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Inflammatory Diseases Unit, Roche Bioscience, Palo Alto, CA 94304
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-TCR
transgenic BALB/c DO11.10 mice, we investigated the ability of
Ag-specific CD4+ T cell activation to induce lung chemokine
production and leukocyte recruitment. Within 1 h of exposure of
DO11.10 mice to OVA aerosol, lung mRNA and protein for the neutrophil
chemokines KC and macrophage inflammatory protein (MIP)-2 were greatly
increased. Accordingly, neutrophils in the airways increased by
>50-fold, and KC and MIP-2 proved to be functional because their
neutralization significantly reduced airway neutrophilia.
CD4+ T cell activation was critical because
CD4+ but not CD8+ T cell depletion reduced KC
production, which correlated well with the previously observed
inhibition of neutrophil influx after CD4+ T cell
depletion. In vitro studies confirmed that OVA-induced KC and MIP-2
production was conditional upon the interaction of CD4+ T
cells with APCs. A likely secondary mediator was TNF-, and a
probable source of these chemokines in the lung was alveolar
macrophages. Thus, Ag-specific CD4+ T cell activation in
the lung leads to rapid up-regulation of neutrophil chemokines and the
recruitment of neutrophils to the site of Ag exposure. This may be a
key early event in the pathogenesis of Ag-induced lung
inflammation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The discovery of the chemokines has been an important step toward our better understanding of cell trafficking during inflammation (9, 10). Chemokine receptors are members of the superfamily of G protein-coupled receptors. Signaling induces rapid phenotypic changes, which enhance the interaction of the chemokine receptor-bearing cell with endothelial adhesion molecules, stimulating firm adhesion and allowing diapedesis into the tissue (9, 11). Chemokines acting on neutrophils in the human are related to the IL-8 family of peptides (12, 13, 14, 15, 16), and although IL-8 has not been detected in the mouse, it is now accepted that the CXC chemokines KC (17) and macrophage inflammatory protein (MIP)3-2 (18) play an important role in neutrophil trafficking in the mouse. IL-8 and related chemokines bind to CXC chemokine receptor (CXCR) 1 and CXCR2 (19, 20). The critical role of CXCR2 in neutrophil migration from the circulation has been demonstrated in CXCR2-deficient mice (21).
Suggestive of a role for chemokines in allergic lung disease, increased chemokine levels are seen in airway tissues from allergic asthmatics (3, 22, 23, 24) and animal models of lung allergy (25). Consequently, it will be useful to understand the mechanisms explaining up-regulation of chemokines in lung allergy. It has recently been shown that segmental allergen challenge in allergic asthmatics rapidly induced neutrophil chemokine production and neutrophil influx (3). In that study, although neutrophil chemokine production was thought to be mast cell independent, the underlying mechanism was unclear. The critical role for CD4+ T cells in human and animal lung allergy (26, 27, 28, 29, 30) led us to postulate a role for these cells as important modulators of lung chemokine levels. Thus, the specific focus of this report is on neutrophil chemokines and the potential that activation of Ag-specific CD4+ T cells had in promoting lung neutrophil chemokine production.
BALB/c DO11.10 mice express an OVA-specific -TCR on their
CD4+ T cells (31). Exposure of naive
DO11.10 mice to OVA aerosol results in a CD4+ T
cell-dependent inflammatory response comprised in part of a rapid
infiltration of neutrophils into the lung (32). In this
report, we characterize the mechanism of lung neutrophil
chemoattraction seen in DO11.10 mice after Ag inhalation. In doing so,
we demonstrate that Ag-specific CD4+ T cell
activation can lead to neutrophil chemokine production in the lung.
This may explain the rapid neutrophil recruitment to the airways seen
after Ag challenge in lung allergy and provide another pathogenic role
for CD4+ T cells in disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For all studies, homozygous -TCR-transgenic BALB/c DO11.10
mice between 8 and 12 wk of age and of either sex were used. Mice were
given access to water and commercial chow ad libitum, housed on site,
and tested negative for pathogens. For Ag exposure, the mice were
placed in a plexiglass box and exposed to aerosols of PBS alone or PBS
with 0.001–5% OVA or 5% BSA (both obtained from Sigma, St. Louis,
MO) generated by a Pari Star nebulizer (Pari-Werk, Starnberg, Germany)
for a 20-min period. At various time points after Ag exposure, mice
were anesthetized (urethane, 
1.5 g/kg i.p.), and a tracheal cannula
was inserted and a BAL was performed with four 0.3-ml aliquots of PBS
containing 0.1% BSA and 0.05 mM EDTA. The total number of leukocytes
in 20 µl of BAL fluid was determined using a Coulter Counter (Coulter
Electronics, Hialeah, FL). Differential leukocyte counts were made by
counting 300 cells on stained (Diff-Quik; Dade Diagnostics, Aguada, PR)
cytospin preparations by light microscopy using standard morphological
criteria.
Detection of KC and MIP-2 mRNA in the lung
Lungs were perfused in situ with 100 U/ml heparinized PBS,
removed, and cleaned of connective tissue before being snap-frozen in
liquid nitrogen. Total RNA was prepared from lungs from experimental
and control mice using Trizol Reagent (Life Technologies, Gaithersburg,
MD) according to manufacturer directions. RNA from at least four mice
per group was pooled. A 10-µg quantity of total RNA was used in the
RNase protection assay. The mCK-5 kit obtained from PharMingen (San
Diego, CA) was used to prepare the
35S-radiolabeled RNA probes. Included in the kit
were probes for the following mouse chemokines: lymphotactin, RANTES,
eotaxin, MIP-1, MIP-1, MIP-2, IP-10, monocyte chemoattractant
protein-1 (MCP-1), TCA-3, and the housekeeping genes GAPDH and L32. To
label, 6 x 105 cpm of probe was added to
each sample, and incubations were performed overnight at 55°C. After
RNase digestion, samples were run in denaturating polyacrylamide gels.
For the Northern blot analysis, 20 µg of total RNA was separated on a
1% agarose gel containing 6.5% formaldehyde. RNA was transferred to a
nylon membrane and hybridized to a 0.5-kb
32P-labeled probe corresponding to most of the
coding sequence of the KC gene plus the 3' untranslated region.
Hybridization was performed at 65°C in Rapid-Hyb buffer (Amersham
International, Amersham, U.K.). Gels were dried and exposed to
autoradiographic films or analyzed in a PhosphorImager (Molecular
Dynamics, Sunnyvale, CA).
Determination of chemokine protein levels in BAL fluid and lung homogenates
BAL samples were centrifuged at 1500 rpm for 10 min at 4°C to pellet cells, and supernatants were removed and stored at -80°C. After BAL, lungs were perfused with heparinized saline, removed, and snap frozen in liquid nitrogen. Lungs were homogenized in 3 ml of PBS and centrifuged to clear debris. KC, MIP-2, eotaxin, and MCP-1 levels were determined in BAL fluid and supernatants from the lung homogenates using commercially available ELISA kits for murine chemokines (R&D Systems, Minneapolis, MN). For KC and MIP-2 ELISA, samples were diluted to ensure they fell within the range of the assay (KC, 15.6–1000 pg/ml; MIP-2, 7.8–500 pg/ml).
Administration of anti-CD4 and anti-CD8 Abs
Mice were given an i.p. injection of either rat anti-mouse CD4 (250 µg; GK1.5) or rat anti-mouse CD8 (125 µg; 53-6.7), which have been shown to deplete these cells in vivo (33) or the same amount of the respective isotype control Ab (rat IgG2a or rat IgG2b; all Abs obtained from PharMingen) at 48 and 24 h before 5% OVA aerosol. BAL was performed at 6 h, and the BAL fluid was stored at -80°C for analysis of chemokine levels by ELISA. In these experiments, CD4 and CD8 depletion was confirmed using FACS analysis based on the expression of the murine T cell surface markers, CD4 and CD8 (data not shown). Briefly, peribronchial lymph nodes were excised into cold DMEM supplemented with 10% FCS. After homogenization and separation over a Lympholyte gradient (Cedarlane Laboratories, Hornby, Ontario, Canada), cells were stained with rat anti-mouse Thy 1.2 in combination with either rat anti-mouse CD4 or rat anti-mouse CD8 (all obtained from PharMingen). Analysis of the lymphocyte population was performed with a FACS Scan flow cytometer (Becton Dickinson, San Jose, CA) and confirmed that selective depletion of either CD4+ or CD8+ cells was obtained under the appropriate conditions.
Administration of KC and MIP-2 neutralizing Abs
At 30 min before the OVA aerosol, mice were lightly sedated by halothane anesthesia and 50 µg of each of rat anti-mouse KC and rat anti-mouse MIP-2 or isotype controls (rat IgG2a for anti-KC and rat IgG2b for anti-MIP-2; all obtained from R&D Systems) were administered intranasally (i.n.) in a total volume of 100 µl. BAL was performed 6 h after the 5% OVA aerosol, and cells were enumerated as already described.
Chemokine production in vitro
Spleens were isolated from naive BALB/c DO11.10 mice (source of
CD4+ T cells) and naive wild-type BALB/c mice
(source of APCs; Charles River Breeding Laboratories, Wilmington, MA)
and placed into ice-cold DMEM supplemented with 2% FCS. Spleens were
strained through a wire mesh filter to disperse cells. Isolation of
CD4+ T cells was performed using positive
selection with magnetic beads (Dynal, Oslo, Norway). Isolated
CD4+ T cells were cultured at a concentration of
2 x 105 cells per well in flat-bottom
96-well plates using one of two protocols. In the first,
CD4+ T cells were cultured for 24 h in the
presence of 2 x 106 irradiated (2600 rad)
spleen cells (APCs) from wild-type mice and 300 nM of specific (OVA) or
nonspecific Ag (hen egg lysozyme, HEL; Sigma). Similar experiments
tested the effects of anti-TNF-, clone MP6-XT3, 10 µg/ml
(PharMingen) and anti-IFN-
, clone XMG1.2, 10 µg/ml
(PharMingen) and the relevant isotype control, IgG1, clone R3-34, 10
µg/ml (PharMingen) on chemokine production. The neutralizing Abs were
present for the entire culture period. In the second protocol,
CD4+ T cells were cultured for 24 h in the
presence of IL-2 (100 U/ml) in control or anti-CD3-coated wells. In
another experiment, alveolar macrophages were recovered aseptically by
BAL from naive DO11.10 mice and cultured for 24 h without
stimulation. DMEM contained -mercaptoethanol (50 µM) and was
supplemented with 10% FCS and L-glutamine. Cultures were
maintained in a 200-µl volume, and supernatants were assayed for KC
and MIP-2 by ELISA as described above. In the case of the
anti-CD3-stimulated CD4+ T cells,
supernatants were also assayed for IFN- (PharMingen).
Data analysis
Results are expressed as mean ± SEM of n observations (mice). Significant differences between treatment groups were determined by ANOVA in conjunction with the Dunnett’s modified t statistic for comparison to a single control group (34). Differences were considered significant when p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Exposure of BALB/c DO11.10 mice to a 5% OVA aerosol induced a BAL
cellular infiltrate, which by 6 h consisted almost entirely of
neutrophils (Fig. 1
). Conversely, a 5%
aerosol of BSA failed to induce any cell infiltrate, demonstrating the
Ag specificity of the neutrophil influx. Interestingly, neutrophils are
selectively recruited in the airways, whereas eosinophils, macrophages,
and lymphocytes are nearly absent. DO11.10 mice were very sensitive to
OVA aerosol, such that there was still significant neutrophil influx
with an OVA aerosol concentration of 0.01% (Fig. 2A). The 5% OVA aerosol
produced an influx of neutrophils, which could be seen as early as
3 h, peaked at 6 h, and although still elevated above
baseline, was resolving by 24 h after OVA aerosol exposure (Fig. 2B). Staining of dispersed lung cells with an Ab specific
for the transgenic TCR (KJ1-26) and for CD3 (Thy 1.2) and subsequent
FACS analysis indicated that the first 6 h of this response to OVA
aerosol was not associated with any preferential recruitment of
Ag-specific or nonspecific T cells to the lung (data not shown).
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Total lung RNA was used to determine the time course of KC and
MIP-2 mRNA expression after OVA aerosol. Northern blot analysis was
used to detect KC expression, and a RNase protection assay was used to
detect MIP-2 expression. KC mRNA was not detected in lungs from naive
mice, but was clearly detected in lungs from mice at 1 h, peaked
at 3 h, and was declining but still detectable by 6 h after
5% OVA aerosol exposure (Fig. 3A). MIP-2 mRNA expression
followed a similar pattern, such that expression peaked at 3 h and
although still detectable was declining by 6 h after 5% OVA
aerosol exposure (Fig. 3B).
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Protein levels for various murine chemokines during the first
24 h after 5% OVA aerosol were determined in BAL fluid and lung
homogenates by ELISA. Levels of KC and MIP-2 in BAL fluid were greatly
increased by OVA aerosol and were maximal by 1 h after aerosol
exposure. Although the elevation in MIP-2 expression was much more
transient than KC, they both had declined to baseline by 24 h
(Fig. 4, A and B).
A similar time course for KC and MIP-2 protein production was observed
in the lung homogenates prepared from 5% OVA aerosol-challenged mice
(Fig. 4, C and D). As an important control, KC
levels in BAL fluid were tested 6 h after 5% BSA aerosol.
Although slightly elevated above baseline, KC levels after BSA aerosol
were much lower (58 ± 19 pg/ml; n = 5;
p < 0.05) than at the corresponding time point in 5%
OVA aerosol-exposed mice (2205 ± 53 pg/ml). Differences in
eotaxin and MCP-1 expression after OVA aerosol were detected but were
much lower (100-fold) than those seen for KC and MIP-2 and occurred
at later time points (Fig. 4, E and F).
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Abs to murine KC and MIP-2 were administered i.n. before OVA
aerosol exposure to establish whether these chemokines were
functionally involved in neutrophil recruitment in vivo. Administration
of anti-KC alone or anti-MIP-2 alone failed to affect
neutrophil numbers in BAL at 6 h after 5% OVA aerosol (data not
shown). However, the combination of these Abs reduced neutrophil influx
seen at 6 h after OVA aerosol by 69%, when compared with the
combined isotype control treatment (Fig. 5).
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We have previously shown that in vivo depletion of
CD4+ T cells but not CD8+ T
cells abrogates the neutrophil influx seen at 6 h in this model
(32). To demonstrate that this effect is indeed related to
reduced chemokine production, KC levels were measured in BAL fluid
after OVA aerosol in mice depleted of CD4+ or
CD8+ T cells. CD4+ T cell
depletion resulted in a 61% reduction in KC levels detected in BAL
fluid measured at 6 h after 5% OVA aerosol (Fig. 6). This correlated with the 66%
reduction in neutrophil influx seen after this treatment
(32). Differences in MIP-2 protein levels due to
CD4+ or CD8+ T cell
depletion could not be determined at the 6-h time point because MIP-2
levels had already returned to near baseline levels (see Fig. 3
B).
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The ability of OVA to stimulate chemokine production upon Ag
presentation to CD4+ T cells was tested in vitro
using CD4+ T cells isolated from the spleens of
BALB/c DO11.10 mice and APCs (irradiated splenocytes) from BALB/c
wild-type mice. ELISA analysis of supernatants harvested after 24-h
culture revealed that the production of KC and MIP-2 was highest in
OVA-stimulated cultures that contained both APCs and
CD4+ T cells (Fig. 7A). Cultures containing both
APCs and CD4+ T cells that were stimulated with
an irrelevant Ag, HEL, produced much less KC and MIP-2. Both APC and
CD4+ T cells were required because OVA-stimulated
cultures of APCs alone or CD4+ T cells alone
clearly produced much lower amounts of KC and MIP-2 protein compared
with cultures in which both cell populations were present (Fig. 7A). Addition of anti-TNF- reduced OVA-induced
production of KC and MIP-2 by 40 and 55%, respectively (Fig. 7B). Although anti-IFN- had some effect on KC
production (22% reduction), it failed to reduce MIP-2 production (Fig. 7B).
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but failed to produce either KC or MIP-2
(Table I). BAL performed on naive DO11.10
mice resulted in the recovery of >95% pure population of alveolar
macrophages. Upon culture for 24 h, even nonstimulated macrophages
produced large amounts of both KC and MIP-2 (data not shown). It
appeared that despite care in handling these cells during the process
of recovery, alveolar macrophages became activated to produce large
amounts of KC and MIP-2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neutrophils and their chemokines in lung inflammation
KC and MIP-2 gene expression and production of their respective proteins was induced very rapidly after OVA aerosol exposure. Consequently, neutrophils were seen in the BAL fluid of DO11.10 mice as soon as 3 h after OVA aerosol exposure. The rapid nature of this response is consistent with the role of these cells as important effector cells in acute inflammation. In the human, IL-8 is thought to be one of the important neutrophil chemoattractants in lung allergy associated with asthma (24, 35). Given the lack of reagents to inhibit the activity of neutrophil chemokines in vivo, this has been difficult to prove definitively. The neutralizing Abs for KC and MIP-2 confirmed that KC and MIP-2 were functional chemoattractants in this mouse model. Even so, the inhibition of neutrophil influx seen with the neutralizing Abs was not complete, suggesting that other chemokines may also be involved. The fact that only the combination of neutralizing Abs for KC and MIP-2 inhibited BAL neutrophil influx suggested that KC and MIP-2 were equally redundant in mediating neutrophil chemoattraction. Consistent with the absence of other leukocytes in the BAL fluid after OVA aerosol, chemokines known to be important in the trafficking of other leukocytes such as eotaxin and MCP-1 (9) were not regulated in a similar time frame or to the same extent as the neutrophil chemokines KC and MIP-2.
The increase in protein for a chemokine seen after an inflammatory insult should be preceded by an increase in expression of its mRNA. Our data suggest that this is the case for KC because increased mRNA for KC was detected by 1 h after OVA aerosol, peaked at 3 h, and was declining by 6 h. Protein levels in both BAL fluid and lung were also elevated by 1 h and remained at this level until at least 6 h after OVA aerosol exposure. However, in the case of MIP-2, there appears to be a less close relationship between mRNA expression and protein production. MIP-2 mRNA expression peaked at 3 h, by which time the protein levels in both the BAL fluid and lung were already declining. Although well below peak levels, MIP-2 protein was still elevated at 6 h after OVA aerosol exposure. The reasons for this aberrant association between MIP-2 protein production and mRNA expression are unclear but there are numerous possibilities. For instance, the inflammatory response might be associated with up-regulation of proteases responsible for the degradation of MIP-2 or else much of the newly synthesized MIP-2 might be truncated to yield a peptide form that could not be detected by the ELISA. N-terminal truncation is commonly seen in chemokines and has been shown to occur for the human neutrophil chemokine granulocyte chemotactic protein-2 (16).
IL-8 and related chemokines are important regulators of neutrophil margination to tissues in many inflammatory settings. It has been shown that the production of neutrophil chemokines correlates with neutrophil influx in LPS (36, 37), particulate dust (38), ozone (39), immune complex (40), bacterial infection (41), and cystic fibrosis (42) models of lung inflammation. Mechanisms explaining up-regulation of the IL-8-related chemokines in allergy are poorly understood. The study of Lukacs and colleagues (43) in a mouse model of lung allergy demonstrated that the neutrophil chemokine ENA-78 played an important role in neutrophil influx into the lung after Ag challenge. In that study, in vitro work indicated that both Ag-specific and nonspecific activation of mast cells caused the release of ENA-78. They propose that IgE-dependent activation of mast cells may be an important pathway in the production of neutrophil chemoattractants. Up-regulation of neutrophil chemokines seen in the current study is clearly independent of IgE because the exposure to OVA aerosol was a primary event and, in any case, serum total IgE was shown to be at baseline in DO11.10 mice both before and after primary OVA aerosol exposure (32).
Ag-specific activation of CD4+ T cells as a mechanism for chemokine production in allergic responses
In support of a critical role for CD4+ T
cells in defining the nature of an inflammatory response in the context
of allergy, in vivo depletion of CD4+ T cells but
not CD8+ T cells was shown to inhibit the airway
neutrophil margination in DO11.10 mice after OVA aerosol exposure
(32). Consistent with CD4+ T cells
playing a part in neutrophil chemoattraction, here we have shown that
CD4+ T cell depletion, but not
CD8+ T cell depletion, inhibited the increase in
neutrophil chemokines seen in the BAL fluid after OVA aerosol. To
verify the notion that Ag-specific CD4+ T cell
activation could directly lead to production of neutrophil chemokines,
we performed in vitro studies using CD4+ T cells
and APCs. CD4+ splenocytes isolated from BALB/c
DO11.10 mice were stimulated with OVA in the presence of irradiated
splenocytes as APCs. This interaction led to an impressive neutrophil
chemokine production that was clearly Ag specific because the
irrelevant but chicken egg-derived Ag, HEL, failed to produce a similar
response. Experiments with anti-CD3-activated
CD4+ splenocytes from DO11.10 mice indicated that
stimulation through the TCR complex results in significant IFN- but
not KC or MIP-2 production by CD4+ T cells.
Therefore, CD4+ T cells themselves seem unlikely
sources for KC and MIP-2. When we cultured alveolar macrophages that
were isolated by BAL, even nonstimulated macrophages produced large
amounts of KC and MIP-2 (data not shown). This is in line with another
study in which cultured alveolar macrophages from humans were shown to
produce neutrophil chemokines in the resting state and after LPS
stimulation (44). Because alveolar macrophages can also
function as APCs, they may be involved in the neutrophil chemokine
production seen in our model. Furthermore, the rapid neutrophil
chemokine expression and neutrophil influx into the lungs suggested
that the lung APCs were able to process OVA and present it to
CD4+ T cells very quickly. In such a short time,
APCs will not have had time to migrate to the regional lymph nodes of
the lung to present Ag to lymph node CD4+ T
cells. Experiments using KJ1-26, an Ab specific for the transgenic
TCR-bearing T cells, in combination with FACS analysis, demonstrated
that there was no preferential recruitment of Ag-specific T cells to
the lung at least within the first 6 h after OVA aerosol exposure.
Therefore, the production of neutrophil chemokines must have been the
result of activation of CD4+ T cells residing at
the lung mucosal surface.
Candidate mediators that may be responsible for the induction of KC and
MIP-2 production as a result of the interaction between
CD4+ T cell and APC included cytokines such as
TNF- and IFN-. TNF- has been shown to induce the production of
neutrophil chemokines, resulting in neutrophil infiltration in a murine
air-pouch model of leukocyte migration (45) and in the
alveolar epithelial cell response to LPS (46). In
addition, IFN- has been shown to be involved in the induction of
lung chemokine production (47). Using ELISA to measure
each of these cytokines in BAL fluid, neither TNF- nor IFN- were
elevated above control levels at 1 or 6 h after OVA aerosol
exposure (data not shown). However, when we tested the effects of
anti-TNF- and anti-IFN- on in vitro production of KC and
MIP-2 by CD4+ T cells, we found evidence for
involvement of TNF- as a secondary mediator. Neutralization of
TNF- markedly reduced the production of both KC and MIP-2 in this
system and TNF- may be an important secondary mediator encouraging
the production of these chemokines in vivo.
Recruitment of neutrophils into the lungs is commonly seen in asthma and animal models of lung allergy (1, 2, 3, 4, 5). Mechanisms to explain increased neutrophil infiltration in asthma and allergy are not well characterized. Since activation of CD4+ T cells has been implicated as a causal mechanism in lung allergy (26, 27, 29), we proposed that CD4+ T cells can play a role in mediating the rapid recruitment of neutrophils to the lung. The model used in this study did not mimic human asthma, as there was no evidence for airway hyperresponsivensss (AHR) in response to either single OVA aerosol challenge or multiple OVA aerosol challenges (32). Using the DO11.10 mouse, our point has been to demonstrate that an early outcome of Ag-specific CD4+ T cell activation in the airways is the up-regulation of neutrophil chemokines that promote the migration of these cells into the lung. These data do not allow us to conclude that neutrophilic inflammation is incapable of inducing AHR, especially because it is likely that there are many factors that might contribute to the development of AHR, including, for instance, the chronicity of the disease. Even so, our findings are particularly interesting in the context of a recent study by Nocker and coworkers using segmental allergen challenge in allergic asthmatics (3). These investigators showed that within 4 h after allergen challenge, IL-8 is produced and neutrophils are recruited to the site of exposure. The lack of an increase in the levels of the mast cell mediator tryptase was suggestive of no mast cell involvement. Thus, CD4+ T cell activation may be a mechanism explaining neutrophil recruitment in response to allergen inhalation.
The ability of CD4+ T cells to respond rapidly to Ag and induce production of chemokines may have significance in occupational asthma in which there is limited evidence for IgE-related immunity but clearly an Ag-specific pathophysiology (48). In these asthmatic patients, although IgE to the sensitizing agent cannot be detected, asthmatic responses can develop quickly upon re-exposure to the Ag (48). In subjects with grain-dust-induced occupational asthma, there was significant IL-8 activity in BAL fluid, and neutrophils predominated in bronchial biopsy specimens (49). Also, in patients with occupational asthma induced by low m.w. compounds, neutrophilic inflammation is dominant over eosinophilic inflammation (50). Therefore, it is conceivable that Ag-specific CD4+ T cell activation resulting in rapid neutrophil chemokine production and neutrophil recruitment may be a pathogenic mechanism in these types of asthma.
In summary, we have shown that Ag-specific activation of CD4+ T cells in the airways of BALB/c DO11.10 mice results in rapid production of functional neutrophil chemokines. This suggests that, similarly to cytokine production, chemokine production may be an important outcome of Ag-specific CD4+ T cell activation. Indeed, CD4+ T cell activation leading to neutrophil chemokine production may explain the neutrophil influx seen commonly in the lungs of allergic asthmatics after Ag challenge. These data extend the role for CD4+ T cells in allergy from cells that regulate tissue cytokine levels to cells that are also players in the production of chemokines that signal recruitment of effector cells to the site of Ag challenge.
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Acknowledgments |
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Footnotes |
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2 Current address: Institut de Recherche Jouveinal/Parke Davis, 3a9 rue de la Loge, 94265 Fresnes Cedex, France
3 Abbreviations used in this paper: MIP, macrophage inflammatory protein; BAL, bronchoalveolar lavage; HEL, hen egg lysozyme; i.n., intranasal(ly); CXCR, CXC chemokine receptor; MCP-1, monocyte chemoattractant protein-1; AHR, airway hyperresponsiveness.
Received for publication October 29, 1999. Accepted for publication October 25, 2000.
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F. J. D. Mennechet, L. H. Kasper, N. Rachinel, W. Li, A. Vandewalle, and D. Buzoni-Gatel Lamina Propria CD4+ T Lymphocytes Synergize with Murine Intestinal Epithelial Cells to Enhance Proinflammatory Response Against an Intracellular Pathogen J. Immunol., March 15, 2002; 168(6): 2988 - 2996. [Abstract] [Full Text] [PDF]
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)
or 5% OVA aerosol (•). BAL was performed 1, 3, 6, or 24 h after
aerosol exposure. Results are expressed as mean ± SEM of data
from four to six mice. Significance (*) was determined in
(A) by ANOVA followed by a Dunnett’s modified
t test to correct for multiple comparisons and in
(B) by Student’s t test and indicates
OVA aerosol treatment significantly different from PBS aerosol-exposed
mice (p < 0.05).
