Effects of eosinophil granule major basic protein on phosphatidylcholine secretion in rat type II pneumocytes

Manabu Okumura1,2, Hirofumi Kai2, Shinya Shinozawa1, Yoichiro Isohama2, and Takeshi Miyata2

1 Department of Pharmacy, Miyazaki Medical College, Miyazaki 889-1692; and 2 Department of Pharmacological Sciences, Faculty of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Eosinophils are involved in inflammatory diseases such as asthma. We previously reported that activated eosinophils increased the phosphatidylcholine (PC) secretion in primary cultures of rat type II pneumocytes. Increased PC secretion was confirmed to be partly mediated by superoxide anions released from activated eosinophils. However, the influence of eosinophil granule proteins on PC secretion is unknown at present. In this study, we determined whether eosinophil major basic protein (MBP) influences PC secretion. MBP dose dependently increased the PC secretion in rat type II pneumocytes without producing any cell damage. The MBP-induced increase in PC secretion was significantly reduced by preadministration of either H-7, a protein kinase inhibitor, or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, a chelator of intracellular Ca2+, but not by H-89, a protein kinase inhibitor. Our results suggest that the MBP-induced increase in PC secretion may provide mechanical stability and protect against lung atelectasis.

asthma; surfactant


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EOSINOPHILS PLAY an important pathological role in allergic diseases such as asthma, and examination of bronchoalveolar lavage fluid of asthmatic patients usually demonstrates a significant increase in eosinophil count (19, 29). Eosinophils release several particles including leukotrienes; platelet-activating factor; various oxygen-derived toxic metabolites such as superoxide anions, H2O2, and hydroxyl radicals (28); and toxic cationic proteins such as major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil peroxidase (EPO) and eosinophil-derived neurotoxin (EDN) (9, 11). The cytotoxic proteins and other mediators cause hyperreactivity of respiratory smooth muscles (7) as well as desquamation of and damage to respiratory epithelial cells including type II pneumocytes.

Type II pneumocytes produce lung surfactant to reduce the surface tension of the alveolar air-liquid interface, thereby providing mechanical stability and preventing alveolar atelectasis (2). Hence examination of the influence of cytotoxic proteins and other mediators released from eosinophils on the secretion of lung surfactant is important for our understanding of normal lung physiology as well as of certain pathological pulmonary conditions. Previous studies (2, 15, 21) have shown an increased secretion of phosphatidylcholine (PC), the predominant component of pulmonary surfactant, by a variety of physiological and pharmacological agents. Furthermore, a recent study by Okumura et al. (22) showed that activated eosinophils increase PC secretion in primary cultures of rat type II pneumocytes. Such an increase was not suppressed by ONO-1078, a selective antagonist of peptide leukotrienes, or TCV-309, an antagonist of platelet-activating factor, but was suppressed by a combination of superoxide dismutase and catalase. However, our results also showed that the combined use of both enzymes did not completely inhibit the secretion of PC. These results suggested that increased PC secretion was partly mediated by superoxide anions released from activated eosinophils and might represent one facet of the defense mechanisms aimed at attenuating cellular damage induced by superoxide anions.

Superoxide anions released from eosinophils participate in the early asthmatic reaction, whereas the eosinophil granule proteins participate in the late asthmatic reactions (LAR). Eosinophil granule proteins increase the secretion of histamine by basophils and mast cells (30) and the generation of superoxide anion by lung macrophages (14). However, to our knowledge, the effect of eosinophil granule proteins on the secretion of lung surfactant has not been previously reported.

Based on the above findings and the results of the previous study by Okumura et al. (22) demonstrating failure of the combined use of superoxide dismutase and catalase to completely inhibit the activated eosinophil-induced increase in PC secretion, we hypothesized in the present study that eosinophil granule proteins might increase the secretion of lung surfactant in patients with asthma during LAR. To test this hypothesis, we examined the effects of MBP, a primary constituent of eosinophil granule proteins, on PC secretion in primary cultures of rat type II pneumocytes.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and chemicals. Rats and guinea pigs were purchased from Kyudo Farm (Fukuoka, Japan), tissue culture medium was from Nissui Pharmaceutical (Tokyo, Japan), and fetal bovine serum was from JRH Bioscience (Lenexa, KS). [Methyl-3H]choline and Aquasol II were obtained from NEN Research Products (Boston, MA). Sephadex G-50 was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). H-7, H-89, and HA-1004 were purchased from Seikagaku (Tokyo, Japan). 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM was from Wako Pure Chemical Industries (Osaka, Japan). Other reagents and biochemicals were purchased from Sigma (St. Louis, MO). The experimental protocol was approved by the Ethics Review Committees for Animal Research of Miyazaki Medical College (Miyazaki, Japan) and the Faculty of Pharmaceutical Sciences, Kumamoto University (Kumamoto, Japan).

Primary cultures of rat type II pneumocytes. Type II pneumocytes were isolated from the lungs of adult specific pathogen-free male Wistar rats (body weight 180-200 g) according to the method of Dobbs et al. (4). This method yields ~107 cells/rat. Cells were suspended at 106 cells/ml in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 74 kBq/ml of [methyl-3H]choline (specific activity 3.0 TBq/mmol), 100 U/ml of penicillin, and 100 µg/ml of streptomycin; plated on 24-well tissue culture plates (Falcon); and then cultured at 37°C in 5% CO2-air for 18 h. Nonadherent cells were removed from the wells by washing before the assay. For cellular identification, the sample was stained with tannic acid-polychrome stain (17) and alkaline phosphatase stain (6). The purity of the type II pneumocytes was 95 ± 7% (SE; n = 8 cultures), and their viability was 97 ± 8% (n = 8 cultures) as confirmed by the trypan blue exclusion test.

Preparation of guinea pig MBP. Eosinophils were isolated from peritoneal exudates of Hartley guinea pigs by a modification of the method originally described by Pincus (24). Briefly, peritoneal eosinophil-rich exudates were produced by weekly intraperitoneal injections of 7,500 U/ml of polymyxin B sulfate for at least 8 wk. Furthermore, 50 ml of Hanks' balanced salt solution containing 20 U/ml of sodium heparin were injected intraperitoneally the day after the last injection of polymyxin B sulfate. The abdomen was massaged gently, and the peritoneal fluid was collected. The fluid was fractionated by centrifugation through solutions of Nycodenz. The purified fraction of eosinophils was collected from the interface between 1.088 and 1.098 g/ml in the gradient. Eosinophils were 96% pure as determined with Litt's stained smears. MBP was purified from isolated guinea pig eosinophils according to the procedure described by Gleich et al. (11). Briefly, suspensions of eosinophils in 0.34 M sucrose were repeatedly pipetted, then centrifuged for 10 min at 400 g at 4°C to remove unbroken cells. The opalescent supernatants were transferred to another tube and centrifuged at 25,000 g for 20 min at 4°C. The pellet was solubilized in 0.02 M acetate buffer, pH 4.3, in 0.01 M HCl and analyzed by gel filtration on 1.2 × 45-cm columns of Sephadex G-50. The purified MBP was homogeneous as confirmed by the presence of a single electrophoretic protein band on SDS-PAGE gels as described previously (10; data not shown). The concentration of MBP was determined as described previously (10, 12) and was adjusted to 100-fold of the indicated final concentration with sodium acetate buffer. Sodium acetate buffer did not alter spontaneous PC secretion.

Metabolic labeling of PC and treatment of cultures. Secretion of PC by cultured type II pneumocytes was determined as follows. The cells were rinsed with fresh serum- and antibiotic-free medium to remove [methyl-3H]choline and unattached cells. MBP was added 30 min after the rinse, followed by incubation for 90 min. At the end of the incubation period, the medium was aspirated, the cells were lysed with 2 ml of ice-cold 0.05% Triton X-100 solution, and the lipids were extracted from both the cells and medium with chloroform-methanol (2:1 vol/vol). PC was separated from other phospholipids by thin-layer chromatography (18), and its radioactivity was measured with a liquid scintillation counter after the addition of 5 ml of Aquasol II to each sample. Secretion is expressed as the amount of [3H]PC in the medium after a 90-min incubation as a percentage of that in the cells plus medium.

Detection of cell membrane damage. The presence or absence of cytoplasmic leakage due to cell membrane damage after MBP treatment was determined by measuring lactate dehydrogenase (LDH) activity in the culture medium with a commercial LDH assay kit (Nippon Shoji). LDH activity released into the medium did not exceed 1% of the total cell content in all experiments (data not shown).

Statistical analysis. The concentration of PC is expressed as mean ± SE. Differences among groups were assessed by Duncan's multiple range test (a nonparametric test). P < 0.05 denoted the presence of a significant difference.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation of eosinophil granule proteins and their effect on PC secretion. Similar to a previous study on eosinophil granule proteins of guinea pigs (30), suspensions containing eosinophil granule proteins (EPO, EDN, ECP, and MBP) showed three separate peak fractions by gel filtration on columns of Sephadex G-50 when each fraction was monitored for changes in absorbance at 277 nm (Fig. 1, top). The first peak (G2) corresponded to the void volume of the column and contained a variety of bands as observed on SDS-PAGE gels. The second peak (G3) contained two slightly separated bands, whereas the third peak (G4) contained one major band with a molecular weight < 14,000. These results indicated that the third peak contained only MBP.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 1.   Top: fraction profile of eosinophil granule protein by gel filtration on columns of Sephadex G-50. Each fraction was monitored for changes in absorbance at 277 nm. G1, fraction preceding void volume; G2, eosinophil peroxidase (EPO) fraction; G3, eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) fraction; G4, major basic protein (MBP). Bottom: effects of each fraction on PC secretion in primary cultures of rat type II pneumocytes. Each fraction was ultrafiltered, concentrated, and resuspended at a granule concentration corresponding to 1 × 106 eosinophils/ml culture medium. Values are means ± SE expressed as amount of [3H]phosphatidylcholine (PC) in medium after 90-min incubation as percentage of that in cells plus medium (% of control) from 6 experiments. [3H]PC secretion after 90 min was 0.75 ± 0.07% in control cultures (n = 5). Significant difference from control value: * P < 0.05; ** P < 0.01.

Next, we examined the effects of various peak fractions, including the fraction preceding the void volume (G1), on PC secretion in primary cultures of rat type II pneumocytes. Each fraction was ultrafiltered, concentrated, and resuspended at a granule concentration corresponding to 1 × 106 eosinophils/ml culture medium. G1 and G3 fractions did not increase PC secretion; in contrast, G2 and G4 fractions significantly increased PC secretion by 21 and 54%, respectively (Fig. 1, bottom).

Effects of MBP on PC secretion. MBP caused an ~1.5-fold increase in PC secretion in primary cultures of rat type II pneumocytes without increasing LDH activity in the culture medium. The MBP-induced increase of PC secretion was concentration dependent, and the peak secretion was observed at an MBP concentration of 8 × 10-9 M (Fig. 2). Furthermore, examination of the kinetics of PC secretion showed that it commenced within 5 min of the addition of MBP (8 × 10-9 M) and then increased progressively throughout the observation period (90 min; Fig. 3). The profile was not different from that of the control culture. In contrast, the addition of terbutaline (1 × 10-6 M), which was used as protein kinase A-related agent, resulted in a steep increase in the first 30 min, but this was followed by a small increase throughout the remaining 60 min (Fig. 3).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2.   MBP concentration-dependent secretion of PC from type II pneumocytes. Isolated pneumocytes were incubated with indicated concentrations of MBP for 90 min. , Value for terbutaline (1 × 10-6 M; positive control). Values are means ± SE from 6 experiments. [3H]PC secretion after 90 min was 0.51 ± 0.04% in control cultures (not incubated with MBP; n = 5).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 3.   Kinetics of MBP-stimulated PC secretion in type II pneumocytes. open circle , Control culture; , MBP (8 × 10-9 M); triangle , terbutaline (1 × 10-6 M). Values are means ± SE from 6 experiments. [3H]PC secretion after 90 min was 0.89 ± 0.03% in control cultures (n = 5).

Effects of several inhibitors on MBP-induced increase in PC secretion. To determine the mechanism involved in MBP-induced increase in PC secretion, we examined the effects of several inhibitors of intracellular pathways. In these experiments, the inhibitor was added 10 min before the application of 8 × 10-9 M MBP. H-7 (1 × 10-5 M), a protein kinase C inhibitor, and BAPTA-AM (5 × 10-6 M), a chelator of intracellular Ca2+, significantly suppressed the MBP-induced increase in PC secretion. However, no synergistic effect was noted when the two inhibitors were added simultaneously. H-89 (6 × 10-6 M), a protein kinase A inhibitor, and HA-1004 (1 × 10-5 M), a control to H-7, did not influence the MBP-induced increase in PC secretion.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The major finding of the present study was that MBP purified from guinea pig eosinophil granules increased PC secretion in primary cultures of rat type II pneumocytes and that such increases were MBP concentration dependent (Fig. 2). Furthermore, we also demonstrated that MBP-stimulated PC secretion was significantly inhibited by H-7, a protein kinase inhibitor, and BAPTA-AM, an intracellular Ca2+ chelator (Fig. 4).


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 4.   Effects of several inhibitors on MBP-induced PC secretion in type II pneumocytes. Each inhibitor was added 10 min before addition of MBP (8 × 10-9 M), followed by further incubation for 90 min. H-89 (6 × 10-6 M), HA-1004 (1 × 10-5 M), H-7 (1 × 10-5 M), and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM (5 × 10-6 M) were used as inhibitors of PC secretion pathways. -, In presence of MBP alone. Values are means ± SE from 6 experiments. [3H]PC secretion after 90 min was 0.88 ± 0.06% in control cultures (n = 5). * Significant difference from MBP alone, P < 0.05.

Eosinophils infiltrate the airways and lungs of asthmatic patients and release many granule proteins, leukotrienes, platelet-activating factor, and various oxygen-derived toxic metabolites. The direct involvement of these substances on lung surfactant has been reported (8, 25). However, the effect of the eosinophil itself on the secretion of surfactant is poorly understood. In a previous study by Okumura et al. (22), activated eosinophils were found to increase PC secretion in primary cultures of rat type II pneumocytes, which was partly mediated by superoxide anion released from these cells. However, we speculated that the granule proteins, in addition to superoxide anions, were probably involved in the increased PC secretion because such secretion was not completely inhibited by pretreatment with superoxide dismutase combined with catalase. To our knowledge, the possible involvement of eosinophil granule proteins on PC secretion has not been previously investigated.

Previous studies (13, 20) have shown that MBP causes desquamation of and damage to respiratory epithelial cells. Furthermore, MBP has been reported to stimulate histamine release from human basophils and rat mast cells (30). Histamine increases the surfactant secretion by a receptor-mediated process (3). Histamine release is an important pathophysiological reaction in asthma, and the increased surfactant secretion induced by histamine may be one of the protective reactions during the early asthmatic reaction. However, our results showed that the concentration of MBP necessary to increase PC secretion was far less than that of MBP to stimulate histamine release. Our results suggest that secretion of lung surfactant may be increased in asthmatic patients by granule proteins released from eosinophils during the early stages of the LAR. Such an increase in pulmonary surfactant secretion may serve as a protective mechanism against cellular damage caused by cytotoxic granule proteins.

MBP is the primary constituent of eosinophil granules (10, 12) and accounts for >50% of the granule proteins in the guinea pig (11, 16). Furthermore, previous studies have shown high serum concentrations of MBP in patients with eosinophilia (1) and in the sputum and bronchoalveolar lavage fluid of asthmatic patients (5, 29). Based on these early findings, we examined, in the present study, the specific effects of MBP. In addition to MBP, eosinophil granules contain three other major cationic proteins, EPO, EDN, and ECP, that have been purified from peritoneal exudates in the guinea pig. In this study, these granule proteins were separated from each other by Sephadex G-50 as described in a previous study (30). The first protein peak contained EPO, and the second peak contained EDN and ECP. The pooled fraction containing EPO increased PC secretion significantly, but the concentration of EPO was lower than that of MBP. In contrast, the pooled fraction containing EDN and ECP did not increase PC secretion. These results suggested that in addition to MBP, EPO may also participate in increasing PC secretion induced by eosinophils in primary cultures of rat type II pneumocytes. However, taking into consideration the relatively low amount of EPO, our results show that the majority of secreted PC was mediated to a large extent by MBP.

PC secretion from type II pneumocytes is regulated via various intracellular pathways (2, 15, 21, 23). The principal secretion pathway is the activation of a cAMP-dependent protein kinase, protein kinase C, and high concentrations of intracellular Ca2+. In basophils, MBP stimulates histamine release, which is inhibited by pertussis toxin (27). Also, in mast cells, histamine release is induced by protein kinase C activation by diacylglycerol through a pertussis toxin-sensitive G protein. In type II pneumocytes, H-7, but not HA-1004, significantly but partly inhibited the MBP-increased PC secretion. H-7 is a relatively selective protein kinase C inhibitor, but it also exerts a slight inhibitory effect on protein kinase A and protein kinase G as well as on Ca2+/calmodulin-dependent kinase. In contrast, the HA-1004 dose not have any inhibitory effect on protein kinase C, although it is inhibitory of other kinases, with a potency equivalent to that of H-7. Therefore, MBP may stimulate PC secretion as well as histamine release in basophils and mast cells through protein kinase C activation. In support of this, the kinetics of MBP-stimulated PC secretion (Fig. 3) is similar to that of 1-oleoyl-2-acetyl-sn-glycerol-stimulated PC secretion previously reported (26). BAPTA-AM also significantly inhibited PC secretion increased by MBP, suggesting that intracellular Ca2+ plays an important role in MBP stimulation. The finding that BAPTA-AM in combination with H-7 did not synergistically or additively inhibit MBP-induced PC secretion (Fig. 4) suggests that intracellular Ca2+ and protein kinase C may act on the same signaling pathway of MBP stimulation of PC secretion. Furthermore, incomplete inhibition of PC secretion by BAPTA-AM and H-7 suggests that other pathways may be involved in MBP-induced PC secretion, although further studies with higher concentrations of H-7 and other protein kinase C inhibitors are needed.

In conclusion, our results showed that MBP, the primary constituent of eosinophil granule proteins, increased PC secretion in primary cultures of rat type II pneumocytes. Such an effect of eosinophils on PC, the predominant component of pulmonary surfactant, may provide mechanical stability and prevent lung atelectasis in asthmatic patients during early LAR. Our results also showed that the MBP-induced increase in PC secretion was mediated, at least in part, by protein kinase C and intracellular Ca2+. Further studies are necessary to identify other pathways that regulate PC secretion.


    ACKNOWLEDGEMENTS

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: T. Miyata, Dept. of Pharmacological Sciences, Faculty of Pharmaceutical Sciences, Kumamoto Univ., 5-1 Oe-Honmachi, Kumamoto 862, Japan. (E-mail: tmiyata{at}gpo.kumamoto-u.ac.jp).

Received 25 August 1998; accepted in final form 19 January 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Butterfield, J. H., K. M. Leiferman, and G. J. Gleich. Nodules, eosinophilia, rheumatism, dermatitis and swelling (NERDS): a novel eosinophilic disorder. Clin. Exp. Allergy 23: 542-547, 1993[Medline].

2.   Chander, A., and A. B. Fisher. Regulation of lung surfactant secretion. Am. J. Physiol. 258 (Lung Cell. Mol. Physiol. 2): L241-L253, 1990[Abstract/Free Full Text].

3.   Chen, M., and L. A. Brown. Histamine stimulation of surfactant secretion from rat type II pneumocytes. Am. J. Physiol. 258 (Lung Cell. Mol. Physiol. 2): L195-L200, 1990[Abstract/Free Full Text].

4.   Dobbs, L. G., R. Gonzalez, and M. C. Williams. An improved method for isolating type II cells in high yield and purity. Am. Rev. Respir. Dis. 134: 141-145, 1986[Medline].

5.   Dor, P. J., S. J. Ackerman, and G. J. Gleich. Charcot-Leyden crystal protein and eosinophil granule major basic protein in sputum of patients with respiratory diseases. Am. Rev. Respir. Dis. 130: 1072-1077, 1984[Medline].

6.   Edelson, J. D., J. M. Shannon, and R. J. Madon. Alkaline phosphatase: marker of alveolar type II cell differentiation. Am. Rev. Respir. Dis. 138: 1268-1275, 1988[Medline].

7.   Flavahan, N. A., N. R. Slifman, G. J. Gleich, and P. M. Vanhoutte. Human eosinophil major basic protein causes hyperreactivity of respiratory smooth muscle. Am. Rev. Respir. Dis. 138: 685-688, 1988[Medline].

8.   Gilfillan, A. M., and S. A. Rooney. Leukotrienes stimulate phosphatidylcholine secretion in cultured type II pneumocytes. Biochim. Biophys. Acta 876: 22-27, 1986[Medline].

9.   Gleich, G. J., and C. R. Adolphson. The eosinophilic leukocyte: structure and function. Adv. Immunol. 39: 177-253, 1986[Medline].

10.   Gleich, G. J., D. A. Loegering, F. Kueppers, S. P. Bajaj, and K. G. Mann. Physiochemical and biological properties of the major basic protein from guinea pig eosinophil granules. J. Exp. Med. 140: 313-332, 1974[Medline].

11.   Gleich, G. J., D. A. Loegering, and J. E. Maldonado. Identification of a major basic protein in guinea pig eosinophil granules. J. Exp. Med. 137: 1459-1471, 1973[Medline].

12.   Gleich, G. J., D. A. Loegering, K. G. Mann, and J. E. Maldonado. Comparative properties of the Charcot-Leyden crystal protein and the major basic protein from human eosinophils. J. Clin. Invest. 57: 633-640, 1976[Medline].

13.   Gleich, G. J., S. Motojima, E. Frigasa, G. M. Kephart, T. Fujisawa, and L. P. Kravis. The eosinophilic leukocyte and the pathology of fatal bronchial asthma: evidence for pathologic heterogeneity. J. Allergy Clin. Immunol. 80: 412-415, 1987[Medline].

14.   John, A. R., H. Peter, and J. A. Steven. The effects of eosinophil-granule major basic protein on lung-macrophage superoxide anion generation. J. Allergy Clin. Immunol. 89: 746-752, 1992[Medline].

15.   Kai, H., Y. Isohama, K. Takaki, Y. Oda, K. Murahara, K. Takahama, and T. Miyata. Both beta 1- and beta 2-adrenoceptors are involved in mediating phosphatidylcholine secretion in rat type II pneumocyte cultures. Eur. J. Pharmacol. 212: 101-103, 1992[Medline].

16.   Lewis, D. M., J. C. Lewis, D. A. Loegering, and G. J. Gleich. Localization of the guinea pig eosinophil major basic protein to the core of the granule. J. Cell Biol. 77: 702-713, 1978[Abstract].

17.   Mason, R. J., S. R. Walker, B. A. Shields, J. E. Henson, and M. C. Williams. Identification of rat alveolar type II epithelial cells with a tannic acid and polychrome stain. Am. Rev. Respir. Dis. 131: 786-788, 1985[Medline].

18.   Miyata, T., H. Kai, K. Furusawa, H. Nakamura, M. Saito, Y. Okano, and K. Takahama. Secretomotor and mucolytic effects of mabuterol, a novel bronchodilator. Arch. Int. Pharmacodyn. Ther. 288: 147-160, 1987[Medline].

19.   Monchy, J. G. D., H. F. Kauffman, P. Venge, G. H. Koeter, H. M. Jansen, H. J. Sluiter, and K. Vries. Bronchoalveolar eosinophilia during allergen-induced late asthmatic reactions. Am. Rev. Respir. Dis. 131: 373-376, 1985[Medline].

20.   Motojima, S., E. Frigas, D. A. Loegering, and G. J. Gleich. Toxicity of eosinophil cationic proteins for guinea pig tracheal epithelium in vitro. Am. Rev. Respir. Dis. 139: 801-805, 1989[Medline].

21.   Okumura, M., M. Tsuruoka, Y. Isohama, H. Kai, K. Takahama, and T. Miyata. Effects of xanthine derivatives on phosphatidylcholine secretion in primary culture of rat type II pneumocytes. Jpn. J. Pharmacol. 67: 165-168, 1995[Medline].

22.   Okumura, M., M. Tsuruoka, Y. Isohama, H. Kai, K. Takahama, and T. Miyata. Activated eosinophils stimulate phosphatidylcholine secretion in primary culture of rat type II pneumocytes. Biochem. Mol. Biol. Int. 38: 569-575, 1996[Medline].

23.   Pian, M. S., G. L. G. Dobbs, and N. Duzgunes. Positive correlation between cytosolic free calcium and surfactant secretion in cultured rat alveolar type II cells. Biochim. Biophys. Acta 960: 43-45, 1988[Medline].

24.   Pincus, S. H. Production of eosinophil-rich guinea pig peritoneal exudates. Blood 52: 127-134, 1978[Abstract].

25.   Roya, F. R., D. R. Hoffman, B. Zhao, and J. M. Johnston. Platelet-activating factor in surfactant preparations. Lancet 341: 858-860, 1993[Medline].

26.   Sano, K., D. R. Voelker, and R. J. Mason. Involvement of protein kinase C in pulmonary surfactant secretion from alveolar type II cells. J. Biol. Chem. 260: 12725-12729, 1985[Abstract/Free Full Text].

27.   Thomas, L. L., L. M. Zheutlin, and G. J. Gleich. Pharmacological control of human basophil histamine release stimulated by eosinophil granule major basic protein. Immunology 66: 611-615, 1989[Medline].

28.   Venge, P., and L. Hakansson. The eosinophil and asthma. In: Asthma, edited by M. A. Kaliner, P. J. Barnes, and C. G. A. Persson. New York: Dekker, 1991, vol. 49, p. 477-502. (Lung Biol. Health Dis. Ser.)

29.   Wardlaw, A. J., S. Dunnette, G. J. Gleich, J. V. Collins, and A. B. Kay. Eosinophils and mast cells in bronchoalveolar lavage in subjects with mild asthma. Am. Rev. Respir. Dis. 137: 62-69, 1988.

30.   Zheutlin, L. M., S. J. Ackerman, G. J. Gleich, and L. L. Thomas. Stimulation of basophil and rat mast cell histamine release by eosinophil granule-derived cationic proteins. J. Immunol. 133: 2180-2185, 1984[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 276(5):L763-L768
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Okumura, M.
Articles by Miyata, T.
Articles citing this Article
PubMed
PubMed Citation
Articles by Okumura, M.
Articles by Miyata, T.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online