The Surfactant System of the Lungs: Morphology and Clinical Significance 9783110860962, 9783110113877

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The Surfactant System of the Lungs

Konrad Morgenroth English Edition Editor: Michael Newhouse

The Surfactant System of the Lungs Morphology and Clinical Significance Final Translation: Carol Newhouse Drawings: Gerhard Pucher

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Walter de Gruyter Berlin • New York 1988

Prof. Dr. m e d . K o n r a d M o r g e n r o t h Ruhr-Universität B o c h u m Institut für P a t h o l o g i e Universitätsstraße 150 D-4630 Bochum-Querenburg Dr. M i c h a e l T. N e w h o u s e Firestone Regional Chest and Allergy Unit St. J o s e p h ' s H o s p i t a l - M c M a s t e r U n i v e r s i t y 5 0 C h a r l t o n A v e n u e East Hamilton, Ontario L 8 N 4 A 6 Canada

Deutsche Bibliothek Cataloging in Publication Data Morgenroth, Konrad: The surfactant system of the lungs : morphology and clin. significance / Konrad Morgenroth. Engl. ed. ed.: Michael Newhouse. Final transl.: Carol Newhouse. Drawings: Gerhard Pucher. - Berlin ; New York : de Gruyter, 1988 Dt. Ausg. u.d.T.: Morgenroth, Konrad: Das Surfactantsystem der Lunge. Span. Ausg. u.d.T.: Morgenroth, Konrad: El sistema surfactante del pulmon ISBN 3-11-011387-2

© Copyright 1988 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means - nor transmitted nor translated into a machine language without written permission from the publisher. Printed in Germany. The quotation of registered names, trade names, trade marks, etc. in this copy does not imply, even in the absence of a specific statement that such names are exempt from laws and regulations protecting trade marks, etc. and therefore free for general use. Typesetting and Printing: Appl, Wemding. - Binding: Lüderitz & Bauer G m b H , Berlin. Cover design: Rudolf Hübler, Berlin.

Preface

Numerous clinical and basic research studies have contributed to our understanding of the function of the secretory system of the alveoli. It has been demonstrated that this system is essential for effective pulmonary ventilation and thus for maintaining one of the most important physiological processes, namely gas exchange. Abnormalities of the surfactant system relate to the pathogenesis of various pulmonary diseases and must thus be taken into account in developing appropriate therapies. Understanding the normal structure and function of the alveolar secretory system and its disorders, may contribute in an important way to a basic understanding of pulmonary disease. The purpose of this volume is to describe the function of the surfactant system in relation to its morphology and to demonstrate its clinical significance. In addition, light and electron micrographs are used to provide a visual impression of the structure/function relationships of this system. Photographs are supplemented by three dimensional reconstructions drawn by Gerhard Pucher which provide a functional correlation to a considerable amount of morphological data. I wish to thank Mr. Pucher, who demonstrates his considerable understanding of morphological details and their significance, for many years of fruitful collaboration. I would also like to thank DeGruyter and company for the excellent design of this volume which has given it its special character. K. Morgenroth

Table of contents

1.

Introduction

1

2. 2.1 2.2 2.3

Structure of the Alveolar System Type I Pneumocytes Type II Pneumocytes Interstitium

2 3 5 6

3. Biosynthesis of Surfactant 3.1 Control of Surfactant Production

10 17

4.

18

Physiology of Surfactant

5.

Morphology of the Surfactant in the Alveolar System 5.1 Surfactant as a Component of Alveolar Defence Mechanisms

6.

Morphology of Surfactant in the Bronchial System

19 28 34

7. Abnormalities of the Surfactant System . . 7.1 Surfactant and Tobacco Smoking 7.2 Respiratory Distress Syndrome of the Newborn (IRDS), Hyaline Membrane Disease 7.3 Adult Respiratory Distress Syndrome (ARDS) 7.4 The Surfactant System in Pneumonia . . . 7.5 The Surfactant System in Pneumoconiosis . 7.6 Disturbances of the Surfactant System as a Component of Mucostasis

47 47

106

Bibliography

109

52 65 80 91

VII

1. Introduction

Since publication of the results of von Neergard's physiological studies in 1929, it has been known that surface active agents are essential for maintaining effective alveolar ventilation. Surfactants assure that alveoli expand readily during inhalation but undergo controlled contraction rather than collapse during exhalation. Numerous clinical investigations and animal studies have shown the clinical importance of the alveolar secretory system of the lung (Farrell 1982, van Golde 1984). It has lately been appreciated that in addition to the physical characteristics of surfactant demonstrated by von Neergard, these substances also play an important role in alveolar and bronchial defense mechanims, a fact that has become of increasing importance in the light of man's increasing exposure to environmental pollutants. Following the publication of the studies of Gil and

Weibel (1970) which characterized surface active agents pathophysiological^ and biochemically, it is appropriate to add a morphological aspect which contributes considerably to our understanding of the action of surfactants in the alveoli. The results of a series of biochemical and pathophysiological studies (Warembourg et al. 1968, Reifenrath 1980) suggest that the surfactants formed in the alveoli also fulfill important functions in the bronchial system. The ability of the system to respond to injury is particularly important since all parts of the lung, both the conducting airways and alveolar regions, are constantly exposed to numerous exogenous, noxious substances contained in the inhaled air. This review concentrates on the morphological basis of these surface active agents and describes the morphology of this system under normal and pathological conditions.

2. Structure of the Alveolar System (Figs. 1 - 4 ) formly lined by a layer of epithelial cells. These cells can be differentiated according to the morphology of their cytoplasm into two types: the type I pneumocyte (Pneumocyte I) and the type II pneumocyte (Pneumocyte II).

The lung can be simply divided into the system of conducting airways and the alveolar system. The alveolar system has a total surface area of 80 m 2 during exhalation and 120 m 2 during inhalation, making it the largest body surface in direct contact with the external environment. The alveoli are uni-

Fig.1: Structure of the Alveolar System of the Lung. View from an alveolar duct into the associated alveoli. A smooth epithelium is seen covering the alveolar spaces. Scanning electron micrograph: x350

2

Fig.2: Structure of Alveoli. The thin alveolar septa contain abundant capillaries filled with erythrocytes (stained blue). The alveolar spaces are lined by flat extensions of the alveolar epithelial cells. Semi-thin section. Basic fuchsin and methylene blue. Scanning electron micrograph: x 6 2 0

2.1 Pneumocytes I ed in the center of the cell. Flat cytoplasmic extensions overlap one another to various degrees and probably slide back and forth with inspiration and expiration. Between adjacent cells there are tight junctions which surround the Pneumocytes II like a collar. The Pneumocyte I cells lie on a continuous basal lamella, which over large areas, fuse with the basal lamellae of the endothelial cells of the alveo-

The Pneumocyte I cells cover almost 95% of the alveolar surface. The diameter of these cells is about 50 |xm, their surface area 2,300 |im 2 . They have very few organelles and are characterized by a flat, broad, cytoplasmic body, a 0.1-0.3 wide reticulum, scattered lysosomes and mitochondria and one golgi complex. At the periphery of the cell are many pinocytotic vesicles. An oval nucleus is locat3

lar capillaries. Thus the epithelial cells, basal lamellae and capillary endothelium form the gas exchange barrier of the alveolar wall. It is assumed that Pneumocytes I cannot reproduce. The repair and replacement of these large surface cells is

probably provided for by the Pneumocytes II and possibly by common, undifferentiated precursor cells. Autoradiographically, mitotic activity is observed only in the Pneumocytes II.

Fig.3: Structure of Alveolar Septa. Cross-section with evenly arranged and uniformly sized capillaries in the interstitium of the alveolar septa. Beyond, the alveolar spaces are lined with uniform alveolar epithelium. Scanning electron micrograph: x 1040

4

Fig. 4: Structure of the Alveolar Wall. This section allows a view into two alveolar capillaries containing erythrocytes. Well seen is the wall of the alveolus (arrows) consisting of the fused alveolar epithelium and capillary endothelium. Scanning electron micrograph: x 6 0 0 0

2.2 Pneumocytes II (Figs. 5 - 1 1 ) The Pneumocytes II are cuboidal with a diameter of about 9 (j.m. They usually occur singly and only occasionally are small groups of two or three cells found in the alveolar epithelium. They are characterized by 0.1 |i.m wide microvilli which lie on their surface. The cytoplasm of these cells contains more organelles than that of the Pneumocytes I. A rough endoplasmic reticulum can be seen. In addition, there are free ribosomes, mitochondria, lysosomes, multi-vesicular bodies and a well developed golgi complex. The nucleus which is located at the center of the cell has a well developed nucleolus. The Pneumocytes II lie flat on the basal lamella. On the

periphery, these cells are overlapped in a collar-like fashion by the extensions of the Pneumocytes I so that they protrude only slightly above the level of the epithelial plane. A particular morphologic characteristic of the Pneumocytes II is the presence of large lamellar bodies (0.2-2 |i.m diameter) which constitutes 18%-24% of the cytoplasm of these cells. These lamellar bodies are thought to function as the substrate for the synthetic processes carried out by these cells and to be of equal importance to the intracytoplasmically formed surface active substances. 5

Fig.5: Biosynthesis of Surfactant in Pneumocytes II. Three dimensional reconstruction. According to autoradiographic studies, the precursors of surfactant are carried to the epithelial cells via the circulatory system and enter the epithelial cells by diffusion through the capillary endothelium. After passage through the golgi complex, synthesis begins in the endoplasmic reticulum of the Pneumocytes II. Surfactant is then formed in a step wise fashion. The contents of the osmiophilic lamellar bodies are secreted into the alveolar spaces by merocrine secretion. 1 = Nucleus 2 = Golgi Complex 3 = Mitochondria 4 = Endoplasmic Reticulum 5 = Osmiophilic Lamellar Bodies 6 = Secretion of the Surfactant Material 7 = Pneumocytes I of the Alveolar Epithelium 8 = Capillary Endothelium

The lamellar bodies which can be seen in electron micrographs and the structure of the alveolar surfactant are similiar to aqueous dispersions of phospholipid or phospholipid-protein combinations. The lamellar bodies are probably formed from multi vesicular bodies which contain similar lysosomal enzymes. It is also likely that the multi vesicular bodies which are closely related to the cisternae of the endoplasmic reticulum, are precursors to the development of the lamellar bodies. The multi vesicular bodies which are formed in the golgi complexes are surrounded by an approximately 90 A thick membrane and contain a vesicle surrounded by a limiting membrane. The substances contained within the lamellar bodies are transported from the cytoplasm into the alveolar space by merocrine secretion. The individual lamellar bodies move towards the cell membrane and attach themselves to

its inner surface. After they come into contact with the cell membrane, it opens above them and the contents of the lamellar body can then be deposited through this opening onto the surface of the cell.

2.3 Interstitium The interstitium is a thin framework of tissue lying between the alveoli, consisting mainly of alveolar capillaries, arranged in a reticular formation around the alveoli. The capillary endothelium is flat except for slight bulges on that part lying opposite to the alveolar wall containing the cell organelles and nucleus. The capillary wall is uniformly covered by a sheet of overlapping thin cytoplasmic extensions of the endothelial cells. The extensions of adjacent endothelial cells overlap and where they join they

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form contact zones that are characterized by fine filamentous cytoplasmic compactions. In the cytoplasm of the endothelial cells lie irregularly distributed pinocytotic vesicles. Where the basal lamella of the endothelium lies immediately adjacent to the basal lamella of the alveolar epithelium they fuse, resulting in a single basal lamella in the region between the alveolar epithelium and capillary endo-

thelium. Between the capillaries are located individual fibrocytes and delicate strands of collagen fibres which form the support of the alveolar structure. In addition, lymphocytes and histiocytes are irregularly distributed. They should be considered a readily available cell pool for local immunological reactions. Occasional nerve endings have also been demonstrated in the interstitium.

< Fig. 6: Structure of Pneumocytes II. The cuboidal alveolar epithelial cells contain a centrally located nucleus and regions of abundant mitochondria. On the surface there is an irregular microvillar pattern. The Pneumocytes I surround the cuboidal epithelial cells like a collar. Transmission electron micrograph: x 19,000

Fig. 7: Surface Structure of the Alveolar Epithelium. In the center of the photograph, in a niche, a Pneumocyte II is seen. On the surface of this cuboidal epithelial cell there is an irregular microvillar pattern. Scanning electron micrograph: x 5000

9

3. Biosynthesis of Surfactant (Fig. 5) Autoradiographic investigations suggest that the components of surfactant are delivered to the Pneumocytes II via the vascular system. Labelled palmitate and choline, for example, can be detected in the endoplasmic reticulum and golgi complexes of the Pneumocytes II only a few minutes after their intravenous injection. In addition to storage, it is possible that there is also ongoing synthesis in the lamellar bodies which increase in size as surfactant is produced. The biosynthesis of surface active substances has been observed after they have been washed out of the lung. In addition, it has been possible to isolate Pneumocytes II and to systematically study the chain of biosynthetic events in vitro (van Golde 1985).

proteins are necessary for the proper function of the surfactant system. The protein fraction probably consists of three proteins: albumin with a molecular weight of 69,000 and two proteins with molecular weights of 35,000 and 10,000. The latter is probably a metabolic product of the 35,000 molecular weight fraction. The presence of carbohydrates has been proven using both electron microscopic and biochemical methods. It is believed that the protein fraction helps to determine the physical state of the phospholipids which alternates constantly between gel and liquid. It has been established that these proteins are neither serum albumins nor gammaglobulins. The total amount of surfactant in the lung is extremely small. About 3% of the blood/air barrier consists of surfactant with a thickness of up to 500 A . There is only about 50 mm 3 surfactant per m 2 of alveolar surface. Recycling of secreted surfactant appears to be an important mechanism for the metabolism of the phospholipids in surfactant. That this recycling takes place, is indicated by studies in which the specific activity was measured after intravascular injection of radiolabeled components of surfactant. After intratracheal instillation of surfactant aerosols and dipalmitylphosphatidylcholine, a very non-homogenous distribution of activity was found in the alveoli. The greatest activity was recorded in the Pneumocytes II after two hours. These data demonstrate that a specific uptake of phospholipids by the

Surfactant is not a chemically pure substance, but rather an emulsion of lipids (phospholipids), proteins, and carbohydrates. The lipids make up about 90% of the total and the proteins about 10%. About 65% of the surfactant consists of lecithins (phosphatidylcholines). Cholesterol and phosphatidylglycerol make up another 10% of the surfactant. Functionally, dipalmatylecithin, which accounts for about 50% of the lipids, is the most important component. The bipolar structure of this material makes it highly surface active. The quaternary ammonium ions of the hydrophilic choline group are immersed in the aqueous phase of the alveolar surface while the hydrophobic C-18 fatty acid chains in the d-position of the glycerides project into the alveolar lumen. Because of the high proportion of lecithins in the total phospholipid content of the lung, surfactant material is able to assume several different physical states. It is the chemical structure of lecithin that determines unusual characteristics of its compounds, namely the ability to form gels, liquid crystals, mono- and oligo-molecular films, micelles and liposomes. Even though the lipids are the most important constituent of surfactant, proteins are also important for the normal functioning of this material.

Fig.8: Structure of the Osmiophilic Lamellar Bodies of the Pneumo- r> cytes II. Individual granules are seen to be surrounded by elementary membranes. Within these are variably sectioned lamellae of osmiophilic membranes (arrows). Between the osmiophilic lamellar bodies can be seen components of the rough endoplasmic reticulum. Adjacent to this are occasional mitochondria with a uniform christate structure. Transmission electron micrograph: x 35,000

For a long time the existence of a protein fraction was controversial. It is however, now apparent that 10

it

Pneumocytes II can take place. The labelled components leave the lung within about 4.3 hours and are subsequently found mainly in the kidney. Studies have shown that about 85% of the components of surfactant are re-utilized so that only small amounts are synthesized de novo. How this recycling takes place is not completely clear. It has been suggested that the individual phospholipid molecules are taken up by the cells. It is possible that resorption and removal affects only those components which do not participate in the recycling process. It is also possible that only certain components such as phospholipids are recycled and that the others are synthesized de novo lJob & Jacobs 1984).

Surfactant

Lipid approx. Lecithin Cholesterol Phosphytidylglycerol

90% 65% 10% 10%

Carbohydrate

Protein 10% Table 1

Fig.9: Between the cisternae of the endoplasmic reticulum (ER) are t> osmiophilic lamellar bodies with densely arranged high contrast phospholipid membranes. To the right is the nucleus of the Pneumocyte II. Transmission electron micrograph: x 29,000

12

13

Fig. 10: Surfactant Spreading onto the Alveolar Surface. Three dimensional reconstruction. The surfactant material that has been formed in the Pneumocytes II and ejected onto the alveolar surface, spreads out as a monomolecular film over the alveolar epithelium. The surfactant smooths the uneveness between the Pneumocytes I and II. Occasional microvilli protrude into the alveolar space through the surfactant film. 1 = Pneumocyte II 2 = Pneumocyte I 3 = Surfactant Layer 4 = Alveolar Capillary

14

16

Fig. 12: Surface Structure of a Pneumocyte II with Secretion of Surfactant (Arrows). In addition to the microvillar structure it is possible to recognize on the cell surface, surfactant material that has been discharged into the alveolar space consisting of complexes of various sizes.

the surface as a monomolecular film (arrows) over a cytoplasmic extension of the Pneumocyte I. In the surfactant film there is a dense basal layer above which a somewhat lighter granular zone can be identified. In the Pneumocyte I are irregularly distributed pinocytotic vesicles. Beneath this is the common basal lamella (BL) between the alveolar epithelium and capillary endothelium. In the associated section of capillary endothelium (EN) are pinocytotic vesicles.

Transmission electron micrograph: x 56,000

Transmission electron micrograph: x 240,000

20

Fig. 15: Structure of Surfactant in an Alveolar Epithelial Niche. The phospholipids are arranged in part as layers and in part as vesicles (arrows). The surfactant evens out recesses in the alveolar surface. Beneath this is seen the interstitium between alveoli containing collagen fiber bundles, an elastic fiber and a capillary (AL = alveolar space: EN = capillary endothelium).

Fig. 16: Reticular Aggregation of Surfactant on the Alveolar Epitheli- > um. Between the net-like phospholipid membranes lie finely granular protein components corresponding to the apoprotein of surfactant. Transmission electron micrograph: x 65,000

Transmission electron micrograph: x 35,000

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The surfactant produced by the Pneumocytes II assumes its position on the energy rich boundary surface hypophase by virtue of its "spreading pressure" since it spreads out like a droplet of oil on the surface of water. This space-saver function of the surfactant is made possible by its amphiphilic structure consisting of a hydrophilic group at one end of the surfactant molecule and a hydrophobic group at the other. The spreading pressure is explained by the presence of the hydrophilic group on the surfactant molecule by which the free energy (the boundary surface energy) of the hypophase is diminished. The surfactant lies with its hydrophilic side adjacent to the hypophase and forms the interface with the alveolar lumen. It is likely that the hypophase is covered by at least two layers of hydrophilic molecules with the hydrophobic molecules between them, thus forming a closed membrane. The surfactant itself assumes its most energy efficient state when it is not spread out. The surfactant material is shed when enough surfactant is spread on the alveolar surface and when there is inadequate room for further spreading. For surfactant, the droplet form is also energetically the most efficient. Under the transmission electron microscope, these surfactant spheres look like circular zones or holes. After serial sections into ultra-thin layers, the surfactant spheres appear as spherical cross sections.

ders) (Schneider 1980). A "pneu" is a drop with an easily deformable surface which encloses a liquid or gas and is itself embedded as a whole in a liquid or gas medium. These characteristics allow fluid mechanical transport of "surfactant spheres" in the bronchial system. Two droplets with the same composition fuse into one larger drop when they have different diameters since they then have different Laplace pressures. This is the result of liquid mechanical flow from the point of higher pressure to that of lower pressure. The alveoli as a whole are as though coated with a large surfactant bubble which forces the hypophase into the nooks and crannies between the alveolar lining cells. Surfactant has the following functions in the alveoli : — Surfactant must achieve the hypophase surface and becomes its boundary. This decreases the surface tension in the alveoli. — Surfactant maintains a residual surface tension which facilitates alveolar emptying without collapse. — The spreading pressure of surfactant forces the hypophase into a thin layer facilitating alveolar gas exchange. — Surfactant protects the alveolar hypophase from dehydration since otherwise the constant gas exchange would quickly dry out the alveoli which could then be more readily injured by exogenous toxic substances. Thus the surfactant has a defence function.

The locations of the hydrophilic and hydrophobic layers are the same as in the spread surfactant. Such surfactant spheres have been called "pneus" (blad-

Fig. 17: Removal of Excess Surfactant by Alveolar Macrophages. Re- > dundant surfactant material in the alveolar space is phagocytosed and incorporated into the cytoplasm of the alveolar macrophages (AL = alveolar space: MA = macrophages). Transmission electron micrograph: x 4300

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