journals

1. Am. J. Respir. Cell Mol. Biol. Vol. 23, pp. 204–212, 2000
Internet address: www.atsjournals.org
A CD36 Synthetic Peptide Inhibits Bleomycin-Induced Pulmonary
Inflammation and Connective Tissue Synthesis in the Rat
Teshome Yehualaeshet, Robert O’Connor, Asher Begleiter, Joanne E. Murphy-Ullrich, Roy Silverstein,
and Nasreen Khalil
Departments of Internal Medicine and Pathology, and the Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg,
Mannitoba; BC Cancer Agency and Vancouver Hospital, Vancouver, British Columbia, Canada; Department of Pathology, University of
Alabama at Birmingham, Birmingham, Alabama; and Division of Hematology/Oncology, Cornell Medical Center, New York, New York
Transforming growth factor (TGF)-␤1 is an important regula-
tor of inflammation and fibrosis. TGF-␤1 is usually secreted as
a biologically latent protein called latent TGF-␤1 (L-TGF-␤1).
L-TGF-␤1 has no biologic effect unless L-TGF-␤1 is converted
to its active form. Using a well-recognized model of lung in-
jury induced by the antineoplastic antibiotic bleomycin (Blm),
we demonstrated that 7 d after intratracheal Blm administration,
total lung TGF-␤ was maximally increased. This induction was
due to TGF-␤1 production by alveolar macrophages that,
when explanted, generated increased quantities of L-TGF-␤1
complexed with the glycoprotein thrombospondin (TSP)-1.
The TSP-1/L-TGF-␤1 complex was associated with CD36, a re-
ceptor for TSP-1. The association of TSP-1/L-TGF-␤1 to CD36
was critical for plasmin-mediated release of mature TGF-␤1. In
this paper we show that, compared with administration of
Blm by itself, when a synthetic peptide of CD36 between
amino acids 93 and 110 is given concomitantly with Blm to
rats, alveolar macrophages generate markedly less active TGF-␤1,
the rats gain weight more rapidly, and there is less inflammation,
collagen I and III, and fibronectin synthesis. These findings
demonstrate a novel in vivo mechanism of activation of L-TGF-␤1
in lung injury and the importance of alveolar macrophage–
derived active TGF-␤1 in the pathogenesis of pulmonary in-
flammation and fibrosis.
Before an increase in connective tissue synthesis after lung
injury, there is an influx of inflammatory cells that are
present not only in the alveoli but also in the interstitium
(1). In inflammatory infiltrates the alveolar macrophages
are activated to produce a number of proinflammatory
and fibrogenic cytokines (2) such as transforming growth
factor (TGF)-␤ (3–6), platelet-derived growth factor (7),
insulin growth factor-1 (7), tumor necrosis factor (TNF)-␣,
interleukin (IL)-1, and IL-5 (4, 5, 8). Of these cytokines,
TGF-␤1 has been demonstrated to regulate not only itself
but also a variety of fibrogenic cytokines (9) that ultimately
are important in the pathogenesis of inflammation and
connective tissue synthesis (2–8).
Although TGF-␤ exists in three isoforms in mammals,
it is the TGF-␤1 isoform that has been most commonly as-
sociated with disorders characterized by inflammation and
fibrosis (10). All the TGF-␤s are initially synthesized as
large precursors that are 390 to 414 amino acids in size
(11). The intracellular and extracellular processing of the
TGF-␤1 isoform has been most extensively studied. Be-
fore the secretion of the TGF-␤1, the intracellular pro-
tease furin cleaves the amino terminal end of the precur-
sor between amino acids 278 and 279 (11), resulting in an
amino terminal peptide called the latency-associated pep-
tide (LAP) and a carboxy terminal protein called the ma-
ture TGF-␤1. However, the cleaved 75-kD amino terminal
portion of the protein noncovalently associates with the
25-kD carboxy terminal portion of the protein (11). When
the carboxy terminal TGF-␤1 peptide is secreted as a com-
plex with the LAP it is called latent TGF-␤1 (L-TGF-␤1),
and in this form L-TGF-␤1 cannot interact with its recep-
tor or have a biologic effect. In vitro, the LAP can be re-
moved from its association with the TGF-␤1 by pH Ͻ 2 or
Ͼ 8, heat of 100ЊC (11), chaotropic agents, proteases such
as plasmin (5, 6, 11), cathepsin A and D, endoglycosidase,
sialic acid, reactive oxygen species, or high concentrations
of glucose (11). Alternatively, L-TGF-␤1 can be activated
without removing the LAP by interacting with the glyco-
protein thrombospondin (TSP)-1 (12) or the integrin ␣v␤6
(13). Although the expression of TGF-␤1 has been associ-
ated with a number of inflammatory and fibrotic diseases,
for TGF-␤1 to be physiologically or pathologically signifi-
cant in these disorders it must be present in a biologically
active form. The in vivo activation of L-TGF-␤1 in these
instances is poorly understood.
We have used a well-recognized model of lung injury
induced by the antineoplastic antibiotic bleomycin (Blm)
(3–6) to describe a novel mechanism of activation of L-TGF-
␤1 (4–6). After a single intratracheal dose of Blm, there is
injury to the alveolar epithelium and endothelium, fol-
lowed by recruitment and activation of inflammatory cells
before epithelial cell regeneration, resolution of inflam-
mation, and enhanced connective tissue synthesis (3–6).
We have demonstrated that 7 d after Blm-induced lung in-
jury and before collagen synthesis, activated alveolar mac-
rophages were the primary source of a 30-fold increase in
total lung TGF-␤ content (3–6). When these alveolar mac-
rophages were explanted they generated active TGF-␤1,
whereas alveolar macrophages from normal saline-treated
rats or those receiving no treatment secreted either no
TGF-␤1 or small amounts of L-TGF-␤1 (4–6). In addition,
7 d after Blm injury alveolar macrophages released maxi-
mal quantities of the serine protease plasmin and the glyco-
protein TSP-1 (5). The presence of ␣2-antiplasmin or aproti-
nin, both inhibitors of plasmin or anti–TSP-1 antibodies,
inhibited the post-translational activation of alveolar mac-
rophage–derived L-TGF-␤1 (5, 6). It was also demonstrated
(Received in original form December 30, 1999 and in revised form March 16,
2000)
Address correspondence to: Dr. Nasreen Khalil, M.D., Div. of Respiratory
Medicine, University of British Columbia, 655 W. 12th Ave., Vancouver,
BC, V5Z 4R4 Canada. E-mail: nasreen.khalil@bccdc.hnet.bc.ca
Abbreviations: analysis of variance, ANOVA; bronchoalveolar lavage,
BAL; bleomycin, Blm; Canadian Council on Animal Care, CCAC; hema-
toxylin and eosin, H&E; latency-associated peptide, LAP; latent TGF-␤1,
L-TGF-␤1; polymorphonuclear leukocyte, PMN; Tris-buffered saline, TBS;
transforming growth factor, TGF; thrombospondin, TSP.

2. Yehualaeshet, O’Connor, Begleiter, et al.: CD36 Peptide Inhibits Pulmonary Fibrosis 205
that before the release of active TGF-␤1 the L-TGF-␤1
that was complexed with the TSP-1 interacted with the
TSP-1 receptor CD36 on the alveolar macrophage (6). The
CD36–TSP-1/L-TGF-␤1 interaction appears critical to the
activation process because in the presence of antibodies to
CD36 that prevent the association of TSP-1 to CD36 the
activation of L-TGF-␤1 was totally abrogated. TSP-1 asso-
ciates with CD36 by interaction of TSP-1 with the
ectodomain of CD36 between amino acids 93 and 110. In
the presence of a synthetic peptide of the ectodomain of
CD36 that mimics the region between amino acids 93 to 110
the activation of L-TGF-␤1 by explanted alveolar mac-
rophages was also inhibited (6).
In this paper we demonstrate that compared with Blm
administration alone the concomitant administration of
Blm and the CD36 synthetic peptide 93-110 significantly
reduces alveolar macrophage secretion of active TGF-␤1,
inflammatory lesions, and collagen I and III and fibronec-
tin synthesis. These findings support the importance of bi-
ologically active TGF-␤1 derived from alveolar macrophages
in the pathogenesis of Blm-induced pulmonary inflammation
and fibrosis. Further, these findings suggest that a TSP-
1–CD36 plasmin–dependent mechanism is involved in the
process of local activation of alveolar macrophage–derived
L-TGF-␤1.
Materials and Methods
Animals
Female Sprague–Dawley rats, free of respiratory disease and weigh-
ing between 250 and 300 g, were obtained from the University of
Manitoba vivarium. In each experiment, all rats were matched
for age and weight. In compliance with the Canadian Council on
Animal Care (CCAC), the numbers of rats used were restricted
to numbers that were as minimal as possible to adequately address
the most relevant issues related to this work.
Reagents
Blm (Blenoxane) was purchased from Bristol-Myers Squibb (Evans-
ville, IN). Neutralizing antibodies to TGF-␤1-3 were obtained from
Genzyme (Cambridge, MA). Antibody to procollagen I and III
(Cedarlane Laboratories Inc., Hornby, ON, Canada) and fibronec-
tin (Sigma, St. Louis, MO) were used for immunoblotting.
Preparation of Synthetic CD36 Peptides
The CD36 peptide YRVRFLAKENVTQDAEDNC (93-110), the
scrambled peptide of 93-110 (RFAYLRKNVTENDEQAVCD),
and the CD36 synthetic peptide (208-224) CADGVYKVFNGKD-
NISKV were synthesized (6), on the basis of the work of Leung and
colleagues (14). The peptides were synthesized with an Applied
Biosystems model 431A peptide synthesizer, using Fmoc (N-[9-
Fluoreny-D-methoxycarbonyl]) chemistry, and were purified by
reverse high-performance liquid chromatography using a C18
column.
Blm and Synthetic Peptide Administration
This procedure is described in detail in References 3–6. Briefly,
rats were given normal saline, 1 ␮g of Blm, 1,600 ␮g of a CD36
synthetic peptide, or 1 ␮g of Blm concomitantly with 1,600 ␮g
of a CD36 synthetic peptide in a total volume of 500 ␮l sterile
normal saline. Rats used as controls received 1,600 ␮g of the
CD36 synthetic peptide 93-110 or a CD36 peptide unrelated to
the site of interaction of CD36 with TSP-1 mimicking the amino
acid 208-224 sequence of the CD36 ectodomain (14). The quan-
tity of 1,600 ␮g of peptide used was based on a pilot study demon-
strating that quantities less than 1,600 ␮g of the peptide did not
affect Blm toxicity in a significant manner. The CD36 synthetic
peptide from amino acids 208 to 224 has previously been demon-
strated not to inhibit activation of L-TGF-␤1 (6). In addition, a
scrambled peptide of the CD36 amino acid 93-110 (sequence
given earlier) was also used. After administration of various
agents, the rats were killed at different time intervals. For some
experiments, 7 d after reagent administration was chosen as the
time to harvest alveolar macrophages, on the basis of our findings
that alveolar macrophages are maximally stimulated at this time
to secrete active TGF-␤1 (5). Weights and appearances of rats
were recorded daily after receiving the various treatments.
Histology and Histochemistry
Fourteen days after Blm administration, a time previously reported
to be associated with increased connective tissue synthesis and in-
flammation (1, 3), we obtained lungs for histology as previously
described (3). At 24 h after fixation in 10% formalin the lungs
were embedded in paraffin, sectioned, and stained with hematox-
ylin and eosin (H&E) for histology and Mason’s Trichrome for dis-
tribution of collagen, as well as Alcian blue for location of pro-
teoglycans. Because Blm-induced lung injury is patchy in nature,
all lung segments were examined from each treatment group. All
slides were blinded and two independent examinations (by au-
thors N.K. and R.O.) were done and then collated. For the extent
of lung involved, the lung sections were examined under low
power and revealed the entire lung section that contained both
normal lung and an inflammatory or fibrotic lesion. The propor-
tion of lung section with inflammation was then reported as a
percentage of the overall lung section. For grading the extent of
staining with Mason’s Trichrome or Alcian blue, 0 designated no
staining, ϩ1 designated detectable color (green for Mason’s
Trichrome and blue for Alcian blue), and ϩ2 designated an ex-
tent of staining between ϩ1 and ϩ3. The grade ϩ3 was reserved
for those instances where there was extensive staining within an
inflammatory and fibrotic lesion.
Differential Cell Count of Cells Obtained by
Bronchoalveolar Lavage
Cells obtained by bronchoalveolar lavage (BAL), as previously
described (4–6), were suspended at a concentration of 1 ϫ 106
cells/ml and were used in a cytospin preparation (4). The cells
were stained using Diff-Quik (Baxter Healthcare Corp., Miami,
FL) fixative and nuclear/cytoplasmic stains (4). Five fields at high
power were used to enumerate and identify cells as macrophages,
polymorphonuclear leukocytes (PMNs), or lymphocytes (4).
Macrophage Cultures
The lungs were lavaged to obtain cells for culture of alveolar mac-
rophages as previously described (4–6). Alveolar macrophages
were maintained in serum-free media containing Gentamicin (4 mg/
100 mls; Roussel, Montreal, PQ, Canada), Fungizone (100 ␮1/
100 mls; GIBCO BRL, London, ON, Canada), and 0.2% clotted
bovine calf plasma (BCP) (National Biological Laboratory Lim-
ited, Dugald, MB, Canada). After 20 h of incubation at 37ЊC, 5%
CO2, the media were collected in the presence of protease inhibi-
tors—leupeptin, 0.5 ␮g/ml, from Amersham, Buckinghamshire, UK;
and aprotinin and pepstatin, 1 ␮g/ml each, both from Sigma
(Oakville, ON, Canada)—frozen at Ϫ80ЊC until ready for TGF-␤
quantitation (4–6).
CCL-64 Mink Lung Epithelial Growth Inhibition Assay
for TGF-␤
The CCL-64 growth inhibition assay to identify and quantitate
TGF-␤ has been described elsewhere (3–6). Briefly, neutral con-

3. 206 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 23 2000
ditioned media or conditioned media that were acidified and
subsequently neutralized were added to subconfluent cells in
␣–Eagle’s minimum essential medium, 0.2% BCP, 10 mM N-2-hy-
droxyethylpiperazine-NЈ-ethane sulfonic acid at pH 7.4, and peni-
cillin (25 ␮g/ml) and streptomycin (25 ␮g/ml), and cultured as 5 ϫ
104
cells/0.5 ml in 24-well Costar dishes (Flow Laboratories, Inc.;
Mississauga, ON, Canada). After 22 h the cells were pulsed with
0.25 ␮Ci of 5-[125
I]iodo 2Ј-deoxyuridine [125
I]UdR (ICN Pharma-
ceutical, Costa Mesa, CA) for 2 to 3 h at 37ЊC, then lysed with 1 ml
of 1 N NaOH for 30 min at room temperature. The [125
I]UdR was
then counted in a ␥ counter (LKB Instruments, Gaithersburg,
MD). A standard curve using porcine TGF-␤1 was included in each
assay. For confirmation of TGF-␤ activity, neutralizing mono-
clonal antibody against TGF-␤1-3 (Genzyme) was added before the
addition of the conditioned media (3–6) and resulted in abrogation
of all TGF-␤ activity.
Protein Extraction for Western Analysis
Untreated rats and rats treated with various agents were killed
and the peripheral blood, heart, and blood vessels were removed
as described earlier (3–6). The lungs were snap-frozen on dry ice
with ethanol and stored at Ϫ80ЊC until protein extraction. Whole-
lung protein extraction was performed as described previously (6).
Briefly, the frozen lungs were pulverized in a chilled mortar and
placed in tissue lysis buffer containing 1 mM phenylmethylsulfonyl
fluoride (Sigma). The samples were further homogenized in the
presence of 0.5% Triton X-100, then centrifuged at 200 mg for 10 min
at 4ЊC. The supernatant was collected and protein levels were de-
termined using a Bio-Rad protein assay (Bio-Rad Laboratories,
Hercules, CA).
Western Analysis
The protein samples (25 ␮l) were electrophoresed on a 10% so-
dium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis in
a Mini-PROTEAN II Electrophoresis Cell (Bio-Rad). Protein
molecular weight markers (Amersham) were run parallel to each
blot as an indicator of the molecular weight. Equal loading of
protein was evaluated using silver staining (not shown). The sep-
arated proteins were transferred at 50 V overnight onto nitrocel-
lulose membrane (GIBCO BRL) in a Mini Trans-Blot chamber
with transfer buffer (25 mM Tris-Cl, 192 mM glycine, and 20%
methanol). The nitrocellulose membrane was blocked for 1 h us-
ing 5% instant skim-milk powder in Tris-buffered saline (TBS). For
detection of procollagen I and III a 1:300 dilution of antibody was
used, whereas a 1:1,000 dilution was used for fibronectin in 1%
instant skim-milk powder. After washing, the nitrocellulose mem-
brane was incubated with horseradish peroxidase linked with the
secondary antibody (goat antimouse immunoglobulin G; Bio-Rad)
as recommended by the manufacturer. Finally the washed blots
were exposed to an enhanced chemiluminescence (ECL) detection
system (Amersham) and recorded on an autoradiograph (Kodak
X-Omat film). Before reprobing, the nitrocellulose membrane
was incubated at 50ЊC for 30 min with a stripping buffer (100
mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl, pH
6.7). The blots were rinsed twice with TBS. To ensure the re-
moval of antibodies, membranes were incubated with the ECL
detection reagents and exposed to film (Kodak). No band was de-
tected, confirming that all antibodies were stripped off the mem-
brane. The same nitrocellulose membrane was blocked using 5%
instant skim-milk powder in TBS for detection of procollagen III
and fibronectin.
Cytotoxicity Assay
LY/5178Y mouse lymphoma cells were incubated with normal
saline, Blm (1 ␮g/500 ␮l of normal saline), CD36 synthetic pep-
tide 93-110 (1,600 ␮g/500 ␮l of normal saline), or Blm (1 ␮g /500 ␮l
of normal saline) plus CD36 synthetic peptide 93-110 (1,600 ␮g)
for 1 h at 37ЊC in media containing horse serum. The cytotoxic ac-
tivity of the agents on the LY/5178Y cells was determined by a
soft agar cloning assay and was expressed as the surviving cell
fraction as described previously (15). Cloning efficiencies ranged
from 35 to 65%. The cytotoxicities of the different treatments in
the cell line were compared statistically by analysis of variance
(ANOVA).
Statistical Analysis
Statistical analysis using ANOVA or Wilcoxon’s rank signed non-
parametric statistical test was done by Dr. Bob Tate, Biostatistical
Unit, University of Manitoba.
Results
Post-Translational Activation of Alveolar
Macrophage–Derived L-TGF-␤1
Our previous work demonstrated that a single intratra-
cheal dose of Blm resulted in generation of active TGF-␤1
by explanted alveolar macrophages that was maximal 7 d
after Blm administration (3–6). TGF-␤2 and TGF-␤3 from
alveolar macrophages remained unchanged (4). Similar to
our previous findings, alveolar macrophages obtained from
rats 7 d after Blm administration secreted increased quantities
of TGF-␤1, which was 61.7 Ϯ 8.6% in the active form (Figure
1A). Alveolar macrophages from rats that had received
Blm concomitantly with the CD36 peptide 93-110 generated
decreased quantities of active TGF-␤1 while the quantity
of total TGF-␤1 released remained unchanged (Figure 1A).
In this group of rats the percent of active TGF-␤ secreted
was only 2.2 Ϯ 2.1% of the total TGF-␤1 released by these
alveolar macrophages (Figure 1A). We reported previously
that activated alveolar macrophages were the primary source
of the 30-fold increase in total lung TGF-␤ 7 d after Blm
administration (3, 4). In the present study total lung TGF-␤
was not quantitated because alveolar macrophages after
CD36 synthetic peptide and Blm administration release
increased quantities of total TGF-␤ compared with normal
saline-treated rats. It was then unlikely that there would
be a significant change in total lung TGF-␤ after Blm and
CD36 peptide administration. Further, the method for
quantitation of total lung TGF-␤ does not distinguish active
from latent TGF-␤ (3, 4) and the quantitation of active TGF-␤
was more pertinent to the current study. The administration
of a scrambled peptide of the amino acids between 93 and
110 (CD36 s93-110) of CD36 concomitantly with Blm did
not affect the generation of active or L-TGF-␤1 (Figure
1B). In addition, the administration of the control CD36
synthetic peptide 208-224 concomitantly with Blm had no
significant effect on generation of active or L-TGF-␤1 or
the percent of active TGF-␤ secreted by alveolar macro-
phages compared with Blm-treated rats (Figure 1B). The
CD36 peptide 93-110, CD36 s93-110, or 208-224 given alone
(data not shown) or normal saline administration did not
induce TGF-␤1 production and had no effect on the gen-
eration of active or L-TGF-␤1.
General Appearance and Weight of the Rats
Rats that had been given normal saline, or one of the syn-
thetic peptides alone, or no treatment appeared healthy
and gained weight with normal aging as observed during

4. Yehualaeshet, O’Connor, Begleiter, et al.: CD36 Peptide Inhibits Pulmonary Fibrosis 207
the course of the experiments (Figure 2) and no rats from
these groups died. Rats that had received Blm looked gen-
erally unwell, characterized by poor ambulation and activity
in the cage. In addition, these rats lost considerable amounts
of weight (Figure 2). The death rate in Blm-treated rats
was approximately 11 to 13%. However, rats that had re-
ceived both Blm and the CD36 synthetic peptide 93-110 con-
comitantly looked generally better, were more active in
their cages, did not lose as much weight, and had a more
prompt weight gain (Figure 2). There were no deaths in
this group. Rats that had received the CD36 peptide 208-
224 concomitantly with Blm had the characteristics of rats
treated with Blm alone, as described earlier (data not
shown). However, rats that had received Blm concomitantly
with the scrambled peptide of CD36 between amino acids 93
and 110 lost weight in excess of rats treated with Blm alone
(data not shown). It is possible that rats treated with Blm
alone or those treated with Blm and the scrambled peptide
would have had a greater weight loss than demonstrated in
Figure 2. However, to comply with the CCAC these rats
were force-fed pureed, high-calorie food and hydrated
with intraperitoneal injections of normal saline when weight
loss of Ͼ 10% from the previous day was observed.
Differential Cell Count in BAL Fluid
Normally the cells retrieved by BAL contain greater than
95% macrophages while PMNs, lymphocytes, and other
cells make up the rest of the cell population (1, 4). After
Blm-induced lung injury there is an increase in not only
PMNs but also lymphocytes, basophils, mast cells, and other
cells (1, 4). The differential cell count in BAL fluid is in
keeping with previous findings in rats treated with normal
saline or the CD36 synthetic peptides where macrophages
were predominantly present while PMNs and lymphocytes
were less in number (Figure 3). After Blm administration
the percent and absolute numbers of PMNs were increased.
However, when the CD36 synthetic peptide 93-110 was
administered with Blm the percent and absolute numbers
of PMNs decreased while the percent and absolute numbers
of macrophages increased (Figure 3). The total number of
Figure 1. Quantity of TGF-␤ secreted by explanted alveolar mac-
rophages obtained from rats after a number of intratracheal
treatments. The quantity of TGF-␤ present in neutral condi-
tioned medial (CM) (filled bars) represents TGF-␤ in an already
active form. TGF-␤ in acidified, then neutralized CM (striped
bars) represents total TGF-␤ of the same sample. The percent of
active TGF-␤ (open bars) in each sample is derived by using the
TGF-␤ content in neutral CM as the numerator and total TGF-␤
as the denominator. (A) The rats were given intratracheally nor-
mal saline, 1 ␮g of Blm, or 1 ␮g of Blm and 1,600 ␮g of the CD36
synthetic peptide 93-110 (CD36 93-110). The quantity of active
TGF-␤ (filled bars) after administration of normal saline com-
pared with that after Blm treatment had a P value р 0.01. The
quantity of total TGF-␤ (striped bars) between these groups had
a P value р 0.02 while the percent active TGF-␤ (open bars) in
these groups had a P value of р 0.0009 (ANOVA). The quantity
of active TGF-␤ (filled bars) after administration of Blm com-
pared with Blm plus CD36 93-110 had a P value р 0.01
(ANOVA), whereas the comparison of percent active TGF-␤
(open bars) between these two groups had a P value р 0.0009
(ANOVA). The quantity of total TGF-␤ (striped bars) after ad-
ministration of Blm compared with Blm plus CD36 93-110 was
not significant (ANOVA). (B) Rats were given intratracheal
Blm, Blm plus the scrambled peptide of CD36 synthetic peptide
93-110 (CD36 s93-110), or Blm and the CD36 synthetic peptide
208-224 (CD36 208-224). Compared with Blm administration
alone, the administration of CD36 s93-110 or CD36 208-224 did
not significantly affect the quantity of active (filled bars), total
(striped bars), or percent active (open bars) TGF-␤ (ANOVA).
All data are the means of experiments done on six to eight rats.
Figure 2. Changes in weight of rats after intratracheal treatment.
Percent changes in weight of rats from baseline after 500 ␮l of
normal saline administration (open circles), 1,600 ␮g of the syn-
thetic peptide 93-110 (filled triangles), 1 ␮g of Blm (filled circles),
and 1 ␮g of Blm plus 1,600 ␮g of the CD36 synthetic peptide 93-
110 (filled squares). The changes in weight between the Blm-
treated or Blm plus CD36 synthetic peptide 93-110–treated
groups compared with normal saline or CD36 synthetic peptide
93-110 alone was statistically significant (P value р 0.0001) at all
time points except Day 1. The changes in weight between the
Blm-treated group compared with that of the Blm plus CD36
synthetic peptide 93-110–treated group was statistically signifi-
cant (P values between 0.003 and 0.0001) at all time points except
Day 1. No statistical difference was observed among groups
treated with normal saline or CD36 synthetic peptide 93-110
(ANOVA).

5. 208 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 23 2000
inflammatory cells after the use of Blm concomitantly with
CD36 synthetic peptide 93–110 was 6.3 ϫ 106
Ϯ 1.2. However,
when Blm alone was used the number of cells retrieved was
8.4 ϫ 106
Ϯ 0.1 (P value for these comparisons is р 0.01).
Histologic Changes
No inflammatory or fibrotic lesions were observed at any
time interval in lungs of rats that had received normal sa-
line or the CD36 synthetic peptides 93-110 (not shown). In
agreement with numerous previous reports (3–6), rats that
had received Blm 14 d earlier had patchy areas of inflam-
mation and fibrosis in several lobes but the most pro-
nounced lesions were in the right lower lobe (Figure 4A).
The patchy lesions were characterized by an increase in in-
terstitial and alveolar inflammatory cells as well as the
presence of interstitial fibroblasts and early granulation
tissue with extensive staining, using Mason’s Trichrome
for collagen and Alcian blue for proteogylcan (Figures 4B
and 4C). The extent of staining with Alcian blue and Ma-
son’s Trichrome in Figures 4B and 4C is representative of
grade ϩ3 (Table 1). However, 14 d after Blm and CD36
synthetic peptide 93-110 administration there was evi-
dence of less inflammation and fibrosis, inasmuch as the
size of lesions was reduced and occupied a smaller volume
of the overall section (Table 1; Figures 4D–4F). In addi-
tion, the number of cells retrieved by BAL was lower 7 d
after administration of Blm and the CD36 synthetic pep-
tide, suggesting that there is less inflammation when the
peptide is administered. After Blm and concomitant CD36
synthetic peptide 93-110 administration, the presence of
Alcian blue and Mason’s Trichrome within the lesion was
minimal and is representative of a grade ϩ1 (Figures 4D
and 4E; Table 1).
Expression of Connective Tissue Proteins
After a single intratracheal dose of Blm there is increased
expression of collagen I and III, decorin, fibronectin, and a
variety of other connective tissue proteins (16). Total pro-
tein extracted from lungs of rats treated with Blm was 2-fold
higher than protein obtained from untreated rats, or from
those treated with normal saline or one of the CD36 syn-
thetic peptides alone. This increase in protein extracted is
expected to represent not only the aforementioned con-
nective tissue proteins but also the protein content of in-
flammatory cells and influx of proteins from the circula-
tion. However, the total protein content of lungs from rats
that had received Blm concomitantly with the CD36 syn-
thetic peptide 93-110 was approximately 50% less than
that obtained when Blm alone was administered. It should
be noted that to comply with the CCAC’s requirement to
use as few rats as possible, the entire time course was not
done for experiments used to determine the effects of the
CD36 synthetic peptide on connective tissue synthesis af-
ter Blm-induced lung injury. Instead, the changes in ex-
pression of procollagen I and III and fibronectin were
done using a time course where rats were killed at regular
time intervals consisting of 4, 7, 14, 21, and 28 d after Blm
or normal saline administration (data not shown). Procol-
lagen III and fibronectin were maximally expressed 7 d af-
ter Blm treatment, whereas procollagen I was increased in
expression 7 and 14 d after Blm administration (data not
shown). To determine the effects of CD36 synethetic pep-
tide 93-110 on collagen III and fibronectin expression, rats
were killed at 4 and 7 d. To detect changes in collagen I
synthesis, rats were killed 7 and 14 d after Blm and CD36
synthetic peptide 93-110 administration (Figure 5A). Fur-
ther, rats that had received Blm concomitantly with scram-
bled peptide CD36 amino acids 93-110 demonstrated ex-
treme morbidity, and experiments on these rats were
abbreviated. The expression of procollagen I was reduced
(Figure 5A) in rats treated with Blm and the CD36 syn-
thetic peptide 93-110 compared with rats that had received
Blm alone or those that had received Blm and the scram-
bled peptide of CD36 93-110 or CD36 peptide 208-224
(Figure 5A). The expression of procollagen III and fi-
bronectin was reduced in rats that received Blm and the
CD36 peptide 93-110 (Figures 5B and 5C).
Cytotoxicity of Blm when Combined with the CD36
Synthetic Peptide 93-110
The intratracheal administration of Blm results in a ran-
dom distribution of the drug (1, 3). The alveolar epithelial
and endothelial cell injury that follows is in the areas of
Figure 3. Differential cell count retrieved by BAL 7 d after in-
tratracheal treatment. (A) Percent of total cells that were identi-
fied as macrophages (open bars), lymphocytes (striped bars), and
PMNs (filled bars). (B) Absolute number of cells ϫ 106
cells iden-
tified as macrophages (open bars), lymphocytes (striped bars),
and PMNs (filled bars). The percent and absolute number of
macrophages after normal saline treatment compared with Blm
treatment has a P value р 0.0005. However, there is no statistical
difference in the percent or absolute number of macrophages af-
ter the normal saline treatment compared with Blm plus CD36
93-110. The percent and absolute number of PMNs after normal
saline treatment compared with Blm or Blm plus CD36 93-110
has a P value р 0.0001. The percent and absolute number of
PMNs after Blm treatment compared with Blm plus CD36 93-110
has a P value р 0.001. The percent and absolute numbers of lym-
phocytes were not statistically significant in any comparison
among the groups (ANOVA). The data are the means of experi-
ments done on six rats.

6. Yehualaeshet, O’Connor, Begleiter, et al.: CD36 Peptide Inhibits Pulmonary Fibrosis 209
Blm deposition (17). When the Blm was combined in the
same syringe with the CD36 synthetic peptide, the deposi-
tion of both substances was likely to be in the same distri-
bution. The combination of Blm and the CD36 synthetic
peptide 93-110 did not affect the potency of Blm toxicity
to Blm-sensitive LY/5178Y lymphoma cells (Figure 6). For
this reason the initial in vivo Blm injury to the alveolar ep-
ithelial and endothelial cells is not likely to be affected
when Blm and the CD36 synthetic peptide 93-110 are ad-
ministered together. It is of note that the administration of
Blm concomitantly with the CD36 synthetic peptide 93-
110 did not totally abrogate the generation of active TGF-
␤1, inflammation, or connective tissue synthesis. There
was, however, a reduction in all these parameters, which
supports the probability that Blm administration leads to
pulmonary toxicity that can be ameliorated by the effects
of the CD36 synthetic peptide 93-110.
Discussion
The presence of CD36 synthetic peptide 93-110 in cultures
of alveolar macrophages obtained after Blm-induced lung
injury prevents the conversion of L-TGF-␤1 to active
TGF-␤1 (6). The present report is the first to describe that
when the same CD36 synthetic peptide is administered to
rats with Blm compared with administration of Blm alone,
there is a reduction of inflammation and connective tissue
synthesis induced by Blm. The administration of the CD36
synthetic peptide 93-110 with Blm results in decreased se-
cretion of active TGF-␤1 by explanted alveolar macrophages.
It is of note that after Blm-induced lung injury the overex-
pression of TGF-␤1 is seen almost exclusively in alveolar
macrophages (3). No TGF-␤1 was observed in alveolar epi-
thelial cells or interstitial inflammatory cells (3). It then
follows that in vivo the effect of the CD36 synthetic peptide
93-110 most likely inhibits the activation of alveolar mac-
rophage–derived L-TGF-␤1. The reduction of active TGF-␤1
from alveolar macrophages may then lead to amelioration
of Blm-induced inflammation and connective tissue synthesis.
The mechanisms by which the in vitro presence of (6)
or in vivo administration of the CD36 synthetic peptide
93-110 with Blm diminishes the release of active TGF-␤ by
explanted alveolar macrophages may be similar. It had
Figure 4. Histologic changes in rat lungs 14 d after intratracheal treatment. After administration of 1 ␮g of Blm, paraffin-embedded
lung sections were stained with: (A) H&E for histology; arrow is in area of fibroconnective tissue (original magnification: ϫ4); (B) Al-
cian blue for proteoglycan distribution; arrow is in an area of proteoglycan expression and is in the same region as identified by an arrow
in A (original magnification: ϫ25); and (C) Mason’s Trichrome for collagen distribution; arrow is in the area of fibroconnective tissue
(original magnification: ϫ25). After administration of 1 ␮g of Blm plus the CD36 synthetic peptide 93-110 (1,600 ␮g), paraffin-embed-
ded lung sections were stained with: (D) H&E; arrow is located at one of the areas of inflammation and fibrosis (original magnification:
ϫ4); (E) Alcian blue; arrow is in the area of proteoglycan expression identified by an arrow in D (original magnification: ϫ25); and (F)
Mason’s Trichrome; arrow is in an area with fibroconnective tissue identified by an arrow in D (original magnification: ϫ25). The histol-
ogy demonstrated is representative of three rats per group.
TABLE 1
Histologic changes after intratracheal treatment
Intratracheal Treatment
Percent of Lung
Involved in
Inflammation
and Fibrosis
Extent of Staining with:*
Mason’s
Trichrome Alcian Blue
Blm (1 ␮g) 40.5 Ϯ 2.2 ϩ2.9 Ϯ 0.01 ϩ3 Ϯ 0
Blm (1 ␮g) plus CD36
synthetic peptide
93-110 (1,600 ␮g) 8.0 Ϯ 2.4 ϩ0.9 Ϯ 0.4 ϩ0.8 Ϯ 0.4
*0: no staining, ϩ1: detectable color of the Mason’s Trichrome or Alcian
blue, ϩ2: staining between ϩ1 and ϩ2, and ϩ3: extensive staining with Mason’s
Trichrome and Alcian blue within the lesion. The percent of lung involved after
Blm compared with Blm plus CD36 synthetic peptide 93-110 has a P value of
р 0.002. The extent of staining with Mason’s Trichrome or Alcian blue after
treatment compared with Blm plus CD36 synthetic peptide 93-110 has a P value
р 0.001. The statistical analysis for this data was done using Wilcoxon’s rank
signed nonparametric statistical test.

7. 210 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 23 2000
previously been demonstrated that the amino acid se-
quence CSVTCG of TSP-1 interacts with the CD36 in the
region of the amino acids 93 to 110 of CD36 (18). The CS-
VTCG region in the type 1 repeats of TSP-1 is also important
for the release of active TGF-␤1 by alveolar macrophages
because the addition of the synthetic TSP-1 peptide CSVTCG
to cultures of activated alveolar macrophages inhibits the
activation of L-TGF-␤1 (N. Khalil and colleagues, unpub-
lished data). It has been demonstrated that the interaction
of the various domains of TSP-1, with the ligands may de-
pend on the conformational state of the TSP-1 molecule
(19). When TSP-1 is soluble the sequence CSTVCG,
present in two sites within the type 1 repeats of TSP-1, is
more exposed than when TSP-1 is associated with a cell
surface or matrix proteins (19). We demonstrated previ-
ously that alveolar macrophages generate TSP-1/L-TGF-␤1
complexes after in vivo Blm injury (6). It is conceivable
that when the CD36 synthetic peptide 93-110 is present ei-
ther in vitro or in vivo, the peptide associates with the CS-
VTCG region of TSP-1 in the TSP-1/L-TGF-␤1 complex.
The association of the CD36 synthetic peptide 93-110 with
the CSVTCG region in TSP-1 of the TSP-1/L-TGF-␤1
complex may interfere with the TSP-1/L-TGF-␤1 interac-
tion with the CD36 receptor on the surface of the alveolar
macrophage (6). Because the association of the TSP-1/L-
TGF-␤1 with the alveolar macrophage CD36 receptor is
critical to the activation of the L-TGF-␤1 by plasmin (5,
6), prevention of this association may diminish the activa-
tion of L-TGF-␤1 in vivo.
The alveoli normally contain greater than 95% mac-
rophages. After Blm administration the number of mac-
rophages remains the same but decreases in percentage
due to an increase in the number of PMNs (1, 4). How-
ever, when the CD36 peptide 93-110 was administered
with Blm, there was a reduction in numbers of PMNs so
that an increase in the percentage of alveolar macrophages
was observed. The reduction in numbers of PMNs could
be due to a number of reasons. TGF-␤1 is a potent
chemoattractant of PMNs (20), and when the CD36 syn-
thetic peptide 93-110 was administered the decrease in ac-
tive TGF-␤1 in the alveolar space could lead to a reduc-
tion in PMN recruitment to the alveoli. The interaction of
TSP-1 with CD36 could also be important in areas of in-
jury in recruitment and activation of PMNs (21, 22). Blm
administration injures endothelial cells (17), and TSP-1
that is released by injured endothelial cells (21, 22) binds
to the same cells, where it functions to recruit PMNs and
localize the PMNs to the endothelial cells and activates
PMNs on endothelial cells to release reactive oxygen in-
termediates (21, 22). Because Blm injures the endothelial
cells (17), this role of TSP-1–mediated recruitment of
PMNs could contribute to lung injury induced by Blm ad-
ministration. The binding of TSP-1 to endothelial cells is
mediated by the association of TSP-1 to CD36, which is lo-
cated on the endothelial cells (23). It is then conceivable
that the presence of the CD36 synthetic peptide 93-110
may prevent the association of TSP-1 to endothelial cells,
resulting in reduction in the recruitment of PMNs.
CD36 is an 88-kD membrane glycoprotein that func-
tions as a receptor for TSP-1 collagen and erythrocytes in-
fected with Plasmodium falciparum (24). The interaction
of CD36 with TSP-1 has been described to be important
Figure 5. Expression of connective tissue proteins after a number
of intratracheal treatments detected by Western analysis. (A)
Procollagen I expression 7 and 14 d after Blm administration
(lanes 1 and 2, respectively), Blm plus CD36 synthetic peptide 93-
110 (CD36 93-110) (lanes 3 and 4, respectively), Blm plus the
scrambled peptide of CD36 amino acids from 93-110 (s93-110)
(lanes 5 and 6, respectively), and Blm plus the CD36 synthetic
peptide 208-224 (lanes 7 and 8, respectively). The numbers 7 and
14 at the top denote the number of days after treatment. (B) Pro-
collagen III expression 4 and 7 d after Blm administration in the
absence of CD36 synthetic peptide 93-110 (left and middle lanes,
respectively) and concomitantly with CD36 synthetic peptide 93-
110 (right lane). The numbers 4 and 7 at the top denote the num-
ber of days after treatment. (C) Fibronectin expression 4 and 7 d
after Blm administration in the absence of CD36 synthetic pep-
tide 93-110 (left and middle lanes, respectively) and concomi-
tantly with CD36 synthetic peptide 93-110 (right lane). The num-
bers 4 and 7 at the top denote the number of days after treatment.
The numbers on the left in B and C denote molecular weights of
the bands (in kD). The immunoblots are representative of exper-
iments done on three or four separate rats.
Figure 6. Survival of LY/5178Y
lymphoma cells determined by
a clonogenic assay was done
in the presence of 500 ␮l nor-
mal saline (open bar), 1,600 ␮g
CD36 synthetic peptide 93-110/
500 ␮l normal saline (striped
bar), 1 ␮g Blm/500 ␮l normal
saline (dark-striped bar), or 1 ␮g
Blm/500 ␮l normal saline plus
1,600 ␮g of the CD36 synthetic
peptide 93-110 (filled bar). The
cytotoxicity of Blm or Blm plus
CD36 synthetic peptide 93-110 compared with that of normal
saline or CD36 synthetic peptide 93-110 alone had a P value of
р 0.001 (ANOVA). The cytotoxicity of Blm compared with that
of Blm plus CD36 synthetic peptide 93-110 was not statistically
significant. The data represent the mean of six separate experi-
ments.

8. Yehualaeshet, O’Connor, Begleiter, et al.: CD36 Peptide Inhibits Pulmonary Fibrosis 211
for a number of functions such as platelet aggregation,
platelet-monocyte adhesion, platelet tumor cell adhesion,
and macrophage uptake of apoptotic cells (24). Of these
functions relevant to Blm lung injury are the observations
that platelet aggregation has been reported to occur in
early wounding (25) and is important in Blm-induced in-
jury and inflammation (26). Further, platelet-aggregation
(27) or platelet monocyte adhesion may result in activa-
tion and release of cytokines (27). The presence of the
CD36 synthetic peptide 93-110 in vitro prevents platelet
aggregation (14). In vivo, the presence of the CD36 syn-
thetic peptide 93-110 could result in inhibition of platelet
aggregation, platelet-monocyte adhesion, and release of
cytokines, and thus less inflammation and fibrosis. These
effects of the CD36 synthetic peptide 93-110 could also
contribute to the amelioration of Blm-induced inflammation
and fibrosis.
Over the past few years a number of treatments, such as
anti–TNF-␣ antibodies (28), antioxidants (29), interferon-␥
(30), and pirfenidone (31), have been demonstrated to ame-
liorate the Blm-induced pulmonary inflammation and fi-
brosis. Nonetheless, Giri and associates confirmed the im-
portance of TGF-␤1 in the pathogenesis of Blm-induced
inflammation and fibrosis in mice when they demonstrated
that administration of TGF-␤1 antibodies and Blm resulted
in less lung injury and fibrosis (32). However, the location
of the neutralizing effects of the anti–TGF-␤1 antibodies
was not apparent from Giri and coworkers’ study (32). On
the basis of the current work and that of others, the admin-
istration of TGF-␤1 antibodies could have neutralized the
TGF-␤1 generated by endothelial cells (33) and/or alveolar
epithelial cells (13), as well as alveolar macrophages (3–6).
Our work has shown that the generation of plasmin in
addition to the cellular localization of L-TGF-␤1 (5, 6), is
important for the activation of alveolar macrophage–derived
L-TGF-␤1. However, there are other mechanisms of acti-
vation of L-TGF-␤1 that are independent of proteases. For
example, reactive oxygen intermediates (11), hyperglycemia
(11), and, in some but not all circumstances (34), TSP-1
can activate L-TGF-␤1 in the absence of proteases (12). In
addition, Munger and associates (13) described another
protease-independent activation of L-TGF-␤1 that may be
important in Blm-induced lung injury (13). It was observed
that the RGD (arginine–glycine–aspartic acid) sequence
in the LAP interacts with the integrin ␣v␤6 on alveolar epi-
thelial cells (13), leading to conformational changes of the
L-TGF-␤1 and exposing the site on TGF-␤1 that interacts
with the TGF-␤ receptor type II (13). It is not known
whether Blm administration to rats, a different species than
mice, induces ␣v␤6 on alveolar epithelial cells and is there-
fore important in the activation of L-TGF-␤1. It is also not
known whether Blm injury induces CD36 expression on
rat alveolar epithelial cells that subsequently binds the TSP-
1/L-TGF-␤1 complex and results in plasmin-mediated activa-
tion of L-TGF-␤1. Regardless of the expression of ␣v␤6 or
CD36 on rat epithelial structures, we have demonstrated
that the reduction of alveolar macrophage–derived active
TGF-␤1 or the potential for interruption of the CD36/
TSP-1 interactions is associated with a decrease in inflam-
mation and fibrosis after Blm-induced lung injury. Collec-
tively, our findings not only suggest a novel in vivo mecha-
nism of activation of L-TGF-␤1 but also support the
importance of alveolar macrophage–derived active TGF-
␤1 in pulmonary fibrosis.
Acknowledgments: The work in this manuscript was supported by a grant from
the Medical Research Council of Canada to one author (N.K.). The authors
thank Dr. Arnold Greenberg for reviewing the manuscript, Dr. Bob Tate for
the statistical analysis, Mrs. Stephanie Moorehouse for her technical assistance,
and Ms. Carolin Hoette for typing the manuscript.
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