ORIGINAL ARTICLE

Expression of transforming growth factor-bb1 and tumour necrosisfactor-aa in bronchoalveolar lavage cells in murine pulmonary

fibrosis after intraperitoneal administration of bleomycin

A MAEDA, S ISHIOKA, Y TAOOKA, K HIYAMA AND M YAMAKIDO

The Second Department of Internal Medicine, Hiroshima University School of Medicine,Hiroshima, Japan

Expression of transforming growth factor-bb1 and tumour necrosis factor-aa in bronchoalveolarlavage cells in murine pulmonary fibrosis after intraperitoneal administration of bleomycinMAEDA A, ISHIOKA S, TAOOKA Y, HIYAMA K, YAMAKIDO M. Respirology 1999; 4: 359–363Objective: We previously observed increased expression of interleukin-1b, platelet-derived growthfactor-A, and insulin-like growth factor-I in bronchoalveolar lavage (BAL) cells during the develop-ment of pulmonary fibrosis after an intraperitoneal administration of bleomycin in mice. Thepurpose of this study was to investigate the roles of tumour necrosis factor (TNF)-a and transform-ing growth factor (TGF)-b1 in this model. Methodology: We investigated the mRNA expression levels of TNF-a and TGF-b1 in BAL cells ofInstitute for Cancer Research mice after 10 days of the intraperitoneal administration of bleomycinwith or without treatment with a specific neutrophil elastase inhibitor, ONO-5046·Na. Results: On day 1 but not on days 15 and 29, the relative amount of TGF-b1 mRNA in thebleomycin-treated mice was significantly decreased compared with control mice. In the mice treatedwith both bleomycin and ONO-5046·Na intermediate values for TGF-b1 were obtained. No signifi-cant differences in TNF-a expression were observed in any of the treatment groups. Conclusions: These results suggest that a reduced expression of TGF-b1 in BAL cells in the earlyphase may be important during the development of murine pulmonary fibrosis induced by anintraperitoneal administration of bleomycin.

Key words: bleomycin, bronchoalveolar lavage, mouse, pulmonary fibrosis, reverse transcription–polymerase chain reaction, transforming growth factor-b, tumour necrosis factor-a.

animals, and animal models of bleomycin-inducedpulmonary fibrosis have been widely used.2–4

Although bleomycin has usually been administeredintratracheally to animals for convenience and eco-nomic reasons,4 the intraperitoneal administration ofbleomycin to mice has been reported to be suitablefor investigating the early stages of pulmonary fibro-sis.3 We previously reported murine pulmonary fibrosis after an intraperitoneal administration ofbleomycin, and this method induces fibrosis mainlyin the subpleural regions as observed in human pul-monary fibrosis.5 We also demonstrated increasedexpressions of cytokines (IL-1b in the early phase andPDGF-A and IGF-I during the development of pul-monary fibrosis) in bronchoalveolar lavage (BAL)cells in this model.5 Activated macrophages were considered to be the main source of these cyto-kines, however, the mechanism of their activationremains unclear in this model. Since evaluation of the cytokine network is considered helpful in

Respirology (1999) 4, 359–363

INTRODUCTION

The pathogenesis of pulmonary fibrosis following a variety of lung injuries is poorly understood.Cytokines such as interleukin (IL)-1, tumour necrosisfactor (TNF)-a, transforming growth factor (TGF)-b,platelet-derived growth factor (PDGF), and insulin-like growth factor (IGF)-I, which are induced invarious types of cells, have been suggested to play arole in pulmonary fibrosis.1 Bleomycin is well knownto induce pulmonary fibrosis in both humans and

Correspondence: Shinichi Ishioka, The SecondDepartment of Internal Medicine, Hiroshima UniversitySchool of Medicine, 1-2-3 Kasumi, Minami-ku,Hiroshima 734-8551, Japan.

Received 25 December 1998; accepted for publication11 May 1999.

understanding the pathogenesis of pulmonary fibro-sis, we investigated the mRNA expression levels of thecytokines, TNF-a and TGF-b1, in BAL cells.

Tumour necrosis factor-a is a pro-inflammatorycytokine produced by macrophages in response toendotoxin exposure.1 Piguet et al. demonstrated anincrease in TNF-a mRNA level in lung tissue fromdays 5 to 15 after a single intratracheal bleomycininstillation in mice.6 Transforming growth factor-bis a multifunctional growth-modulating cytokinecapable of inhibiting the proliferation of certainresponder cells, augmenting the proliferation ofothers, and controlling multiple aspects of connectivetissue turnover, as well as influencing numerouscontrol points in the immune system.7 Transforminggrowth factor-b1 is the predominant isoform of thethree mammalian TGF-b isoforms (TGF-b1, -b2, and-b3). Previous studies have demonstrated increases inTGF-b production8 and TGF-b mRNA9,10 in rodentlung tissues after an intratracheal instillation ofbleomycin.

We previously found that a newly developed specific neutrophil elastase inhibitor, ONO-5046·Na,11 suppressed the mRNA expressions of IL-1b, macrophage inflammatory protein (MIP)-2,PDGF-A, and IGF-I in BAL cells and ameliorated pul-monary fibrosis in murine pulmonary fibrosisinduced by an intraperitoneal administration ofbleomycin.12 With respect to these findings, wehypothesized that if TNF-a and/or TGF-b1 play rolesin the pathogenesis of this pulmonary fibrosis model,the administration of ONO-5046·Na would suppresschanges in the expression of these cytokines. In thepresent study, we compared the expression of thesecytokines in ONO-5046·Na-treated and -untreatedmice administered (because of simultaneous admin-istration) of bleomycin.

MATERIALS AND METHODS

Sample preparation

Institute for Cancer Research mice were purchasedfrom Charles River Japan (Yokohama, Japan). First,bleomycin (Bleo™; Nippon Kayaku, Tokyo, Japan)was dissolved in 200 µL of a saline solution andinjected intraperitoneally at a dose of 7.5 mg/kgbodyweight into 10-week-old mice daily on 10 con-secutive days as previously described (BLM micegroup, n = 30).5 Control mice received an equiva-lent amount of the saline solution for 10 days (SLNmice group, n = 30). ONO-5046·Na (sodium N-[2-[4-(2,2-dimethylpropionyloxy)phenylsulfonylamino]benzoyl]aminoacetate tetrahydrate) (ONO Pharma-ceutical, Osaka, Japan) was dissolved in 200 µL salinesolution and injected subcutaneously into 10-week-old mice for 10 days at a dose of 100 mg/kg body-weight with a simultaneous administration ofbleomycin as in BLM group (BLM-ONO mice group,n = 15). As the controls for the BLM-ONO group, 200 µL of the saline solution was injected subcuta-neously to half of the animals of the above BLM group(BLM-SLN mice group, n = 15) and SLN group (SLN-

SLN mice group, n = 15). Animals were killed by cervi-cal dislocation under ether anaesthesia on days 1, 15,and 29 after the last administration of bleomycin,ONO-5046·Na, and/or saline. BronchoalveolarLavage was performed as previously described.5 TheBAL cells were immediately centrifuged at 250 g for10 min. The cell pellet was preserved at –80°C untiluse.

Evaluation of cytokine expression by reversetranscription–polymerase chain reaction

mRNA was directly extracted from the cell pellet withthe use of a kit (Quick Prep™ Micro mRNA Purifica-tion kit; Pharmacia Biotech, Tokyo, Japan) accordingto the manufacturer’s recommendations. One-fourthof the poly(A)+ RNA solution was reverse transcribedinto cDNA in a 20 µL reaction mixture containing 1.2 µmol/L oligo(dT)18 primer (Sigma, St Louis, MO,USA), 500 µmol/L of each deoxynucleotide (TaKaRa,Kyoto, Japan), 50 mmol/L Tris-HCl (pH 8.3), 75 mmol/L KCl, 3 mmol/L MgCl2, 10 mmol/L DTT, 20 units RNase inhibitor (RNasin™; Promega,Madison, WI, USA), and 200 units Molony murineleukaemia virus RNase H-reverse transcriptase(Superscript™; GIBCO/BRL, Gaithersburg, MD,USA). The reaction mixture was incubated at 37°C for60 min and then at 95°C for 5 min to inactivate thereverse transcriptase. Oligonucleotide primer sets for TNF-a (692 b.p.), TGF-b1 (472 b.p.), and b-actin(540 b.p.) as an internal control were purchased fromCLONTECH (Palo Alto, CA, USA). The optimalnumber of PCR cycles for each primer set was deter-mined in preliminary experiments so that the reac-tion was carried out during the course of exponentialamplification as previously described.5 A coincidentamplification (co-amplification) of cDNA for eachcytokine and b-actin was then carried out in one tube.Polymerase chain reaction amplifications were per-formed with 1 µL of cDNA solution in a 100 µL reac-tion mixture containing 0.5 µmol/L of each primer,200 µmol/L of each deoxynucleotide, 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2,0.001% gelatin, and 2.5 units Taq DNA polymerase(AmpliTaq™; Perkin-Elmer, Foster City, CA, USA).The PCR conditions were: denaturation at 95°C for 1 min, annealing at 63°C (TGF-b1) or 65°C (TNF-a) for 1.5 min, and extension at 72°C for 2.5 min, using a Program Temp Control System PC-700™ (ASTEC,Fukuoka, Japan). Aliquots of the PCR reaction prod-ucts (8 µL) were electrophoresed on a 2% agarose geland stained with ethidium bromide. The intensity ofethidium bromide luminescence for each PCRproduct was measured by a charge-coupled device(CCD) imaging system (Densitograph™ AE-6900MF;ATTO, Tokyo, Japan). The cytokine/b-actin ratio of theintensity of ethidium bromide luminescence for eachPCR product was calculated. The cytokine/b-actinratio of each experimental mouse was then divided bya mean of those in control mice killed on the sameday to evaluate the relative amount of cytokine mRNAin each mouse.

360 A Maeda et al.

Statistical analysis

The expression levels of TNF-a and TGF-b1 at 1, 15,and 29 days after treatment were compared betweenBLM mice and SLN mice as well as among BLM-ONOmice, BLM-SLN mice, and SLN-SLN mice using the Mann–Whitney U-test for non-parametric data. A probability value of less than 0.05 was consideredsignificant.

RESULTS

While no significant differences were observed in theTNF-a expression levels between the BLM and SLNmice groups on days 1, 15, and 29, the level of TGF-b1 mRNA on day 1 after the last administration wasdecreased to approximately 60% in the BLM groupcompared with that in the SLN group (P < 0.01).However, no significant differences were found inTGF-b1 expression on days 15 and 29 between the twogroups. Although no significant differences wereobserved in the TGF-b1 and TNF-a mRNA levelsbetween the BLM-ONO and BLM-SLN or SLN-SLNmice groups, the level of TGF-b1 mRNA on day 1 inthe BLM-ONO group was between those in BLM-SLNand SLN-SLN groups (Fig. 1).

DISCUSSION

In the present study, the level of TGF-b1 mRNA in BALcells was decreased on day 1 and returned to normalon days 15 and 29 after an intraperitoneal adminis-tration of bleomycin. The findings also suggested thatthe mRNA expression level of TNF-a in BAL cells isunlikely to change in the present model.

Current concepts suggest that the cytokinesinduced in various cell types have important roles in

the pathogenesis of pulmonary fibrosis.1 Previousstudies reported an increased expression and/orsecretion of TNF-a and TGF-b1 in a bleomycin-induced rodent model of pulmonary fibrosis.6,8–10

Because our findings did not support this, the sig-nificance of these findings should remain equivocaluntil further studies are undertaken. However, thedecreased expression of TGF-b1 at 1 day afterbleomycin treatment was probably restored by the administration of ONO-5046·Na (Fig. 1), andwould support the significance of the decreased TGF-b1 expression in the early phase of the presentmodel.

Transforming growth factor-b is a multifunctionalgrowth-modulating cytokine thought to be one of themost important factors in pulmonary fibrosis.1,7

Increases in TGF-b production8 and TGF-b mRNA9,10

in lung tissues have been demonstrated after intra-tracheal instillation of bleomycin in rodents, while inthe present study, the level of TGF-b1 mRNA on day 1after 10 days of bleomycin treatment was decreasedto approximately 60% of controls and had returned tothe control level on days 15 and 29. The apparent dis-crepancy between the previous findings and oursmay have several explanations. First, we extractedmRNA directly from the total BAL cell sampleswithout purification of the macrophages so that arti-ficial stimuli would not affect the findings. Althoughprevious studies have demonstrated that many typesof cells in the lungs (including mononuclear cells,fibroblasts, bronchial epithelial cells, and endothelialcells) have the potential to express TGF-b,13–15 acti-vated alveolar macrophages are considered the pre-dominant source of TGF-b in bleomycin-inducedpulmonary fibrosis.8,16–18 Because the proportion ofmacrophages among the BAL cells was always 90% or more in the present model,5,12 the mRNA of TNF-aor TGF-b1 is likely to be derived from alveolarmacrophages. In situ hybridization studies identify-

TGF and TNF in murine pulmonary fibrosis 361

Figure 1 Relative amounts of cytokine mRNA in bronchoalveolar lavage cells from intraperitoneal bleomycin-treated micewith ( , BLM-ONO mice) or without (�, BLM-SLN mice) treatment with ONO-5046·Na, and saline solution-treated controlmice (�, SLN-SLN mice) groups. The data are mean ± SEM of the values for each mice group (n = 5) on days 1, 15, and 29after treatment, respectively. (a) Tumour necrosis factor-a; (b) transforming growth factor-b1. * P < 0.05.

ing cell types/per cent positives, or purificationstudies to determine the source factors should bepursued to provide an explanation.

Second, in most of these studies, bleomycin wasadministered intratracheally,4,6,8–10,14–18 because it wasconvenient and economical to do so. In contrast, weadministered bleomycin to mice intraperitoneally,since this method was reported to be suitable forinvestigating the early stages of pulmonary fibrosis.3

This method enabled us to induce fibrosis mainly inthe subpleural regions, as observed in human pul-monary fibrosis,5 while the intratracheal instillationof bleomycin induces fibrosis mainly in peribronchi-olar regions.19 Harrison and Lazo developed a murinemodel of pulmonary fibrosis induced by a constantsubcutaneous infusion of bleomycin.20 They reportedthat the subcutaneous infusion of bleomycin pro-duced a progressive, confluent, subpleural, fibroticlesion that resembled the human disease and causedfibrosis at 4 weeks after treatment for 1 week.20 Similarcharacteristics and time course were observed in ourmodel, suggesting that the pathogenesis in these twomodels of pulmonary fibrosis may be similar. Hoytand Lazo reported the total pulmonary content ofTGF-b mRNA in a C57Bl/6 mice model of pulmonaryfibrosis 1, 2, 3, and 4 weeks after a constant subcuta-neous infusion of bleomycin for 1 week.21 Althoughthey did not observe it at day 1, the ratio of TGF-bmRNA to poly(A)+ RNA was increased approximatelyfive-fold 1 week after the treatment and had returnedto control levels at 2, 3, and 4 weeks.21 This suggeststhat the initial decreased TGF-b1 might be followedby over expression towards the end of the first week,followed by return to normal. Alternatively, chronicintraperitoneal dosing may not be associated withover expression of TGF-b1 at any point. Similarfactors (i.e. the intraperitoneal administration ofbleomycin and extraction of mRNA without purifica-tion of macrophages) may be involved in the appar-ent discrepancy in the findings of the expression ofTNF-a between our study and others.6,22

The role of the reduced expression of TGF-b1 inalveolar macrophages in the early phase of this modelis unclear. Because TGF-b inhibits the proliferation ofendothelial cells, lymphocytes, and certain fibro-blasts,7 a reduced secretion of TGF-b might suppressthe inhibitory effects on these cells. The degree ofinhibition is, however, critically dependent on variousfactors, such as culture conditions, cell density, TGF-b concentration, and the presence of other cytokines.7

In addition, the role of TGF-b in vivo is not well under-stood. Further studies are necessary to understandthe role of this and other cytokines in pulmonaryfibrosis induced by an intraperitoneal administrationof bleomycin.

In conclusion, the alterations of TGF-b expressionobserved in the early phase may be important in thepathogenesis of murine pulmonary fibrosis inducedby an intraperitoneal administration of bleomycin.Further investigations are necessary to clarify thepathophysiological differences in bleomycin-inducedpulmonary fibrosis generated by different routes ofbleomycin administration, providing insight into thepathogenesis of pulmonary fibrosis, which occursafter a variety of lung injuries.

ACKNOWLEDGEMENTS

This study was supported in part by a grant-in-aidfrom the Japanese Ministry of Health and Welfare anda grant from the Smoking Research Foundation.

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