9
Jnurnol ofDruR Targeting. 1998. Vol. 5, No 6, pp. 471479 Repnnts available directly from the publisher Photocopying permitted by license only 0 1998 OPA (Overseas Publishers Association) N.V. Published by license under the Hanuood Academic Publishers imprint. pan of The Gordon and Breach Publishing Group Printed in Malaysia. Smaller Sized Particles are Preferentially Taken up by Alveolar Type I1 Pneumocytes M. GRIESE' and D. REINHARDT The Lung Research Group, Kinderpoliklinik, Ludwig-Maximilians- Universiry. Pettenkofer Strasse 8a, 80336 Munich, Germany (Received 4 June 1997: Revised 16 September 1997; in final form 27 September 1997) The uptake of both lung surfactant and other particles from the alveolar space plays an essential role in surfactant metabolism, host defense and may be of relevance for targeting drugs into alveolar cells. To better understand the effect of particle size on the uptake by type I1 pneumocytes, rat type I1 cells in primary culture were investigated. We observed that inert latex particles of 15 nm were taken up to a much greater extent than bigger particles. No strong size dependency was observed in the range from about 200 to lo00 nm. A similar observation was made with a natural lipid extracted lung surfactant which was taken up to a greater extent when prepared at particles of about 100 nm than a preparation with a particle size range from about 200-2000 nm. Alveolar type I1 cells take up smaller particles better than larger ones, but the size selectivity is rather limited. These type I1 cell properties may contribute to a preferential elimination of the smaller particle fractions from the alveolar space. Keywords: Pulmonary surfactant, latex particles Classificution Categories DPPC, dipalmitoyl-phosphatidylcholine INTRODUCTION Type I1 pneumocytes are key cells in the alveolar space of the lungs, performing a variety of functions like the regulation of pulmonary surfactant meta- bolism, ion transport and alveolar repair in response to tissue injury. Surfactant particles are taken up at a high rate by these cells for lipid recycling (Wright 1990). The mechanisms of the uptake process of pulmonary surfactant into type I1 cells are not well understood (Fisher et al. 1987, Chander et al. 1983, Muller et al. 1995, Chinoy et al. 1994, Griese et al. 1991, Horowitz et al. 1996). Endocytotic pathways clearly play a role, one appearing to be clathrin dependent, the other actin dependent (Muller et al. 1995). The surfactant liposomes are believed to be taken up intact (Chander et al. 1983, Muller et al. 1995), but little is known about the kind of surfactant particles which enter type I1 cells, for example, their size and structure. These features are believed to be of 'Corresponding author. Phone: ++49 89 5 160 3715, Fax: ++49 89 5 160 4733 e-mail: [email protected] 47 1 Journal of Drug Targeting Downloaded from informahealthcare.com by Mcgill University on 12/17/14 For personal use only.

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Page 1: Smaller Sized Particles are Preferentially Taken up by Alveolar Type II Pneumocytes

Jnurnol ofDruR Targeting. 1998. Vol. 5, No 6, pp. 471479 Repnnts available directly from the publisher Photocopying permitted by license only

0 1998 OPA (Overseas Publishers Association) N.V. Published by license under the

Hanuood Academic Publishers imprint. pan of The Gordon and Breach

Publishing Group Printed in Malaysia.

Smaller Sized Particles are Preferentially Taken up by Alveolar Type I1 Pneumocytes

M. GRIESE' and D. REINHARDT

The Lung Research Group, Kinderpoliklinik, Ludwig-Maximilians- Universiry. Pettenkofer Strasse 8a, 80336 Munich, Germany

(Received 4 June 1997: Revised 16 September 1997; in final form 27 September 1997)

The uptake of both lung surfactant and other particles from the alveolar space plays an essential role in surfactant metabolism, host defense and may be of relevance for targeting drugs into alveolar cells. To better understand the effect of particle size on the uptake by type I1 pneumocytes, rat type I1 cells in primary culture were investigated. We observed that inert latex particles of 15 nm were taken up to a much greater extent than bigger particles. No strong size dependency was observed in the range from about 200 to lo00 nm. A similar observation was made with a natural lipid extracted lung surfactant which was taken up to a greater extent when prepared at particles of about 100 nm than a preparation with a particle size range from about 200-2000 nm. Alveolar type I1 cells take up smaller particles better than larger ones, but the size selectivity is rather limited. These type I1 cell properties may contribute to a preferential elimination of the smaller particle fractions from the alveolar space.

Keywords: Pulmonary surfactant, latex particles

Classificution Categories DPPC, dipalmitoyl-phosphatidylcholine

INTRODUCTION

Type I1 pneumocytes are key cells in the alveolar space of the lungs, performing a variety of functions like the regulation of pulmonary surfactant meta- bolism, ion transport and alveolar repair in response to tissue injury. Surfactant particles are taken up at a high rate by these cells for lipid recycling (Wright 1990). The mechanisms of the uptake process of pulmonary surfactant into type I1 cells are not well

understood (Fisher et al. 1987, Chander et al. 1983, Muller et al. 1995, Chinoy et al. 1994, Griese et al. 1991, Horowitz et al. 1996). Endocytotic pathways clearly play a role, one appearing to be clathrin dependent, the other actin dependent (Muller et al. 1995). The surfactant liposomes are believed to be taken up intact (Chander et al. 1983, Muller et al. 1995), but little is known about the kind of surfactant particles which enter type I1 cells, for example, their size and structure. These features are believed to be of

'Corresponding author. Phone: ++49 89 5 160 3715, Fax: ++49 89 5 160 4733 e-mail: [email protected]

47 1

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472 M. GRIESE and D. REINHARDT

especial importance because they might help the type I1 cells to identify which surfactant material should be reinteralized from the alveolar space. Although the involvement of pulmonary epithelial cells in the uptake of mineral particles inhaled into the lungs is important (Churg 1996), the role of particle size in the process is again unclear. Some evidence suggests that smaller (“ultrafine”, about 15 nm) particles are more rapidly taken up than larger by tracheal epithelial cells. In a similar way, type I alveolar epithelial cells take up “fine” (about 100 nm) particles better than bigger particles (1 pm) (Churg 1996). The behaviour of type I1 cells has not been examined in detail. The goals of this study were therefore, firstly, to determine to what extent particle uptake is dependent on size; secondly, to generate natural surfactant fractions of different size, and thirdly, to characterize their uptake by type I1 pneumocytes.

MATERIALS AND METHODS

Chemicals and drugs

Dulbecco’s modified Eagle’s medium (DMEM), Ear- le’s balanced salt solution (EBS) and fetal bovine serum were purchased from GIBCO (Karlsruhe, Germany), porcine pancreatic elastase from Elastin Products (Owensville, MO, USA), L-a-(2 palmitoyl- 9, 10-3H(N) dipalmitoyl-phosphatidylcholine ([”I- DPPC) from New England Nuclear-Du Pont (Frank- furt, Germany), and Optifluor from Packard Instruments (Gronningen, Netherlands). Rat immuno- globulin G was purchased from Sigma Chemical Company (Deisenhofen, Germany). All other bio- chemicals were from Merck (Darmstadt, Germany) or Serva (Heidelberg, Germany) at highest purity availa- ble. Size defined latex particles (FluoSpheres, carboxylate-modified) were from Molecular Probes Europe, Leiden, Netherlands.

Isolation and culture of type 11 cells

Type I1 cells were isolated from the lungs of adult male Sprague-Dawley rats (200-300 g; Charles River, Sulzfeld, Germany) by the elastase digestion and

immunoglobulin G panning method. The freshly isolated cells were plated at a density of 2-3.5 X lo6 cells per dish on 35-mm plastic dishes and cultured in 1.5 ml Dulbecco’s modified Eadle’s medium (DMEM) containing 10% fetal bovine serum, strepto- mycin (10 mg/ml), and penicillin (100 unitdml) for 18-20 h at 37°C in humidified atmosphere of 95% air- 5% C02. At least 95% of the cells were identifiable as type I1 cells after this period of culture by staining with phosphine 3R (Griese et al. 1991).

Surfactant preparation

When surfactant was to be prepared, an aliquot of the lyophilised, lipid extracted, bovine natural surfactant (Alveofacf, Thomae Biverach, Germany) was dis- solved in chloroform and [”I-DPPC added to a final specific activity of 1.5 Ci/mol. The mixture was again dried under nitrogen, suspended in EBS (contains NaCl 117 mM, CaC12 1.8 mM, KCl 5.3 mM, MgS04 0.8 mM, NaH2P0, 1 mM, Glucose 5.5 mM, phenol red 0.01 g/l and 25 mM N-2-hydroxyethylpiperazine- N-2-ethanesulfonic acid (HEPES), pH 7.4) or water at a concentration of 1 pg DPPC/pI by gentle vortexing, and sonicated (1 X or 7 X 1.5-min bursts) at 50°C with a probe sonifier HD800 equipped with a MS73D tip (Bandelin, Berlin, Germany) at 20% maximum output. When assessed by thin layer chromatography ~ 0 . 5 % of the DPPC was degraded under these conditions, even when the sonication was continued for 20 min. Alveofact contains phosphatidylcholine (80% of total phospholipids (wtlwt) of which 85% is dipalmitoylphosphatidylcholine (n = 3)) and the hydrophobic surfactant proteins SP-B and SP-C (about 2% in relation to total phospholipid mass, Seeger et al. 1993). After overnight storage at 4”C, the surfactant preparation was centrifuged at 1,200 g for 20 min., at 4°C to remove aggregates ( ~ 2 % of initial phospholipid mass) and titanium particles from the probe sonicator. The supernatant contained the surfac- tant particles, which were warmed to 37°C and characterised by laser light scattering (Lo-Sizer, Malvern, Seefeld, Germany) and measurements of optical density or used in the uptake experiments.

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Page 3: Smaller Sized Particles are Preferentially Taken up by Alveolar Type II Pneumocytes

PARTICLE UPTAKE BY PNEUMOCYTES 413

Surfactant uptake

After the overnight culture, the cells were rinsed three times with fresh EBS without serum or antibiotics but containing 25 mM HEPES buffer (pH 7.4) and incubated for 30 rnin at 37°C in 95% air-5% COz. Then the surfactant or latex particles were added. The dish was briefly shaken and the incubation continued for 90 min. To stop the reaction, the medium was rapidly removed, the cells were washed five times with cold phosphate buffered saline (PBS), and PBS containing 0.25% trypsin and 0.54 mM EDTA at 37°C was added. After 5 min at room temperature, the cells and medium were separated by centrifugation for 10 min at 800Xg, 4°C. The cell pellet was resuspended in fresh PBS and a new tube and the centrifugation was repeated. An aliquot of the cells was transferred to a scintillation vial and incubated in 0.1 N NaOH (0.6 ml) for 30 min at 50°C to solubilize the protein. After cooling to room temperature, Optifluor (10 ml) was added and radioactivity measured in a liquid scintillation counter. The uptake of liposomes was expressed in nanomoles DPPC per unit of type I1 cell protein determined in an other aliquot. In the experiments with the latex particles, uptake was quantitated on suspended cells in a fluorometer at exitation 490 nm and emission at 5 15 nm (LS 50 B, Perkin-Elmar, Uberlingen, Germany). In all the experiments, culture dishes incubated overnight with DMEM containing serum and antibiot- ics but no cells were treated in parallel exactly as described above. Radioactivity or fluorescens recovered from these dishes was routinely less than 10% of those of the dishes with cells and was substracted from the trypsin-resistant cell-associated marker.

Surfactant aggregation

Aggregation of the surfactant suspension was investi- gated by turbidity measurements at 540 nm on a Beckman Model 24 spectrophotometer. Surfactant was incubated at 37°C and a concentration of 50 pM in EBS (pH 7.4) and the change in optical density (OD) was recorded.

Determination of surfactant particle size

The size of surfactant particles was determined by photon correlation spectroscopy using a laser equipped Lo-Sizer (Malvern, Seefeld, Germany). A multimodal exponential sampling algorithm was employed for the calculation of mean vesicle diame- ters and peak width, after 10 min of measurements at an angle of 90" and a temperature of 37°C.

Microscopy

Cells were viewed and photographed (ASA 400, Fuji, Tokyo, Japan) at identical exposure times and camera settings using an IMT-2 inverse phase contrast microscope with an IB-filter block (Olympus, Ham- burg, Germany).

Lactate dehydrogenase assay

The rate of lactate dehydrogenase release into medium was monitored to assess cellular integrity. The cells were cultured as in the uptake experiments, after which lactate dehydrogenase activity in the cells and medium was assayed spectrophotometrically by following the disappearance of NADH at 340 nm. No toxicity by the liposomes or latex particles were noted.

Data analysis

Type I1 cells were isolated from the pooled lungs of three to four rats and distributed among the various treatment groups. Two dishes with cells and two blank dishes without cells per group were used in each uptake experiment. Each dish was processed separately and the values averaged to yield a single data point per group per experiment. All data are means f SE from the number of individual experi- ments indicated. The data were analyzed statistically as indicated in the RESULTS.

RESULTS

Uptake of latex particles

Latex particles sized 15 nm were taken up much more avidly by type I1 cells than larger ones. There was no

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Page 4: Smaller Sized Particles are Preferentially Taken up by Alveolar Type II Pneumocytes

474

75c cn w

M. GRIESE and D. REINHARDT

0 0 500 1000

SIZE OF PARTICLES (nm) FIGURE 1 significantly better than those of larger size (p < 0.001, Wilcoxon-test. 2-sided). Date are mean i SEM from 6 experiments.

Uptake of fluorescent latex particles of defined size by type I 1 pneumocytes cultured on plastic. 15 nm particles were taken up

clear dependency on size in the range of 100- 1000 nm (Figure 1). In addition to these data obtained with the fluorometric assay, the same was also demonstrated by fluorescense microscopy (Figure 2) . The distribu- tion of the fluorescent label clearly indicated the intracellular localisation of the cell associated latex particles.

Uptake of surfactant made in water

Surfactant particles were prepared by resuspending a natural, lipid-extracted bovine lung surfactant in water. As indicated in the upper part of Table 1, vortexing resulted in a rather homogenous particle preparation with more than 90% of the particles having a diameter of around 100 nm. These prepara- tions were further refined by sonication (Table 1, upper part). With incubation at 37°C for up to 2 hours no significant changes occured in size (Figure 3, left

panels) and state of aggregation (Figure 4). The uptake of these surfactant preparations by type I1 pneumocytes however, did not significantly vary with the change in size, as induced by vortexing or additional ultrasonications (Table 2, upper part). The uptake process was temperature sensitive and did not merely represent binding, as indicated by reduction of uptake upon fixation of the cells by glutaraldehyde (Table 2, upper part). In agreement with earlier studies (Griese et al. 1991), usage of fluorescently labelled lipids clearly showed the intracelluar local- isation of the lipid label (data not shown).

Uptake of surfactant made in EBS

Surfactant particles prepared by resuspension of the natural, lipid-extracted, lung surfactant in EBS were substantially bigger (Table 1, lower part, Figure 3, right panels) than those made in water. Vortexing

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Page 5: Smaller Sized Particles are Preferentially Taken up by Alveolar Type II Pneumocytes

PARTICLE UPTAKE BY PNEUMOCYTES

a) b)

475

FIGURE 2 Type 11 cells uptake of fluorescent latex particles of defined size (15 nm in a, band 500 nm in c, d). Phase contrast images (right hand panels) and fluorescent images (left hand panels). Bar indicates 100 pm. Cells were incubated with the particles for 90 min, the dishes were washed extensively and observed under the microscope (a-d). Additionally, to mimic the conditions of the other uptake experiments, and to reduce non specific background and surface binding, cells were dislodged with trypsin/EDTA from the dish and washed 2 times by centrifugation (inserts in c and d). This eliminated the background staining completely. similar as quantitated by fluorometry and assessment of radioactivity. (see color plate I)

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Page 6: Smaller Sized Particles are Preferentially Taken up by Alveolar Type II Pneumocytes

476 M. GRIESE and D. REINHARDT

TABLE 1 Characterization of size and state of aggregation of bovine lipid extracted lung surfactant

diameter f peak aggregation

A = 540 nm

Method of Peak size preparation number % distribution width (nm) optical density at

(by number)

Preparation of surfactant in water, incubation in EBS

2 8 442 f 169 3

128 f 44 0.412 f 0.014 vortex only 1 92

1 X sonication

7 X sonication

vortex only

1 X sonication

7 X sonication

1 2

1 2 3

1 2 3 1 2 3 1 2 3

99 147 f 37

3 100 90 f 38

Preparation of surfactant in EBS, incubation in EBS 75 587 f 139 21 1131 f 248 10 2240 f 432 76 238 f 74 16 630 f 142 30 1104 f 334 95 117 f 41 5 522 f 123 2 1012 f 321

0.31 1 f 0.020

0.328 0.035

0.728 f 0.035

0.306 f 0.071

0.242 f 0.065

Size measurements of the liposomal surfactant mixture were made by quasi laser light scattering. Relative frequency (% distribution) of certain size classes and their respective size is given. The state of aggregation was estimated from measurements of OD in a spectrophotometer. The bovine lipid extracted surfactants were dispearsed either in the presence of water or EBS by vortexing or additional ultrasonications. Measurements were performed immediately thereafter. Data are means f SEM or mean peak width from 3 to 5 experiments.

alone resulted in relatively huge particles (500-2000 nm diameter) and aggregates (increased O.D.). With sonication the size and aggregates were reduced, they were however, always higher than those made in the presence of water. No significant changes in size and state of aggregation occured with incubation at 37°C for up to 2 h (Figures 3,4). The uptake of these bigger surfactant preparations by the type I1 cells was significantly lower than that of the two smaller particles made in water. Although the preparation with the biggest particles had the lowest uptake, again no consistent differences between vortexed and sonicated preparations were observed (Table 2, lower Part).

DISCUSSION

Our data support the hypothesis that particle uptake by type I1 pneumocytes is dependent on the particle

size. In the case of both inert latex particles and of particles prepared from a lipid-extracted pulmonary surfactant, the uptake appeared to be the better the smaller the particles were. It is also clear from the evidence that the dependency on size was not very strong, e.g. the size selectivity was rather low over the range from about 100-1000 nm particles. Temperature dependency of the uptake, resistance to trypsin and EDTA and the microscopic observation of the distribution pattern of the label taken up, clearly indicated internalized particles rather than extrac- ellularly bound material.

When cells are exposed to the same mass of particles, e.g. a particular lipid concentration or fluorescence intensity, the particle number or surface area of the particles is greater when smaller particles are used. The basic question connected with this, namely whether particle size alone or particle number or both factors together determine the increase in uptake has yet to be definitely solved (Churg 1996).

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PARTICLE UPTAKE BY PNEUMOCYTES 471

400.

200.

0-

However, as shown in Figure 2, the larger particles were far less likely to be internalized, even when the cells were exposed to large number of particles. In view of the fact that the composition of the liposomes may also affect uptake (Allen et al. 1991, Lee et al. 1992, Chander et al. 1983), we preferred a natural lipid-extracted surfactant preparation, which is cur- rently widely used in neonatology for our studies of the behaviour of liposomal particles of different sizes. The structural organisation of these complex mixtures which contain lipids and the hydrophobic surfactant proteins SP-B and SP-C, is difficult to control. SP-A which may directly perturb surfactant lipid organisa-

9.6 t 2

88.6 f 5

,

tion (Haagsman et al. 1990) and which might also be involved in binding and lipid uptake into type I1 cells (Fisher et al. 1987, Wright 1990, Griese et al. 1991, Horowitz et al. 1996) was omitted from the surfactant preparation in the present investigation to permit a clearer study of the effect of particle size. Separate experiments with SP-A will be necessary to clarify its precise role.

The generation of surfactant fractions from these complex mixtures with identical composition but different sized particles, is rather difficult to accom- plish. We first tried molecular sieving by gel filtration and filtration through filters of a defined pore size.

low-

800.

600

400-

F + 1 x sonication

-

~-4-i!--4-$ 99.4 f 4

kJ: ti-

,OOt

0 30 60 90 120 TIME (min)

3000r

2500-

2000 - I @ 8

1000.

vortex only

T

0 30 60 90 120

3000

t 1 x sonication

T 2000

o t , 0 30 60 90 120

3 l5O0- 1000.

500- 2 f

7 t 7 x sonicati

0 30 60 90 120 TIME (min)

Ralatlve frequency

(XI

3 f l 22 f 4 67 f 3

1912

21 f 2 m i 2

7 f 2

7 f 2

89 f 3

FIGURE 3 Particle size of the surfactant preparation and its changes over time. A bovine lipid extracted surfactant was either dispearsed in the presence of water (left sided panels) or in Earle's balanced salt solution (EBS) (right sided panels) by vortexing or additional ultrasonications with a probe. Size measurements were made at the indicated time points by quasi laser light scattering. Data are means It peak width from 6 experiments. Up to 3 size classes were detected in a sample. Their relative frequency distribution (%) did not change significantly with time and is also given. The data are mean f SEM of 6 determinations and as such do not exactly add up to 100%.

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Page 8: Smaller Sized Particles are Preferentially Taken up by Alveolar Type II Pneumocytes

478 M. GRIESE and D. REINHARDT

However, the particle yield in the ultrafine size range was insufficient relative to the huge amounts of starting material. Mechanical suspension by vortexing and sonication turned out to be the most reliable way of reproducibly generating sufficient particles which remained stable during the incubations performed. We took advantage of the marked effect which the initial medium for resuspension has on the organisa- tion of the lipid mixture. The usage of a salt solution containing high concentrations of Ca++ instead of pure , water has been shown to generate particle populations that were bigger (Budger et al. 1992). Thus, surfactant liposomes which had an identical lipid and protein composition were generated and then suspended and incubated with the cells in the same medium. This makes it likely that the differences observed in uptake

n E c 0 d- In

-P a

d 0 W

Z 0 I-

fY 0 v>

<

H

n

m

0.8

0.6

0.4

0.2

0.0

between the surfactant preparations are merely due to the differences in size and state of aggregation of the surfactant preparations which we observed. A direct effect from the presence of EBS during liposome preparation on uptake cannot however be completely ruled out. Within the limited size range that could be generated by vortexing and sonication, no significant impact on uptake was noticed. This is in agreement with earlier data which also show only a minor effect on uptake of a natural surfactant preparation by sonication or vortexing (Fisher et al. 1987). Similarly, bone marrow macrophages were shown to take up liposomes of less than 100 nm better than those in the range of 200 to 800 nm. Similarly, in the afore- mentioned range, no differences in uptake were observed (Allen et al. 1991).

I I € I I

0 30 60 TTMF (rnin)

90 120

FIGURE 4 The state of aggregation of the surfactant suspcnbion wab estiinahxl from meaSurements of optical density (OD) at 540 nm. N o changes over time were observed. Squares represent vortexed samples, circles those with 1 additional sonication and triangles those with 7 additional sonications. Closed symbols are surfactant prepared in water and incubated in EBS, open symbols represent surfactant prepared in EBS and incubated in EBS. Data are means i SEM from 5 independent experiments.

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PARTICLE UPTAKE BY PNEUMOCYTES 479

TABLE 2 Effect of the method of preparation on uptake of lung surfactant dipalmitoylphosphatidylcholine (DPPC) by type I1 cells.

Method of surfactant preparation Conditions of incubation

Surfactantphospholipid uptake (nmol DPPC/mg protein)

37°C 4°C glutaraldehyde, 37°C

vortex only 1 X sonication 7 X sonication

vortex only 1 X sonication 7 X sonication

Preparation of surfactant in water, incubation in EBS

1.36 i 0.41 (6)§ 1.27 i 0.38 (6)s 1.01 i 0.18 (7)5

0.47 i 0.24 (4)* 0.95 i 0.12 (2) 0.27 i 0.13 (5)*

Preparation of surfactant in EBS, incubation in EBS

0.35 f 0.07 (8) 0.39 * 0.09 (7) 0.47 i 0.15 (6)

0.21 i 0.09 (6)* 0.17 i 0.05 (3) 0.10 i 0.01 ( 5 )

0.34 i 0.01 (2) 0.28 0.11 (3)

0.13 (1)

0.04 * 0.02 (3) 0.11 1: 0.01 (2) 0.08 i 0.01 (4)

The surfactants were prepared by vortexing or additional ultrasonications either in the presence of water or EBS. Type I1 cells were isolated from lungs of adult rats, cultured for 18 h and incubated in Earl’s balanced salt solution (EBS) with the r3H]-DPPC labelled surfactants at a concentration of 30 p M phospholipid for 90 min, washed and treated briefly with trypsidEDTA to remove surface bound material. Surfactant uptake was then measured in the centrifugation assay as described in METHODS. Data are means SEM from the number of experiments indicated in brackets. §Uptake at 37°C of surfactant prepared in the presence of water was significantly higher than that prepared in EBS (P < 0.0005, two-tailed Wilcoxon test). *Values at 37 and 4°C were statistically different where indicated, P < 0.05, corrected for multiple comparisons.

In the alveolar space of the lungs uptake of surfactant lipids is mediated by clathrin-dependent, possibly receptor mediated processes, and by recep- tor-independent adsorptive endocytosis (Muller et al. 1995). Size dependent uptake may in particular play a significant role in adsorptive endocytosis and thus contribute to the preferential elimination of smaller sized (el00 nm) surfactant or other particles from the alveolar space by type II epithelial cells.

Acknowledgements

The technical assistance of J. Gnielka and J. Khachani and the help of J. Biirgermeister M.A. in the preparing the English manuscript are appreciated. This work was supported by grants of the DFG (Gr 970/3-1 and 3-2).

References

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Budger V.G., Kiseleva H.V., Serehryakova M.V., Sokoloff A.V. (1992) Ca2+-mediated interaction between negatively charged and neutral liposomes. FEBS Letts, 313, 169-172.

Chander A., Claypool W.D., Strauss J.F., Fisher A.B. (1983) Uptake of liposomal phosphatidylcholine by granular pneumo- cytes in primary culture. Am. J. Physiol., 245, C397-C404.

Chinoy M.R., Dodia C., Fisher A.B. (1993) Increased surfactant internalization by rat type II cells cultured on microporous membranes. Am. J. Physiol., 264, L300-L307.

Churg A. (1996) The uptake of mineral particles by pulmonary epithelial cells. Am. J. Resp. Crif. Care Med., 154, 1124-1 140.

Fisher A.B., Chander A., Reicherter J. (1987) Uptake and degradation of natural surfactant by isolated rat granular pneumocytes. Am. J. Physiol., 253, C792-C796.

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