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in the Inflammatory Component of Allergic Airway Disease
Millennium Pharmaceuticals, Inc., Cambridge, MA 02139.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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/ß (SDF-1/ß) is
phylogenetically a primitive chemokine widely expressed in a variety of
tissues and cell types. This expression is detectable in the absence of
stimuli provided by bacterial or viral infections and allergic or
autoimmune disorders. Based on these and other findings, SDF-1 has
not been considered an inflammatory chemokine, but, rather, has been
believed to be involved in certain homeostatic processes, such as
leukocyte recirculation. SDF-1 is a potent chemoattractant for
lymphocytes and monocytes that mediates its activity via the chemokine
receptor CXCR4. Study of the role of SDF-1/CXCR4 in vivo during
inflammation has been limited by the fact that transgenic mice that
have been made deficient in either molecule die early in life due to
developmental defects. The present study was aimed at evaluating the
functional relevance of the SDF-1/CXCR4 axis during an inflammatory
process. Neutralizing Abs to CXCR4 reduced lung eosinophilia
(bronchoalveolar lavage fluid and interstitium) by half, indicating
that CXCR4-mediated signals contribute to lung inflammation in a mouse
model of allergic airway disease (AAD). This reduction in inflammation
was accompanied by a significant decrease in airway
hyper-responsiveness. SDF-1 neutralization resulted in similar
reduction in both lung allergic inflammation and airway
hyper-responsiveness. Retroviral delivery of a CXCR4 cDNA to leukocytes
resulted in greater inflammation when transduced mice were subjected to
a mouse model of AAD. These results highlight that, although considered
a noninflammatory axis, the involvement of CXCR4 and SDF-1 is
critical during AAD, and this receptor and its ligand are potentially
relevant in other inflammatory processes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ß
(SDF-1/ß;2 also
known as pre-B cell growth stimulating factor), the only chemokine
described to bind CXCR4 (fusin, LESTR, npy3r) (1, 2), is
expressed constitutively in a wide variety of tissues and is also a
powerful chemoattractant for T lymphocytes, among other cell types
(3). Mice genetically deficient for SDF-1/ß exhibit a
severe defect in cardiac development and die perinatally
(4). CXCR4 is expressed on human PBLs, monocytes,
neutrophils, and CD34+ hemopoietic progenitors
and has been described as the major coreceptor for HIV-1/2 on T
lymphocytes (1, 2, 3, 5, 6). Similar to mice lacking
SDF-1/ß, CXCR4-deficient mice die in utero (7, 8),
complicating analysis of the contribution of this chemotactic axis
in vivo. Lung inflammation and other disorders associated with asthma are dependant on the actions of chemokines and their specific receptors (9, 10, 11, 12, 13, 14, 15, 16). During lung allergic inflammation, chemokine-chemokine receptor interactions direct both the recruitment of different infiltrating cell types to the lung and the production of inflammatory mediators that intensify pulmonary damage (10, 12, 15, 16, 17, 18, 19, 20).
Signals delivered through the chemokine receptor CXCR4 upon interaction
with its ligand, SDF-1/ß, result in the most efficacious
chemoattraction of T lymphocytes detected to date (1, 2, 3, 21, 22). Most recently, other axes, such as macrophage inflammatory
protein-3ß/CCR7 has been described to participate in naive T cell
chemoattraction as well as dendritic cell/recirculating T cell
interactions (23, 24).
T lymphocyte-derived products such as IL-4 and IL-5 are critical players in the development and progression of lung eosinophilia and airway hyper-responsiveness (25, 26, 27, 28, 29, 30). In fact, the in vivo depletion of T lymphocytes in mice or the lack of lymphocytes in genetically deficient mice prevents this pathological response (12, 26, 31). In humans, postmortem examination of the airway or bronchial biopsies of asthmatic patients reveal large T lymphocyte infiltrates (32).
The constitutive expression of SDF-1 in a wide variety of tissues,
including lung, together with the lack of modulation of its expression
during inflammation suggest a putative noninflammatory role of this
chemokine, possibly related to the maintenance of normal leukocyte
recirculation (3, 33). However, because 1) chemokine
expression in the lung promotes the recruitment of inflammatory
leukocytes to this organ; 2) lymphocytes are absolutely required for
the development of lung allergic reactions; and 3) SDF-1 is a very
efficient chemoattractant for lymphocytes, we decided to analyze the
relevance of CXCR4-SDF-1/ß interactions during inflammation,
specifically on leukocyte recruitment to the lung and the subsequent
impact during allergic airway disease (AAD). To assess the role of
CXCR4/SDF-1 in inflammation we used a mouse model of lung
inflammation based on the repeated exposure of mice to aerosolized OVA
(34). In this report we 1) studied the effects of the
blockage of CXCR4 on three end points of AAD: leukocyte accumulation in
interstitium and airway lumen as well as airway hyper-responsiveness;
2) evaluated AAD progression after CXCR4 overexpression in leukocytes
following retroviral gene transduction; and 3) examined the correlation
between the accumulation of specific leukocyte types and the expression
of CXCR4 in the inflamed lungs. The findings presented here establish
an important role for CXCR4 and SDF-1 during the development of AAD
and suggest a potential relevance of this receptor and its ligand in
other lymphocyte-mediated inflammatory reactions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rabbit polyclonal Abs against murine CXCR4 were prepared according to standard methods (35). This Ab was generated against the 16-aa peptide corresponding to aa 181–196 in the mCXCR4 peptide sequence. The sequence of the peptide was QGDISQGDDRYICDRL. This 16-aa peptide shares 85 and 69% homology with its rat and human orthologue genes, respectively, and does not appear in any other known gene described to date. The ability of the neutralizing Abs raised against the second extracellular domain of human CXCR4 to block T cell infection by certain HIV-1 and HIV-2 strains is consistent with the role of this second extracellular loop in the coreceptor activity of CXCR4 (36, 37). Rabbit serum was passaged over a protein A column, and then anti-CXCR4 Abs were purified from the flow-through on an affinity column using the same peptide (Research Genetics, Huntsville, AL).
Generation of the HEK-293/mCXCR4 cell line
The full coding sequence of mCXCR4 was subcloned into the
mammalian cell expression vector pcDNA3.1 (Invitrogen, San Diego, CA).
Stable cell lines were generated following transfection of the
expression vector into HEK-293 cells using Lipofectamine (Life
Technologies, Gaithersburg, MD) and selected with G418 (0.8 mg/ml; Life
Technologies). Clones expressing high levels of mCXCR4 were identified
by binding to 125I-labeled SDF1- (Amersham,
Arlington Heights, IL), selected, and expanded for the experiments
described here.
Cell culture, immunofluorescent staining, and FACS analysis
The mouse cell lines BAF-3 and L1.2 were grown in RPMI 1640 supplemented with 10% FCS. IL-3 (5 ng/ml) was added to cultured BAF-3 cells (38). Murine CD4+ T splenocytes were purified using the R&D Systems separation kit (Minneapolis, MN) and were cultured in RPMI 1640 supplemented with 10% FCS.
Cells (1 x 105) were resuspended in staining buffer (0.1% BSA, 0.02% sodium azide, and PBS) incubated with 10 µg/ml (1/50) of purified anti-mouse CD16/CD32 (FcR; PharMingen, San Diego, CA) for 20 min at 4°C, and then incubated with 10 µg/ml of either anti-mouse CXCR4 Abs or normal rabbit IgG for 30 min at 4°C. Cells were washed and stained with FITC-conjugated goat anti-rabbit IgG (Dako, Glostrup, Denmark) for 20 min at 4°C. The stained cells were washed twice, collected by FACScan flow cytometer, and analyzed by CellQuest software (Becton Dickinson, Mountain View, CA). Murine bone marrow, blood, or spleen cells were stained with FITC/PE-labeled mAbs specific for CD3, CD4, CD8, B220, MAC-1, and GR-1 (PharMingen, San Diego, CA) following the protocol described above.
In vitro chemotaxis
The in vitro migration of BAF-3 cells and
CD4+ T splenocytes to different concentrations of
SDF-1 (R&D Systems) was evaluated in duplicate using Costar
Transwells (Cambridge, MA) as previously described (34).
In the blocking experiments cells were preincubated with either 10
µg/ml of anti-mCXCR4 Ab or control Ab at 4°C for 30 min before
their addition to the Transwell inserts.
Calcium flux assay
Cells were labeled with the fluorochrome fluo-3/AM (Molecular Probes, Eugene, OR) according to the manufacturer’s recommendations. Briefly, 50 µg of fluo-3 AM was dissolved in 44 µl of DMSO and diluted to 10 µM with modified Gay’s buffer (MGB; 5 mM KCl, 147 mM NaCl, 0.22 mM KH2PO4, 1.1 mM Na2HPO4, 5.5 mM glucose, 0.3 mM MgSO4-7H2O, 1 mM MgCl2, 10 mM HEPES (pH 7.4), and 0.1% BSA). Cells (107) were resuspended in 1 ml of MGB and incubated with an equal volume of 10 mM fluo-3 mix for 30 min at room temperature. Excess dye was removed by centrifugation, and cells were resuspended at a concentration of 2–106/ml in MGB buffer and 1.5 mM CaCl2. Calcium influx was measured on the FACScan by analyzing FL1 (linear scale) vs time. In the blocking experiments cells were preincubated with either 10 µg/ml of anti-mCXCR4 Ab or control Ab at 4°C for 30 min before evaluation of Ca2+ mobilization.
In vivo induction of AAD
Eight- to 10-wk-old C57BL/6J mice were purchased from The
Jackson Laboratory (Bar Harbor, ME) and kept in a specific
pathogen-free mouse facility (Millennium Pharmaceuticals, Cambridge,
MA). The mouse model of lung inflammation used here consists of a
sensitization phase (OVA, 0.1 mg/mouse i.p. on day 0; Sigma, St. Louis,
MO) and an induction of the response phase (2% OVA for 5 min i.n. on
day 8 and 1% OVA for 20 min. i.n. on days 15–21; Fig. 3
A).
PBS (i.p. and/or i.n.) was administered to mice as a negative control.
For the blocking experiments, mice also received 10 µg/mouse of
neutralizing polyclonal Abs against either CXCR4 (described here) or
SDF-1 (R&D Systems). These Abs were administered i.v. and 30 min
before OVA provocation on day 8 and days 15–21 or on days 20–21 (Fig. 3A). OVA-treated control mice were injected with the same
amount of control Ab at the same time points indicated during
treatment. Rabbit Ig fraction (Dako, Santa Barbara, CA) was used as a
control for the CXCR4 Ab. Three hours after OVA administration on day
21, mice were sacrificed by CO2 asphyxiation and
analyzed for leukocyte accumulation and AHR.
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Immunohistochemical phenotyping and quantitation of leukocytes
Total BAL cell counts were performed, and aliquots (5 x 105 cells/slide) were pelleted onto glass slides by cytocentrifugation. To determine the number of eosinophils and neutrophils, slides were stained with Wright-Giemsa (Fisher Diagnostics, Pittsburgh, PA). T lymphocytes, B lymphocytes, and mononuclear phagocytes were identified by Thy 1.2 (53-2.1; PharMingen, San Diego, CA), IgM (II/41; PharMingen, San Diego, CA), and Moma-2 (BioSource, Camarillo, CA) staining, respectively, as previously described (12). The percentages of eosinophils, lymphocytes, neutrophils, and macrophages were determined by counting their number in eight high power fields (x40 magnification; total area, 0.5 mm2) per area randomly selected and dividing this number by the total number of cells per high power field. To obtain the absolute number of each leukocyte subtype in the lavage, these percentages were multiplied by the total number of cells recovered from the BAL fluid.
Lung sections from the different experimental groups of mice were prepared as previously described (12). Sections (4 µm) were stained with hematoxylin/eosin according to standard protocols. An estimation of the percentage of each leukocyte subtype within the infiltrate in OVA- plus Ab-treated mice or OVA- plus rabbit Ig-treated controls was made by counting 200 cells in one randomly selected peribronchiolar infiltrate and determining the numbers of eosinophils, monocytes, and lymphocytes present.
Measurement of CXCR4 mRNA expression by multiprobe RNase protection assay
Total RNA from the lungs of OVA-treated mice or control littermates at different time points was extracted by a single-step method using RNA STAT-60 (Tel-Test, Friendswood, TX). CXCR4 mRNA expression was determined by Multiprobe RNase protection assay as previously described (16). A 403-bp mCXCR4 probe was derived by PCR using the following primers: 5'-GTAATACGACTCACTATAGGGAACGCTGCTGTAGAGGTTGAC-3' and 5'-GTAACCACCACGGCTGTA-3'. The identity and quantity of each mRNA species in the original RNA sample were determined based on the signal intensities given by the appropriately sized, protected probe fragment bands. Values were created by expressing mCXCR4 up-regulation relative to its expression in normal tissue. The sample loading was normalized by the housekeeping gene, GAPDH, which is included in each template set.
Assessment of mCXCR4 and SDF-1 protein by immunohistochemistry
Protein expression was determined in noninflamed and inflamed
mouse lung tissue samples with either polyclonal rabbit anti-mCXCR4
Ab or polyclonal goat anti-mouse SDF1 (R&D Systems) followed by
a modified avidin/biotin staining method as previously described
(12). Peripheral blood smears from control mice (BM-mock
RV) and CXCR4 overexpressing littermates (CXCR4 RV) were fixed in
acetone and immunostained by overnight incubation at 4°C with the
anti-mouse CXCR4 Ab diluted in PBS/0.05% Tween. Slides were washed
in PBS, then incubated for 30 min at room temperature in PBS/Tween
containing a 1/200 dilution of FITC-labeled goat anti-rabbit IgG
(Southern Biotechnology Associates, Birmingham, AL) and a 1/30 dilution
of PE-labeled mouse anti-CD45.1 (Ly5.2). Slides were washed twice
in PBS/Tween and mounted in Fluoromount-G (Southern Biotechnology
Associates) for microscopy. Control slides were stained with an
isotype-matched negative control Ab instead of primary Ab, or
biotinylated anti-rabbit or goat Ig or streptavidin complex was
selectively omitted.
Generation of retrovirally transduced leukocytes
The open reading frame of CXCR4 cDNA was cloned into pMSCVneoEB
(39). Clones containing sense orientation of inserts were
identified and then purified by MaxiPrep (Qiagen, Valencia, CA) and
transfected into 293 Ebna (Invitrogen) using Lipofectamine (Life
Technologies) reagent along with pN8e gagpol and pN8e EnvE wobble.
Viral supernatants were harvested through 0.2-mm pore size filter and
then used to infect tunicamycin-pretreated 
E cells (50 mg/ml) with
polybrene (8 mg/ml; Sigma) to generate pools of stable virus-producing
cells under G418 selection (1 mg/ml; Life Technologies).
For donor BM cells, BL6.SJL (ly5.1) mice were injected with 5-fluorouracil (15 mg/kg), and after 4 days BM cells were harvested (recovery being around 2 x 106 cells/mouse). BM cells (4 x 106 cells) were cocultured for 72 h in a 100-mm dish with mitomycin C-treated CXCR4 producer cells at 80% confluence in RPMI containing 10% FCS, 1% penicillin-streptomycin, 1% essential amino acids, and the following growth factors and cytokines at 0.2 µg/ml: IL-3, SCF, and IL-6. At the time of transplant, lethally irradiated C57/B6 mice (950 rad) were injected i.v. with 1 x 105 virally infected bone marrow cells. Mice were monitored at 12 days and 5 wk posttransplant in the peripheral blood for evidence of reconstitution and gene expression before analysis at 9 wk. At this time mesenteric lymph nodes and bone marrow were phenotyped to demonstrate that neither viral infection nor expression of transgene affected the normal distribution of leukocyte/lymphocyte subsets. Nine weeks after reconstitution, expression studies and migration studies were performed on retrovirally transduced mice following the protocol explained above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because CXCR4-deficient mice die in utero (7, 8),
neutralizing Abs against CXCR4 were designed to study the contribution
of this chemokine receptor to inflammatory allergic reactions in vivo.
Affinity-purified anti-mCXCR4 Abs that reacted positively with the
peptide against which they were prepared gave positive immunostaining
with a variety of mouse hemopoietic cells. Anti-mCXCR4 Abs strongly
immunostained K293 cells transfected with a cDNA encoding the full
coding sequence of the mCXCR4 gene, B cell lines such as BAF-3 and
L1.2, monocytic cell lines such as RAW287 and J774, T cell lines such
as AE-7 and Dorris, as well as CD4+ purified
splenocytes, blood mononuclear cells, and blood neutrophils (Fig. 1, A and B, and
data not shown). All these cell types also show CXCR4 mRNA expression
(data not shown). Eosinophils from IL-5 transgenic mice, CHO cells, and
mock-transfected K293 cells that did not show CXCR4 mRNA expression by
PCR were not stained by the same Ab (Fig. 1, A and
B, and data not shown). Similarly, K293 cells transfected
with a mouse and/or human cDNA encoding CCR1, CCR2, CCR3, CCR4, CCR6,
CCR7, CCR9, D6, and CXCR2 were not stained by the anti-mCXCR4 Abs
(data nor shown), indicating that cross-reactivity with other chemokine
receptors can be excluded.
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C) and was almost completely blocked by
preincubating BAF-3 cells with SDF-1 (Fig. 1C).
The ability of the anti-CXCR4 Abs to neutralize the action of
SDF-1 on different cell lines expressing CXCR4 was analyzed. BAF-3
cells were found to migrate (25–30%) in response to SDF-1 (Fig. 2A). This migration was
completely abrogated after preincubation of these cells with
anti-mCXCR4 Abs (Fig. 2A). Blockage of SDF-1-induced
BAF-3 cell migration was achieved by the anti-mCXCR4 Abs in a
dose-dependent manner (data not shown). Eotaxin-induced migration of
eosinophils from IL-5 transgenic mice was not affected by the
anti-mCXCR4 Abs, confirming the specificity of these Abs for CXCR4
(Fig. 2A).
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, as shown by Ca2+
influx (Fig. 2B and data not shown). Anti-mCXCR4 Abs (10
µg/ml) also blocked the SDF-1-induced Ca2+
influx of L1.2 cells (Fig. 2B).
Taken together, these results demonstrate the ability of the
anti-mCXCR4 Abs to neutralize functional responses induced by
SDF-1 via CXCR4 in a specific manner.
Effect of CXCR4 neutralization on the accumulation of inflammatory leukocytes in the lung
Monocytes and lymphocytes are central to the inflammatory process
that results in eosinophilia and AAD (12, 16, 26, 27, 28, 29).
These cell types also respond functionally to SDF-1 via CXCR4 in
chemotaxis assays in vitro (40). In vivo blockage
experiments of this chemokine receptor during AAD were performed using
the specific neutralizing anti-CXCR4 Abs that were characterized
above (Figs. 1 and 2). The mouse model of OVA-induced inflammatory
response studied here induces the accumulation of monocytes and
macrophages in both lung interstitium and airways that becomes maximal
at early stages (sensitization phase) of the response (3 h after OVA
challenge on day 15) and an increasing interstitium and airway
accumulation of eosinophils and T lymphocytes that reaches its maximum
at later stages (challenge phase) of the response (12, 16)
(3 h after OVA challenge on day 21; Fig. 3). Development of airway
hyper-responsiveness is another feature of this model, reaching its
maximum at later stages of the response (12, 16).
Neutralizing anti-CXCR4 Abs (10 µg/mouse/day) were delivered i.v.
daily either during the whole OVA i.n. treatment (days 8 and 15–21, 30
min before OVA challenge) or exclusively at very late stages of the
response (days 20–21; Fig. 3A) to evaluate the involvement
of CXCR4 in the sensitization phase of the phenotype as well as during
the challenge phase. In either case, analysis was performed 3 h
after OVA challenge on day 21. Because the location of infiltrating
cells within the lung correlates strongly with the severity of the
inflammatory response and AHR (41, 42, 43), leukocyte
enumeration was performed in the airway lumen (BAL fluid) as well as in
the interstitium (lung sections) after OVA treatment.
CXCR4 neutralization during OVA treatment affected lymphocytes and eosinophils in both BAL fluid and pulmonary interstitium
Effect of CXCR4 neutralization on the pulmonary recruitment
of lymphocytes and monocytes. Mononuclear cell numbers were
reduced by 50% in the BAL fluid of OVA-treated mice following CXCR4
neutralization (Fig. 3B). Because 90% of these mononuclear
cells are CD4+ T lymphocytes (12),
this cell subset is clearly affected by the CXCR4 blockage. A decrease
in mononuclear cell infiltration in the lung interstitium of these mice
was also detected compared with that in OVA-treated control littermates
(Fig. 4, A and B,
and Table I). In fact, when sections were
examined blindly and assigned a morphological score based upon the
extent and size of peribronchiolar infiltrates, CXCR4 blockage was seen
to decrease the average score to 2.1 ± 0.6 compared with a mean
score of 4.5 ± 0.5 in control mice (Fig. 4 and Table I). Resident
macrophage numbers in the lung of OVA-treated mice were not affected by
the CXCR4 blockage (Fig. 3B and 4). No neutrophil
infiltration was detected in the lung in these groups of experimental
mice (Fig. 4 and data not shown).
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B and 4 and Table I). Because eosinophils
do not express CXCR4 or respond to SDF-1, the observed decrease in
eosinophilia is likely to be a secondary consequence of the CXCR4
neutralization-induced reduction of mononuclear cell (monocyte and/or
lymphocyte) accumulation in the lung.
In fact, monocytes and lymphocytes have been described to promote
eosinophil infiltration in this organ (12, 13, 14, 15, 16, 26, 27, 28, 29).
This interpretation was supported by experiments in which
neutralization of CXCR4 was performed exclusively in the last 2 days of
the treatment (days 20 and 21). Under these conditions, only a small
reduction (<10%, which did not reach statistical significance) in
OVA-induced eosinophil and mononuclear cell infiltration (in both the
airway lumen and lung interstitium) was detected after CXCR4
neutralization (Fig. 3B and data not shown).
The effect of neutralization of the CXCR4 ligand, SDF-1, was also
assessed in these studies. A similar reduction in the total number of
eosinophils and mononuclear cells was detected in both the BAL fluid
and lung interstitium of OVA-treated mice following SDF-1
neutralization compared with that induced by OVA following CXCR4
neutralization (Fig. 4 and Table II). As
observed in the CXCR4 neutralization experiments, this reduction was
only achieved when anti-SDF-1 Ab was administered from early
stages of the inflammatory response (data not shown).
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. These Abs were administered i.v.
and 30 min before OVA provocation on day 8 and days 15–21. Rabbit Ig
fraction was used as control for the CXCR4 Ab. AHR was evaluated 3
h after the last OVA administration on day 21. Fig. 5 shows that after methacholine
provocation on day 21, the experimental group of mice that was
subjected to CXCR4 neutralization showed a reduction of AHR of 30–35%
compared with that detected in OVA control-treated littermates at the
same time point. Similarly, SDF-1 neutralization reduced OVA-induced
AHR to a similar extent (Table II). This reduction was observed at the
point of maximal lung leukocyte accumulation (day 21, 3
h).
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Nine weeks after transplantation, CXCR4 protein expression was
demonstrated in 100% of CXCR4 retrovirally transduced donor derived
(Ly5.2+) peripheral blood cells (Fig. 6). Conversely, low endogenous CXCR4
expression was detected when an empty virus was used as a control (Fig. 6). No differences in total cell numbers and phenotype were detected
when bone marrow, blood, thymus, peripheral lymphoid organs, and BAL
fluid from the CXCR4-transduced mice were compared with their
equivalent organs in the control littermates (data not shown). This
suggests that the overexpression of CXCR4 does not alter leukocyte
differentiation and normal distribution of these cells in tissues.
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). Mononuclear cell numbers (lymphocytes
and monocytes) were also increased in the BAL fluid of OVA-treated
CXCR4-transduced mice (337 x 103plus 26.7 x 103) compared with that
observed in the control mice (241.6 x 103
plus 29.1 x 103; Fig. 7). Increased
eosinophilia in the lung interstitium of these mice was also detected
compared with that observed in OVA-treated control littermates (Fig. 8). The morphological scores of
peribronchiolar, perivascular, and interstitial infiltrates (extent and
size) showed that CXCR4 overexpression increases the average score to
4.5 ± 0.3 compared with a mean score of 3.5 ± 0.2 in
OVA-treated control mice (Fig. 8). Macrophage numbers were similar in
the lungs of both groups of mice (Figs. 7 and 8). It is important to
note that no neutrophil infiltration was detected in the lung of
CXCR4-transduced mice (Figs. 7 and 8 and data not shown). These data
indicate that overexpression of CXCR4 on leukocytes results in an
increased response to inflammatory stimuli.
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expression in the lung during the course of
AADThe results presented above show that modulation of the levels of CXCR4 on leukocytes (decreased levels by Ab neutralization or increased levels by gene transduction) correlates well with the intensity of the inflammatory response in the lung following OVA challenge in this particular mouse model. Therefore, we tested whether the detection of CXCR4-positive cells parallels the course of the inflammatory response during AAD.
This is specially relevant, because its ligand, SDF-1, is expressed
in the lung even under noninflammatory conditions, and no significant
change in its already high expression levels is observed during the
course of this AAD model (see Fig. 10 and data notshown). The main
source of SDF-1 protein before or after challenge is alveolar and
bronchial epithelium (see Fig. 10).
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). Although there was little CXCR4 mRNA
expression on day 0, by day 15 there was a 2-fold increase in
expression, which peaked on day 21. Day 15 represents the time of peak
infiltration of monocytes in this model (12). Cellular
localization of CXCR4 protein within inflamed lungs was then determined
by immunohistochemistry using anti-CXCR4 Abs described
above. Concomitant with the RNA
expression pattern, there was minimal expression of CXCR4 in normal
lungs (Fig. 9Bi), but a significant increase in expression
was evident on day 21 (Fig. 9Bii). The main cell type
expressing CXCR4 was mononuclear cells infiltrating the tissue
and mostly T lymphocytes, which make up the majority of infiltrating
mononuclear cells at this time point (data not shown)
(12).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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axis during AAD. This model, which has been used in both the mouse
(12, 16, 34) and the guinea pig (10), is
based on the observation that the administration of OVA induces massive
eosinophil infiltration and AHR (10, 12, 28, 29, 31, 34, 44). These are the two principal parameters of lung disease
analyzed. Even though most of the models of OVA-induced AAD combine the
OVA Ag in alum to induce a stronger response, preimmunization with
higher doses of OVA alone (0.1 mg) and subsequent aerosolized challenge
induce similar monocyte, CD4+ T cell, and
eosinophil lung infiltration, Th2 cytokine production, and AHR
(10, 12, 28, 29, 31, 34, 44). However, it is also known
that the mechanisms involved in the induction of the disease are
influenced by the route and dose of administration of the Ag. In fact,
depletion of T cells abrogates eosinophil infiltration by modulating
eotaxin expression in some AAD models (44) or without
affecting chemokine expression in some others (12). The
mouse strain used is also critical. Different components, such as IL-4
or IL-5, are central or secondary players in the induction of the
inflammatory response, which is influenced by the mouse genetic
background (28, 29). As indicated, mouse C57BL/6.SJL bone
marrow progenitor cells were infected with an MSCV retrovirus
containing the CXCR4 cDNA. Lethal irradiation chimeras were generated
using a standard protocol based on the use of these congenic mice with
a different allotype in the Ly5 locus. For comparison of the OVA
response in the bone marrow reconstitution experiments with CXCR4 or
SDF-1 neutralization experiments, the C57BL/6J strain was chosen.
C57BL/6J mice show a relatively modest OVA-induced AHR response
compared with that observed in BALB/c mice. However, 1) the OVA
protocol used here has been previously assessed in both BALB/c and
C57BL/6J strains of mice with very similar results (12, 34); 2) significant OVA-induced lung allergic inflammation
reactions have been induced in genetically mice made deficient for cell
types, cytokines, or adhesion receptors in a C57BL/6J background
(12); and 3) BALB/c and C57BL/6J mice subjected to CXCR4
or SDF-1a neutralization during OVA treatment showed similar phenotype
(data not shown).
Taken together, our results indicate that despite the high levels of
SDF-1 in the lung before or after the induction of an inflammatory
response, the CXCR4/SDF-1 axis plays a critical role in the
recruitment of inflammatory leukocytes into the lung and the subsequent
induction of pathophysiological manifestations characteristic of
asthmatic reactions. It is conceivable that the expression of a
particular chemokine is necessary, but not sufficient, for the
induction of cell recruitment. Eotaxin is highly expressed in the mouse
thymus under noninflammatory conditions, and yet no eosinophilia has
been detected in this particular organ (34). Thus,
SDF-1 is constitutively expressed in the lung, but presumably other
events must be required, such as up-regulation of its receptor CXCR4,
adhesion receptor overexpression, and production of other inflammatory
mediators, to achieve the inflammatory response.
Monocytes and eosinophils have been described to promote lung
eosinophilia in this particular mouse model (16). Because
CXCR4 is not expressed on eosinophils, and SDF-1 does not induce
eosinophil migration in vitro, the CXCR-4-SDF-1 interaction could be
indirectly regulating the accumulation of eosinophil by acting through
its target cell types, monocytes and T lymphocytes. In this model of
AAD, monocyte accumulation occurs at early stages of the inflammatory
response and precedes the development of eosinophilia
(12). T lymphocyte recruitment in the lung parallels
eosinophil accumulation (12). Accordingly, neutralization
of CXCR4-mediated interactions should be more critical at early stages
of the inflammatory response. No effect or few effects are detected
when CXCR4 signals were neutralized late in the induction of the
disease (days 20 and 21; Fig. 3). One could argue that the longer
administration of the anti-CXCR4 Abs could induce a systemic
depletion of CXCR4-expressing leukocytes. Spleen and bone marrow
cellularity have been evaluated during anti-CXCR4 blockage. The
number and phenotype of cells from both organs were not affected by the
Ab compared with those in control littermates (OVA or OVA plus rabbit
Ig; data not shown).
In conclusion, we provide three lines of evidence to support the
crucial role of CXCR4-SDF1 interactions during AAD: 1) there is a
correlation between the influx of CXCR4+ cells
(supported by an increase in CXCR4 mRNA in the lungs) and the
progression of the inflammatory phenotype; 2) the neutralization of
CXCR4-mediated signals with either anti-CXCR4 or anti-SDF-1
Abs leads to a reduced inflammatory and AHR response to OVA; and 3) the
overexpression of CXCR4 by retroviral delivery of a CXCR4 cDNA in
leukocytes augments lung eosinophilia. On the basis of these findings
we now propose that, although not considered a typical" inflammatory
chemokine, the CXCR4/SDF-1 axis plays a very significant role in the
inflammatory component of AAD.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: SDF-1/ß, stromal cell-derived factor-1/ß; mCXCR4, mouse CXCR4; AAD, allergic airway disease; MGB, modified Gay’s buffer; i.n., intranasally; BAL, bronchoalveolar lavage; AHR, airway hyper-responsiveness; MSCV, murine stem cell virus.
Received for publication December 10, 1999. Accepted for publication April 14, 2000.
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in bronchoalveolar lavage fluid of allergic asthmatic patients. Am. J. Respir. Crit. Care Med. 153:1398.[Abstract]
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). Eotaxin-induced eosinophil migration in vitro (
) was not
affected by the anti-CXCR4 Abs (
). Normal rabbit Ig was used as
the control Ab (open symbols). B, SDF-1
8 wk posttransplant. Cells were double stained for CXCR4 (FITC) and
the donor-specific leukocyte surface marker Ly5.2 (PE). CXCR4 was
detected at high levels in Ly5.2-positive granulocytes and lymphocytes
in the CXCR4 RV mice and at lower (endogenous) levels in Ly5.2-negative
lymphocytes.
