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* Department of Allergy and Clinical Immunology, Faculty of Medicine, Imperial College, National Heart and Lung Institute, London, United Kingdom; and
Department of Pharmacology, School of Pharmacy, Faculty of Medicine, Hebrew University of Jerusalem-Hadassah Medical School, Jerusalem, Israel
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1 and IL-13 are temporarily associated with myofibroblast
formation and deposition of tenascin and procollagen I. Biopsies were
taken from atopic volunteers at 1, 3, 6, 24, 48, and 72 h after
intradermal allergen challenge and were examined by
immunohistochemistry. Following the peak of the late-phase reaction (6
h) there were persisting TGF-1+ eosinophils, 
-smooth
muscle actin+ myofibroblasts, tenascin immunoreactivity,
and procollagen-I+ cells 24–48 h postchallenge. Direct
evidence of generation of repair markers was obtained by coculture of
eosinophils and fibroblasts. This resulted in -smooth muscle actin
immunoreactivity that was inhibitable by neutralizing Abs to TGF- as
well as production of tenascin transcripts and protein product.
TGF-1 and IL-13 also induced tenascin expression. We conclude that
TGF-1 and IL-13, provided partially by eosinophils, contribute to
repair and remodeling events in allergic inflammation in human atopic
skin. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1, and in coculture systems
this cell type stimulates fibroblast proliferation, collagen synthesis,
and lattice contraction (5, 6, 7). In addition, the Th2-like
cytokines IL-4 and IL-13, also expressed by eosinophils, up-regulate
fibroblast chemokine and matrix protein expression (8, 9)
and weakly induce a myofibroblastic phenotype (9).
Furthermore, the selective overexpression of IL-13 in murine clara
cells causes subepithelial matrix deposition (10).
Eosinophils also express other growth factors and cytokines that
modulate mesenchymal cells, including fibroblast growth factor-2
(FGF-2)3 (11),
nerve growth factor (12), vascular endothelial growth
factor (13), and IL-4 (14).
Collagen deposition and remodeling events are usually considered to be
chronic processes that, in the context of asthma, are the result of
long term exposure to Ag. However, Gizycki et al. (15)
observed an increase in myofibroblast formation 24 h after
inhalational challenge in atopic asthmatics, suggesting that fibroblast
activation can occur shortly after allergen exposure, possibly as a
result of eosinophil-derived TGF-1. Thus, further in vivo evidence
in man is required regarding the relationship among eosinophil
infiltration, allergic inflammation, and specific allergen-induction of
markers of repair. For these reasons we have tested the hypothesis that
in allergic inflammation in vivo in man, eosinophil infiltration is
temporarily associated with fibroblast activation (as shown by the
formation of -smooth muscle (-SM) actin+
myofibroblasts) as well as the deposition of procollagen I and the
matricellular protein tenascin.
We have used the allergen-induced cutaneous late phase reaction (LPR)
as our experimental model because this has been used extensively to
study the cellular and molecular characteristics of allergic
inflammation. Biopsies have been taken at time points after intradermal
injection of allergen and examined by immunohistochemistry. In addition
we have used fibroblast cultures to show that eosinophil-associated
TGF-1 and IL-13 induce the formation of tenascin over a similar time
course as that observed in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ten atopic subjects who were not receiving oral corticosteroids were recruited from the Allergy Clinic of the Royal Brompton Hospital, Imperial College staff, or by advertisement. No subject had taken aspirin within the week before the study. Inclusion criteria were as follows: 1) age of 18–55 years, 2) history of seasonal and/or perennial allergic rhinitis and/or asthma, 3) absence of any other illness, and 4) positive skin prick test (weal diameter, >5 mm) to grass pollen, house dust mite extract, or cat dander (all from ALK, Horsholm, Denmark) in the presence of a positive histamine and negative vehicle control. All patients had a positive radioallergosorbent test, and the median total serum IgE concentration was 398.5 IU/ml (range, 65–1102). All subjects gave informed consent, and the study was approved by the Royal Brompton and Harefield Trust ethics committee.
Study design and processing of specimens
All injections were performed with a 29-gauge needle and a 0.5-ml plastic syringe. Using this method, 30 biological units of allergen (mixed grass, cat dander, or house dust mite) was injected intradermally into individual sites on the extensor aspect of the forearms of each subject. An additional site was injected with a similar volume of diluent. Macroscopic responses were measured at 1, 3, 6, 24, 48, and 72 h by evaluating skin induration by resistance to the movement of a sharpened pencil point with which the reaction was outlined. Permanent sticky tape records of the outlines of the responses at all time points were then made. A 4-mm disposable biopsy punch was used to take a biopsy from the center of each reaction after using 1% plain lignocaine for local anesthesia. The control site injected with diluent was biopsied at 24 h. In this way each patient served as his/her own control. Tissue biopsies were immediately fixed in 4% paraformaldehyde and were washed in 15% PBS-buffered sucrose (Sigma-Aldrich, Poole, U.K.), embedded in OCT (optimal cutting temperature; Miles, Elkhart, IN), and snap-frozen in isopentane precooled in liquid nitrogen. Cryostat sections (<8 µm) were cut from biopsies, mounted onto SuperFrost Plus slides, dried overnight at 37°C, and stored with silica gel at -80°C until use (all from VWR, Dagenham, U.K. unless otherwise stated).
Histochemistry and immunohistochemistry
Eosinophil accumulation was determined by Congo red, an elective
stain for eosinophils (16), in tissue sections as
described previously (17). Briefly, sections were washed
in PBS for 5 min, then incubated in 0.5% Congo red (Sigma-Aldrich) in
ethanol/0.1 M glycine (1/1) for 5 min at room temperature. The slides
were rinsed in 70% ethanol until the background became clear, then
mounted in Glycergel (DAKO, Cambridge, U.K). The alkaline
phosphatase/anti-alkaline phosphatase (APAAP) technique was used to
enumerate cells immunoreactive to an mAb against -SM actin (DAKO;
clone 1A4), IL-13 (R&D Systems, Oxford, U.K.), and tenascin (Clone
T2H5; Caltag-Medsystems, Towcester, U.K.). The APAAP technique was
performed as described previously (18). Briefly, sections
were pretreated with 0.1% saponin (Sigma-Aldrich)/PBS for 30 min and
were incubated overnight at room temperature in a humidified chamber
with relevant primary mouse mAb. Sections were washed in PBS, incubated
with rabbit anti-mouse Ig (DAKO) for 30 min, then washed again. A
third layer of soluble complexes of AP and mouse APAAP (DAKO) was
incubated for an additional 30 min and developed with Fast Red
(Sigma-Aldrich) as chromogen for signal visualization (DAKO). To
enumerate procollagen I-positive cells, sections were incubated with a
rat anti-procollagen-I (MAB 1912; Chemicon, Harrow, U.K.) at 200
ng/ml for 2 h. Sections were washed extensively in PBS, then
developed using an alkaline phosphatase (AP)-conjugated rabbit
anti-rat system according to the manufacturer’s instructions
(Vector Laboratories, Peterborough, U.K.). Cells were counterstained
with Harris’ hematoxylin (VWR) and mounted in Glycergel. Positive
cells stained red after development with Fast Red. Substitution of the
primary Ab with an irrelevant isotype-matched Ab of the same species
was used as a negative control. One biopsy section from each time point
was evaluated from each patient. The whole biopsy section was counted,
and the total number of single-positive cells was expressed as the
number of cells per square millimeter of biopsy. To enumerate the
effect of eosinophil coculture on myofibroblast formation, fibroblast
cytospins were immunostained with -SM actin as described above.
Fields were chosen at random, and a minimum of 250 cells was counted.
Results were expressed as the percentage of positive cells. To
determine the role of TGF-, fibroblast cultures were pretreated for
1 h with a neutralizing Ab to active TGF- (all isoforms; clone
1D11; R&D Systems) before addition of eosinophils.
Double immunohistochemistry
The relative contribution of eosinophils to the expression of
TGF-1 was examined by double immunohistochemistry. Sections were
incubated with a chicken anti-TGF-1 Ab (R&D Systems) at 10
µg/ml for 2 h in a humidity chamber. Sections were then
incubated with a biotinylated horse anti-chicken Ab (Vector
Laboratories) for 1 h, followed by an AP-conjugated streptavidin
label (DAKO). Following development with Fast Blue (Vector
Laboratories) as the chromogen for signal visualization, sections were
stained with Congo red as described above. Although Fast Blue is
alcohol-soluble, signal intensity was unaffected. After development,
TGF-1+ cells stained blue, and eosinophils
stained orange. Double-positive cells stained for both colors. Whole
sections were counted to enumerate double-positive eosinophils and the
total number of TGF-1 immunoreactive cells. No
immunoreactive-positive cells were visualized following substitution
with an irrelevant chicken IgY Ab.
Purification of peripheral blood eosinophils
Eosinophils were purified from the peripheral blood of atopic subjects as previously described using a combination of density gradient centrifugation and immunoselection (19). Briefly, peripheral venous blood was collected into syringes containing sodium citrate as the anti-coagulant. Following dextran (6%, w/v; VWR), sedimentation to remove erythrocytes, the leukocyte-rich plasma layer was transferred onto Histopaque-1.083 (Sigma-Aldrich) before being centrifuged at 400 x g for 15 min at 20°C. The resultant PBMC and basophil band was removed, and the granulocyte pellet was collected. Contaminating erythrocytes were removed by hypotonic lysis using ice-cold water for 30 s, and the pellet was washed in HEPES-modified RPMI medium containing 0.5% BSA and 5 mM EDTA (all from Sigma-Aldrich). Granulocytes were incubated with anti-CD16 beads (Miltenyi Biotech, Bisley, U.K.) for 30 min at 4°C. To deplete contaminating T cells and monocytes, 20 µl anti-CD3 and anti-CD14 beads (Miltenyi Biotech) were added. Eosinophils were passed through a magnetic field and collected by negative selection in the eluent. Eosinophils were >98% pure as determined by Kimura staining and >99% viable as determined by trypan blue (Sigma-Aldrich). Cells were cultured in fibroblast medium supplemented with IL-5 (10 pM; R&D Systems).
Fibroblast culture
Primary human dermal fibroblasts were obtained from biopsies of
normal skin from informed consent volunteers who underwent minor skin
surgery. The biopsies were placed as explants, and fibroblasts were
obtained and cultured as described (7). The human fetal
lung fibroblasts (MRC-5) were obtained from European Collection of Cell
Cultures (Ref. no. 97112601; Salisbury, U.K). Fibroblasts were
routinely maintained in DMEM supplemented with 10% (v/v) FBS, 2 mM
L-glutamine, 100 U/ml penicillin, and 100 µg/ml
streptomycin (all from Sigma-Aldrich) at 37°C in a humidified
environment containing 5% CO2. Cells were
passaged by trypsinization using 0.05% trypsin/0.02% EDTA
(Sigma-Aldrich) and seeded into slide chambers, 12-well plates, or
96-well microtiter plates (VWR) at 5 x 103,
10 x 103, or 2 x
103 cells/well, respectively. Fibroblasts were
cultured for 3 days until 
70% confluent before treatment with human
eosinophils or exogenous cytokine as described below. Following
treatment, fibroblasts were washed with PBS and stored at -80°C in
situ, collected as cytospins or cell pellets, or lysed for RNA
extraction.
Immunocytofluorescence
To determine whether eosinophils induce myofibroblast formation,
dermal fibroblasts were grown on slide chambers (ICN, Basingstoke,
U.K.) in the absence or the presence of eosinophils for 24 h, then
washed with PBS and fixed with 4% paraformaldehyde. Following
permeabilization, slides were stained with anti--SM actin Ab
diluted in PBS containing 0.1% saponin and 10% normal human serum for
2 h at room temperature. After extensive washing, slides were
incubated with a BODIPY-conjugated goat anti-mouse Ab (0.5 µg/ml;
Molecular Probes, Cambridge, U.K.) for 1 h at room temperature.
Fibroblasts were mounted in fluorescent mounting medium (DAKO), and
images were acquired using a Leica TCS SP confocal microscope
(Heidelberg, Germany).
Western immunoblotting
The degree of -SM actin expression in response to coculture
with human eosinophils or TGF-1 was analyzed by immunoblotting.
Fibroblast pellets were resuspended in RIPA lysis buffer (PBS, 1%
Nonidet P-40, 1% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA)
containing 0.1% SDS, 1 mM PMSF, 1 µg/ml aprotinin, 5 µg/ml
leupeptin, and 1 mM sodium orthovanadate on ice for 30 min (all from
Sigma-Aldrich). Cell lysates were collected by microcentrifugation
(Eppendorf, Sunderland, U.K.), mixed with an equal volume of
sample buffer (50 mM Tris, 400 mM glycine, 0.2% SDS, and 10%
glycerol, pH 8.3), and boiled for 5 min at 95°C. Rainbow m.w. markers
and cell extracts (15 x 103 cells/lane)
were loaded onto a 10% SDS gel and electrophoresed for 1 h at 150
V in a mini-gel system. Separated proteins were transferred onto a
polyvinylidene difluoride membrane in transfer buffer containing 20%
methanol for 1 h. Membranes were blocked for 1 h in blocking
buffer (5% fat-free milk powder in PBS/Tween (0.05%)), then incubated
overnight at 4°C with anti--SM actin Ab (250 ng/ml) in
blocking buffer under constant agitation. After three 5-min washes, the
membrane was incubated with HRP-conjugated goat anti-mouse (1/4000
dilution; DAKO) in blocking buffer for 30 min at room temperature and
subsequently developed with ECL detection reagent (Amersham, Little
Chalfont, U.K.) for 5 min. X-ray film (Calumet, Milton Keynes, U.K.)
was exposed to chemiluminescence reaction, and the film was developed
(all reagents from Bio-Rad unless stated).
Semiquantitative RT-PCR for tenascin-C
Total RNA was extracted from the MRC-5 fibroblasts using RNeasy
minicolumns (Qiagen, Crawley, U.K.) and eluted in
diethylpyrocarbonate-treated dH2O. RNA quality
and quantity were assessed by ethidium bromide-agarose gel
electrophoresis and by relative absorbance at 260 vs 280 nm. cDNA was
synthesized from 200 ng total RNA. The gene-specific primers were as
follows: tenascin-C: forward primer,
5'-TGAACAAAATCACAGCCCAG-3'; reverse primer,
5'-CAGTGGAACCAGTTAACGCC-3'; and -actin: forward primer,
5'-TCCTGTGGCATCCACGAAACT-3'; reverse primer,
5'-GAAGCATTTGCGGTGGACGAT-3'. Tenascin-C primers showed 100%
alignment with human mRNA for tenascin-C only as checked with GenBank
and EMBL sequence databases. The predicted sizes of tenascin-C and
-actin DNA products were 333 and 315 bp, respectively. The PCR
amplification mixture consisted of 10x PCR buffer, 1.25 mM
MgCl2, 1 U Taq polymerase (Promega,
Southampton, U.K.), 200 µM deoxyribonucleosides (dATP, dGTP, dCTP,
and dTTP), 0.3 µM of each primer, and appropriate dilution of cDNA,
made up to 50 µl with sterile distilled water. Amplification was
conducted for 37 cycles in a DNA thermal cycler (Hybaid, U.K.) under
the following reaction conditions: 94°C for 5 min for first cycle,
then 35 cycles at 94°C for 1 min, 55°C for 1 min, and 72°C for 1
min, and the last cycle at 72°C for 10 min. PCR products (15
µl) were separated by electrophoresis on a 2% agarose gel containing
ethidium bromide. For visualization of the bands, the gel was
photographed under UV light. The photographs were scanned, and bands
were quantified using Gel Works Intermediate software (Ultra-Lum,
Claremont, CA). Results are expressed as arbitrary units.
In situ cell ELISA
Fibroblasts grown in a 96-well microtiter plate were treated to
various concentrations of TGF-1 (100 pM), IL-4 (10–1000 pM), IL-5
(100 pM), IL-13 (10–1000 pM), or FGF-2 (1000 pM; all human recombinant
proteins; R&D Systems) for 24 h, then washed three times with PBS
and left to air-dry overnight. Eight replicates were performed for each
treatment group. Wells were blocked with PBS containing 2% BSA and
0.05% Tween 20 for 2 h at 37°C, then incubated with 100 µl
anti-tenascin Ab at 10 µg/ml in PBS containing 0.1% saponin and
10% normal human serum overnight at room temperature. After labeling,
the plate was washed three times for 5 min each time with PBS/Tween and
incubated with an AP-conjugated goat anti-mouse Ab (DAKO) for
2 h. After extensive washing, the chromogenic substrate,
p-nitrophenyl phosphate (5 mM, pH 10.0; DAKO), diluted in
100 mM sodium bicarbonate/carbonate buffer (pH 10), was applied to
produce color, and absorbance was read at 405 nm after 10-min
development in a microtiter plate reader (Titer-Tek Multiskan;
Labsystems, Basingstoke, U.K.). Absorbance values obtained with
an irrelevant mouse IgG1 monoclonal were similar to those found for
wells treated with substrate only.
Statistical analysis
Data were analyzed using a statistical software package (Minitab Release 13.1; Minitab, State College, PA). The Wilcoxon signed rank test was used to analyze changes in the numbers of immunoreactive-positive cells in response to allergen. Data from in vitro studies were analyzed by Student’s t test. A value of p > 0.05 was accepted as nonsignificant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All subjects (n = 10) exhibited an
allergen-induced, but not diluent-induced, cutaneous LPR, with mean
diameters (SEM) of 34.7 ± 2.5 (1 h), 45.1 ± 5.3 (3 h),
62.1 ± 7.5 (6 h), 47.5 ± 8.2 (24 h), 30.4 ± 6.7 (48
h), and 8.8 ± 3.8 (72 h) mm (Fig. 1
). At 72 h the LPR had completely
subsided in five of the 10 volunteers.
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) compared with diluent challenge. Unlike the LPR, which had
resolved, all subjects had tissue eosinophilia at the 72 h point.
No significant correlation was found between eosinophil numbers and
size of LPR.
TGF-1 and IL-13
The kinetics of TGF-1 expression showed a pattern similar to
that observed for eosinophils (Fig. 2A). At diluent-challenged
sites very few cells were immunoreactive for TGF-1 (17.7 ± 3.4
cells/mm2), whereas after allergen challenge the
numbers of TGF-1+ cells increased markedly at
1 h (120.1 ± 20.7 cells/mm2), was
maximal at 6 h (204.7 ± 47.1
cells/mm2), and then declined progressively.
TGF-1+ cells were still evident at 72 h
(30.6 ± 13.6 cells/mm2), but at levels
similar to that found at the diluent site. At 1, 3, 6, 24, 48, and
72 h the percentages of eosinophils that were
TGF-1+ were 30, 25, 37, 48, 30, and 22%,
respectively. Thus at 24 h eosinophils represented approximately
one-half of the inflammatory cells found to be
TGF-1+. Other cellular sources of TGF-1
included neutrophils (earlier) and macrophages (later time points; data
not shown). The allergen-induced LPR was also associated with an
increase in the number of IL-13+ cells (Fig. 2B). This was apparent even at 1 h (21.5 ± 6.2
cells/mm2; p = 0.014) and peaked
at 24 h (39.8 ± 6.3 cells/mm2;
p = 0.006). Significant numbers persisted up to 72
h (9.6 ± 1.5 cells/mm2; p =
0.008). Double staining with Congo red revealed that appreciable
numbers of IL-13+ cells were eosinophils and
ranged from 8.5 ± 2.7% (48 h) to 19.5 ± 7.8% (3 h; data
not shown). The relative contribution of eosinophils to the total
number of IL-13-immunoreactive cells was considerable given the high
number present within the tissue and the comparatively low numbers of
IL-13+ cells. Other inflammatory cells
expressing IL-13 were neutrophils (earlier time points) and lymphocytes
(particularly at 24 h). Examples of Congo
red+ eosinophils and
TGF-1+ cells are shown in Fig. 3, A and B.
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There was a small, but significant, increase in the numbers of
single -SM actin myofibroblast-like cells at 24 and 48 h when
allergen challenge sites were compared with diluent
(p < 0.02; Fig. 2C). This persisted
to 72 h. Fig. 3C also shows an example of
immunohistochemistry of -SM actin in a cutaneous LPR. These
single -SM actin+ cells were independent of
any vascular or glandular structure and were found to reside mostly in
the lower dermis. Pericytes localized around blood vessels were also
-SM actin+, but were excluded from cell
counts. A moderate increase in the numbers of cells expressing
procollagen I was observed at 24 h compared with
diluent-challenged sites (6.6 ± 1.7
cells/mm2) after allergen challenge (10.0 ±
2.5 cells/mm2), peaked significantly at 48 h
(16.0 ± 4.2 cells/mm2; p =
0.006), and declined slightly at 72 h (9.4 ± 3.2
cells/mm2; Fig. 2D). At
diluent-challenged sites a small number of immunoreactive cells was
also observed, indicating low level turnover of collagen. Other
structures, such as eccrine sweat glands, blood vessels, smooth muscle,
and hair follicles, were likewise immunoreactive for -SM actin and
procollagen I in both control and allergen-challenged sites.
Tenascin expression
In diluent-challenged sites very few single cells were found to be
positive for tenascin-C. Similarly, at 1 and 3 h following
allergen challenge little or no immunoreactivity was evident. At 6
h a number of fibroblast-like cells, fusiformic in shape and
distributed predominantly in the lower dermis, were immunoreactive for
tenascin. In biopsies obtained at 24 and 48 h after challenge, the
number of tenascin+ cells was significantly
increased compared with that at 3 h (p <
0.01) and was still elevated at 72 h (Fig. 4A). The temporal pattern of
tenascin expression at both the endothelial and eccrine sweat gland
basement membrane and in and around the arrector pilorum muscle closely
matched that observed at the single-cell level (Fig. 4, B
and C). Tenascin expression was significantly up-regulated
around basement membranes at 24 and 48 h compared with that at
3 h (p < 0.01).
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Culture of primary human dermal fibroblasts (Fig. 5A) with human eosinophils
(Fig. 5B) resulted in a marked increase in -SM
actin+ cells (green signal). These cells showed
the hallmark morphological features of myofibroblasts, appearing larger
in size with irregular projections and bundles of actin filaments and
stress fibers. When light was collected from both the red and green
channels of the confocal microscope, eosinophils appeared orange,
indicating strong autofluorescence (Fig. 5C). To quantify
the effect of eosinophils on myofibroblast formation, fibroblasts were
harvested by trypsinization and stained for -SM actin expression.
Eosinophils obtained from various donors (n = 3–8)
induced the formation of -SM actin+ cells in a
time-dependent manner (Fig. 6A). Myofibroblast formation
was observed only after 6 h and peaked at 24 h, at which time
numbers had increased 4-fold. -SM actin+ cells
remained elevated out to 3 days, indicating that fibroblast
differentiation by eosinophils led to a stable myofibroblast phenotype.
No effect on fibroblast -SM actin expression was observed following
IL-5 (10 pM) treatment alone (data not shown).
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B shows the effects of various eosinophil
concentrations from various subjects (n = 3–8) on
myofibroblast formation after a 3-day culture. Cultures were not
examined beyond this time. Even at relatively low eosinophil
concentrations, i.e., one eosinophil to every two fibroblasts, there
was a significant increase in the number of myofibroblasts
(p < 0.01). This effect was maximal at a 1:1
ratio between the two cell types. Increasing the concentration of
eosinophils to fibroblasts resulted in a decrease in myofibroblast
formation in a cell-dependent manner in all experiments, although an
effect was still apparent at this ratio (1:16). Fibroblast viability,
as determined by trypan blue, was >90% at all points.
Effect of anti-TGF-
To investigate the eosinophil-derived factor responsible for
eosinophil-induced myofibroblast formation, cultures were incubated in
the presence or the absence of anti-TGF- Ab at 0.1, 1, and 10
µg/ml. Fibroblasts cultured in the presence of eosinophils showed a
marked up-regulation in the level of -SM actin expression
(lane 2), in contrast to fibroblasts grown in medium
alone, where a band was barely visible (lane 1).
Treatment with the neutralizing Ab to active TGF- significantly
inhibited eosinophil-induced fibroblast -SM actin expression in a
dose-dependent manner as determined by both Western blot (Fig. 7A) and immunocytochemistry
(Fig. 7B; p < 0.01). To control against a
nonspecific Ab effect, fibroblasts were incubated with an irrelevant
IgG2a Ab (lane 4). This control Ab was without
effect. Eosinophils alone did not express -SM actin (data not
shown).
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MRC-5 fetal lung fibroblasts were treated with eosinophils to
determine the effect on the expression of tenascin gene and gene
product. MRC-5 fibroblasts constitutively expressed low levels of
tenascin mRNA. As observed with -SM actin expression, relatively low
eosinophil concentrations were found to up-regulate tenascin gene
expression (Fig. 8A), with a
1:1 ratio between the two cell types inducing the most marked
up-regulation. At this ratio, eosinophils up-regulated tenascin
transcripts at 3 h (Fig. 8B) and significantly
up-regulated protein after a 24-h coculture (Fig. 8C;
p < 0.02). Eosinophils alone did not express either
gene or gene product for tenascin (data not shown).
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Dermal fibroblasts were incubated in the absence or the presence
of various concentrations of IL-4, IL-5, IL-13, TGF-1, and FGF-2 for
24 h. Supernatants were removed, and fibroblasts were examined for
tenascin expression in situ by ELISA. Under control conditions,
fibroblasts constitutively expressed tenascin (Fig. 9). Treatment with IL-13 induced a marked
up-regulation in the expression of tenascin in a dose-dependent manner.
Both IL-4 and TGF-1 (100 pM) also up-regulated the expression of
tenascin compared with medium alone. In contrast, IL-5 had no effect at
100 pM, whereas FGF-2 decreased basal tenascin expression at 1000
pM.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-immunoreactive cells (6 h after allergen challenge) preceded
the formation of myofibroblast-like cells and their associated matrix
proteins, which were prominent at later time points when allergic
inflammation (i.e., the LPR) was resolving (24 and 48 h). The
function of eosinophils in these events was further evaluated in a
coculture system using primary dermal fibroblasts. Eosinophils induced
the differentiation of fibroblasts to myofibroblasts, which was
associated with an up-regulation of tenascin gene expression and gene
product. Thus, IgE-mediated hypersensitivity induced features of the
remodeled phenotype. We speculate that the formation of
myofibroblast-like cells and the activation of fibroblasts may be
partly under the control of infiltrating
TGF-1+/IL-13+
leukocytes, particularly the eosinophil.
The apparent dissociation between the magnitude of the LPR and tissue
eosinophils at later time points after allergen challenge (Fig. 1) led
us to hypothesize that eosinophils may play a role in repair and tissue
remodeling during resolution of the inflammatory reaction. Several
reports have suggested a role for eosinophils in repair processes
associated with allergic tissue injury. Thus, TGF-1 expression was
prominent in nasal polyps and increased in airway biopsies from
asthmatic subjects (20, 21, 22). TGF-1, through specific
binding to TGF- receptor type II (23, 24), is a
predominant differentiation factor of the myofibroblast phenotype
(25, 26). Fibroblast activation by Th2 cytokines may be
TGF- dependent, since IL-4 up-regulated TGF-1 mRNA and product by
eosinophils, and animal models of IL-13-induced fibrosis suggest that
these effects are mediated indirectly via activation of a TGF-
pathway (27, 28).
Of interest was the discordance between the total eosinophils and
TGF-1+ eosinophils at the different time
points, i.e., more eosinophils were TGF-1+ at
24 and 48 h than at 1, 3, or 6 h (Fig. 2A). Thus,
tissue eosinophils appear to up-regulate their TGF-1 expression in
situ. A similar finding was observed by Wong and colleagues
(3) in a hamster model of cutaneous wound healing. In
contrast, IL-13 expression was significantly elevated at all time
points, but peaked at 24 h, confirming our earlier findings (Fig. 2B) (29). However, in the present study with
sampling at earlier time points, we observed significant increases in
the numbers of infiltrating IL-13+ leukocytes as
early as 1 h following allergen challenge. Moreover, the
percentage of eosinophils positive for IL-13 protein ranged between 8
and 20% (data not shown), suggesting that eosinophils represent a
substantial source of IL-13. Other cellular sources of IL-13 included
mast cells, neutrophils, and lymphocytes. Thus, the expression for both
TGF-1 and IL-13 preceded the formation of myofibroblasts (Fig. 2C), suggesting that the allergen-induced up-regulation of
these potent differentiation factors may induce the transformation of
fibroblasts to myofibroblasts in vivo.
We have also shown that intradermal allergen challenge induces the
formation of -SM actin-immunoreactive myofibroblast-like cells.
Myofibroblasts have also been reported after allergen-induced late
asthmatic reactions (15) and may contribute to the
thickened subepithelial reticular basement membrane (RBM) evident
within the airways of asthmatics, since there is a correlation between
the thickness of the RBM and the number of myofibroblasts beneath it
(30). Our skin model allowed us to study allergen-induced
myofibroblast formation over a prolonged time course (Fig. 2C). We found that cell numbers increased at 24 h and
were still significantly elevated at 48 h compared with diluent
control. Similarly, we observed a significant increase in the numbers
of fibroblast-like procollagen-I+ cells at
48 h (Fig. 2D) suggesting that active remodeling and
fibroblast activation persist several hours after resolution of the
LPR. In the context of allergic inflammation in the lung, our data
indicate that this structural cell may persist long after the height of
the LPR and may be responsible for the increased deposition of new
matrix proteins.
Tenascin, a highly regulated member of the matricellular family, is
expressed during development and growth and in response to injury
(31, 32). We have observed a significant up-regulation in
the expression of tenascin within fibroblast-like cells, in and around
bundles of smooth muscle and at the vascular smooth muscle basement
membrane in response to allergen challenge (Fig. 4). The temporal
pattern of tenascin expression at all three sites was identical and
increased prominently at 24 h, at which time the gross LPR was
declining. Single tenascin-immunoreactive cells, predominantly located
in the lower dermis and fusiformic in shape, were evident at 6 h,
peaked at 24 h, and remained elevated out to 72 h. The
increased expression of tenascin at the basement membrane of eccrine
glands and blood vessels may affect cell trafficking. Tenascin has been
demonstrated to act as a permissive substrate to prevent or allow cell
migration (33), and tenascin knockout mice exhibit
prolonged influx and retention of polymorphonuclear leukocytes
(34). We suggest that tenascin may function to coordinate
the inflammatory cell infiltrate, since the peak expression of tenascin
around blood vessels was observed at 24 h, when the numbers of
tissue granulocytes at the site of inflammation were in decline
compared with 6 h (35). Moreover, the temporal
expression of tenascin suggests that 1) repair mechanisms are in place
and become activated to control the allergic inflammation; and 2) these
processes may result from interactions between eosinophils and other
inflammatory cells with mesenchymal cells. In support of these
findings, we have shown that the selective depletion of
allergen-induced tissue eosinophilia following treatment with
anti-IL-5 led to a significant decrease in tenascin formation in
the cutaneous LPR (manuscript in preparation). In addition, these
remodeling processes occurred in response to acute inflammation. It is
a misconception that remodeling only occurs slowly in response to
chronic inflammation, since there is a dynamic state of cell and ECM
protein turnover in both diseased and healthy tissues. In
isocyanate-induced asthma, for example, only a relatively short period
of exposure to an occupational sensitization was required for
remodeling to occur (36), and there was a decrease in the
thickness of the RBM and the numbers of subepithelial fibroblasts after
cessation of exposure.
To support our in vivo findings we developed an in vitro coculture
system and showed by use of immunofluorescence and immunoblot that
human eosinophils induced the formation of myofibroblasts from primary
human dermal fibroblasts. These cells showed the hallmark morphological
features of myofibroblasts, appearing larger in size with irregular
projections and bundles of -SM actin filaments (Fig. 5). Similar
results were obtained with lung fibroblasts (data not shown). The
effect was inhibitable by anti-TGF- as previously observed by
others (Fig. 7, A and B) (37, 38).
Eosinophils at relatively low concentrations (1:1 ratio; 50 x
103/ml), without the addition of other factors,
induced a 4-fold increase in the number of myofibroblasts (Fig. 6B), an effect that occurred rapidly (within 24 h; Fig. 6A), similar to that found in vivo (Fig. 2C)
(15). The low numbers of eosinophils required for
myofibroblast formation support the view that this cell is involved in
thickening of the reticular basement membrane in asthma. Indeed, in a
retrospective analysis both airway eosinophilia and epithelial RBM
thickening have been demonstrated to be apparent in children who later
go on to develop asthma up to 4 yr before asthma is clinically
expressed (39). Higher eosinophil/fibroblast ratios
inhibited myofibroblast formation, possibly via release of FGF-2. FGF-2
is known to be expressed by blood (our unpublished observation)
and tissue eosinophils (11) and to be an inhibitor of
TGF--induced myofibroblast formation (37).
In an attempt to confirm our in vivo findings, we determined whether
eosinophil coculture induced the expression of fibroblast tenascin. As
observed in the diluent-challenged sites in vivo, fibroblasts
constitutively expressed tenascin. However, eosinophil treatment led to
a 2- to 3-fold increase in tenascin mRNA (Fig. 8A) and
significantly increased tenascin protein at 24 h (Fig. 8B). Eosinophil coculture also increased transcripts for
procollagen I and significantly up-regulated procollagen protein (data
not shown). A number of eosinophilic cytokines may be responsible for
this effect, with TGF-1, IL-4, and IL-13 known to stimulate
fibroblast collagen production (10). Consistent with
previous reports, we showed that TGF-1 (40, 41) and
IL-4 (42) increased tenascin expression and for the first
time demonstrated that addition of exogenous IL-13 induced a marked and
dose-dependent increase in the expression of tenascin (Fig. 9).
Furthermore, FGF-2 reduced basal tenascin expression. We have yet to
ascertain whether FGF-2 inhibits TGF-1- or IL-4-induced tenascin
expression. However, Davidson et al. (43) have shown FGF-2
to be an inhibitor of TGF-1-mediated collagen production in skin
fibroblasts.
In conclusion, we propose that following allergen-induced,
IgE-dependent mast cell degranulation there is infiltration of IL-13-
and TGF-1-positive eosinophils and other leukocytes, which leads to
myofibroblast formation. This is followed by an increase in the
expression of associated matrix proteins, procollagen I and tenascin.
We speculate that resident structural cells become activated in a
spatial and sequential pattern to produce matrix proteins such as
tenascin and collagens, which modulate the inflammatory response,
leading to its resolution.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. A. Barry Kay, Department of Allergy and Clinical Immunology, Faculty of Medicine, Imperial College, National Heart and Lung Institute, Dovehouse Street, London, U.K. SW3 6LY. E-mail address: a.b.kay{at}ic.ac.uk
3 Abbreviations used in this paper: FGF-2, fibroblast growth factor; AP, alkaline phosphatase; APAAP, alkaline phosphatase/anti-alkaline phosphatase; LPR, late phase reaction; RBM, reticular basement membrane; -SM, -smooth muscle.
Received for publication May 30, 2002. Accepted for publication August 5, 2002.
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, p < 0.02; 
,
TGF-
