Bleomycin-Stimulated Hamster Alveolar Macrophages Release ...

3 downloads 112 Views 1MB Size Report
ing bleomycin-instilled hamsters, the FP activity of. AM culture supernatants was increasedsignificantlyon days 1, 5, and 10 after instillation ofbleomycin. The.
American Journal of Pathology, Vol. 132, No. 3, September 1988 Copyright e American Association of Pathologiats

Bleomycin-Stimulated Hamster Alveolar Macrophages Release Interleu kin-i From the First Department ofInternal Medicine,* the First Department ofPathology, t and the Department ofParasitology, t Yamagata University School ofMedicine, Yamagata,Japan

AKIRA SUWABE, MD,* KEIJI TAKAHASHI, MD,* SHOJI YASUI, MD,* SHIGERU ARAI, MD,t and FUJIRO SENDO, MD*

The capacity of alveolar macrophages (AM) of bleomycin-instilled hamsters to proliferate mouse thymocytes (interleukin-1 activity) and hamster fibroblasts (fibroblast proliferation (FP) activity was studied. Using bleomycin-instilled hamsters, the FP activity of AM culture supernatants was increased significantly on days 1, 5, and 10 after instillation of bleomycin. The interleukin-1 (IL-1) activity, however, was increased

significantly on day 1 only as compared with salinetreated hamsters. Next, normal AM were stimulated in vitro by bleomycin. After being fractionated by chromatography, their culture supernatant showed IL-1 activity, which also indicated FP activity. These results suggest that bleomycin directly stimulates AM to release IL-1 in the fibrogenic responses. (Am J Pathol 1988, 132:512-520)

RECENTLY, MANY RESEARCHERS have begun to study the role of alveolar macrophages (AM) in the development of pulmonary fibrosis, and many reports have been published regarding the soluble factors derived from AM that cause fibroblast proliferation. With this view, some reporters'-7 have sressed that the AM in humans release an AM-derived growth factor (MDGF) in vitro, whereas others8-'3 have reported that the human AM inhibit lung fibroblast proliferation. In considering these confficting results, many issues regarding the role of soluble factors on the development of pulmonary fibrosis obviously remain to be clarified. It is well known that bleomycin induces diffuse lung injuries and consequential pulmonary fibrosis in experimental laboratory animals.3-5'9"10"4'417 Kovacs and coworkers4'5 have reported that AM from rats instilled with bleomycin release MDGF after in vitro incubation without stimulation, and that the excess MDGF is recovered from the intra-alveolar spaces. This research suggests the role that AM may play during the injury ofthe lungs. In addition to MDGF, interleukin1 (IL- 1) has been reported as a potential regulator of fibroblast proliferation.'8 Many investigators'9123 have shown that macrophages release IL-I when they are activated by certain stimulators including silica particles or endotoxin (lipopolysaccharide:LPS). Others,2'24 however, have reported that IL-I and MDGF are distinct entities.

When searching for the relation between IL-I and MDGF, it would seem more sophisticated to use AM samples obtained from injured lungs, but such an approach has not yet been performed. In this research, the authors first assessed the time course of mouse thymocyte proliferation (IL- 1) activity and hamster fibroblast proliferation (FP) activity detected in the culture supernatants of AM lavaged from hamsters instilled with bleomycin. The in vitro effect of bleomycin on normal AM also were investigated to investigate the direct effect of bleomycin on AM.

512

Materials and Methods Experimental Animals Disease-free male Syrian golden hamsters and C3H/HeJ mice were purchased from Shizuoka Laboratory Animal Co. (Hamamatsu, Japan). In vivo Instillation of Bleomycin Four-week-old hamsters (body weight, 50 to 100 g) were anesthetized intraperitoneally with pentobarbiAccepted for publication April 25, 1988. Address reprint requests to Keiji Takahashi, MD, The First Department of Internal Medicine, Yamagata University School of Medicine, Zao-Iida, Yamagata, 990-23, Japan.

Vol. 132 * No. 3

BLEOMYCIN-STIMULATED AM RELEASE IL-1

tal (Somnopentil; Pitman-Moore Inc., Washington Crossing, NJ) and given a single intratracheal instillation of bleomycin chloride (5 units/kg body weight; Nippon Kayaku Co., Tokyo, Japan) dissolved in sterile saline, as previously reported.25'26 This dose of bleomycin induces pulmonary fibrosis about 30 days after its instillation.25 As a sham control, the other hamsters were instilled in the same manner with sterile saline instead of bleomycin. The instilled hamsters were then anesthetized and killed on days 1, 5, 10, and 20 after instillation of the bleomycin and saline. Each bleomycin group consisted offour hamsters, and each control group consisted ofthree hamsters.

was calculated by counting the total cell number of nonadherent cells. After a 24-hour incubation of adherent cells without stimulation, the supernatant was recovered and preserved as AM supernatant in the same manner as BALF supernatant. In this assay system, the recovered supernatants (except for the supernatants obtained from in vitro stimulation with bleomycin [see below]), were not dialyzed because there was a possibility that low molecular inhibitory factors, such as the prostaglandine E2, might be dialyzed from the supernatant. To examine the ability of normal AM to induce IL1 production or fibroblast proliferation, adherent cells from alveolar cells of normal hamsters were prepared as described above. Two million adherent cells/2 ml/ well were incubated in RPMI5% in both the presence and absence of 0.2 mg/well of silica particles (particle size: 0.007 g, surface area: 400 ± 20 sq m/g; Sigma Chemical Co., St. Louis, MO). After a 24-hour incubation, culture supernatants were recovered, filtrated, and preserved.

Preparation of Supernatants At various points in time, the lungs of the hamsters instilled with the bleomycin and saline were lavaged using the standard procedure.27'28 Two kinds of supernatants were prepared from the lungs of each hamster, the bronchoalveolar lavage fluid supernatant, and the AM culture supernatant. The first lavage was performed with 3 to 5 ml of RPMI- 1640 medium (GIBCO, Chagrin Falls, OH) supplemented with 25 mM of HEPES (Dojin Labo Co. Ltd., Kumamoto, Japan), 100 IU/ml of penicillin, 100 ug/ml of streptomycin, and 5% heat-inactivated fetal calf serum (FCS; GIBCO) (this medium was abbreviated as RPMI5%). After the recovered fluid was centrifuged at 1 ,500g for 5 minutes at 4 C, the supernatant was recovered and passed through a Millex-HA filter (pore size: 0.45 g; Millipore Corp., Bedford, MA) and preserved as "BALF supernatant at -70 C until assay (see below). To collect alveolar cells, lavage was continued with calcium- and magnesium-free sterile phosphatebuffered saline (PBS; 0.15 M NaCl, 0.02 M phosphate, pH 7.2) until 50 ml of BALF was recovered. After the recovered BALF was centrifuged at 400g for 5 minutes at 4 C, the cell pellets obtained were washed twice with PBS and suspended in a supplemented RPMI1640 medium containing 10% FCS (RPMIIO%). The total count ofthis cell suspension was determined using a hemocytometer and the viability was assessed by trypan blue exclusion. The differential count was determined from the cytocentrifuged cells. Ninetyfive to 100% of the cells were viable as determined by the trypan blue exclusion test. One million cells/ml/ well were seeded to a culture plate (Micro Test II 3040 plate; Becton Dickinson and Co., Oxnard, CA), and incubated at 37 C in 5% CO2 and 95% air. After incubation for 1 hour, nonadherent cells were removed by a vigorous washing two times with FCS-free medium (RPMIO%). One milliliter of fresh RPMI5% was then added to adherent cells. The number ofadherent cells

513

Fibroblast Proliferation (FP) Assay

The fibroblasts maintained after separation from embryonal hamsters were used in this assay. The maintenance of these fibroblasts was procured by doing a medium exchange at an interval ofevery 3 days, and performing their passage once a week. The fibroblasts with passage numbers between 2 and 5 were used in this assay. Before the FP assay, fibroblast samples collected by trypsinization were suspended with RPMIo% and adjusted to 5 X 104 cells/ml. One hundred microliters ofthe cell suspension was mixed with 100 Al of the tested supernatants in a 96-well flat-bottom sterile tissue culture plate (Nunc, Roskilde, Denmark). The final number of fibroblasts was 5 X 103 cells/well and the final FCS concentration was 2.5%. After incubation for 48 hour, 0.5 ,uCi of tritiated thymidine (3H-TdR; Amersham, Arlington Height, IL) was added to each well. Reaction mixtures were then incubated for another 24 hours. At the end of the incubation, the supernatants were aspirated, and the fibroblasts were trypsinized for 15 minutes and harvested on a Labomush filter paper by use of a Labomush cell harvester LM 101-10 (Laboscience Co. Ltd., Tokyo, Japan). This filter paper was added to scintillation liquid (ACS II; Amersham), and the radioactivity ofthis paper was then counted by a scintillation counter (Aloka Liquid Scintillation system, LSC-75 1; Aloka, Tokyo, Japan). A triplicated assay was made in each of the tested supernatants.

514

SUWABE ET AL

AJP * September 1988

Table 1 -Serial Changes of Total Cell Yield and Cell Populations of Nucleated Cells Lavaged from The Lungs Instilled with Bleomycin

Days afer instillation Nontreated (N = 4) Day 1 Saline (N = 3) Bleomycin (N = 4) Day 5 Saline (N = 3) Bleomycin (N = 4) Day 10 Saline (N = 3) Bleomycin (N = 4) Day 20 Saline (N = 3) Bleomycin (N = 4)

Polymorphonuclear cells/head (X106)

Total cell yield/head (X1 06)

Mononuclear cells/head (X1 06)

1.25 ± 0.26

1.22 ± 0.25

0.33 ± 0.04

2.86 ± 0.23 7.70 ± 0.89*

1.18 ± 0.25 1.16 ± 0.45

1.68 ± 0.53 6.03 ± 1.34t

2.68 ± 0.48 8.12 ± 2.761

2.62 ± 0.48 3.84 ±1.66

0.03 ± 0.02 4.28 ± 1.1 1 t

3.20 ± 0.71 5.82 ± 1.00

2.61 ± 0.12 5.02 ± 0.73t

0.48 ± 0.48 0.56 ± 0.32

1.50 ±1.70 4.89 ± 1.39

1.49 ±1.70 4.86 ± 1.34*

0.01 ± 0.01 0.12 ± 0.07

Values are mean ± SE of each group. Significantly different from corresponding control value. *P SC D

501

U I

1

10

20

25

Fraction Number

lyzed with PBS, which resulted in the greater inhibition of thymocyte and fibroblast synthesization by the dialyzed supernatant (data not shown). To separate the active substance(s) from bleomycin supernatant completely, the supernatant sample was then fractionated with Sephadex G-50. Elution patterns of fractionated supernatants for both IL- 1 and FP activity are shown in Figure 5. Fractionated supernatants were separated into two groups by protein contents; fractions A and B. Fraction A contained large protein contents and also showed both IL- I and FP activity, although their peaks were not identical. On the other hand, fraction B showed a small peak absorbance at 280 nm. It was suggested that fraction B might contain bleomycin, because bleomycin has an absorbance peak at 290 nm, which is very close to 280 nm, and because both activities of fraction B were below that of medium control. This result indicated that bleomycin supernatant contained factors showing both IL-I and FP activity. Both peaks were not identical, however, possibly because both thymocytes and fibroblasts showed the different sensitivity to the cytotoxicity of bleomycin.

To clarify the relationship between IL- 1 and FP activity in bleomycin supernatant, a large sample was produced. After undergoing a concentration process, bleomycin supernatant was fractionated with Sephadex G-200 (Figure 6). The elution pattern showed that the IL- 1 activity had a single peak, with its molecular weight being approximately 15,000. The control supernatant did not show any IL- 1 activity after going the same fractionation process as mentioned above (data not shown). The FP activity showed three peaks-A, B, and C-with the highest peak (peak C) being identical to the IL- 1 peak. The other peaks (peaks A and B) were thought to be derived from FCS components, because the elution pattern of control supernatant had the peaks A and B, but not C (data not shown). Therefore, it was likely that the FP activity in bleomycin supernatant was caused by IL- 1. Discussion

The present study periodically examined IL- 1 and FP activity in the supernatant obtained from AM la-

Vol. 132 * No.3

BLEOMYCIN-STIMULATED AM RELEASE IL-1

vaged from hamsters instilled with bleomycin. Significantly increased IL-I activity was detected only on day 1 of the instillation, whereas FP activity continued to be increased up to day 10. It has been reported previously that in in vitro systems IL-1 has an FP activity,'8 that its in vivo relevance is still unknown. The experimental results suggested that IL- 1 could play a certain role in in vivo fibrogenesis especially in early phases after bleomycin instillation. The fact that IL-1 was detected only in the early phase suggested that FP activity in later days was originated from growth factor(s) other than IL-1. It has been reported previously that MDGF is a fibroblast growth factor unrelated to IL- 1.224 Kovacs et al reported that MDGF was released from AM obtained from the rat lungs instilled with bleomycin.4'5 Considering both of these views, FP activity detected on day 5 and 10 may be ascribed to the MDGF released from AM. Estes et al reported that IL- 1 not only showed FP activity itself but also augmented the FP activity of MDGF.24 In the present experimental system, IL-1 and MDGF were released from AM at different phases after bleomycin instillation. Thus, synergism of IL-I and MDGF may not have occurred. However, in idiopathic pulmonary fibrosis in humans, fibrogenic substance(s) may exist in the lungs for a long period of time and continuously stimulate AM. Under such stimulation, IL-I may augment the FP activity of MDGF. That AM obtained from the hamsters lungs instilled with bleomycin spontaneously produced IL-I in vitro suggests the possibility of direct stimulation of AM with bleomycin. To examine this possibility, AM obtained from normal hamsters were first in vitro incubated in the presence of bleomycin, and then their IL-I production was examined. It was found that the supernatant obtained from the stimulated AM contained IL-1. However, a significant increase of MDGF activity was not observed from bleomycin stimulation. This in vitro result, taken together with the in vivo finding that FP activity other than IL-I was detected in the AM supernatant up to day 10, suggested that MDGF was released from AM by indirect stimulation with bleomycin. Namely, bleomycin may act first on pulmonary cells other than AM including alveolar epithelial cells (type I and II), endothelial cells, lymphocytes, and polymorphonuclear cells, and thereafter these cells may activate the AM to release MDGF. In this study, AM stimulated by bleomycin in vivo released IL-I in the early phase of the instillation, but MDGF in the later phase. In consideration of the pathogenesis of pulmonary fibrosis, it was likely that MDGF might be more important than IL-1. How-

ever, because IL- I has been reported to have multiple function, such as lymphocyte proliferation, it is suggested that IL- 1 also may play critical roles in the early phase of the development of pulmonary fibrosis. There are conflicting results about the AM-fibroblast interaction in fibrogenic responses. Some investigators8'13 have reported that AM inhibited the fibroblast growth through the products of arachidonic acids including prostaglandine E2. In the present system, however, the inhibitory effect of AM on the fibroblast was not observed at any stage. These conflicting results could be explained by considering the differences in the species, the source of fibroblasts, the conditions of the FP assay,34 and the timing of lavage performance.

519

References 1. Leibovich SJ, Ross R: A macrophage-derived growth factor that stimulates the proliferation of fibroblasts in vitro. Am J Pathol 1976, 84:501-514 2. Bitterman PB, Rennard SI, Hunninghake GW, Crystal RG: Human alveolar macrophage growth factor for fibroblasts: Regulation and partial characterization. J Clin Invest 1982, 70:805-822 3. Bigby TD, Allen D, Leslie CG, Henson PM, Cherniac RM: Bleomycin-induced lung injury in the rabbit: Analysis and correlation of bronchoalveolar lavage, morphometrics, and fibroblast stimulating activity. Am Rev Respir Dis 1985, 132:590-595 4. Kovacs EJ, Kelly J: Secretion of macrophage-derived growth factor during acute lung injury induced by bleomycin. J Leukocyte Biol 1985, 37:1-14 5. Kovacs EJ, Kelly J: Intra-alveolar release of a competence-type growth factor after lung injury. Am Rev Respir Dis 1986, 133:68-72 6. Lemaire I, Beaudoin H, Masse S, Gromdin C: Alveolar macrophage stimulation of lung injury. Am Rev Respir Dis 1986, 133:68-72 7. Phan SH, Kunkel SL: Inhibition of bleomycin-induced pulmonary fibrosis by nordihydroguaretic acid: The role of alveolar macrophage activation and mediator production. Am J Pathol 1986, 124:343-352 8. Harington JS, Ritchie M, King PC, Miller K: The in vivo effects of silica-treated hamster macrophages on collagen production by hamster fibroblasts. J Pathol 1973,109:21-36 9. Phan SH, Thrall RS: The role of soluble factors in bleomycin-induced pulmonary fibrosis. Am J Pathol 1981, 106:156-164 10. Clark JA, Kostal KM, Marino BA: Bleomycin-induced pulmonary fibrosis in hamsters: An alveolar macrophage product increases fibroblast prostandin E2 and cyclic adenosine monophosphate and suppresses fibroblast proliferation and collagen production. J Clin Invest 1983, 72:2082-2091 11. Lugano EM, Dauber JH, Elias JA, Bashey RI, Jimenz SA, Daniele RP: The regulation of lung fibroblast proliferation by alveolar macrophage in experimental silicosis. Am Rev Respir Dis 1984, 129:767-771

520

SUWABE ET AL

12. Elias JA, Zurier RB, Rossman MD, Berube M-L, Daniele RP: Inhibition of lung fibroblast growth by human lung mononuclear cells. Am Rev Respir Dis 1984, 130: 810-816 13. Elias JA, Rossman MD, Zurier RB, Daniele RP: Human alveolar macrophage inhibition of lung fibroblast growth: A prostandin-dependent process. Am Rev Respir Dis 1985, 131:94-99 14. Wesselius U, Catanzaro A, Wasserman SI: Neutrophil chemotactic activity generation by alveolar macrophages after bleomycin injury. Am Rev Respir Dis

1984,129:485-490 15. Thrall RS, Phan SH, McCormick JR, Ward PA: The development of bleomycin induced pulmonary fibrosis in neutrophil-dependent and complement-depleted rats. Am J Pathol 1981, 105:76-81 16. Clark JG, Kuhn C III: Bleomycin-induced pulmonary fibrosis in hamsters: Effect of neutrophil depletion on lung collagen synthesis. Am Rev Respir Dis 1982, 126: 737-739 17. Thrall RS, Barton RW, D'Amato DA, Sulavic SB: Differential cellular analysis o bronchoalveolar lavage fluid obtained at various stages during the development of bleomycin-induced pulmonary fibrosis in the rat. Am Rev Respir Dis 1982,126:488-492 18. Schmidt JA, Mizel SB, Cohen D, Green I: Interleukin 1, a potential regulator of fibroblast proliferation. J Im-

munol 1982,128:2177-2182 19. Koretzky GA, Elias JA, Kay SL, Rossman MD, Nowell PC, Daniele RP: Spontaneous production of interleukin-I by human alveolar macrophages. Clin Immunol Immunopathol 1983, 29:443-450 20. Lamontagne L, Gauldie J, Stadnyk A, Richards C, Jenkins E: In vivo initiation of unstimulated in vitro interleukin-1 release by alveolar macrophages. Am Rev Re-

spirDis 1985,131:326-330 21. Hunninghake GW: Release ofinterleukin-l by alveolar macrophages of patients with active pulmonary sarcoidosis. Am Rev Respir Dis 1984, 129:569-572 22. Wewers MD, Rennard SI, Hance AJ, Bitterman PB, Crystal RG: Normal human alveolar macrophages obtained by bronchoalveolar lavage have a limited capacity to release interleukin- 1. J Clin Invest 1984,74:22082218 23. Eden E, Turino GM: Interleukin-l secretion by human alveolar macrophages stimulated with endotoxin is augmented by recombinant immune (gamma) interferon. Am Rev RespirDis 1986,133:455-460 24. Estes JE, Pledger WJ, Gillespie GY: Macrophage-derived growth factor for fibroblasts and interleukin-l are distinct entities. J Leukocyte Biol 1984, 35:115-129

AJP

A Sepember 1988

25. Snider GL, Celli BR, Goldstein RH, O'Brrien JJ, Lucey EC: Chronic interstitial pulmonary fibrosis produced in hamster by endotracheal bleomycin: Lung volumes, volume-pressure relations, carbon monoxide uptake, and arterial blood gas studies. Am Rev Respir Dis 1978, 117:289-297 26. Snider GL, Hayes JA, Korthy AL: Chronic interstitial pulmonary fibrosis produced in hamsters by endotracheal bleomycin. Am Rev Respir Dis 1978, 117:10991108 27. Myrvic QN, Leake ES, Fariss B: Studies on pulmonary alveolar macrophages from the normal rabbit: A technique to procure them in a high state of purity. J Immunol 1961, 86:128-132 28. Reynolds HY, Newball HH: Analysis of proteins and respiratory cells obtained from human lungs by bronchial lavage. J Lab Clin Med 1974,84:559-573 29. Conlon PJ, Henney CS, Gillis S: Cytokine-dependent thymocyte response: Characterization of IL-I and IL-2 target subpopulations and mechanism of action. J Immunol 1982, 128:797-801 30. Reisner Y, Linker-Isreli M, Sharon N: Separation of mouse thymocytes into two subpopulations by the use of peanut agglutinin. Cell Immunol 1976,25:129-134 31. Kaelin RM, Center DM, Bernardo J, Grant M, Snider LG: The role of macrophage-derived chemoattractant activities in the early inflammatory events of bleomycin-induced pulmonary injury. Am Rev Respir Dis 1983, 128:132-137 32. Hunninghake GW, Gadek JE, Fales HM, Crystal RG: Human alveolar macrophage-derived chemotactic factor for neutrophil. J Clin Invest 1980,66:473-483 33. Ross R, Nist C, Kariya B, Rivest MJ, Raines E, Callis J: Physiological quiescence in plasma-derived serum: Influence of platelet-derived growth factor on cell growth in culture. J Cell Physiol 1978,97:497-508 34. Gritter HL, Adamson YR, King GM: Modulation of fibroblast activity by normal and silica-exposed alveolar macrophages. J Pathol 1986, 148:263-271

Acknowledgment The authors thank Drs. Shigemi Fuyama and Youichi Fujii, The First Department of Pathology, Yamagata University School of Medicine, for their critical comments, and Dr. Takao Yamashita, The Department of Parasitology, Yamagata University School of Medicine, for donating standard IL-1, and Shinobu Sato, The First Department of Internal Medicine, Yamagata University School of Medicine, for his critical comments.