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Regulation of pulmonary inflammation and fibrosis through expression of integrins ╨Ю┬▒V 3 and ╨Ю┬▒V 5 on pulmonary T lymphocytes.

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Vol. 60, No. 5, May 2009, pp 1530–1539
DOI 10.1002/art.24435
© 2009, American College of Rheumatology
Regulation of Pulmonary Inflammation and Fibrosis
Through Expression of Integrins ␣V␤3 and ␣V␤5 on
Pulmonary T Lymphocytes
Irina G. Luzina,1 Nevins W. Todd,1 Natalia Nacu,2 Virginia Lockatell,2 Jung Choi,2
Laura K. Hummers,3 and Sergei P. Atamas1
of neutralizing anti–integrin ␣V antibody or a genetic
deficiency of integrin ␤3 in the CCL18 overexpression
model significantly attenuated CCL18-driven pulmonary lymphocytic infiltration and collagen accumulation. Jurkat T cells overexpressing integrin ␣V␤3 or
integrin ␣V␤5 in cocultures with primary pulmonary
fibroblasts stimulated collagen accumulation and
Smad2 nuclear translocation. Neutralizing anti-TGF␤
antibody attenuated the profibrotic effect of integrinexpressing T cells.
Conclusion. Pulmonary infiltrating T lymphocytes may express integrins ␣V␤3 and ␣V␤5 that are
necessary for lymphocytic infiltration and T cell–
associated TGF␤ activation and collagen accumulation.
Objective. Pulmonary diseases associated with
fibrosis, including scleroderma lung disease, are characterized by the accumulation of T cells in the lungs.
These cells are thought to facilitate lung fibrosis, but the
exact mechanisms of their profibrotic action are not
clear. Several ␣V-containing integrins, including ␣V␤3
and ␣V␤5, have been shown to directly activate transforming growth factor ␤ (TGF␤) and promote collagen
accumulation. The aim of this study was to investigate
whether pulmonary T cells express profibrotic integrins
and regulate collagen accumulation.
Methods. Expression of integrins was assessed by
immunohistochemical analysis of lung tissue, by flow
cytometry using bronchoalveolar lavage fluid from patients with systemic sclerosis (SSc), and in a CCL18
overexpression animal model of pulmonary T cell infiltration. Experiments in cell cultures were performed to
determine whether integrin-expressing T cells are profibrotic in cocultures with pulmonary fibroblasts and, if
so, through what possible mechanism.
Results. Lymphocytes and integrin-positive cells
were present in the lungs, and pulmonary T cells
expressed integrins ␣V␤3 and ␣V␤5 in patients with
SSc and in the animal model. Systemic administration
Pulmonary fibrosis, or excessive accumulation of
connective tissue in the lungs, is a severe and even
deadly complication that occurs in a variety of diseases,
such as the idiopathic interstitial pneumonias, the systemic connective tissue diseases, sarcoidosis, graftversus-host disease, occupational or environmental lung
diseases, and some rare genetic diseases (1). The exact
causes of pulmonary fibrosis remain poorly understood,
but the mechanisms of this devastating condition appear
numerous and diverse, including inflammation-related
processes and processes unrelated to inflammation.
An important commonality among various fibrotic diseases of the lungs is the frequent association
with excessive pulmonary accumulation of T lymphocytes. The T cells constitute a relatively minor population in a normal lung; this population expands numerically and undergoes phenotypic changes in association
with lung inflammation and fibrosis (2). It remains
unclear whether the infiltrating T lymphocytes promote
fibrosis, accumulate in a futile attempt to counter it, or
are innocent bystanders in an ongoing response to
Supported by the NIH (grants R01-HL-054163 and R03-AR054946) and the Maryland Chapter of the Arthritis Foundation.
Irina G. Luzina, MD, PhD, Nevins W. Todd, MD, Sergei P.
Atamas, MD, PhD: University of Maryland School of Medicine, and
Baltimore VA Medical Center, Baltimore, Maryland; 2Natalia Nacu,
MD, Virginia Lockatell, BSc, Jung Choi, BSc: University of Maryland
School of Medicine, Baltimore; 3Laura K. Hummers, MD: Johns
Hopkins University Medical Institutions, Baltimore, Maryland.
Address correspondence and reprint requests to Sergei P.
Atamas, MD, PhD, University of Maryland School of Medicine,
Department of Medicine, Division of Rheumatology and Clinical
Immunology, 10 South Pine Street, MSTF 8-34, Baltimore, MD 21201.
Submitted for publication May 14, 2008; accepted in revised
form January 10, 2009.
pulmonary injury (2). Extensive data from animal models and limited observations in humans suggest that
depending on specific phenotypic features of the infiltrating pulmonary T cells, their contribution may indeed
be either profibrotic or antifibrotic (2). Pulmonary infiltration of T lymphocytes mediated by overexpression of
a selective chemotactic factor, CCL18, causes a moderate T lymphocyte–dependent accumulation of collagen
(3), whereas in combination with bleomycin injury, the
same CCL18-mediated T lymphocytic infiltration has a
partially protective antifibrotic effect (4).
It is likely that the infiltrating lymphocytes mediate their profibrotic effect on pulmonary fibroblasts
through cytokines, particularly the most potent profibrotic cytokine, transforming growth factor ␤ (TGF␤),
as well as Th2/Tc2 cytokines, chemokines, CD40 ligation, Fas/FasL, and perforin–granzyme pathways (2,5–
7). However, T lymphocytes of the “proinflammatory”
(tumor necrosis factor ␣–expressing) or Th1 phenotype may also be protective and act antifibrotically (2).
We and other investigators have previously shown that
T lymphocytes accumulate in the lungs of patients with
scleroderma lung disease, and that these T cells appear
to be activated and express a profibrotic pattern of
cytokines, chemokines, and cell surface molecules (6,7).
Pulmonary lymphocytic infiltration and collagen accumulation in patients with scleroderma lung disease may
be driven by CCL18, which is a selective chemoattractant of T cells but not other cell types (3,4,8–11). It is
noteworthy that the infiltrating pulmonary T lymphocytes in patients with scleroderma lung disease express
various integrin chains, including integrin ␣V, when
compared with scleroderma patients with no pulmonary
involvement or healthy control subjects (7).
Recently, a novel integrin-dependent mechanism
of fibrosis has been discovered that depends on TGF␤
activation by integrin ␣V␤6; epithelium-restricted ␤6⫺/⫺
mice showed only a minor fibrotic response in the lung
to bleomycin administration compared with wild-type
mice (12). Integrins are heterodimers, with 8 ␤ subunits
and 18 ␣ subunits that associate into 24 known integrins.
They mediate cell adhesion and play an important role
in a variety of cellular and extracellular processes,
including survival, proliferation, and migration (13). It
appears that not only integrin ␣V␤6 but also other
␣V-containing integrins, including ␣V␤1, ␣V␤3, ␣V␤5,
and ␣V␤8, may also activate latent TGF␤ and act
profibrotically (14–16). The activation of TGF␤ may
occur through binding of the RGD motif of the latencyassociated peptide (LAP) (13), whereas integrin ␣V␤8
may also activate latent TGF␤ by membrane type 1
matrix metalloproteinase–dependent degradation of
LAP (16). The expression of integrins ␣V␤3 (17) or
␣V␤5 (18,19) on scleroderma but not normal fibroblasts
has recently been implicated as a contributing factor
maintaining the profibrotic autocrine TGF␤ signaling
loop in scleroderma.
We considered the possibility that pulmonary
infiltrating T lymphocytes may express integrins in association with fibrosis (7), and that such expression may be
a driving force contributing to prolonged T lymphocyte
infiltration (3) and/or connective tissue accumulation in
the lungs. We previously reported that expression of
integrin ␣V␤6 heterodimers on the surface of pulmonary T cells is minimally increased in patients with
scleroderma (7). Therefore, this study focused on the
potential pathophysiologic role of T cells that express
other profibrotic integrins, ␣V␤3 and ␣V␤5. Based on
observations in patients with diffuse parenchymal lung
disease, in an animal model of pulmonary fibrosis, and
in in vitro experiments, we report that the expression of
integrins ␣V␤3 and/or ␣V␤5 on pulmonary T lymphocytes may regulate the extent of lymphocytic infiltration
and the degree of pulmonary fibrosis, whereas T lymphocytes that do not express integrins may be not
involved in the fibrotic regulation process.
Patients and samples. Patients with systemic sclerosis
(SSc) or idiopathic pulmonary fibrosis (IPF) were recruited
from the University of Maryland Medical Center, the Baltimore VA Medical Center, and Johns Hopkins School of
Medicine. The study was approved by the institutional review
boards at the University of Maryland and Johns Hopkins
Explant lungs were obtained during transplantation
procedures from a patient with SSc and from 2 patients with
IPF. The explant lungs were immediately delivered to the
Department of Pathology, sectioned, and 1 ⫻ 1 ⫻ 1–cm cubes
were excised from each lobe for histologic and immunohistochemical analyses. Also, 2 explant lungs from separate normal
donors, which were initially planned but ultimately not used for
transplantation, were sectioned in a similar manner to obtain
tissue samples. An additional 5 sections of lung tissue from
patients with SSc were kindly provided by Dr. Elena Tourkina
(Medical University of South Carolina).
The bronchoalveolar (BAL) procedures with differential cell counts in 25 patients with SSc and 5 healthy volunteers
were performed as previously described (6). The BAL samples
were immediately delivered on ice to the research laboratory
and processed. When indicated, CD3⫹ T cells were purified
from BAL fluid samples using positive selection with nonactivating magnetic beads, as previously described (7). Flow
cytometry assays confirmed ⱖ93% purity of these cells; the
remaining cells were mostly alveolar macrophages.
In vivo CCL18 overexpression murine model. Wildtype C57BL/6 mice or integrin ␤3–deficient mice (The Jackson
Laboratory, Bar Harbor, ME) were infected intratracheally
with replication-deficient adenoviral constructs that either did
or did not (as a control) encode CCL18, as previously described (3,4). The mice were thus challenged with pulmonary
overexpression of CCL18 for 14 days to attract T cells to the
lungs (3,4). Expression of CCL18 was confirmed by enzymelinked immunosorbent assay (ELISA) of BAL fluids and
lung homogenates. When indicated, mice were treated daily
from day 3 to day 13 with intraperitoneal injections of neutralizing antibody against integrin ␣V (BD PharMingen, San
Diego, CA) at a dose of 30 ␮g/injection. Control CCL18overexpressing mice were similarly treated with isotypematching control immunoglobulin. Procedures for obtaining
mouse lung tissue samples and BAL fluid were previously
described (3,4).
Primary pulmonary fibroblast cultures and Jurkat T
cells. Normal human primary pulmonary fibroblast cultures
derived from adult donors were purchased from Cambrex
(Walkersville, MD). Fibroblast cultures were maintained in
T75 culture flasks in a humidified atmosphere of 5% CO2 at
37°C in high-serum tissue culture medium (Dulbecco’s modified Eagle’s medium supplemented with 2 mM glutamine,
2 mM sodium pyruvate, 50 mg/liter gentamicin, and 10%
bovine calf serum; all from Invitrogen, Carlsbad, CA). The cell
cultures used in the experiments were preincubated for 24
hours in similar conditions, using low-serum medium (0.5%
dialyzed bovine calf serum with no TGF␤ detectable by
ELISA) supplemented with 0.28 mM ascorbic acid and 0.2 mM
␤-aminopropionitrile (Sigma, St. Louis, MO) in addition to the
aforementioned reagents. The cell culture medium for all
experiments was the same low-serum medium. In all experiments, fibroblast cell lines were tested in passages 3–7. For the
experiments, fibroblasts were plated at 3.5 ⫻ 105 cells/well in
6-well Costar plates, incubated overnight in 3 ml/well of
high-serum medium, and then for 24 hours in low-serum
The Jurkat T cell line (American Type Culture Collection, Manassas, VA) was cultured as recommended by the
supplier and cotransfected with human integrin ␣V (ITGAV)–
and integrin ␤5 (ITGB5)–encoding plasmids (OriGene, Rockville, MD) or was transfected with control noncoding plasmid
using the Amaxa electroporation system (Amaxa, Gaithersburg, MD). In the coculture experiments, 2 ⫻ 106 transfected
T cells/well were cocultured with primary fibroblasts in lowserum medium for 48 hours.
Histologic and immunohistochemical analyses. Histologic and immunohistochemical analyses of human and mouse
lung tissues were performed as previously described (3,4).
Sections (5 ␮m) of the explant lung were paraffin-embedded
and stained with hematoxylin and eosin or with Masson’s
trichrome to reveal collagen. Antibodies to human integrins
␣V and ␤3 were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA), and those to human ␣V␤5 heterodimers
were obtained from Chemicon (Temecula, CA) (high-quality
anti-human antibodies for immunohistochemical analyses of
integrin ␣V␤3 heterodimer expression were not commercially
available). Antibodies to mouse integrins ␣V, ␤3, and ␤5 were
obtained from Santa Cruz Biotechnology (anti-mouse antibod-
ies for immunohistochemical analyses of integrin ␣V␤3 or
␣V␤5 heterodimers were not commercially available).
Flow cytometric analyses. For flow cytometric analyses, BAL cells from humans or mice were obtained as described above and stained with fluorescence-labeled antibodies
or corresponding isotype control antibodies (BD PharMingen).
Cellular fluorescence was measured with a FACScan flow
cytometer (Becton Dickinson, Franklin Lakes, NJ). Expression
of human integrin ␣V␤3 (ITGAVB3), ␣V␤5 (ITGAVB5), or
␣V␤6 (ITGAVB6) heterodimers (US Biological, Swampscott,
MA) was measured on BAL CD3⫹CD4⫹ or CD3⫹CD8⫹
cells from individual patients (with no sample pooling). BAL
samples from patients and control subjects were also characterized by flow cytometry for the expression of CD45RA,
CD45RO, CD69, CD25, and HLA–DR on CD3⫹CD4⫹
or CD3⫹CD8⫹ cells. Many of the commercially available
antibodies tested in our laboratory for mouse ITGAVcontaining integrin heterodimers did not allow for high-quality
specific resolution of expressing cells in flow cytometry assays
(data not shown). Therefore, the expression of monomer
integrins ␣V (ITGAV), ␤3 (ITGB3), or ␤5 (ITGB5) was
measured on pulmonary CD3⫹ lymphocytes from the BAL
fluid of individual (no sample pooling) control or CCL18overexpressing mice, using antibodies obtained from Santa
Cruz Biotechnology.
Collagen assays. Collagen levels were measured in cell
culture supernates using Western blotting or 14C-proline incorporation assays, as previously described (3,4,9–11). Equal
numbers of fibroblasts in all cultures in each experiment were
confirmed with CellTiter 96 AQueous assays (Promega, Madison, WI).
Nuclear translocation of Smad2. In some experiments,
fibroblasts were transfected with a Smad2–green fluorescent
protein (GFP) construct (a kind gift from Dr. Andrew
Chantry, University of East Anglia, UK), using the Amaxa
electroporation system. The intracellular localization of the
construct was assessed by confocal or fluorescence microscopy.
Nuclear and cytoplasmic fractions of fibroblasts were prepared
as previously described (10), and total Smad2/3 protein was
evaluated by Western blotting, using an antibody obtained
from Cell Signaling Technology (Danvers, MA).
Statistical analysis. Data were processed using Statistica software (StatSoft, Tulsa, OK). Groups were compared
using Student’s 2-tailed t-test, the Mann-Whitney U test, or
analysis of variance (ANOVA), as indicated in the text.
Categorical measures were compared using chi-square statistics. Pearson’s correlation analyses were used to examine
pairwise associations between variables. For all analyses, P
values less than or equal to 0.05 were considered significant.
Expression of integrins on pulmonary T lymphocytes. To begin addressing the issue of the possible
contribution of integrin-expressing T cells in the lungs of
patients with interstitial lung disease, initial immunohistochemical experiments were performed. Histologic
Figure 1. Histochemical and immunohistochemical analyses of paraffin-embedded sections of healthy control lung (Ctrl) and an explant lung from
a patient with scleroderma lung disease (SSc). A, The sections were stained with hematoxylin and eosin (H&E), Masson’s trichrome (Trichr), or
immunohistochemically (brown) for CD3, CD4, CD8, integrins ␣V and ␤3, and ␣V␤5 heterodimers, as indicated. Note the profound inflammatory
infiltration in association with deposition of extracellular matrix, the presence of T lymphocytes, and the presence of cells expressing integrins.
Randomly selected panels are shown. (Original magnification ⫻ 200.) B, Confocal microscopic images of lung sections from patients with systemic
sclerosis, stained for CD3 (fluorescein isothiocyanate–conjugated) or integrin ␤3 (trichrome-conjugated), and merged images.
examination of healthy lungs revealed few, if any, T
lymphocytes in the interstitium or alveolar space,
whereas numerous cells positive for CD3, CD4, and
CD8 were present in the lungs of patients with SSc
(Figure 1A) or IPF (additional data are available at
pdf). Also, cells positive for integrins ␣V or ␤3, as well as
cells positive for integrin ␣V␤5 heterodimers, were
present in the lungs of 6 patients with SSc and 2 patients
with IPF (Figure 1; additional data are available at
pdf). No staining was observed with the matching isotype control antibodies; rare T cells (maximum 1–2 per
microscopic field) were observed in healthy lungs. These
observations suggest that T cells and integrin-expressing
cells are present in the lungs of patients with interstitial
lung diseases. Confocal microscopy of the SSc lung
sections revealed coexpression of CD3 and integrin ␤3
on numerous cells (Figure 1B).
The subsequent experiments were performed to
evaluate whether integrins are indeed expressed on the
surface of pulmonary lymphocytes, as suggested by our
previous analyses of messenger RNA (mRNA) expres-
sion in these cells (7). Flow cytometry assays of BAL
cells from 25 patients with scleroderma and 5 healthy
nonsmoking control volunteers were performed.
BAL cells were stained for integrin ␣V␤3, ␣V␤5,
or ␣V␤6 heterodimers, as well as for markers of T cells
(CD3, CD4, CD8). No expression of these integrins was
observed on T cells from healthy control subjects (Figures 2A and B). Integrins ␣V␤3 and ␣V␤5, but not
␣V␤6, were expressed on as many as 50% of T cells from
the lungs of patients with scleroderma (Figures 2A and
B). In 14 of 25 patients with scleroderma, more than
10% of BAL fluid T cells were positive for integrins
␣V␤3 or ␣V␤5. Similar flow cytometry assays revealed
that integrins ␣V␤3 and ␣V␤5 were not expressed on
blood T cells from healthy control subjects or patients
with scleroderma. Regression analyses of the percent
positive BAL fluid CD4⫹␣V␤3⫹, CD4⫹␣V␤5⫹,
CD8⫹␣V␤3⫹, and CD8⫹␣V␤5⫹ cells showed strong
positive pairwise correlations (P ⬍ 0.01 for each regression; additional data are available at http:// but
no correlation with CD4⫹ or CD8⫹ cells positive for
HLA–DR, CD25, CD45RA, CD45RO, or CD69 (P ⬎
Figure 2. Expression of integrins in pulmonary T lymphocytes. A and B, Flow cytometry assays for integrin ␣V␤3 and ␣V␤5 heterodimers in
bronchoalveolar lavage (BAL) fluid T cells (gated on CD3⫹ cells) from a patient with systemic sclerosis (SSc; red) and a healthy volunteer (blue).
Staining with isotype control antibody is shown in black. C and D, Quantitative reverse transcription–polymerase chain reaction (Q-PCR) of RNA
from BAL fluid T cells from 2 patients with SSc (red) and 2 healthy volunteers (blue), using specific primers for 18S ribosomal RNA (rRNA;
reference) or integrin ␤3 mRNA (C) or integrin ␤5 mRNA (D). Negative controls with the primers for corresponding integrins are shown in black.
Close overlap of 18S rRNA amplification curves indicates equal total RNA concentration in all samples. Amplification of integrin mRNA occurred
earlier in the samples from patients with SSc than in the samples from healthy volunteers, suggesting higher steady-state levels of mRNA.
0.05; r2 ⱕ 0.08 in all cases). No correlation was observed
between the percent integrin-positive BAL fluid T cells
and the percentage of macrophages, lymphocytes, neutrophils, or eosinophils in BAL fluid (P ⬎ 0.05; r2 ⱕ 0.15
in all cases). There was no correlation with the percent
predicted forced vital capacity (P ⬎ 0.05; r2 ⱕ 0.01 in all
cases) or the percent predicted diffusing capacity for
carbon monoxide (P ⬎ 0.05; r2 ⱕ 0.04 in all cases).
ANOVAs showed that patient groups did not differ (P ⬎
0.05) in the percent integrin-positive BAL fluid T cells
when analyzed according to sex, race, age, or combinations of these characteristics.
To validate selected results of the flow cytometry experiments described above, real-time reverse
transcriptase–polymerase chain reaction assays for integrin ␤3 mRNA were performed, using mRNA purified
from BAL fluid T cells that were enriched by positive
selection with nonactivating magnetic beads, as previously described (7). The levels of integrin ␤3 mRNA
were significantly higher in BAL fluid T cells from
patients with SSc compared with those from healthy
volunteers (Figure 2C). Thus, we determined that pulmonary T lymphocytes express integrins ␣V␤3 and
␣V␤5 in patients with SSc but not in healthy individuals.
Expression of integrins by BAL T cells in the
CCL18 overexpression animal model. We then considered that pulmonary levels of the T cell–selective chemokine CCL18, as well as pulmonary levels of T cells,
are significantly elevated in the lungs of patients with
SSc or other pulmonary fibrotic diseases (for review, see
ref. 2). Our group recently introduced a new animal
model based on adenoviral delivery of the CCL18 gene
to the lungs in vivo (3,4). The overexpression of CCL18
in vivo manifests as pulmonary T lymphocyte infiltration, T cell–dependent activation of TGF␤, and accumulation of collagen (2–4). This model is more suitable
for the current study than is a more traditional model of
bleomycin-induced pulmonary fibrosis, because the
bleomycin model is known to be T cell independent
Similar to what was observed in humans with
interstitial lung disease (Figures 1 and 2; additional data
are available at
supplfigures2009ar.pdf), the infiltrating cells in the
CCL18 overexpression animal model expressed integrins
␣V, ␤3, and ␤5 (Figure 3A). Stainings were performed
for individual integrin chains, because high-quality antibodies against mouse integrins ␣V␤3 or ␣V␤5 were not
commercially available. To confirm that such expression
occurs specifically on T cells, flow cytometry assays of
BAL fluid cells were performed (Figures 3B–D). Again,
commercially available antibodies against mouse ␣V–
containing integrin heterodimers did not allow for highquality specific resolution of expressing cells in flow
cytometry assays; therefore, staining was performed for
individual integrin chains. Thus, we determined that
Figure 3. Lung histology and T cell flow cytometry in the CCL18
animal overexpression model. A, Trichrome (Trichr) staining of lung
sections showed infiltration (nuclei stain in purple) and collagen fibers
(blue; arrows) in response to CCL18 overexpression. Immunohistologically (brown), isotype control antibody did not stain tissue sections,
whereas infiltrates that consist predominantly of T cells stained for
integrins ␤2 (used as positive control), ␣V, ␤3, and ␤5. Minimal or no
changes were observed in control mice infected with a similar noncoding adenovirus. (Original magnification ⫻ 200.) B and C, Isotype
control antibodies for integrins ␤3, ␤5, or ␣V did not stain pulmonary
T cells, whereas CD4⫹ and CD8⫹ T cells stained for integrins ␤3
(ITGB3) and ␤5 (ITGB5). D, Integrins ␤3 and ␣V were coexpressed
on pulmonary T cells. Gating was on CD3⫹ (CyChrome-labeled
antibody) cells; the percent double-positive cells is indicated in the
upper right quadrants. Most of the T cells in the CCL18overexpressing mice were double positive, suggesting that both integrin ␣V and integrin ␤3 are coexpressed on the same T cells, consistent
with the notion that these chains may form heterodimers on T cells.
PE ⫽ phycoerythrin; FITC ⫽ fluorescein isothiocyanate.
pulmonary T lymphocytes express integrins not only in
patients with interstitial lung disease but also in the
animal model of CCL18-mediated pulmonary T lymphocyte infiltration and fibrosis.
Role of integrin expression in lymphocyte infiltration and collagen accumulation. To determine
whether integrin expression may be a determining factor
in T lymphocyte infiltration and collagen accumulation
in the lungs, 2 types of experiments were conducted.
First, CCL18-overexpressing mice were treated systemically with integrin ␣V–blocking antibody. Such treatment significantly attenuated lymphocytic infiltration
and collagen accumulation compared with that in
CCL18-overexpressing mice treated with isotype control
antibody (Figure 4). Second, adenovirus-mediated
CCL18 overexpression was initiated in integrin ␤3–
deficient mice. In contrast to wild-type mice, the
Figure 4. Effects of treatment with neutralizing anti–integrin ␣V
(ITGAV) antibody or integrin ␤3 (ITGB3) deficiency in the lungs of
CCL18-overexpressing mice. A, Trichrome staining of histologic sections showing collagen in blue. Wild-type (WT) or ITGB3-knockout
(KO) mice challenged with AdNull virus were indistinguishable from
healthy control mice (Ctrl). Overexpression of CCL18 in wild-type
mice caused lymphocytic infiltration and collagen accumulation (arrows). ITGB3-knockout mice overexpressing CCL18 showed minimal,
if any, cellular infiltration or collagen accumulation. Wild-type mice
overexpressing CCL18 and treated with anti-ITGAV neutralizing
antibodies (Ab) had significantly attenuated infiltration and collagen
accumulation (arrowhead). B, Percentage of lymphocytes in the bronchoalveolar lavage (BAL) fluid of wild-type or ITGB3-knockout mice
instilled with the Null noncoding adenovirus or the CCL18-encoding
virus and treated with anti-ITGAV antibody or corresponding isotype
Ig control. The majority of other BAL fluid cells were macrophages,
with occasional neutrophils (not exceeding 1.5%) or bronchial epithelial cells (not exceeding 3%). C, Total lung collagen (measured as
hydroxyproline [Hyp] content) in mice treated as described above. All
experiments were repeated on 2 independent occasions, with consistent results. Values in B and C are the mean and SD results from 5–6
mice per group.
Figure 5. Overexpression of integrins ␣V␤3 or ␣V␤5 on Jurkat cells. A, Flow cytometry with the antibodies against the
indicated heterodimers after transfection with plasmids encoding the indicated integrin chains (black line) or after
transfection with noncoding plasmid (gray histogram). B, Western blotting for type I collagen in fibroblast cultures (Fib),
or cocultures with Jurkat cells transfected with the noncoding plasmid (Fib⫹blank) or transfected to express the
indicated integrins (Itg) (in duplicate for each heterodimer). The bars in the densitograms match the individual Western
blotting bands shown in the gels below. C, Metabolic incorporation of 14C-proline as a surrogate measure of new
collagen synthesis. Fibroblasts were cultured in medium (Med) without or with anti–transforming growth factor ␤
(anti-TGF␤) monoclonal antibody 1D11, as indicated. Alternatively, fibroblasts were cocultured with Jurkat T cells
transfected to overexpress integrin ␣V␤5, without (␣V␤5) or with anti-TGF␤ antibody (␣V␤5⫹1D11). All experiments
were performed on at least 3 independent occasions, with consistent results for both integrin ␣V␤3 and integrin ␣V␤5.
Values are the mean and SD results from triplicate cultures. isot. ⫽ isotype.
ITGB3⫺/⫺ mice had minimal, if any, accumulation of
lymphocytes or collagen in their lungs (Figure 4). Thus,
we determined that the expression of integrins is necessary for T cell infiltration into the lungs and associated
collagen accumulation in CCL18-overexpressing mice.
Effects of integrin ␣V␤3- or ␣V␤5-overexpressing
Jurkat cells on primary fibroblast cultures. To directly
establish whether T cells expressing integrin ␣V␤3 or
integrin ␣V␤5 may act profibrotically, human integrin
chains ␣V and ␤3 or integrin chains ␣V and ␤5 were
overexpressed in a T cell line (Jurkat). Flow cytometry
assays for ␣V␤3 or ␣V␤5 heterodimers confirmed overexpression (Figure 5A). Such an approach was used
because the absolute amounts of primary pulmonary T
cells derived from patient BAL fluid samples are usually
limited (6–8). Obtaining the necessary amounts of
integrin-expressing primary T cells for cell culture assays
would be technically challenging. Pulmonary fibroblasts
were cultured alone, with integrin-encoding constructtransfected Jurkat cells, or with Jurkat cells transfected
with the equivalent amounts of blank, noncoding plasmids (Figure 5B). Cell viability assays revealed that such
transfections did not cause a cytotoxic effect in Jurkat
cells. Western blot assays of fibroblast culture supernates for type I collagen revealed that integrinexpressing but not mock-transfected T lymphocytes
cocultured with fibroblasts stimulated an increase in
collagen levels (Figure 5B).
TGF␤-mediated effect of integrin-expressing T
cells on collagen production in cell cultures. The upregulation of collagen in the cocultures of integrintransfected Jurkat cells and primary fibroblasts was
confirmed in 14C-proline metabolic incorporation assays, reflective of new collagen production rates by
fibroblasts (Figure 5C). Jurkat cells were transfected to
express integrin ␣V␤5 and stimulated collagen upregulation in cocultures with primary fibroblasts (Figure
5C). Importantly, anti-TGF␤ blocking antibody (clone
1D11) at the recommended dose of 1 ␮g/ml had a
minimal effect on collagen production in fibroblast
cultures alone but significantly attenuated the upregulated collagen levels in cocultures with Jurkat cells
transfected to overexpress integrin ␣V␤5 (Figure 5C).
This observation suggested that the effect of integrinexpressing T cells on collagen production in fibroblasts is
dependent on TGF␤. However, ELISAs revealed no
integrins ␣V␤3 or ␣V␤5 (Figures 6A and B; confocal
microscopy was used to definitively confirm nuclear
translocation of Smad2, whereas fluorescence microscopy provided comparable quality of resolution and was
used for quantitative purposes). In parallel, nontransfected primary pulmonary fibroblasts were similarly
cocultured with Jurkat cells overexpressing these integrins, and the relative levels of Smad2/3 in the nucleus
and cytoplasm were assessed by Western blotting (Figure 6C). Increased nuclear accumulation of Smad2
suggests that TGF␤ is indeed involved in such regulation
(Figure 6). Additionally, expression of ␣-smooth muscle
actin was increased in fibroblasts cocultured with integrinoverexpressing Jurkat cells, which is indicative of fibroblast activation (additional data are available at http://
Figure 6. Nuclear translocation of Smad2 in primary adult lung
fibroblasts cocultured with Jurkat cells overexpressing integrins ␣V␤3
or ␣V␤5, or transfected with blank noncoding plasmid. A, Confocal
microscopy of primary fibroblasts transfected with a Smad2–green
fluorescent protein construct and cocultured with Jurkat cells. Fibroblasts cocultured with the blank plasmid-transfected Jurkat cells (top
row) remained quiescent, were not distinguishable from fibroblasts
cultured alone, and showed Smad2 either lacking in the nuclei (white
arrows) or evenly distributed between the nucleus and cytoplasm. In
contrast, fibroblasts cocultured with Jurkat cells that overexpressed
integrins ␣V␤3 or ␣V␤5 showed numerous nuclei with predominant
accumulation of Smad2 (yellow arrows). B, Percent Smad2-positive
nuclei. Fibroblasts cocultured with integrin-overexpressing Jurkat cells
had significantly increased numbers of Smad2-positive nuclei. One
hundred randomly selected cells were counted in duplicate for each
condition. Values are the mean and SD results from 2 independent experiments. C, Western blotting for total Smad2/3,
␤-actin, or histone H1 in the nuclear (N) and cytoplasmic (C) fractions
of fibroblasts stimulated with recombinant human transforming growth
factor ␤ (rhTGF␤) (positive control) or cocultured with primary Jurkat
cells transfected with the constructs indicated. The experiments were
repeated on 2 separate occasions, with consistent results. ITGAVB5 ⫽
anti–integrin ␣V␤5.
increase in total TGF␤ and no detectable freely diffusible active TGF␤ in these cocultures (results not shown).
The latter observation is consistent with the notion that
integrin-mediated activation of TGF␤ is a surfaceassociated event (13), and that activated TGF␤ may be
cell-surface bound or become internalized by fibroblasts.
Therefore, the experiments were aimed at determining
whether intracellular signaling characteristic of TGF␤
may be activated in fibroblasts in cocultures with
integrin-expressing Jurkat T cells.
Adult primary lung fibroblasts were transfected
with a functional Smad2–GFP construct (22) and cocultured for 48 hours with Jurkat cells overexpressing
We report here that pulmonary T lymphocytes
express integrins ␣V␤3 and ␣V␤5 in humans with
interstitial lung disease, particularly those with scleroderma (see Figures 1 and 2), and in the animal model of
CCL18-mediated infiltration and T cell–dependent collagen accumulation (see Figures 3 and 4). Although
expression of integrins ␣V␤3 and ␣V␤5 on subpopulations of T cells was discovered more than a decade ago
(23–26), its role in the disease processes, particularly in
regulating inflammation and the balance of connective
tissue, remains unclear. The results of our study suggest
that expression of these integrins on pulmonary lymphocytes may explain their previously described persistent
infiltration in the lungs (3). We report that antibodymediated neutralization or a genetic deficiency of these
integrins abrogates lymphocyte infiltration and related
collagen accumulation in the lungs (see Figure 4). In
vitro experiments revealed that T cells overexpressing
these integrins are directly profibrotic on cultured primary human pulmonary fibroblasts, likely through a
TGF␤-dependent mechanism (see Figures 5 and 6). Consistent with such a notion, pulmonary infiltration of
T cells in the CCL18 overexpression model is associated with local TGF␤ activation and collagen accumulation (3).
A possibility was considered that the mechanism
of TGF␤ activation and collagen accumulation may be
attributable to the regulatory nature of the infiltrating T
cells. However, immunohistochemical analyses and flow
cytometry assays in the CCL18 overexpression model
revealed that FoxP3-positive cells were present in the
infiltrates and BAL fluid, but they were rare (mean ⫾
SD 7 ⫾ 1% of BAL fluid T cells, as assessed by flow
cytometry), with no percent-wise difference between
CCL18 overexpression alone or in combination with
models of bleomycin-induced fibrosis (results not
shown). Thus, the regulation of fibrosis by T cells likely
occurs in no apparent association with FoxP3 expression.
These results suggest that FoxP3-negative pulmonary T cells that do not produce TGF␤ (not classic Treg
or Th3 phenotypes) may act by expressing integrins and
activating latent TGF␤. Based on this observation, such
cells perhaps may be broadly classified as Treg cells, with
an implied antiinflammatory effect. Overall, the effects
of T lymphocyte infiltration in the lungs are likely
diverse and depend on the specific nature of the local
inflammatory milieu as well as the specific T cell phenotype. Data in humans and in animal models suggest
that pulmonary T cells may facilitate the accumulation
of collagen, attenuate pulmonary fibrosis, or be nonparticipating bystanders in the fibrotic process (2). Our data
suggest that in patients with diffuse parenchymal lung
disease, particularly patients with scleroderma, T lymphocytes may accumulate in the lungs in a futile attempt
to attenuate the autoimmune process and inflammation.
They express integrins ␣V␤3 and ␣V␤5 to ensure adhesion, a persistent presence in the lungs, and TGF␤
activation. The latter factor (TGF␤), a potent immunosuppressive and antiinflammatory regulator, is also the
most potent profibrotic cytokine. As a result, integrinexpressing T cells likely exert a profibrotic effect on the
lung. One implication of this would be that systemic or
local depletion of pulmonary T cells may have a therapeutic antifibrotic effect, but a side effect of such
hypothetical therapy may be an undesired attenuation of
the antiinflammatory action of T cells.
In conclusion, pulmonary infiltrating T lymphocytes may express integrins ␣V␤3 and ␣V␤5. Such
expression likely explains pulmonary lymphocyte infiltration, the persistence of T cells in the lungs, and T
cell–associated TGF␤ activation and collagen accumulation.
Drs. Luzina and Atamas had full access to all of the data in
the study and take responsibility for the integrity of the data and the
accuracy of the data analysis.
Study design. Luzina, Atamas.
Acquisition of data. Luzina, Todd, Nacu, Lockatell, Choi, Hummers.
Analysis and interpretation of data. Luzina, Todd, Atamas.
Manuscript preparation. Luzina, Todd, Hummers, Atamas.
Statistical analysis. Atamas.
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2. Luzina IG, Todd NW, Iacono AT, Atamas SP. Roles of T
lymphocytes in pulmonary fibrosis. J Leukoc Biol 2008;83:237–44.
3. Luzina IG, Papadimitriou JC, Anderson R, Pochetuhen K, Atamas SP. Induction of prolonged infiltration of T lymphocytes and
transient T lymphocyte–dependent collagen deposition in mouse
lungs following adenoviral gene transfer of CCL18. Arthritis
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by CCL18. Am J Pathol 2007;171:428–37.
5. Atamas SP, Luzina IG, Dai H, Wilt SG, White B. Synergy between
CD40 ligation and IL-4 on fibroblast proliferation involves IL-4
receptor signaling. J Immunol 2002;168:1139–45.
6. Atamas SP, Yurovsky VV, Wise R, Wigley FM, Goter Robinson
CJ, Henry P, et al. Production of type 2 cytokines by CD8⫹ lung
cells is associated with greater decline in pulmonary function in
patients with systemic sclerosis. Arthritis Rheum 1999;42:1168–78.
7. Luzina IG, Atamas SP, Wise R, Wigley FM, Choi J, Xiao HQ, et
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8. Luzina IG, Atamas SP, Wise R, Wigley FM, Xiao HQ, White B.
Gene expression in bronchoalveolar lavage cells from scleroderma
patients. Am J Respir Cell Mol Biol 2002;26:549–57.
9. Atamas SP, Luzina IG, Choi J, Tsymbalyuk N, Carbonetti NH,
Singh IS, et al. Pulmonary and activation-regulated chemokine
stimulates collagen production in lung fibroblasts. Am J Respir
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10. Luzina IG, Tsymbalyuk N, Choi J, Hasday JD, Atamas SP.
CCL18-stimulated upregulation of collagen production in lung
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J, et al. The integrin ␣v␤6 binds and activates latent TGF ␤1: a
mechanism for regulating pulmonary inflammation and fibrosis.
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growth factor ␤. Cancer Metastasis Rev 2005;24:395–402.
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between growth factors and integrins: latent forms of transforming
growth factor-␤ are ligands for the integrin ␣v␤1. Mol Biol Cell
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DOI 10.1002/art.24496
Clinical Images: Podoconiosis: foot edema resulting from regional geochemistry
The patient, a 14-year-old boy, presented with bilateral lower leg elephantiasis (A) and lichenification of the overlying skin, with a
mossy appearance over the toes (B). The lesions had begun to appear 5 years prior and were accompanied by edema and itching
of the feet. The boy had lived his entire life in the Guraghe Zone, a region of Ethiopia located 200 kilometers southwest of Addis
Ababa. This area of the country is above 2,500 meters in altitude and is characterized by alkaline volcanic soil and high levels of
rainfall. Due to poor socioeconomic conditions, the patient had remained barefooted throughout his life. Giemsa staining of thick
blood film from a capillary blood sample revealed no microfilariae. Based on this result and on clinical and demographic findings,
podoconiosis (endemic nonfilarial elephantiasis) was diagnosed. Techniques for improving venous and lymphatic drainage were
demonstrated to the patient, who was also given protective footwear. Foot hygiene was also prescribed. Although nearly unknown
outside of endemic regions, podoconiosis is a major public health problem in tropical Africa, Central America, and northern India.
In Ethiopia, at least 1 million individuals are affected and often come from age groups most able to work (i.e., neither too young
nor too old). Podoconiosis is the result of progressive obliterating endolymphangitis, which is induced by the absorption of ultrafine
silica particles through the skin of the feet when a person is exposed to red clay soil derived from alkaline volcanic rock. The early
signs and symptoms of podoconiosis are recurrent episodes of burning and edema of one foot or of the lower leg following periods
of intense physical activity. The increase in the diameter of the leg persists over time. Established lymphedema can progress to
severe elephantiasis. The disease is bilateral, but asymmetric, and the affected area is almost always below the knee. The increased
size of the legs and the stiffness of the skin result in patient disability.
Giuseppe Indolfi, MD
Silvia Fontanazza, MD
Francesco Silenzi, MD
Saint Gabriel Clinique
Guraghe Zone, Ethiopia
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expressions, inflammation, pulmonaria, integrins, fibrosis, regulation, lymphocytes
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