rats, intratracheal challenge with bacterial Ag has been shown to
result in an increase in the frequency of dendriform cells that express
high levels of class II Ag (24, 25). In the course of our experiments,
the number of dendritic cells, detected using the NLDC-145 Ab, was
<1% in LMC preparations of both naive and challenged DO11.10 animals.
The increase in the number of F4/80+ cells in the lungs of
our animals is likely to be a consequence of recruitment and
differentiation of circulating monocytes at the site of inflammation.
The macrophages found in the lungs of aerosol-challenged mice are
phenotypically similar to interstitial macrophages, which are known to
be functionally different from those present in the alveolar spaces
(5). Interstitial macrophages express higher levels of class II and are
more effective at Ag presentation when compared with alveolar
macrophages. The latter cells are highly phagocytic, secrete large
amounts of reactive oxygen radicals, nitric oxide, TNF-, and
IFN-. A role for alveolar macrophages in down-regulating T cell
responses has been demonstrated by other laboratories (26, 27).
Alveolar macrophages can inhibit T cell responses by producing
cytostatic mediators such as PGs (28) and nitric oxide (29, 30, 31), or
alternatively by producing immunomodulatory cytokines such as TGF-ß
(32) and IL-12 (33). Little is known of the relative importance of
these agents during pulmonary inflammation. IFN-, particularly in
combination with IL-2, and or TNF-, has been implicated in eliciting
nitric oxide production by alveolar macrophages (34, 35), a process
that has been shown to be CD40-dependent (36). Consequently, T cell
activation is typically a prerequisite for nitric oxide release.
However, nitric oxide production is inhibited by IL-4, GM-CSF, and
TNF-, and Th2 cells are resistant to its action (37, 38, 39). Therefore,
nitric oxide would be expected to be less effective at inhibiting Th2
responses. It is known that nitric oxide allows T cell activation to
progress up to and including the production of IL-2 and its receptor
(40). Consequently, the actions of nitric oxide are typically
associated with an increase the levels of IL-2 present in cultures of
activated T cells because the cytokine produced cannot be consumed (31, 40). Consistent with this phenomena is the recent demonstration that
nitric oxide induces growth arrest by reducing tyrosine phosphorylation
of jak3 and STAT5 in T cells, which are events downstream of IL-2
signaling through its receptor (19). The growth arrest that we have
observed following OVA inhalation is invariably associated with the
loss of IL-2 production but continued production of IL-4, IL-5, and
IFN-. Consistent with this observation, we found that the inhibition
of nitric oxide production did not restore proliferative responses.
This leads us to conclude that during the onset of Th1- and
Th2-mediated inflammation the expansion of T cells is prevented by a
mechanism not involving nitric oxide production.
Separation of the parenchymal T cells from the adherent cells by a
0.4-µm filter prevented the inhibition of parenchymal T cell
responses. Moreover, supernatants from cultures of LMCs were
ineffective at inhibiting these T cell responses, thus demonstrating
that the immune modulation required cell-cell contact. Neutralizing Abs
to TNF-, TGF-ß, GM-CSF, IFN-, IL-4, IL-10, or IL-12 did not
restore responses. CTLA-4 has been shown to down-regulate T cell
responses (41); however, parenchymal T cells did not express CTLA-4
before or following activation. Similarly, blocking Abs to CD40, CD40
ligand, CD80, CD86, CD28, Fas ligand, VCAM-1, CD31, and CD44 failed to
restore responses. Interestingly, the addition of the ECCD-1 Ab to
E-cadherin partially restored proliferative responses and was found to
be expressed predominantly on low numbers of epithelial cells in the
dispersed LMCs. Given the importance of E-cadherin in the
interaction between T cells and the epithelial cells, its involvement
is noteworthy.
Our studies have demonstrated that following Ag inhalation the responses of lung parenchymal T cells, as measured by proliferation and IL-2 production, were severely attenuated. The unresponsiveness was tissue-specific and a consequence of active inhibition of T cell function by interstitial macrophages. We speculate that this immunoregulation is representative of the normal mucosal T cell response that develops following inhalation of innocuous aeroantigens. Whether such events regulate T cell responses to pathogens entering the lung is unclear (42). Consistent with our findings, other groups have also shown that the induction of T cell responses following aerosol exposure of nontransgenic mice are typically transient despite continued exposure to Ag (14, 15, 22). Such immunoregulatory events are potentially important in maintaining the homeostasis of the normal lung mucosa. Dysregulation of such immunomodulatory function could, conceivably, result in chronic lung inflammation. The presence of activated T cells in the airways of patients with allergic inflammatory conditions such as asthma is well documented (43, 44). The possibility that such chronic inflammatory processes are a manifestation of a failure in immune regulation, rather than persistent activation of allergen-specific T cells, is worthy of investigation.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 S.-C.L and Z.H.J. contributed equally to this manuscript.
3 Address correspondence and reprint requests to Dr. Kevan Roberts, University Medicine, Level D, Centre Block, Southampton General Hospital, Southampton, SO16 6YD, U.K. E-mail:
4 Abbreviations used in this paper: PLN, peripheral lymph node; LMCs, lung mononuclear cells; BAL, bronchoalveolar lavage; EPO, eosinophil peroxidase; PI, propidium iodide.
Received for publication June 18, 1998. Accepted for publication March 11, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
/ T cells. Science 265:1869./+ T cells or interferon (IFN)- in a murine model of allergen stimulation. J. Exp. Med. 187:721.ß-transgenic model. J. Exp. Med. 182:1579. mediates recruitment of neutrophils and eosinophils during inflammation. J. Immunol. 154:5411.[Abstract]
in the induction of nitric oxide synthesizing pathway. J. Immunol. 147:144.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  Z. Jaffar, M. E. Ferrini, M. C. Buford, G. A. FitzGerald, and K. Roberts Prostaglandin I2-IP Signaling Blocks Allergic Pulmonary Inflammation by Preventing Recruitment of CD4+ Th2 Cells into the Airways in a Mouse Model of Asthma J. Immunol., November 1, 2007; 179(9): 6193 - 6203. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Loke, I. Gallagher, M. G. Nair, X. Zang, F. Brombacher, M. Mohrs, J. P. Allison, and J. E. Allen Alternative Activation Is an Innate Response to Injury That Requires CD4+ T Cells to be Sustained during Chronic Infection J. Immunol., September 15, 2007; 179(6): 3926 - 3936. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Donnelly, S. M. O'Neill, M. Sekiya, G. Mulcahy, and J. P. Dalton Thioredoxin Peroxidase Secreted by Fasciola hepatica Induces the Alternative Activation of Macrophages Infect. Immun., January 1, 2005; 73(1): 166 - 173. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Jaffar, T. Sivakuru, and K. Roberts CD4+CD25+ T Cells Regulate Airway Eosinophilic Inflammation by Modulating the Th2 Cell Phenotype J. Immunol., March 15, 2004; 172(6): 3842 - 3849. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. J. Wright, H. Cherwinski, M. Foster-Cuevas, G. Brooke, M. J. Puklavec, M. Bigler, Y. Song, M. Jenmalm, D. Gorman, T. McClanahan, et al. Characterization of the CD200 Receptor Family in Mice and Humans and Their Interactions with CD200 J. Immunol., September 15, 2003; 171(6): 3034 - 3046. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Wohlleben, J. Muller, U. Tatsch, C. Hambrecht, U. Herz, H. Renz, E. Schmitt, H. Moll, and K. J. Erb Influenza A Virus Infection Inhibits the Efficient Recruitment of Th2 Cells into the Airways and the Development of Airway Eosinophilia J. Immunol., May 1, 2003; 170(9): 4601 - 4611. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P'n. Loke and J. P. Allison PD-L1 and PD-L2 are differentially regulated by Th1 and Th2 cells PNAS, April 29, 2003; 100(9): 5336 - 5341. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Jaffar, K.-S. Wan, and K. Roberts A Key Role for Prostaglandin I2 in Limiting Lung Mucosal Th2, But Not Th1, Responses to Inhaled Allergen J. Immunol., November 15, 2002; 169(10): 5997 - 6004. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. C. Tsitoura, V. P. Yeung, R. H. DeKruyff, and D. T. Umetsu Critical role of B cells in the development of T cell tolerance to aeroallergens Int. Immunol., June 1, 2002; 14(6): 659 - 667. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. H. Falcone, P'n. Loke, X. Zang, A. S. MacDonald, R. M. Maizels, and J. E. Allen A Brugia malayi Homolog of Macrophage Migration Inhibitory Factor Reveals an Important Link Between Macrophages and Eosinophil Recruitment During Nematode Infection J. Immunol., November 1, 2001; 167(9): 5348 - 5354. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Wilder, D. S. Collie, D. E. Bice, Y. Tesfaigzi, C. R. Lyons, and M. F. Lipscomb Ovalbumin aerosols induce airway hyperreactivity in naive DO11.10 T cell receptor transgenic mice without pulmonary eosinophilia or OVA-specific antibody J. Leukoc. Biol., April 1, 2001; 69(4): 538 - 547. [Abstract] [Full Text]
|
|
|
|
|
|
A. Wangoo, T. Sparer, I. N. Brown, V. A. Snewin, R. Janssen, J. Thole, H. T. Cook, R. J. Shaw, and D. B. Young Contribution of Th1 and Th2 Cells to Protection and Pathology in Experimental Models of Granulomatous Lung Disease J. Immunol., March 1, 2001; 166(5): 3432 - 3439. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
H. Hakonarson, C. Kim, R. Whelan, D. Campbell, and M. M. Grunstein Bi-Directional Activation Between Human Airway Smooth Muscle Cells and T Lymphocytes: Role in Induction of Altered Airway Responsiveness J. Immunol., January 1, 2001; 166(1): 293 - 303. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. G. KNOTT, P. R. GATER, and C. P. BERTRAND Airway Inflammation Driven by Antigen-specific Resident Lung CD4+ T Cells in alpha beta -T Cell Receptor Transgenic Mice Am. J. Respir. Crit. Care Med., April 1, 2000; 161(4): 1340 - 1348. [Abstract] [Full Text]
|
|
|
|
|
|
Z. H. Jaffar, L. Stanciu, A. Pandit, J. Lordan, S. T. Holgate, and K. Roberts Essential Role for Both CD80 and CD86 Costimulation, But Not CD40 Interactions, in Allergen-Induced Th2 Cytokine Production from Asthmatic Bronchial Tissue: Role for {alpha}{beta}, But Not {gamma}{delta}, T Cells J. Immunol., December 1, 1999; 163(11): 6283 - 6291. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
