[総説］The roles of alveolar type II epithelial cells in acute lung injury and repair: 沖縄地域学リポジトリ

Title

[総説］The roles of alveolar type II epithelial cells in acute

_{lung injury and repair}

Author(s)

Sugahara, Kazuhiro; Nakamura, Seiya; Teruya, Kohji

Citation

琉球医学会誌 = Ryukyu Medical Journal, 20(4): 161-166

Issue Date

2001

URL

http://hdl.handle.net/20.500.12001/3452

The roles of alveolar type II epithelial cells in acute lung injury and repair

Kazuhiro Sugahara, Seiya Nakamura and Kohji Teruya

Department of Anesthesiology,

University of the Ryukyus Faculty of Medicine

207 Uehara, Nishihara, Okinawa 903-0215, Japan

ABSTRACT

Regeneration of alveolar epithelial cells is one of the important repair processes in many types of lung injury. Alveolar type II cells have two major functions, one is to serve as the site of surfactant production and the second is to serve as a progenitor cell for

alveo-lar type I cells, particualveo-larly during re-epithelialization after lung injury. We have examined

effect of several soluble factors on cultured alveolar type II ce】Is in vitro, and also examined the sequential analysis of histopathology and gene expressions of surfactant proteins in al-veolar epithelial cells of rats with lipopolysaccharide (LPS主, bleomycin-, and/or KGF-treated lungs. We have found that KGF is a specific stimulator for alveolar epithelial cells, and KGF instillation prevents bleomycin induced lung injury. KGF may play an important role in maintaining alveolar epithelium and repairing the damaged epithelium after injury. This review will give an overview of the roles of alveolar type II cells in the repairing

proc-esses of acute lung injury and fibrosis. Ryukyu Med. J., 20(4)16ト166, 2001

Key words: acute lung injury, surfactant protein, wound healing, alveolar epithelial cells, in situ hybridization

INTRODUCTION

Though the lung is the organ which carries out the gas exchange, it is in the anatomically and physiologi-cally unique position where it meets air and the whole blood. One of the primary functions of the lung is to provide a large expanse of thin membrane between air and blood, across which efficient exchange of oxygen and carbon dioxide can occur. The lung is also increas-ingly recognized as a complex organ, with metabolic, vasoregulatory, and immunologic functions. The lung's alveolar surface and capillary bed is vast, -300,000,000 alveoli, each supplied by 1,000 capillary segments, form-ing an alveolar surface area of 100-150 m

Though more than 40 cell types exists in the lung, the alveolar epithelium is composed of only two cell types of epithelial cells, alveolar type I cells and alveo-lar type II cells. The majority of the surface area, more than 90% of the alveolus is covered by the squamous alveolar type I cell which is apparently an extremely thm cell and very susceptible to injury. The other cell type is the cuboidal cell, alveolar type II cell, which comprises about 15 % of the cells in the distal lung and is known primarily for its ability to synthe-size, storage, secrete and recycle pulmonary surfactant. Other important functions of alveolar type II cells are a) to serve as a progenitor cell for alveolar type I cells,

particularly during re-epithelia】ization of the alveolus after lung injury, b) to transport sodium from the veolar space into the interstitium so as to minimize al-veolar fluid and thereby to maximize gas exchange, and c) to modify the inflammatory response by

secret-ing a variety of growth factors and cytokines.

SURFACTANT PROTEINS

Pulmonary surfactant, produced by alveolar type II cells, consists of a complex of 90 % lipids and 10 proteins. Specific four pulmonary proteins such as sur-factant protein (SP)-A, SP-B, SP-C and SP-D have been identified (Table 1) and are thought to be important, not only for surfactant function, but also for the host defense of the lung. The hydrophilic surfactant proteins consist of SP-A and SP-D, which are water soluble and collagenous glycoproteins. Both are calcium-dependent lectins and are important components of antibody-independent pulmonary host defense system or innate immunity. More attention has been placed on the roles of SP-A and SP-D as a host defense molecule and their interactions with pathogen and phagocytic cells. In ad-dition, both molecules have been measured in the sys-temic circulation and may be useful biomarkers of human lung disease. The hydrophobic proteins are designated SP-B and SP-C, and are soluble in chloroform/

162 _{The roles of alveolar type II epithelial cells in acute lung injury and repair}

Table 1 Functions of surfactant proteins

Function SP-A SP-B SP-C SP-D Contribute to biophysical properties of surfactant

Inhibition of surfactant secretion

Facilitate turnover of surfactant phosphohpids Facilitate phagocytosis of opsomzed particles

+    +    +

十    一    一

+   +   +

十   一   一    +

methanol. These proteins play important roles in the formation and recycling of the surfactant monolayer. Especially, both are essential for surfactant activity.

Isolation and culture of alveolar type II cells Since alveolar type II cells comprise only 15 % of the cells of the distal lung, investigation of the bio-chemical basis for their function requires a means of isolating alveolar type II cells from other lung cells (Fig. 1). Alveolar type工I cells can be identified by a variety of techniques, such as (a) the modified Papanicolaou stain (Fig. 2), (b) alkaline phosphatase,

(c) tannic acid staining, (d) immunocytochemistry, and (e) electron microscopy to recognize the presence of

lamellar bodies. When alveolar type II cells adhere to

plastic in primary culture, they rapidly spread and lose their ability to synthesize and secrete pulmonary sur-factant. They appear to need to maintain their cuboidal shape, which can be maintained by culturing alveolar type II cells on extracellular matrix.

Transepithehal transport

Alveolar type II cells have the ability to transport sodium from the alveolar to the basolateral space, and chloride and water follow passively. Transport is stimulated by agents that increase cyclic AMP and is blocked by amiloride, Na channel blocker and ouabain, a Na-K-ATPase inhibitor4' The fluid transport has been estimated to be 1.5 L/day in humans. This fluid clearance from the alveolar space is critical in the reso-lution of inflammatory lung injury, and alveolar type II cells may have an important role in this process.

Alveolar type II cells and acute lung injury Regeneration of alveolar epithelial cells is one of the important repair processes in many types of lung

injury. We hypothesize that stimulation of alveolar type

II cells with cell proliferation, surfactant production and transport of alveolar fluid absorption may prevent

lungユnjury and promote lung healing. The pulmonary

responses to acute lung injury and the subsequent repair process can be studied in the following stages; first, an early exudative phase and then prohferative phase, fol-lowed finally by a diffuse fibrotic phase. To identify

Isolation of alveola「type II cells

二

trypsin, elastase, collagenase

2) separation from other cells differential sedimentation

(density gradient) centrifugal e山triation ・引ow cytometry

3) differential attachment panning : IgG-coated dish

4) identification of type II cells morphologiesI bi°chemical

surfactan川ipid, p「ote叫

Fig. 1 Schema of a method of isolation of alveolar type II

cells.

e

Fig. 2 Cultured alveolar type II cells from rat lungs. There are many lamellar inclusion bodies in the cytoplasm around nucleus.

●●

噂

m

mm

・・ ・・ mm mm 無間

・・ mm

,5;

5旨

Lヱ__ユ

轡夢β-actir

5 品

」当 CD

6hr  12hr   24hr   48hr

Fig. 3 Time course of the response of cultured alveolar type II cells to KGF. Cells were cultured for the first 4 days with control medium or medium containing 10 ng/ml KGF. Levels of mRNAs for both SP-A and SP-B were increased after 6 h treatment with KGF, and continued through 12 and 24 h, at which point they appeared maximal (Sugahara et al. 7 .

an important stimulating factor on alveolar type II cells, we have examined the several soluble factors on DNA synthesis and surfactant protein gene expression in primary cultures of alveolar type II cells on Engelbreth-Holm-Swarm (EHS) matrix. Furthermore, to identify the differences of the repair process among lung- injuries, we have examined sequential analysis of histopathology and gene expressions of surfactant proteins in alveolar epithelial cells of rats with lipopolysaccharide (LPS)-, bleomycin-, and/or keratinocyte growth factor (KGF)- treated lungs.

Proliferation of alveolar type II cells and growth fac-tors in vitro: In the in vitro experiments, alveolar type II cells were isolated from the lungs of specific patho-gen free adult male Sprague-Dawley rats with elastase, and a gradient centrifugation. The cells were cultured and soluble factors were tested over a given day.

In the in vitro experiments, we have found that the interaction of soluble factors and extracellular ma-trix components has a strong impact on alveolar type II cell proliferation . Among a panel of hormones, growth factors and cytokines, keratinocyte growth fac-tor (KGF) induced a strong stimulation of SP-A and SP-B mRNAs, with both dose and time dependent man-ners (Fig. 3)7'

Proliferation of alveolar type II cells and surfactant proteins in vivo: In the in vivo experiments, adult rats were anesthetized and a small dose of LPS, bleomycin, KGF or saline was injected intratracheally. After a given period, rat lungs were processed for histology, immunohistochemistry, in situ hybridization and

wv

^畢華華._:貰

Iff

叫細

きi--.

i-;寺津ヰ

Fig.4 Light micrographs (a, b) and immunohistochemical stainings for SP-A (c, d) in the control rat (a, c) and a LPS-administered rat (b, d) 4 days after LPS treatment. Note the increased alveolar type II cells with a number of dark brown immunoreactive products of SP-A (d) (Sugahara et al. (8)). Bar - 20〝m.

Northern blot analysis.

In a small dose of LPS-treated lungs, the alveolar septa in the lobar lesions were trucking with the in-creased number of alveolar type II cells and SP-A production (Fig. 4), which correlated well with overexpression of SP-A, SP-B and SP-C mRNAs (Fig. 5). These changes gradually decreased, and lung struc-ture was returned to almost normal 2 to 4 weeks after LPS treatment , which suggested that proliferation and differentiation of alveolar epithelial cells may play important roles in the repair process of the damaged alveoli after acute lung injury.

In bleomycm-treated lungs, there was increased accumulation of the proteins and mRNA expressions of collagen types I and III (Fig. 6), and the decreased expression of surfactant protein mRNAs in the hbrotic

lesions .

In KGF-treated lungs, alveolar cells with immunoreactive products of SP-A and l〕rdU-positive nu-clei were apparently increased in the lungs 2-3 days after intratracheal instillation (Fig. 7)9

Taken from these results, marked proliferation of alveolar type II cells with increased surfactant protein secretion may promote the repair process after lung in-jury. Therefore, we hypothesized KGF would prevent

bleomycin-induced lung fibrosis, and examined this

hy-pothesis. We have demonstrated the KGF treatment 48

h before and 24 h after bleomycin instillation appar-ently prevented the loss of body weight and the reduc-tion in total lung capacity (TLC) due to bleomycin (Fig. 8) and markedly attenuated the accumulation of

164 The roles of alveolar type II epithelial cells in acute lung injury and repair *1 SB ;. ヽ∴ -卜 1 _ い l . L L H 1

tや*/- -I

*>

V

_&

*

:

t

-孟

¥産

Fig. 5 Autoradiographs of in situ hybridization of SP-A (a, b), SP-B (c, d) and SP-C (e, f) mRNAs in alveolar epithe-lium from a control (a, c, e) and a LPS-administered rat (b, d, f) 4 days after instillation. Note a large number of silver grains for SP-A, SP-B and SP-C mRNAs over alveo-lar type II cells in the LPS-administered lung (Sugahara et al. (8)). Bar - 20/xm.

proteins and mRNA expressions of collagen types I and III (Fig. 6)9). Further, KGF treatment amelio-rated the decreased expression of SP-A, SP-B and SP-C mRNAs in the fibrotic lesions of the bleomycin-treated lungs91.

KGF, which was first purified from the M426

human embryonic lung fibroblast cell line , has been demonstrated to increase DNA synthesis in alveolar type II cells in vitro and to stimulate the prolifera-tion of these cells in vivo . It has been demonstrated that KGF is found only in the mesenchymal cells, and that KGF receptor mRNA is restricted to the epithelial cells in the developing lung . Recently KGF and KGF receptor in the lung have been shown to exhibit severe retardation of the lung growth and to develop pulmo-nary malformationl

Taken from our results and these reports, KGF may play an important role in maintaining alveolar epithelium and in repairing the damaged epithelium after lung injury, and provide new therapeutic vehicles for acute lung injury.

、

Y

′

.

_.P

_、

_′

_L

_h

Fig.6 Immunohistochemical staining using anti-collagen types III (a, e, g) and I (c), and autoradiographs of in situ hybridization of collagen types III (b, f, h) and I (d) mRNAs in paraffin sections of the lungs in a control rat (a, b), a b】eomycin-treated rat (c, d, e, f), and a KGF/bleomycin-treated rat (g, h). Note the 】ack of accu-mulation of collagen type III (g) and type III mRNA (h) in the KGF/bleomycin-treated rat lungs (Sugahara et al. (9)). Bars - 20〝m.

Role of alveolar epithelial cells in repair of inflamma,tory injury

Replacement of damaged alveolar epithelial cells is an important component of successful lung repair. Al-veolar type II cells serve as the progenitor cell for the alveolar surface, and repair of denuded basement mem-brane. Alveolar type II cells also have active transport to remove excess fluid from the alveolar space, which may promote edema clearance.

More recently alveolar type II cells synthesize and

secrete a variety of cytokines, growth factors and other peptide mediators. There are GM-CSF, TGF-fl, MCP-1, RANTES, IL3, IL-8 and complement components (C2, C4, C3, C5). Alveolar epithelial cells also generate arachidonic acid metabolites (PGE2, PGI2) that can modulate pulmonary inflammation. Furthermore, alveolar type II cells express class II major histocompatibihty molecules on the surface, which may influence mflam-matory reactions by modulating the activity of immune

Fig.7 Immunohistochemical staining for SP-A in the lungs from a control rat (a), a KGF-treated rat 1 day (b) and 3 days (c) after KGF instillation. Note the proliferation of alveolar type II cells containing strongly immunoreactive products of SP-A (b, c) (Sugahara et al. (9)). Bar - 20 jiim.

cells. Thus, alveolar epithelial cells may play a critical role in defining the inflammatory environment within the lung by contributing to the initiation, augmenta-tion, resolution and repair of inflammatory processes.

ACKNOWLEDGEMENTS

This work was supported in part by Mitsui Life

Welfare Foundation, and by the Grants-in Aid for

entific Research from the Ministry of Education,

Sci-ence, Sports and Culture, Japan.

REFERENCES

1 ) Langleben D. and Jones R.C. Organization and

role of lung cells : Anesthesia: Biologic foundations

(Yaksh T. et al), ppl281-1301.

2) Mason R. J. and Shannon J.M. Alveolar type II

cells. : The lung: Scientific foundations, 2nd edn.,

Lippincott-Raven Publishers, Philadelphia,

pp543-555, 1997.

3 ) Kuroki Y. and Voelker D.R. Pulmonary surfactant

proteins. J Biol Chem 269: 25943-25946, 1994.

4) Cott G.R., Sugahara K. and Mason R.J.

Stimula-tion of net active ion transport across alveolar type II monolayers. Am J Physiol 250: C222-C227, 1986. 5) Brigham K. L. and Meyrick B. Endotoxin and lung injury. Am Rev Respir Dis 133: 913-927, 1986. 6) Sugahara K., Mason R.J. and Shannon J.M. Ef-fects of soluble factors and extracellular matrix on DNA synthesis and surfactant gene expression in primary cultures of rat alveolar type II cells. Cell Tissue Res 291: 295-303, 1998.

7) Sugahara K., Rubin J.S., Mason R.J., Aronsen E.L.

Fig.8 Macroscopic photographs of the lungs and light micrographs with Azan stain of the vertical section of the whole lung in a bleomycin-treated rat (a, c) and a KGF/bleomycin-treated rat (b, d) 7 days after the treat-merit. Note the almost normal appearance of the lungs in the KGF/bleomycin-treated rat (b, d), compared to the bleomycin-treated rat lungs with many patchy bleeding spots (a), and the shrunken lungs with fibrosis (c) (Sugahara et al. (9)). Bars - 5 J皿

and Shannon J.M. Keratinocyte growth factor in-creases mRNAs for SP-A and SP-B in adult rat al-veolar type II cells in culture. Am J Physiol 269: L344-L350, 1995.

8) Sugahara K., Iyama K., Sano KH Kuroki Y., Akino T. and Matsumoto M.: Overexpression of surfac-tant protein SP-A, SP-B, and SP-C mRNA in rat lungs with lipopolysaccharide-induced injury. Lab Invest 74: 209-220, 1996.

9) Sugahara K., Iyama K., Kuroda M.J. and Sano K. Double intratracheal instillation of keratmocyte growth factor prevents bleomycin-induced lung fi-brosis in rats. J Pathol 186: 90-98, 1998.

10) Rubin J. S., Osada H., Finch P.W.,, Taylor W.G.,

Rudikoff S. and Aaronson P.A. Purification and

characterization of a newly identified growth factor

specific for epithelial cells. Proc Natl Acad Sci

DNA 86: 802-806, 1989.

ll) Panos R. J., Bak P.M., Simonet W.S., Rubin J.S.

and Smith L.J. Intratracheal instillation of keratinocyte growth factor decreases hyperoxia-induced mortality in rats. J Clin Invest 96:

2026-166 The roles of alveolar type II epithelial cells in acute 一ung injury and repair

2033, 1995.

12) Mason I.J., Fuller-Pace F., Smith R. and Dickson C. FGF-7 (keratinocyte growth factor) expression during mouse development suggests roles in myogenesis, forebrain regionalisation and epithelial

mesenchymal interactions. Mech Dev 45: 15-30, 1994.

13) Urtreger A. 0. Development localization of the

splicing alternatives of fibroblast growth factor

receptor-2 (FGFR2). Dev Biol 158: 475-486, 1993.

14) Peters K., Werner S., X. Liao X., Wert S., Whitsett

J. and William L. Targeted expression of a domi-nant negative FGF receptor blocks branching morphogenesis and epithelial differentiation of the

mouse lung. EMBO J 13: 3296-3301, 1994.

15) Simonet W. S., DeRose M.L., Bucay N., Nguyen

H.Q., Wert S.E., Zhou L., Ulich T.R., Thomason A.,

Danilenko D.M., Whitsett J.A. Pulmonary