2 - Embryology of the Lungs

Editors: Shields, Thomas W.; LoCicero, Joseph; Ponn, Ronald B.; Rusch, Valerie W.

Title: General Thoracic Surgery, 6th Edition

Copyright 2005 Lippincott Williams & Wilkins

> Table of Contents > Volume I - The Lung, Pleura, Diaphragm, and Chest Wall > Section I - Anatomy of the Chest Wall and Lungs > Chapter 4 - Cellular and Molecular Biology of the Lung

Chapter 4

Cellular and Molecular Biology of the Lung

Steven J. Mentzer

The primary function of the lung is gas exchange to maintain aerobic metabolism. To sustain an active human adult, oxygen must be adsorbed and carbon dioxide removed. This process requires a mechanism for the ventilation of large volumes of respiratory gases. The upper airways function to conduct gas into and out of the lung. When the respiratory gases reach the alveoli, a large alveolar surface area facilitates the efficient exchange of oxygen and carbon dioxide.

The ventilation of respiratory gases results in the exposure of the lung to a variety of airborne pathogens. A complex defense system uses mechanical and immunologic mechanisms to protect the host from biological pathogens. The importance of mechanical mechanisms of mucociliary clearance is illustrated by the heritable disease cystic fibrosis. The abnormal mucus in cystic fibrosis leads to lung hyperinflation and recurrent infections. In addition to biological threats, the ventilation of gases means that the lung is also exposed to a variety of environmental toxins. Cigarette smoke is a toxin that has effects on both gas exchange (emphysema) and the cells that line the airways (bronchial carcinoma). Similar environmental exposures are responsible for other acquired diseases of the lung.

LARGE CONDUCTING AIRWAYS

A variety of cells and their products ensure optimal gas exchange and limit the impact of airborne pathogens on lung function. The tracheobronchial tree is characterized by airway epithelium specialized for conductance and mucociliary clearance. A mixed population of epithelial cells lines the trachea and bronchi (Table 4-1). These epithelial cells include basal cells, goblet cells, and ciliated columnar epithelium. Characteristic of the proximal airways is the presence of ciliated columnar epithelial cells and goblet cells. Basal cells and so-called intermediate cells are also present. The function of basal cells is unclear, but these cells may give rise to both goblet cells and ciliated epithelial cells. Specialized lymphoid tissues are located along the main airways and especially at the bifurcation of airways where particles and pathogen deposition is concentrated.

Mucous and Serous Glands

Mucous and serous glands are present in the large airways down to the bronchiolar level. These glands are located between the muscle and the cartilage layers of the large airways. The glands are composed of both serous and mucous tubules. Although there may be some mixture, separate areas of the gland are composed of either serous or mucous tubules. These tubules end in collecting and ciliated ducts.

The mucous cells are primarily restricted to mucous tubules. The myoepithelial cells that line part of the mucous glands are responsible for expelling mucous contents into the airway lumen. The mucous glands are supplied by the sympathetic nervous system. Serous cells can be found in serous tubules and have been identified to the level of the bronchioles. The main function of serous cells is to produce lysozyme and possibly aid in the transport of immunoglobulin A (IgA) across the glandular epithelium. IgA is produced by plasma cells, which are found in the region of bronchial glands.

Goblet Cells

The goblet cell is a surface mucus-secreting cell present throughout the bronchial airways. Goblet cells produce mucus. Respiratory airway mucus is composed of glycoproteins with unique viscoelastic properties. These glycoproteins are collectively called mucins. Mucins are heterogeneous macromolecules that have domains for the passive clearance of both proteins and lipids. Mucins may also actively bind microorganisms. The mucin glycoproteins may function as ligands for lectin-like surface receptors of microorganisms. The role of the goblet cell in mucin production is particularly apparent after airway injury. Exposure to cigarette smoke, for

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example, leads to goblet cell metaplasia as well as glandular hypertrophy. Goblet cell metaplasia may also be observed in a variety of other inhalational injuries or in many conditions characterized by a chronic cough.

Table 4-1. Epithelial Cells

Cells Location Function
Basal Tracheobronchial airways Barrier, progenitor
Columnar secretory Tracheobronchial airways Mucus production
Ciliated Tracheobronchial airways Mucociliary clearance
Clara Bronchioles Secretory, progenitor
Alveolar type I Alveoli Air blood barrier
Alveolar type II Alveoli Surfactant, progenitor

Ciliated Columnar Cells

The percentage of ciliated columnar epithelial cells is generally higher in the large airways than in the peripheral airways. About 50% of the cells in the trachea are ciliated epithelium, whereas only 15% of cells in the fifth-generation airways are ciliated. In humans, the ratio of ciliated cells to goblet cells is about 5:1. Ciliated cells propel mucus through the airways.

About 250 cilia are located on the luminal surface of each ciliated cell. The cilia are composed of an array of longitudinal microtubules called an axoneme. The axoneme is arranged with a central doublet surrounded by nine outer doublets. A sliding movement of the microtubules past each other generates the movement of the cilia. The 9 + 2 arrangement of microtubules in human cilia is similar to the structure of axonemes in other plants and animals. The clinical importance of cilia is illustrated by the syndrome primary ciliary dyskinesia (PCD). PCD can involve bronchiectasis, chronic rhinosinusitis, and poorly motile spermatozoa. Kartagener's syndrome, which includes the diagnosis of situs inversus in addition to cilia dysfunction, is a subset of PCD.

The normal airway cilia function to propel both water and mucus. When the cilia propel mucus, the tip of the cilia penetrates the mucus and claws the mucus forward. At the end of the propulsive stroke, the cilia tip leaves the mucus and moves backward beneath the mucus in a recovery stroke. The average beat frequency of cilia ranges from 12 to 15 beats/min but is sensitive to both clinical and pharmacologic factors. Neurohormonal control of ciliary beat frequency appears to be regulated by a -adrenergic mechanism. Ciliated epithelial cells also appear to be sensitive to environmental injury. Cigarette smoke is associated with a loss of ciliated epithelium and replacement with squamous metaplasia. Ischemic injury of the airway, as seen after lung transplantation, is also associated with the loss of ciliated epithelium and an increase in squamous metaplasia.

Effective mucociliary clearance is critical for lung defense (Table 4-2). When ciliary activity is inadequate to remove all secretions from the airway, the physical presence of the mucus initiates neural reflexive cough. The cough generates a high shear force that dislodges the mucus and expels it from the airway. In healthy people, mucus transport in the airways does not require a cough. In contrast, when excess mucus resides in the airway, high expiratory air velocity can play an important role in the clearance of secretions.

Effective mucociliary clearance also depends on the viscoelastic properties of the mucus. In general, the viscosity and elasticity of the mucus varies inversely with the water content. When the water content of the mucus is high, the mucus is effectively cleared without a cough. When the water content of the mucus is low, the mucus is thick and tenacious. Mucus with high viscosity and elasticity can be effectively cleared by a cough. The clinical problem of high viscosity and elasticity occurs in the patient with a poor cough or impaired airflow. For example, the patient with a paretic vocal cord or chronic obstructive lung disease may not be able to generate sufficient airflow to expel the mucus with high viscoelasticity. The inability to clear the airway mucus may result in decreasing airflow and mucus impaction.

SMALL CONDUCTING AIRWAYS

The terminal bronchiole represents the most distal purely conducting portion of the tracheobronchial tree. Although there is some anatomic variation, bronchioles are generally distinguished from bronchi by the fact that bronchi contain cartilage in their walls, whereas bronchioles do not. Terminal bronchioles, like the proximal airways, have an epithelial lining that is specialized for conductance and mucociliary clearance.

Table 4-2. Factors Influencing Mucociliary Clearance

Factor Effect on Mucus Effect on Cilia
Smoke Increase quantity Decrease
Lidocaine No effect No effect
-Adrenergic agonists No effect Increase
Expectorants Mucolytic effect No effect
Gravity No effect No effect
Hydration Decrease viscoelasticity No effect

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The transitional zone between gas exchange areas and the conducting airways is termed the bronchiolar zone. Bronchioles are the most distal conducting airways proximal to the pulmonary acinus. The epithelial lining of bronchioles is largely composed of Clara cells. These nonciliated and nonsquamous epithelial cells constitute 70% to 90% of the cells throughout the transitional bronchiolar zone.

Clara Cells

Clara cells appear to have multiple metabolic functions. The ultrastructure of the Clara cell is characterized by extensive apical projections into the airway lumen and prominent endoplasmic reticulum. A variety of functional studies have established Clara cells as a primary site of xenobiotic metabolism. The Clara cell may also function as a secretory cell for the terminal airways. Clara cell secretory granules may be a source of surfactant apoproteins in the human lung. Clara cells may also serve as a source of arachidonic acid metabolites and antileukoproteases.

The turnover of epithelial cell populations in the bronchiolar region of the lung is very low. With injury, however, there is a dramatic increase in epithelial proliferative activity. Clara cells appear to function as a progenitor of themselves as well as ciliated cells in the bronchioles. The bronchiolar ciliated cell appears to be a principal target of oxidant gases. As bronchiolar ciliated cells are injured, there is proliferation of bronchiolar Clara cells. The hyperplasia of Clara cells may effectively increase the number of respiratory bronchioles by several airway generations. The relationship of Clara cell hyperplasia to bronchiolitis and obliterative small airway processes is unknown.

ALVEOLAR CELLS

The terminal bronchioles give rise to respiratory bronchioles. The respiratory bronchioles not only are conducting airways but also give rise to alveolar ducts that are studded with alveoli. Alveoli are the true gas-exchange surfaces of the lung. The alveoli are composed of specialized epithelial and endothelial cells separated by an interstitial matrix. Associated with these cells are alveolar macrophages. Alveolar macrophages are pivotal regulatory cells in the host defense of the distal airway.

Alveolar Type I Cells

The alveolar type I cells are the dominant component of a continuous layer of alveolar epithelium. The alveolar type I cell forms a thin membrane over 90% of the alveolar surface. The alveolar type I cell is broad and flat with highly branched cytoplasmic processes. Ultrastructural studies have shown that the alveolar type I cells have small nuclei and very few mitochondria. This simplified cellular machinery is believed to be associated with terminal differentiation. Because of their inability to divide, alveolar type I cells are dependent on alveolar type II cells for their replacement.

The alveolar type I cells provide an important barrier to the leakage of water and solutes out of the blood and into the air spaces. This function is the result of tight junctions between alveolar cells. The alveolar tight junctions form a continuous seal between the luminal and abluminal compartments. In addition to serving a barrier function, tight junctions may regulate the polarity of the cell membrane.

Because the alveolar type I cell is incapable of mitosis and repair, these cells are very sensitive to injury. In most models of acute lung injury, alveolar type I cells are the first cells to be damaged. Damaged alveolar type I cells detach from the epithelium, leaving behind a denuded basement membrane. The basement membrane alone provides a poor mechanical barrier. The consequence of a loss of alveolar type I cells is edema and hemorrhage into the alveolar spaces. Subsequent impairment in gas exchange persists until the proliferation of the alveolar type II cells can replace the lost cell population.

Alveolar Type II Cells

Alveolar type II cells constitute about 15% of the cells in the distal lung. Alveolar type II cells have a distinctive appearance by light microscopy. In contrast to the squamous alveolar type I cells, alveolar type II cells are cuboidal in shape. The intracellular stores of surface-active material give the alveolar type II cells a distinctive granular appearance. This distinctive appearance has led to the description of alveolar type II cells as granular pneumocytes.

The primary function of alveolar type II cells is the synthesis and secretion of surface-active material. Alveolar type II cells contain unique organelles called lamellar bodies. Lamellar bodies contain layers of surfactant phospholipids surrounded by a limiting membrane. Lamellar bodies also contain lysosomal enzymes and surfactant proteins. The lipid contained in the lamellar bodies is secreted at the cellular apex. The lamellar body fuses with the apical cell membrane, and the surfactant is released into alveolar space. After the release of surfactant lipids, the spheroid lamellar bodies appear to reorganize into a structure called tubular myelin. Tubular myelin may function to aid in adsorption and facilitate the distribution of surfactant along the alveolar surface.

Alveolar type II cells may also play an important role in the maintenance of the alveolar epithelium by their ability to differentiate into alveolar type I cells. The repair of injured alveolar epithelium occurs by the proliferation of alveolar type II cells. The proliferating type II cells appear to be capable of differentiating into either new alveolar type II cells or squamous alveolar type I cells. The signals that regulate

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differentiation of the proliferating alveolar type to cells appear to be related to the extracellular matrix. The connective tissue that supports the alveolar epithelium may provide the signals the regulate differentiation.

Alveolar type II cells also appear to be important in disease. Alveolar type II cells are morphologically hyperplastic after lung injury. The appearance of alveolar type II cells by light microscopy has led to the term reactive pneumocytes. Reactive pneumocytes also express more class I and II major histocompatibility complex (MHC) antigens. The expression of increased levels of MHC suggests that alveolar type II cells may have an immunologic function or play a role in local autoimmune processes.

Surfactant

Surfactant plays an important role in modulating the surface forces in the alveolus. Surfactant forms a film at the surface of the alveolar lining fluid. The effect of surfactant on surface tension is particularly important at low lung volumes. In early inspiration, surfactant promotes lung expansion by reducing alveolar surface tension. At end expiration, the reduction in surface tension at low transpulmonary pressures prevents atelectasis and lung collapse.

Surfactant is composed of several heterogeneous phospholipid-rich lipoproteins. In the alveolus, surfactant includes the surface phospholipid monolayer, tubular myelin, as well as an apoprotein component. The dominant component of surfactant is phospholipids (Table 4-3). Phospholipids are amphipathic molecules with a polar head attached to a glyceryl backbone. Acyl chains of variable length are attached to the glycerol backbone. In an aqueous environment, such as the alveolus, the phospholipids generally exist as a closed bilayer. Although the exact phospholipid composition can vary, the general characteristic of phospholipid mixtures is that they spontaneously form a surface film at an air fluid interface. The formation of this surface film significantly lowers surface tension. When the surface area decreases, as in expiration, the phospholipid molecules are packed more tightly, further lowering surface tension.

The protein composition of surfactant appears to play an important role in surfactant function. The most abundant surfactant protein is SP-A. SP-A is a large collagen-like glycoprotein that makes up about 4% of the total mass of isolated surfactant. Surfactant is produced in the alveolar type II cells and perhaps is also synthesized in Clara cells. The chemical interaction between SP-A and surfactant lipids is complex. SP-A may play a role in regulating the secretion and turnover of surfactant. Two additional surfactant apoproteins, SP-B and SP-C, have unusual chemical properties in that both are remarkably hydrophobic. Because of their hydrophobicity, these two apoproteins are often referred to as surfactant proteolipids. Both of these proteins are thought to play a role in the formation of the surfactant film.

Table 4-3. Components of Surfactant

Component Percentage Function
Lipids 95  
   Phospholipids 78 Modify surface tension
   Neutral lipids 10 Modify surface tension
Proteins 5 10
   Serum proteins 0 5 Variety of functions
   Apoproteins 5 Regulate turnover

The functional role of surfactant system is illustrated in several pathologic conditions. The acute respiratory distress syndrome is respiratory failure secondary to atelectasis that accompanies premature birth. Premature birth is associated with a deficiency of the surfactant system. When these infants are treated with exogenous surfactant, there is a dramatic improvement in the mechanical properties of the lung. The reduction in surface tension associated with the exogenous surfactant results in a pulmonary inflation and a dramatic improvement in ventilation.

ENDOTHELIAL CELLS

Endothelial cells in the lung form a continuous, nonfenestrated vascular lining extending from the pulmonary arteries to the pulmonary veins, with an intervening capillary meshwork. The blood vessels in the lung are unique vessels in the body because they are low-resistance vessels that carry deoxygenated blood on the arterial side and oxygenated blood on the venous side of the circulation. Endothelial cells compose 40% of all lung cells. The endothelial cells of the lung form a continuous sheet with an area of 130 m3. In the alveolar capillaries, endothelial cells have specialized organelle-free cytoplasm to facilitate gas exchange. These endothelial cells have a thin avesicular zone that is only 35 to 55 nm thick.

Endothelial cells generally orient in the long axis of the vessel, suggesting a morphologic response to existing shear forces. Similar to epithelial cells, endothelial cells have both luminal and abluminal domains to the cell membrane. These cell membrane domains are separated by intercellular tight junctions. Luminal domains have distinct functional characteristics. Proteins that regulate a variety of metabolic functions are expressed on the luminal surface (Table 4-4) and frequently associated as rafts in the lipid

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membranes. In addition, luminal domains appear to direct the secretion of cellular products, including von Willebrand's factor. The abluminal cell membranes interact with extracellular matrix and direct transport of plasma molecules toward the interstitium.

Table 4-4. Endothelial Cell Surface Proteins and Enzymes

Angiotensin-converting enzyme
Nucleotidases
Lipoprotein lipase
Thrombin
Fibrinolytic factors
Antifibrinolytic factors

The luminal membrane of endothelial cells is covered by a fuzzy coat, or glycocalyx, composed of glycosaminoglycans, oligosaccharide moieties of glycoproteins, glycolipids, and sialoconjugates. The cell membrane and its glycocalyx regulate a variety of cell functions. The luminal cell membrane mediates all cellular interactions and regulates recruitment of leukocytes into the lung. Enzymes such as angiotensin-converting enzyme, lipoprotein lipase, and receptors for insulin and low-density lipoproteins are expressed at the blood interface. Plasma proteins such as immunoglobulin, fibrinogen, fibrin, 2-macroglobulin, and albumin can be temporarily associated with the cell surface.

ALVEOLAR MACROPHAGE

The most common immune cell in the lung is the alveolar macrophage. The alveolar macrophage is 5 to 10 times more common in the lung than are T lymphocytes. Alveolar macrophages appear to have multiple functions in the lung. A primary function is their ability to scavenge particles and remove debris from the lung parenchyma. The ability of alveolar macrophages to phagocytize microorganisms provides an important defense against airborne pathogens. The alveolar macrophage also appears to play an important role in the repair and maintenance of lung parenchymal tissue.

Macrophages are far more common in the lower respiratory tract than in the tracheobronchial tree. Alveolar macrophages are believed to be derived from blood monocytes that migrate from pulmonary capillaries into the lung. Alveolar macrophages are thought to have a limited potential to divide and proliferate. The significant increase in alveolar macrophage concentration in some conditions, such as granulomatous lung disease, suggests either active recruitment of blood monocytes or the ability of alveolar macrophages to the divide in situ.

Although alveolar macrophages can have several different phenotypes, subpopulations of alveolar macrophages have not been clearly defined. The reason for this discrepancy is that alveolar macrophages can exist at a variety of different activation states. The activation states of alveolar macrophages appear to regulate the capacity to phagocytize, kill target cells, migrate, and release a broad range of secretory products. The activation signals for alveolar macrophages can include such diverse signals as the phagocytosis of inert particles, receptor binding of immunoglobulin, or exposure to cytokines.

Alveolar macrophages play an important role in maintaining the sterility of the airway. Alveolar macrophages are an important defense against airborne bacteria. The bacterial pathogens can be phagocytized as inert particles or by specific surface receptors on the alveolar macrophage membrane. Surface receptors may include membrane-bound immunoglobulin or receptors for terminal mannose sugars. Once the pathogen is phagocytized, the phagosome fuses with lysosome, and the organism is killed by an oxidative burst. Alveolar macrophages also use nonoxidative mechanisms, including proteases, lysozymes and a variety of other bacteriocidal proteins. An understanding of macrophage-dependent mechanisms of bactericidal activity holds the promise of novel antibacterial therapies.

The effectiveness of alveolar macrophages to eliminate microorganisms is varied: some microorganisms are susceptible to alveolar macrophages, whereas others are resistant. Some common bacterial pathogens, such as Staphylococcus aureus, are readily eliminated by alveolar macrophages. In contrast, Pseudomonas aeruginosa and Klebsiella pneumoniae are relatively resistant to alveolar macrophages and require the presence of neutrophils for elimination. The antimicrobial selectivity of alveolar macrophages can be of clinical importance. For example, patients who have neutropenia from chemotherapy have enhanced susceptibility to macrophage-resistant bacteria. Other resistant infectious agents include organisms such as Mycobacterium tuberculosis and Toxoplasma gondii. These organisms can continue to grow within alveolar macrophages. Activation of alveolar macrophages, with cytokines such as interferon- , can be effective in enhancing the growth suppression of these pathogens.

Alveolar macrophages also play an important role in eliminating damaged lung tissue and airway debris. The role of the alveolar macrophage in maintaining the normal structure of the lung is illustrated by several clinical examples. Alveolar proteinosis is a disease characterized by hypoxemia from large amounts of proteinaceous material found within the alveolar air spaces. The alveolar macrophages in these cases are filled with surfactant-like material. The normal macrophage regulation of surfactant turnover appears to be impaired in these patients. Another example is the anthracosis observed in long-term smokers. The anthracotic material found in bronchoalveolar lavage specimens, as well as in the histologic examination of the peripheral lung, is commonly found within alveolar macrophages. The macrophage appears to be the primary mode of elimination of airway debris.

Alveolar macrophages may also play an important role in directly modulating lung function. Alveolar macrophages have been found to secrete a variety of products that have a direct effect on pulmonary blood flow and vascular permeability. An example of this activity is the ability of alveolar macrophages to secrete nitric oxide. Alveolar macrophages may also secrete a variety of substances that effect airway resistance and hyperreactivity. Examples of these mediators include thromboxane A2, platelet-derived growth factor, and platelet-activating factor.

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LUNG LYMPHATICS

Individual lymphocytes can be located in the alveolar walls and on the epithelial surfaces of the airways. These airway-associated lymphocytes are recovered in the bronchoalveolar lavage specimens along with alveolar macrophages. In the normal bronchoalveolar lavage specimen, the mononuclear cells include lymphocytes, alveolar macrophages, and epithelial cells. The normal lavage lymphocyte composition is about 60% T cells, 10% B cells, and 30% null cells. Clinical conditions in which large numbers of T lymphocytes are obtained from bronchoalveolar lavage specimens include sarcoidosis and acute lung transplant rejection. The study of these T lymphocytes holds the promise of a minimally invasive diagnostic test for a variety of lung diseases.

More organized lymphoid tissue, composed of lymphoid aggregates and nodules, can be found along the bronchial tree. These so-called bronchus-associated lymphoid tissues (BALTs) are located beneath the airway epithelium and are most common at the bifurcation of airways. The lymphocytes found in BALTs are the B lymphocytes associated with humoral immunity. Although BALT tissues are common in experimental animals, the normal human lung has only rudimentary BALT tissues.

The anatomic distribution of lymphoid cells in the lung can be illustrated by lymphomatous involvement of the lung. Lymphoma tissue in the lung generally predominates along the airways and subpleural surfaces of the lung. Recent work in lymphocyte trafficking suggests that lymphocytes home to these tissues. The predominance of lymphocytes along the airways appears to be a pathologic reflection of the normal trafficking of lymphocytes to mucosa-associated lymphoid tissue (MALT).

The airway-associated lymphoid tissues play an important role in the immune response to inhaled antigens. Lymph nodes play a more important role in the response to inhaled antigens in humans than in experimental animals. Most of the lymph node tissue is located in the hilum of the lung and in the mediastinum. Inhaled antigens are delivered to these lymph nodes through peribronchial afferent lymphatics. The course of these lymphatic channels is illustrated by the embolic spread of lung malignancies. Tumor emboli sequentially appear in hilar and mediastinal lymph nodes. Interruption of these hilar lymphatic channels can be of clinical importance in lung transplantation and in sleeve resections of the lung. Submucosal lymphatics do not recanalize for more than 3 weeks after interruption and can be associated with increased lung water and impaired immunity.

The lymph node provides the scaffolding that facilitates the interaction of the B and T lymphocytes as well as the antigen-presenting cells of the immune system. T lymphocytes are cells that constantly recirculate throughout the body. Recirculation provides a mechanism for distributing immune cells throughout the body as well as ensuring antigen-reactive diversity. B lymphocytes can be found leaving the antigen-stimulated lymph node, but generally are not found in the unstimulated efferent lymph. When the lymph node is stimulated by antigen, the cell output in the lymph transiently decreases, and the size of the lymph node increases dramatically. This type of antigen-induced lymph node enlargement is commonly observed in a variety of infectious conditions. The increase in lymph node size is due to the rapid recruitment of lymphocytes from the blood. The lymphocytes recirculating in the blood bind to specialized lymph node endothelium called high endothelial venules (HEVs). HEVs are plump endothelial cells found only in specialized lymphatic tissue such as the lymph node. The recruited lymphocytes migrate to specific compartments within the lymph node. T cells are found in the paracortical regions, whereas B cells and associated germinal centers are found in the cortex of the lymph node. The enlarged lymph node provides the ideal cellular and chemical microenvironment for the generation of antigen-specific immune responses. Ultimately, these activated lymphocytes are released into the efferent lymph and eventually into the bloodstream.

GENETIC REGULATION IN THE LUNG

Genetic control of lung cells has important implications for the normal growth and development of the lung. Some diseases of the lung, such as cystic fibrosis and 1-antitrypsin deficiency, have a clear hereditary association. Genetic changes or mutations in these diseases occur in the germline. Because the genetic changes are inherited, the genetic abnormalities exist in every cell of the body.

More commonly, the genetic associations in lung diseases are sporadic. For example, most of the genetic changes leading to cancer in lung cells occur in cells that would otherwise be considered genetically normal. Environmental toxic or infectious exposures can lead to acquired or somatic mutations. These genetic changes exist only in the affected cells.

Multiple Genetic Hits of Carcinogenesis

The potential interaction of inherited and acquired genetic mutations was initially described by studying the epidemiology of retinoblastoma. Patients with retinoblastoma were found to have either a positive family history of the disease (inherited retinoblastoma) or no apparent family history (sporadic retinoblastoma). Statistical analysis by Knudson (1971) suggested that more than one genetic mutation (or hit ) is required for either inherited or sporadic retinoblastoma. People born with a germline retinoblastoma (Rb) gene mutation already have one hit. Any retinal cell that acquires a second hit in the Rb gene can develop into a

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retinoblastoma tumor. People with this germline mutation are therefore more likely to develop cancer than those without this mutation. In the setting of this genetic predisposition, retinoblastoma tends to occur earlier in life and is commonly associated with other malignancies.

In contrast, retinal cells in people born without the Rb germline mutation must acquire two mutations to develop into the retinoblastoma tumor. Consistent with these enhanced requirements for tumorigenesis (see Fig. 4-1), patients with a sporadic form of retinoblastoma manifest the disease later in life, and the disease is less likely to be associated with other cancers. These observations suggest that the hits necessary to transform a cell toward malignancy can be both acquired and inherited.

The basic concept of multiple mutational hits has been supported with studies of the Rb tumor suppressor gene. Cloning of the Rb gene has shown that in Rb tumors, both alleles of the Rb gene are inactivated, consistent with the two hits necessary for tumorigenesis. The mutational hits described in tumorigenesis generally mean a change in the normal sequence of nucleotide base pairs. This change can be as small as the deletion of one nucleotide base pair or as large as the elimination of the entire gene or group of genes. In non small cell carcinoma of the lung, absent or abnormal Rb proteins have been observed in up to 30% of tumors. There may also be a correlation between the level of abnormal Rb protein expression and the stage of the non small cell lung cancer. In one study, Xu and associates (1991) found that abnormal Rb expression was present in 20% of stage I and II patients and in 60% of stage III and IV patients.

There are several potential mechanisms for the development of genetic mutations. In the normal cells, the DNA of the 46 chromosomes is stably replicated during each mitotic cycle. When damage occurs in the DNA of the somatic cells, there are several DNA repair mechanisms to ensure the fidelity of DNA replication. Disruptions in the DNA repair mechanisms are observed naturally in aging. This genetic instability is also associated with prolonged exposure to occupational and environmental carcinogens. Cigarette smoke, for example, may play a role in disrupting DNA repair mechanisms.

A common feature of lung cancer is an abnormal number or arrangement of chromosomes in the tumor cells. Chromosomal aneuploidy is a gross manifestation of genetic instability. Aneuploidy is often detected by cytogenetic analysis or flow cytometry. Large segments of genetic information can be inverted, duplicated, deleted, or translocated onto another chromosome. These arrangements frequently result in the disruption of genes that can be associated with malignancy. The most common chromosomal abnormality identified in lung cancer is the loss of chromosome 3p. The loss of 3p has been observed in more than 90% of small cell lung cancers and in about 50% of non small cell lung cancers. Zabarovsky and associates (2002) have noted that the loss of 3p may contribute to the development of lung cancers because as many as three tumor suppressor genes reside on the 3p chromosome.

Another mechanism for the mutation of somatic genes is the insertion of viral DNA. DNA viruses incorporate themselves into genomic DNA. The presence of viral DNA frequently leads to cell death. On occasion, however, viral DNA can convert normal cells into cancer cells. Examples of viral induction include Epstein-Barr virus associated lymphomas and SV-40 associated malignant mesothelioma.

Oncogenes

Oncogenes are a class of genes that are expressed in normal cells. The overexpression or mutation of these genes, however, can be associated with uncontrolled growth and tumorigenesis (Table 4-5). In general, oncogenes are phenotypically dominant; a single mutation in one of the paired alleles is sufficient to promote carcinogenesis. An operational definition of an oncogene is a gene whose introduction into a cell results in the transformation of the cell; that is, the cell takes on some of the phenotypic and growth characteristics of a cancer cell.

The original description of oncogenes came from the study of cancer-associated viruses. These viruses were associated with cellular transformation in animals such as monkeys, chickens, rodents, and cats. The classic definition of an oncogene is a cancer-causing gene carried by an acute transforming retrovirus that has a normal counterpart (homolog) referred to as a protooncogene. Work by Cordell and colleagues (1978), and Bishop (1985) showed that the oncogene in the Rous sarcoma virus was not a genuine viral gene, but a preexisting cellular gene, as identified by Stehelin and colleagues (1976), that was copied and modified by an ancestor of the Rous sarcoma virus. The copied and modified gene was used by the virus to transform animal cells. Oncogenes were identified, and are generally named, based on the virus in which they were originally carried. For example, RAS is an oncogene from the rat sarcoma virus, and SRC is an oncogene from the Rous sarcoma virus. Although the research into RNA tumor viruses has found no causal link to human cancer, research on animal retroviruses has provided pivotal insights into the identity of cancer-causing genes.

Oncogenes have generally been found to encode proteins involved in signal transduction. Signal transduction proteins are responsible for the transmission of signals from

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the cell membrane to the replication machinery within the cell nucleus. The involvement of oncogenes in the transduction of these signals provides clues to the understanding of normal growth and control as well as tumorigenesis. When functioning normally, protooncogenes promote cell growth and division. The mutations that transform these protooncogenes into oncogenes lead to a loss in normal cellular regulation and uncontrolled cell growth. The growth-promoting function of oncogenes in signal transduction explains the genetic dominance of the mutation. A mutated oncogene will have a growth-promoting effect on the cell irrespective of the function of the other allele.

Table 4-5. Common Oncogenes

Name Tumor Associations
erb-b2, neu Breast, ovary, gastric
MYC Lymphomas, carcinomas
RET Thyroid carcinoma
K-ras Lung, colon
H-ras Bladder
NMYC Neuroblastoma

Table 4-6. Oncogenes associated with Human Lung Cancer

Name Frequency (%)
K-ras 30
MYC 10 40a
erb-2 25
bcl-2 25
a Expression of myc varies from 10% in non small cell lung cancers to up to 40% in some series of small cell lung carcinomas.

Of the 80,000 genes in the human genome, only about 50 genes have been found to transform cells in vitro. Even fewer genes have been found in mutant form in human cancers (Table 4-6). Only 20 of the protooncogenes capable of cellular transformation have actually been found in human tumors, and even fewer have been associated with thoracic malignancies. An example of a clinically relevant oncogene is K-ras, homologous to Kirsten murine sarcoma virus oncogene. K-ras mutations are found in up to 30% of adenocarcinomas. Rosell and co-workers (1993) have shown that ras mutations and increased ras expression have been correlated with decreased survival in patients with non small cell lung cancer.

Tumor Suppressor Genes

A commonsense approach to cellular regulation would suggest that there are growth-suppressing as well as growth-promoting signals within the cell. The finding that only about 20% of human tumors are associated with oncogenes suggests that the mutation or loss of growth-suppressing genes might also be important in tumorigenesis. The discovery of the retinoblastoma (RB) gene has provided a model for this type of dysregulation. The normal RB gene product (Rb) serves to constrain cell growth and division. When both alleles are mutated or lost, the normal cellular control mechanism is lost. The genes with these features have been collectively referred to as antioncogenes or tumor suppressor genes (Table 4-7).

Tumor suppressor genes are present in all normal cells. When these genes are missing or inactivated by mutation, the cells exhibit uncontrolled growth. This observation has led to the conclusion that tumor suppressor genes normally function to restrain cell growth. In general, one normal allele of a tumor suppressor gene pair is sufficient to prevent malignant transformation. The loss of all or part of the chromosome containing the tumor suppressor gene is commonly referred to as the loss of heterozygosity (LOH). If both copies of the gene are mutated or deleted, the function of the tumor suppressor gene is lost.

Table 4-7. Common Tumor Suppressor Genes

Name Tumor Association
DCC Colon
APC Colon, familial polyposis
BRCA-1, BRCA-2 Hereditary breast, ovarian
p53 Leukemia, multiple carcinomas
Rb Retinoblastoma
WT1 Wilms' tumor

In cancers with an inherited component, one copy of the tumor suppressor gene is mutated at birth. With the acquired mutation of the remaining allele, cellular growth is no longer suppressed, and tumorigenesis can occur (Fig. 4-1). As heritable forms of retinoblastoma have demonstrated, people who inherit a mutated allele have a much higher risk for developing cancer because they have only one functioning gene in reserve. The molecular data from a variety of malignancies suggests that tumor suppressor gene mutations contribute to the development of many cancers.

The most common tumor suppressor gene mutations have been observed in the p53 gene. Mutant versions of the p53 gene have been found in DNA samples from more than half of human tumors examined. The reason that p53 is so common in human tumors is partly related to its mode of action. When the p53 gene is mutated, the mutated gene

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loses its ability to suppress cell growth. The mutated p53 gene also acquires the ability to disrupt actively the function of the remaining intact gene. The consequence of this ability is that only one mutated gene copy is required to interfere with growth suppression, an effect described as a dominant negative mode of action. Levine and associates (1991) have shown that the p53 gene also appears to have several other unique functions that promote cellular proliferation and inhibit cell death (apoptosis).

Table 4-8. Tumor Suppressor Genes in Human Lung Cancer

Name Frequency (%)a
3p Chromosome deletion 50 90
RB 15 90
p53 50 80
p16 60
a In almost all series, the lower frequency reflects findings in non small cell lung cancer and the higher frequencies reflect small cell lung cancer.

Fig. 4-1. Schematic of the Rb gene and the two-hit hypothesis. A. An individual who inherits a mutation of the Rb gene requires only one additional hit for the development of retinoblastoma. B. In contrast, an individual without a germline mutation of the Rb gene requires mutational events affecting both genes to develop retinoblastoma.

In lung cancer, p53 is associated with a point mutation. This point mutation appears to be related to chemicals from cigarette smoking. The overall frequency of p53 mutations in non small cell lung cancer is about 50%, and is 80% in small cell lung cancer. The expression of p53 mutations has not yet been correlated with prognosis in lung cancer (Table 4-8).

As more is learned about the function of these genes, tumor suppressor genes will probably be found to encode proteins involved in transducing negative signals. Tumor suppressor gene products will be involved in the receiving and processing signals from the cell membrane, that is, proteins that normally function to inhibit the replication machinery within the cell nucleus. An example of this processing is the growth-suppressing signal provided by transforming growth factor- (TGF- ). When TGF- binds to the cell membrane, most cells stop growing. In contrast, when cells lose the Rb gene function, they lose the ability to respond to TGF- . These cells grow unrestrained even when exposed to high doses of TGF- .

Fig. 4-2. Schematic of the multiple-hit hypothesis of carcinogenesis. The specific mutations that lead to human lung cancer are unknown at this time. Based on observed abnormalities in lung cancer tumors, a schematized sequence of events is the following: Step 1. The deletion of the 3p chromosome results in the loss of several tumor suppressor genes, leading to dysregulated bronchial epithelial growth and possibly to adenoma. Step 2. The loss of the tumor suppressor gene p53 results in cytologic changes consistent with carcinoma in situ. Step 3. The overexpression of the ras oncogene results in the development of invasive bronchogenic carcinoma.

The clinical relevance of any given tumor suppressor gene most likely will depend on the condition of the entire tumor cell genome. The multiple-step model of carcinogenesis argues that a mutation or alteration in any given gene only serves to push a cell down the pathway to full-blown malignancy. The evolution of a clinically relevant cancer requires the presence of multiple successive changes in distinct genes (Fig. 4-2). The collective effect of these changes is required to push the cell from abnormal to malignant.

Cystic Fibrosis

Cystic fibrosis is a common genetic disease, with 5% of white Americans carrying the mutant version of the gene. About 1 in 2,500 children of European descent carries two defective copies of the gene. These children have the disease of cystic fibrosis. The disease of cystic fibrosis causes impairment of the pancreas, intestines, and liver. Frequently, the most devastating consequence of the disease is the persistent infection in the lung and the subsequent damage to the airways.

For many years, clinicians recognized that children with cystic fibrosis have excessive salt in their sweat. This clinical observation reflected both the pathogenesis and genetic basis for cystic fibrosis. Even the test for cystic fibrosis, the measurement of chloride content in the child's perspiration, remains the cornerstone of the clinical diagnosis. The observation of the excessive salt secretion of children with the disease also provides an important clue to its genetic orgin.

In 1989, a large group of collaborators announced the identification of the gene responsible for cystic fibrosis. The product of this gene was called the cystic fibrosis transmembrane conductance regulator (CFTR) because of its probable regulation of chloride secretion. Sequencing of the gene revealed a mutation that was present in 70% of all cystic fibrosis patients. This gene is frequently referred to as the F508 mutation. The mutation involves the deletion of three nucleotides from the gene, with the resultant loss of a single amino acid (phenylalanine) at position 508 in the CFTR protein.

The CFTR protein appears to form a chloride-permeable channel in the outer membrane of many cells. The movement of chloride through the pore is regulated by the protein, depending on the metabolic condition of the cell. When the gene is mutated, the protein product is retained within the endoplasmic reticulum of the cell and is never expressed at the cell surface. If a normal gene exists, sufficient quantities of the CFTR reach the cell membrane to facilitate relatively normal chloride movement. Consistent with the recessive inheritance pattern of cystic fibrosis, the clinical disease is apparent only when both genes are mutant.

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Although the genetic defect of cystic fibrosis is clear, the pathogenesis of the clinical syndrome remains unclear. The submucosal glands in the conducting airways appear to express a large amount of the CFTR protein. How the absence of normal CFTR leads to the hyperinflated lungs and recurrent Pseudomonas aeruginosa infections is unknown. Further studies of the function of this protein may provide important clues regarding normal ventilation and host defense of the lung.

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General Thoracic Surgery. Two Volume Set. 6th Edition
General Thoracic Surgery (General Thoracic Surgery (Shields)) [2 VOLUME SET]
ISBN: 0781779820
EAN: 2147483647
Year: 2004
Pages: 203

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