Am. J. Respir. Cell Mol. Biol. Vol. 23, pp. 204–212, 2000Internet 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)-

b

1 is an important regula-tor of inflammation and fibrosis. TGF-

b

1 is usually secreted asa biologically latent protein called latent TGF-

b

1 (L-TGF-

b

1).L-TGF-

b

1 has no biologic effect unless L-TGF-

b

1 is convertedto 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-

b

was maximally increased. This induction wasdue to TGF-

b

1 production by alveolar macrophages that,when explanted, generated increased quantities of L-TGF-

b

1complexed with the glycoprotein thrombospondin (TSP)-1.The TSP-1/L-TGF-

b

1 complex was associated with CD36, a re-ceptor for TSP-1. The association of TSP-1/L-TGF-

b

1 to CD36was critical for plasmin-mediated release of mature TGF-

b

1. Inthis paper we show that, compared with administration ofBlm by itself, when a synthetic peptide of CD36 betweenamino acids 93 and 110 is given concomitantly with Blm torats, alveolar macrophages generate markedly less active TGF-

b

1,the rats gain weight more rapidly, and there is less inflammation,collagen I and III, and fibronectin synthesis. These findingsdemonstrate a novel

in vivo

mechanism of activation of L-TGF-

b

1in lung injury and the importance of alveolar macrophage–derived active TGF-

b

1 in the pathogenesis of pulmonary in-flammation and fibrosis.

Before an increase in connective tissue synthesis after lunginjury, there is an influx of inflammatory cells that arepresent not only in the alveoli but also in the interstitium(1). In inflammatory infiltrates the alveolar macrophagesare activated to produce a number of proinflammatoryand fibrogenic cytokines (2) such as transforming growthfactor (TGF)-

b

(3–6), platelet-derived growth factor (7),insulin growth factor-1 (7), tumor necrosis factor (TNF)-

a

,interleukin (IL)-1, and IL-5 (4, 5, 8). Of these cytokines,TGF-

b

1 has been demonstrated to regulate not only itselfbut also a variety of fibrogenic cytokines (9) that ultimatelyare important in the pathogenesis of inflammation andconnective tissue synthesis (2–8).

Although TGF-

b

exists in three isoforms in mammals,it is the TGF-

b

1 isoform that has been most commonly as-sociated with disorders characterized by inflammation andfibrosis (10). All the TGF-

b

s are initially synthesized as

large precursors that are 390 to 414 amino acids in size(11). The intracellular and extracellular processing of theTGF-

b

1 isoform has been most extensively studied. Be-fore the secretion of the TGF-

b

1, the intracellular pro-tease furin cleaves the amino terminal end of the precur-sor between amino acids 278 and 279 (11), resulting in anamino terminal peptide called the latency-associated pep-tide (LAP) and a carboxy terminal protein called the ma-ture TGF-

b

1. However, the cleaved 75-kD amino terminalportion of the protein noncovalently associates with the25-kD carboxy terminal portion of the protein (11). Whenthe carboxy terminal TGF-

b

1 peptide is secreted as a com-plex with the LAP it is called latent TGF-

b

1 (L-TGF-

b

1),and in this form L-TGF-

b

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-

b

1 by pH

,

2 or

.

8, heat of 100

8

C (11), chaotropic agents, proteases suchas plasmin (5, 6, 11), cathepsin A and D, endoglycosidase,sialic acid, reactive oxygen species, or high concentrationsof glucose (11). Alternatively, L-TGF-

b

1 can be activatedwithout removing the LAP by interacting with the glyco-protein thrombospondin (TSP)-1 (12) or the integrin

a

v

b

6(13). Although the expression of TGF-

b

1 has been associ-ated with a number of inflammatory and fibrotic diseases,for TGF-

b

1 to be physiologically or pathologically signifi-cant in these disorders it must be present in a biologicallyactive form. The

in vivo

activation of L-TGF-

b

1 in theseinstances is poorly understood.

We have used a well-recognized model of lung injuryinduced by the antineoplastic antibiotic bleomycin (Blm)(3–6) to describe a novel mechanism of activation of L-TGF-

b

1 (4–6). After a single intratracheal dose of Blm, there isinjury to the alveolar epithelium and endothelium, fol-lowed by recruitment and activation of inflammatory cellsbefore 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 intotal lung TGF-

b

content (3–6). When these alveolar mac-rophages were explanted they generated active TGF-

b

1,whereas alveolar macrophages from normal saline-treatedrats or those receiving no treatment secreted either noTGF-

b

1 or small amounts of L-TGF-

b

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

a

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-

b

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 RespiratoryMedicine, 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-

b

1,L-TGF-

b

1; polymorphonuclear leukocyte, PMN; Tris-buffered saline, TBS;transforming growth factor, TGF; thrombospondin, TSP.

Yehualaeshet, O’Connor, Begleiter,

et al.

: CD36 Peptide Inhibits Pulmonary Fibrosis 205

that before the release of active TGF-

b

1 the L-TGF-

b

1that was complexed with the TSP-1 interacted with theTSP-1 receptor CD36 on the alveolar macrophage (6). TheCD36–TSP-1/L-TGF-

b

1 interaction appears critical to theactivation process because in the presence of antibodies toCD36 that prevent the association of TSP-1 to CD36 theactivation of L-TGF-

b

1 was totally abrogated. TSP-1 asso-ciates with CD36 by interaction of TSP-1 with theectodomain of CD36 between amino acids 93 and 110. Inthe presence of a synthetic peptide of the ectodomain ofCD36 that mimics the region between amino acids 93 to 110the activation of L-TGF-

b

1 by explanted alveolar mac-rophages was also inhibited (6).

In this paper we demonstrate that compared with Blmadministration alone the concomitant administration ofBlm and the CD36 synthetic peptide 93-110 significantlyreduces alveolar macrophage secretion of active TGF-

b

1,inflammatory lesions, and collagen I and III and fibronec-tin synthesis. These findings support the importance of bi-ologically active TGF-

b

1 derived from alveolar macrophagesin the pathogenesis of Blm-induced pulmonary inflammationand fibrosis. Further, these findings suggest that a TSP-1–CD36 plasmin–dependent mechanism is involved in theprocess of local activation of alveolar macrophage–derivedL-TGF-

b

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 ofManitoba vivarium. In each experiment, all rats were matchedfor age and weight. In compliance with the Canadian Council onAnimal Care (CCAC), the numbers of rats used were restrictedto numbers that were as minimal as possible to adequately addressthe most relevant issues related to this work.

Reagents

Blm (Blenoxane) was purchased from Bristol-Myers Squibb (Evans-ville, IN). Neutralizing antibodies to TGF-

b

1-3 were obtained fromGenzyme (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), thescrambled 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 andcolleagues (14). The peptides were synthesized with an AppliedBiosystems model 431A peptide synthesizer, using Fmoc (

N

-[9-Fluoreny-

D

-methoxycarbonyl]) chemistry, and were purified byreverse high-performance liquid chromatography using a C18column.

Blm and Synthetic Peptide Administration

This procedure is described in detail in References 3–6. Briefly,rats were given normal saline, 1

m

g of Blm, 1,600

m

g of a CD36synthetic peptide, or 1

m

g of Blm concomitantly with 1,600

m

gof a CD36 synthetic peptide in a total volume of 500

m

l sterilenormal saline. Rats used as controls received 1,600

m

g of theCD36 synthetic peptide 93-110 or a CD36 peptide unrelated tothe site of interaction of CD36 with TSP-1 mimicking the aminoacid 208-224 sequence of the CD36 ectodomain (14). The quan-

tity of 1,600

m

g of peptide used was based on a pilot study demon-strating that quantities less than 1,600

m

g of the peptide did notaffect Blm toxicity in a significant manner. The CD36 syntheticpeptide from amino acids 208 to 224 has previously been demon-strated not to inhibit activation of L-TGF-

b

1 (6). In addition, ascrambled peptide of the CD36 amino acid 93-110 (sequencegiven earlier) was also used. After administration of variousagents, the rats were killed at different time intervals. For someexperiments, 7 d after reagent administration was chosen as thetime to harvest alveolar macrophages, on the basis of our findingsthat alveolar macrophages are maximally stimulated at this timeto secrete active TGF-

b

1 (5). Weights and appearances of ratswere recorded daily after receiving the various treatments.

Histology and Histochemistry

Fourteen days after Blm administration, a time previously reportedto be associated with increased connective tissue synthesis and in-flammation (1, 3), we obtained lungs for histology as previouslydescribed (3). At 24 h after fixation in 10% formalin the lungswere 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. Allslides were blinded and two independent examinations (by au-thors N.K. and R.O.) were done and then collated. For the extentof lung involved, the lung sections were examined under lowpower and revealed the entire lung section that contained bothnormal lung and an inflammatory or fibrotic lesion. The propor-tion of lung section with inflammation was then reported as apercentage of the overall lung section. For grading the extent ofstaining with Mason’s Trichrome or Alcian blue, 0 designated nostaining,

1

1 designated detectable color (green for Mason’sTrichrome and blue for Alcian blue), and

1

2 designated an ex-tent of staining between

1

1 and

1

3. The grade

1

3 was reservedfor those instances where there was extensive staining within aninflammatory and fibrotic lesion.

Differential Cell Count of Cells Obtained by Bronchoalveolar Lavage

Cells obtained by bronchoalveolar lavage (BAL), as previouslydescribed (4–6), were suspended at a concentration of 1

3

10

6

cells/ml and were used in a cytospin preparation (4). The cellswere stained using Diff-Quik (Baxter Healthcare Corp., Miami,FL) fixative and nuclear/cytoplasmic stains (4). Five fields at highpower 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 macrophageswere maintained in serum-free media containing Gentamicin (4 mg/100 mls; Roussel, Montreal, PQ, Canada), Fungizone (100

m

1/100 mls; GIBCO BRL, London, ON, Canada), and 0.2% clottedbovine calf plasma (BCP) (National Biological Laboratory Lim-ited, Dugald, MB, Canada). After 20 h of incubation at 37

8

C, 5%CO

2

, the media were collected in the presence of protease inhibi-tors—leupeptin, 0.5

m

g/ml, from Amersham, Buckinghamshire, UK;and aprotinin and pepstatin, 1

m

g/ml each, both from Sigma(Oakville, ON, Canada)—frozen at

2

80

8

C until ready for TGF-

b

quantitation (4–6).

CCL-64 Mink Lung Epithelial Growth Inhibition Assayfor TGF-

b

The CCL-64 growth inhibition assay to identify and quantitateTGF-

b

has been described elsewhere (3–6). Briefly, neutral con-

206

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 23 2000

ditioned media or conditioned media that were acidified andsubsequently neutralized were added to subconfluent cells in

a

–Eagle’s minimum essential medium, 0.2% BCP, 10 mM

N

-2-hy-droxyethylpiperazine-

N

9

-ethane sulfonic acid at pH 7.4, and peni-cillin (25

m

g/ml) and streptomycin (25

m

g/ml), and cultured as 5

3

10

4

cells/0.5 ml in 24-well Costar dishes (Flow Laboratories, Inc.;Mississauga, ON, Canada). After 22 h the cells were pulsed with0.25

m

Ci of 5-[

125

I]iodo 2

9

-deoxyuridine [

125

I]UdR (ICN Pharma-ceutical, Costa Mesa, CA) for 2 to 3 h at 37

8

C, then lysed with 1 mlof 1 N NaOH for 30 min at room temperature. The [

125

I]UdR wasthen counted in a

g

counter (LKB Instruments, Gaithersburg,MD). A standard curve using porcine TGF-

b

1 was included in eachassay. For confirmation of TGF-

b

activity, neutralizing mono-clonal antibody against TGF-

b

1-3 (Genzyme) was added before theaddition of the conditioned media (3–6) and resulted in abrogationof all TGF-

b

activity.

Protein Extraction for Western Analysis

Untreated rats and rats treated with various agents were killedand the peripheral blood, heart, and blood vessels were removedas described earlier (3–6). The lungs were snap-frozen on dry icewith ethanol and stored at

2

80

8

C until protein extraction. Whole-lung protein extraction was performed as described previously (6).Briefly, the frozen lungs were pulverized in a chilled mortar andplaced in tissue lysis buffer containing 1 mM phenylmethylsulfonylfluoride (Sigma). The samples were further homogenized in thepresence of 0.5% Triton X-100, then centrifuged at 200 mg for 10 minat 4

8

C. 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

m

l) were electrophoresed on a 10% so-dium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis ina Mini-PROTEAN II Electrophoresis Cell (Bio-Rad). Proteinmolecular weight markers (Amersham) were run parallel to eachblot as an indicator of the molecular weight. Equal loading ofprotein 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 chamberwith 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). Fordetection of procollagen I and III a 1:300 dilution of antibody wasused, 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 thesecondary antibody (goat antimouse immunoglobulin G; Bio-Rad)as recommended by the manufacturer. Finally the washed blotswere exposed to an enhanced chemiluminescence (ECL) detectionsystem (Amersham) and recorded on an autoradiograph (KodakX-Omat film). Before reprobing, the nitrocellulose membranewas incubated at 50

8

C for 30 min with a stripping buffer (100mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl, pH6.7). The blots were rinsed twice with TBS. To ensure the re-moval of antibodies, membranes were incubated with the ECLdetection 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 IIIand fibronectin.

Cytotoxicity AssayLY/5178Y mouse lymphoma cells were incubated with normalsaline, Blm (1 mg/500 ml of normal saline), CD36 synthetic pep-tide 93-110 (1,600 mg/500 ml of normal saline), or Blm (1 mg /500 ml

of normal saline) plus CD36 synthetic peptide 93-110 (1,600 mg)for 1 h at 378C in media containing horse serum. The cytotoxic ac-tivity of the agents on the LY/5178Y cells was determined by asoft agar cloning assay and was expressed as the surviving cellfraction as described previously (15). Cloning efficiencies rangedfrom 35 to 65%. The cytotoxicities of the different treatments inthe cell line were compared statistically by analysis of variance(ANOVA).

Statistical AnalysisStatistical analysis using ANOVA or Wilcoxon’s rank signed non-parametric statistical test was done by Dr. Bob Tate, BiostatisticalUnit, University of Manitoba.

ResultsPost-Translational Activation of AlveolarMacrophage–Derived L-TGF-b1

Our previous work demonstrated that a single intratra-cheal dose of Blm resulted in generation of active TGF-b1by explanted alveolar macrophages that was maximal 7 dafter Blm administration (3–6). TGF-b2 and TGF-b3 fromalveolar macrophages remained unchanged (4). Similar toour previous findings, alveolar macrophages obtained fromrats 7 d after Blm administration secreted increased quantitiesof TGF-b1, which was 61.7 6 8.6% in the active form (Figure1A). Alveolar macrophages from rats that had receivedBlm concomitantly with the CD36 peptide 93-110 generateddecreased quantities of active TGF-b1 while the quantityof total TGF-b1 released remained unchanged (Figure 1A).In this group of rats the percent of active TGF-b secretedwas only 2.2 6 2.1% of the total TGF-b1 released by thesealveolar macrophages (Figure 1A). We reported previouslythat activated alveolar macrophages were the primary sourceof the 30-fold increase in total lung TGF-b 7 d after Blmadministration (3, 4). In the present study total lung TGF-bwas not quantitated because alveolar macrophages afterCD36 synthetic peptide and Blm administration releaseincreased quantities of total TGF-b compared with normalsaline-treated rats. It was then unlikely that there wouldbe a significant change in total lung TGF-b after Blm andCD36 peptide administration. Further, the method forquantitation of total lung TGF-b does not distinguish activefrom latent TGF-b (3, 4) and the quantitation of active TGF-bwas more pertinent to the current study. The administrationof a scrambled peptide of the amino acids between 93 and110 (CD36 s93-110) of CD36 concomitantly with Blm didnot affect the generation of active or L-TGF-b1 (Figure1B). In addition, the administration of the control CD36synthetic peptide 208-224 concomitantly with Blm had nosignificant effect on generation of active or L-TGF-b1 orthe percent of active TGF-b secreted by alveolar macro-phages compared with Blm-treated rats (Figure 1B). TheCD36 peptide 93-110, CD36 s93-110, or 208-224 given alone(data not shown) or normal saline administration did notinduce TGF-b1 production and had no effect on the gen-eration of active or L-TGF-b1.

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 healthyand gained weight with normal aging as observed during

Yehualaeshet, O’Connor, Begleiter, et al.: CD36 Peptide Inhibits Pulmonary Fibrosis 207

the course of the experiments (Figure 2) and no rats fromthese groups died. Rats that had received Blm looked gen-erally unwell, characterized by poor ambulation and activityin the cage. In addition, these rats lost considerable amountsof weight (Figure 2). The death rate in Blm-treated ratswas 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 intheir cages, did not lose as much weight, and had a moreprompt 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 ratstreated with Blm alone, as described earlier (data notshown). However, rats that had received Blm concomitantlywith the scrambled peptide of CD36 between amino acids 93and 110 lost weight in excess of rats treated with Blm alone(data not shown). It is possible that rats treated with Blmalone or those treated with Blm and the scrambled peptidewould have had a greater weight loss than demonstrated inFigure 2. However, to comply with the CCAC these ratswere force-fed pureed, high-calorie food and hydratedwith intraperitoneal injections of normal saline when weightloss of . 10% from the previous day was observed.

Differential Cell Count in BAL Fluid

Normally the cells retrieved by BAL contain greater than95% macrophages while PMNs, lymphocytes, and othercells make up the rest of the cell population (1, 4). AfterBlm-induced lung injury there is an increase in not onlyPMNs but also lymphocytes, basophils, mast cells, and othercells (1, 4). The differential cell count in BAL fluid is inkeeping with previous findings in rats treated with normalsaline or the CD36 synthetic peptides where macrophageswere predominantly present while PMNs and lymphocyteswere less in number (Figure 3). After Blm administrationthe percent and absolute numbers of PMNs were increased.However, when the CD36 synthetic peptide 93-110 wasadministered with Blm the percent and absolute numbersof PMNs decreased while the percent and absolute numbersof macrophages increased (Figure 3). The total number of

Figure 1. Quantity of TGF-b secreted by explanted alveolar mac-rophages obtained from rats after a number of intratrachealtreatments. The quantity of TGF-b present in neutral condi-tioned medial (CM) (filled bars) represents TGF-b in an alreadyactive form. TGF-b in acidified, then neutralized CM (stripedbars) represents total TGF-b of the same sample. The percent ofactive TGF-b (open bars) in each sample is derived by using theTGF-b content in neutral CM as the numerator and total TGF-bas the denominator. (A) The rats were given intratracheally nor-mal saline, 1 mg of Blm, or 1 mg of Blm and 1,600 mg of the CD36synthetic peptide 93-110 (CD36 93-110). The quantity of activeTGF-b (filled bars) after administration of normal saline com-pared with that after Blm treatment had a P value < 0.01. Thequantity of total TGF-b (striped bars) between these groups hada P value < 0.02 while the percent active TGF-b (open bars) inthese groups had a P value of < 0.0009 (ANOVA). The quantityof active TGF-b (filled bars) after administration of Blm com-pared with Blm plus CD36 93-110 had a P value < 0.01(ANOVA), whereas the comparison of percent active TGF-b(open bars) between these two groups had a P value < 0.0009(ANOVA). The quantity of total TGF-b (striped bars) after ad-ministration of Blm compared with Blm plus CD36 93-110 wasnot significant (ANOVA). (B) Rats were given intratrachealBlm, Blm plus the scrambled peptide of CD36 synthetic peptide93-110 (CD36 s93-110), or Blm and the CD36 synthetic peptide208-224 (CD36 208-224). Compared with Blm administrationalone, the administration of CD36 s93-110 or CD36 208-224 didnot significantly affect the quantity of active (filled bars), total(striped bars), or percent active (open bars) TGF-b (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 ml ofnormal saline administration (open circles), 1,600 mg of the syn-thetic peptide 93-110 (filled triangles), 1 mg of Blm (filled circles),and 1 mg of Blm plus 1,600 mg 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–treatedgroups compared with normal saline or CD36 synthetic peptide93-110 alone was statistically significant (P value < 0.0001) at alltime points except Day 1. The changes in weight between theBlm-treated group compared with that of the Blm plus CD36synthetic peptide 93-110–treated group was statistically signifi-cant (P values between 0.003 and 0.0001) at all time points exceptDay 1. No statistical difference was observed among groupstreated with normal saline or CD36 synthetic peptide 93-110(ANOVA).

208 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 23 2000

inflammatory cells after the use of Blm concomitantly withCD36 synthetic peptide 93–110 was 6.3 3 106 6 1.2. However,when Blm alone was used the number of cells retrieved was8.4 3 106 6 0.1 (P value for these comparisons is < 0.01).

Histologic Changes

No inflammatory or fibrotic lesions were observed at anytime interval in lungs of rats that had received normal sa-line or the CD36 synthetic peptides 93-110 (not shown). Inagreement with numerous previous reports (3–6), rats thathad 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 thepresence of interstitial fibroblasts and early granulationtissue with extensive staining, using Mason’s Trichromefor collagen and Alcian blue for proteogylcan (Figures 4Band 4C). The extent of staining with Alcian blue and Ma-son’s Trichrome in Figures 4B and 4C is representative ofgrade 13 (Table 1). However, 14 d after Blm and CD36synthetic peptide 93-110 administration there was evi-

dence of less inflammation and fibrosis, inasmuch as thesize of lesions was reduced and occupied a smaller volumeof the overall section (Table 1; Figures 4D–4F). In addi-tion, the number of cells retrieved by BAL was lower 7 dafter administration of Blm and the CD36 synthetic pep-tide, suggesting that there is less inflammation when thepeptide is administered. After Blm and concomitant CD36synthetic peptide 93-110 administration, the presence ofAlcian blue and Mason’s Trichrome within the lesion wasminimal and is representative of a grade 11 (Figures 4Dand 4E; Table 1).

Expression of Connective Tissue Proteins

After a single intratracheal dose of Blm there is increasedexpression of collagen I and III, decorin, fibronectin, and avariety of other connective tissue proteins (16). Total pro-tein extracted from lungs of rats treated with Blm was 2-foldhigher than protein obtained from untreated rats, or fromthose treated with normal saline or one of the CD36 syn-thetic peptides alone. This increase in protein extracted isexpected 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 ratsthat had received Blm concomitantly with the CD36 syn-thetic peptide 93-110 was approximately 50% less thanthat obtained when Blm alone was administered. It shouldbe noted that to comply with the CCAC’s requirement touse as few rats as possible, the entire time course was notdone for experiments used to determine the effects of theCD36 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 weredone using a time course where rats were killed at regulartime intervals consisting of 4, 7, 14, 21, and 28 d after Blmor 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 inexpression 7 and 14 d after Blm administration (data notshown). To determine the effects of CD36 synethetic pep-tide 93-110 on collagen III and fibronectin expression, ratswere killed at 4 and 7 d. To detect changes in collagen Isynthesis, rats were killed 7 and 14 d after Blm and CD36synthetic 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 wereabbreviated. 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 receivedBlm 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 theCD36 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 epithelialand 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), andPMNs (filled bars). (B) Absolute number of cells 3 106 cells iden-tified as macrophages (open bars), lymphocytes (striped bars),and PMNs (filled bars). The percent and absolute number ofmacrophages after normal saline treatment compared with Blmtreatment has a P value < 0.0005. However, there is no statisticaldifference in the percent or absolute number of macrophages af-ter the normal saline treatment compared with Blm plus CD3693-110. The percent and absolute number of PMNs after normalsaline treatment compared with Blm or Blm plus CD36 93-110has a P value < 0.0001. The percent and absolute number ofPMNs after Blm treatment compared with Blm plus CD36 93-110has a P value < 0.001. The percent and absolute numbers of lym-phocytes were not statistically significant in any comparisonamong the groups (ANOVA). The data are the means of experi-ments done on six rats.

Yehualaeshet, O’Connor, Begleiter, et al.: CD36 Peptide Inhibits Pulmonary Fibrosis 209

Blm deposition (17). When the Blm was combined in thesame 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 syntheticpeptide 93-110 did not affect the potency of Blm toxicityto Blm-sensitive LY/5178Y lymphoma cells (Figure 6). Forthis reason the initial in vivo Blm injury to the alveolar ep-ithelial and endothelial cells is not likely to be affectedwhen Blm and the CD36 synthetic peptide 93-110 are ad-ministered together. It is of note that the administration ofBlm concomitantly with the CD36 synthetic peptide 93-110 did not totally abrogate the generation of active TGF-b1, inflammation, or connective tissue synthesis. Therewas, however, a reduction in all these parameters, whichsupports the probability that Blm administration leads topulmonary toxicity that can be ameliorated by the effectsof the CD36 synthetic peptide 93-110.

DiscussionThe presence of CD36 synthetic peptide 93-110 in culturesof alveolar macrophages obtained after Blm-induced lunginjury prevents the conversion of L-TGF-b1 to activeTGF-b1 (6). The present report is the first to describe thatwhen the same CD36 synthetic peptide is administered torats with Blm compared with administration of Blm alone,there is a reduction of inflammation and connective tissuesynthesis induced by Blm. The administration of the CD36synthetic peptide 93-110 with Blm results in decreased se-cretion of active TGF-b1 by explanted alveolar macrophages.It is of note that after Blm-induced lung injury the overex-

pression of TGF-b1 is seen almost exclusively in alveolarmacrophages (3). No TGF-b1 was observed in alveolar epi-thelial cells or interstitial inflammatory cells (3). It thenfollows that in vivo the effect of the CD36 synthetic peptide93-110 most likely inhibits the activation of alveolar mac-rophage–derived L-TGF-b1. The reduction of active TGF-b1from alveolar macrophages may then lead to ameliorationof 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 peptide93-110 with Blm diminishes the release of active TGF-b byexplanted alveolar macrophages may be similar. It had

Figure 4. Histologic changes in rat lungs 14 d after intratracheal treatment. After administration of 1 mg of Blm, paraffin-embeddedlung sections were stained with: (A) H&E for histology; arrow is in area of fibroconnective tissue (original magnification: 34); (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 arrowin A (original magnification: 325); and (C) Mason’s Trichrome for collagen distribution; arrow is in the area of fibroconnective tissue(original magnification: 325). After administration of 1 mg of Blm plus the CD36 synthetic peptide 93-110 (1,600 mg), 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:34); (E) Alcian blue; arrow is in the area of proteoglycan expression identified by an arrow in D (original magnification: 325); and (F)Mason’s Trichrome; arrow is in an area with fibroconnective tissue identified by an arrow in D (original magnification: 325). The histol-ogy demonstrated is representative of three rats per group.

TABLE 1

Histologic changes after intratracheal treatment

Intratracheal Treatment

Percent of LungInvolved in

Inflammationand Fibrosis

Extent of Staining with:*

Mason’sTrichrome Alcian Blue

Blm (1 mg) 40.5 6 2.2 12.9 6 0.01 13 6 0Blm (1 mg) plus CD36

synthetic peptide93-110 (1,600 mg) 8.0 6 2.4 10.9 6 0.4 10.8 6 0.4

*0: no staining, 11: detectable color of the Mason’s Trichrome or Alcianblue, 12: staining between 11 and 12, and 13: extensive staining with Mason’sTrichrome and Alcian blue within the lesion. The percent of lung involved afterBlm compared with Blm plus CD36 synthetic peptide 93-110 has a P value of< 0.002. The extent of staining with Mason’s Trichrome or Alcian blue aftertreatment compared with Blm plus CD36 synthetic peptide 93-110 has a P value< 0.001. The statistical analysis for this data was done using Wilcoxon’s ranksigned nonparametric statistical test.

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 theregion of the amino acids 93 to 110 of CD36 (18). The CS-VTCG region in the type 1 repeats of TSP-1 is also importantfor the release of active TGF-b1 by alveolar macrophagesbecause the addition of the synthetic TSP-1 peptide CSVTCGto cultures of activated alveolar macrophages inhibits theactivation of L-TGF-b1 (N. Khalil and colleagues, unpub-lished data). It has been demonstrated that the interactionof 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, ismore exposed than when TSP-1 is associated with a cellsurface or matrix proteins (19). We demonstrated previ-ously that alveolar macrophages generate TSP-1/L-TGF-b1complexes after in vivo Blm injury (6). It is conceivablethat 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-b1 complex.The association of the CD36 synthetic peptide 93-110 with

the CSVTCG region in TSP-1 of the TSP-1/L-TGF-b1complex may interfere with the TSP-1/L-TGF-b1 interac-tion with the CD36 receptor on the surface of the alveolarmacrophage (6). Because the association of the TSP-1/L-TGF-b1 with the alveolar macrophage CD36 receptor iscritical to the activation of the L-TGF-b1 by plasmin (5,6), prevention of this association may diminish the activa-tion of L-TGF-b1 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 percentagedue to an increase in the number of PMNs (1, 4). How-ever, when the CD36 peptide 93-110 was administeredwith Blm, there was a reduction in numbers of PMNs sothat an increase in the percentage of alveolar macrophageswas observed. The reduction in numbers of PMNs couldbe due to a number of reasons. TGF-b1 is a potentchemoattractant of PMNs (20), and when the CD36 syn-thetic peptide 93-110 was administered the decrease in ac-tive TGF-b1 in the alveolar space could lead to a reduc-tion in PMN recruitment to the alveoli. The interaction ofTSP-1 with CD36 could also be important in areas of in-jury in recruitment and activation of PMNs (21, 22). Blmadministration injures endothelial cells (17), and TSP-1that is released by injured endothelial cells (21, 22) bindsto the same cells, where it functions to recruit PMNs andlocalize the PMNs to the endothelial cells and activatesPMNs on endothelial cells to release reactive oxygen in-termediates (21, 22). Because Blm injures the endothelialcells (17), this role of TSP-1–mediated recruitment ofPMNs could contribute to lung injury induced by Blm ad-ministration. The binding of TSP-1 to endothelial cells ismediated by the association of TSP-1 to CD36, which is lo-cated on the endothelial cells (23). It is then conceivablethat the presence of the CD36 synthetic peptide 93-110may 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 interactionof CD36 with TSP-1 has been described to be important

Figure 5. Expression of connective tissue proteins after a numberof 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 thescrambled peptide of CD36 amino acids from 93-110 (s93-110)(lanes 5 and 6, respectively), and Blm plus the CD36 syntheticpeptide 208-224 (lanes 7 and 8, respectively). The numbers 7 and14 at the top denote the number of days after treatment. (B) Pro-collagen III expression 4 and 7 d after Blm administration in theabsence 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 dafter 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 ofthe bands (in kD). The immunoblots are representative of exper-iments done on three or four separate rats.

Figure 6. Survival of LY/5178Ylymphoma cells determined bya clonogenic assay was donein the presence of 500 ml nor-mal saline (open bar), 1,600 mgCD36 synthetic peptide 93-110/500 ml normal saline (stripedbar), 1 mg Blm/500 ml normalsaline (dark-striped bar), or 1 mgBlm/500 ml normal saline plus1,600 mg of the CD36 syntheticpeptide 93-110 (filled bar). Thecytotoxicity of Blm or Blm plus

CD36 synthetic peptide 93-110 compared with that of normalsaline or CD36 synthetic peptide 93-110 alone had a P value of< 0.001 (ANOVA). The cytotoxicity of Blm compared with thatof Blm plus CD36 synthetic peptide 93-110 was not statisticallysignificant. The data represent the mean of six separate experi-ments.

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 thesefunctions relevant to Blm lung injury are the observationsthat platelet aggregation has been reported to occur inearly 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 theCD36 synthetic peptide 93-110 in vitro prevents plateletaggregation (14). In vivo, the presence of the CD36 syn-thetic peptide 93-110 could result in inhibition of plateletaggregation, platelet-monocyte adhesion, and release ofcytokines, and thus less inflammation and fibrosis. Theseeffects of the CD36 synthetic peptide 93-110 could alsocontribute to the amelioration of Blm-induced inflammationand fibrosis.

Over the past few years a number of treatments, such asanti–TNF-a antibodies (28), antioxidants (29), interferon-g(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-b1 in the pathogenesis of Blm-inducedinflammation and fibrosis in mice when they demonstratedthat administration of TGF-b1 antibodies and Blm resultedin less lung injury and fibrosis (32). However, the locationof the neutralizing effects of the anti–TGF-b1 antibodieswas not apparent from Giri and coworkers’ study (32). Onthe basis of the current work and that of others, the admin-istration of TGF-b1 antibodies could have neutralized theTGF-b1 generated by endothelial cells (33) and/or alveolarepithelial cells (13), as well as alveolar macrophages (3–6).

Our work has shown that the generation of plasmin inaddition to the cellular localization of L-TGF-b1 (5, 6), isimportant for the activation of alveolar macrophage–derivedL-TGF-b1. However, there are other mechanisms of acti-vation of L-TGF-b1 that are independent of proteases. Forexample, reactive oxygen intermediates (11), hyperglycemia(11), and, in some but not all circumstances (34), TSP-1can activate L-TGF-b1 in the absence of proteases (12). Inaddition, Munger and associates (13) described anotherprotease-independent activation of L-TGF-b1 that may beimportant in Blm-induced lung injury (13). It was observedthat the RGD (arginine–glycine–aspartic acid) sequencein the LAP interacts with the integrin avb6 on alveolar epi-thelial cells (13), leading to conformational changes of theL-TGF-b1 and exposing the site on TGF-b1 that interactswith the TGF-b receptor type II (13). It is not knownwhether Blm administration to rats, a different species thanmice, induces avb6 on alveolar epithelial cells and is there-fore important in the activation of L-TGF-b1. It is also notknown whether Blm injury induces CD36 expression onrat alveolar epithelial cells that subsequently binds the TSP-1/L-TGF-b1 complex and results in plasmin-mediated activa-tion of L-TGF-b1. Regardless of the expression of avb6 orCD36 on rat epithelial structures, we have demonstratedthat the reduction of alveolar macrophage–derived activeTGF-b1 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-b1 but also support theimportance of alveolar macrophage–derived active TGF-b1 in pulmonary fibrosis.

Acknowledgments: The work in this manuscript was supported by a grant fromthe Medical Research Council of Canada to one author (N.K.). The authorsthank Dr. Arnold Greenberg for reviewing the manuscript, Dr. Bob Tate forthe statistical analysis, Mrs. Stephanie Moorehouse for her technical assistance,and Ms. Carolin Hoette for typing the manuscript.

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