Pulmonary Host Defenses

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Chapter 22 Pulmonary Host Defenses

Theo J. Moraes, Chung-Wai Chow, Gregory P. Downey

The epithelial surface of the lung is continuously exposed to a variety of potentially pathogenic microorganisms, allergens, particulate pollutants, and other noxious agents. An intricate defense system has evolved over time to protect the lungs from these potentially harmful entities while preserving homeostasis and lung function (Figure 22-1). This system of defense mechanisms has two components: an innate (nonspecific) response and an adaptive or acquired (specific) response.

Figure 22-1 Pulmonary host defense. The pulmonary host defense system is composed of multiple components, including physical barriers such as the nose and mucous layer that lines the airways. Mechanisms also exist to remove offending microbial pathogens or noxious particles, either directly by phagocytosis or endocytosis or indirectly by mediator-regulated responses. Activation of the immune and inflammatory responses usually will facilitate resolution of the injury and clinical recovery of the patient. However, if these immune and inflammatory responses are excessive or unregulated, tissue injury and disease ensue.

The innate immune response is evolutionarily conserved to provide immediate (occurring over seconds to minutes) host defense in a broad, nonspecific manner. Only vertebrates have an additional, adaptive immune system, which is directed at specific pathogens or molecules. Although the two systems work in concert to protect the host, each has several distinctive features. With rare exceptions, the innate component of the immune system depends on proteins and signaling pathways that exist in a fully functional form, does not require priming, and is not strengthened with subsequent exposures. By contrast, the adaptive immune response requires additional time (days to weeks) (see Figures 22-2 and 22-3) to ramp up to full capacity, is specific to the pathogen (and even to specific molecular determinants of the pathogen), and has memory to provide for stronger responses with subsequent attacks (“anamnestic response”).

Figure 22-2 Timeline of pulmonary host defense mechanisms—immediate, early (seconds to hours), and late (hours to days)—that serve to protect the lung.

Figure 22-3 Interactions among the different pulmonary host defense mechanisms—immediate, early, and late. These defense mechanisms augment the immune and inflammatory responses. TLR, Toll-like receptor.

Together, these immune responses provide a formidable force to combat invading pathogenic microbes, as indicated by the rarity with which healthy humans succumb to lung infections. Specific components of the innate and adaptive host defense mechanisms are reviewed in this chapter.

Structural Defenses

With a surface area in adults of 70 m², which comes into contact with roughly 10,000 L of air a day, the lung is confronted with constant threats from microbes. In addition to inhaled pathogens, high bacterial concentrations are present in oropharyngeal secretions (10⁸/mL), and aspiration of these secretions also may pose a serious risk for invasive infection (aspiration pneumonia). Historically, the lung has been considered to be a sterile environment; recent developments in molecular analysis have provided evidence for the presence of a microbial flora of considerable diversity in the lung, which moreover is altered in disease states. Accordingly, the respiratory tract has developed a series of structural barriers that are designed both to minimize the number of pathogenic microbes entering the lungs and to hasten their clearance before an infection can be established (Table 22-1).

Table 22-1 Structural Defenses of the Airway

Structure	Functions
Nose	Filters air Warms and humidifies air Sediments particles Olfaction Sneeze reflex

Glottis

Protects from gastrointestinal and nasopharyngeal contamination

Cough reflex

Mucociliary escalator

Traps foreign particles

Facilitates physical removal of particles

Epithelium

Barrier to microbes

Mucociliary escalator

Production of antimicrobial factors

Cytokine production

Adhesion molecule expression

Particle size is an important factor determining the degree of penetration into the airways (Table 22-2). Very large particles are filtered by vibrissae (nasal hairs). Particles approximately 30 µm in diameter are removed in the nasal passages, where turbulent airflow results in prolonged air-mucosa contact, with subsequent particle impaction. Most particles between 10 and 30 µm in diameter also will be deposited on the turbinates and nasal septum, carina, or within the larger bronchi. The branching nature of the airways provides two additional mechanisms of protection: (1) the secondary, tertiary, and quaternary carinae force particles to embed in the airway mucosa, thereby preventing further penetration into the lung, and (2) reduced airflow with increased airway branching allows gravity to sediment most particles larger than 2 µm. Particles less than 0.2 to 0.5 µm across tend to stay suspended as aerosols and are exhaled. Much smaller particles (less than 0.1 µm) may be deposited as a result of brownian motion (bombardment with gas molecules).

Table 22-2 Effect of Particle Size on Penetration into Airways

Particle Size	Fate
>>>30 µm	Filtered by vibrissae
>30 µm	Nasopharyngeal impaction
10 to 30 µm	Nasopharyngeal, tracheal, and large bronchial impaction
2 to 10 µm	Sedimentation in airways
0.2 to 2 µm	Reach alveoli
0.2 to 0.5 µm	Exhaled
<0.2 µm	Exhaled or deposited (brownian motion)

Thus, only particles roughly between 2 and 0.2 µm in diameter will reach the alveoli. Unfortunately, most bacteria are from 0.5 to 2 µm in size and, when inhaled, may reach the terminal bronchioles, where they have the potential to establish an infection. Some exceptionally pathogenic bacteria require exceedingly small numbers (e.g., only 2 to 50 organisms) to establish infection.

In addition to particle size, other factors such as shape, charge, and state of hydration may influence the depth of penetration. For example, the grass heads of timothy grass, or Alternaria, can penetrate deeper into the lung than would be expected from their physical size.

Nares

The efficiency of the nose in filtering inspired air and trapping aerosolized particles has been highlighted by inhaled drug delivery studies that demonstrate better pulmonary drug deposition with mouth inhalers than with mask inhalers. Nasal congestion and increased mucus production also contribute to trapping of unwanted particles or microbes, preventing them from going further into the airways.

In addition to filtration, the nose functions to warm and humidify inspired air, thus protecting cells from physical stress and ensuring optimal performance of the mucociliary system. Olfaction also is protective, because sniffing allows potentially harmful gases to be detected before they contact the lower airways.

As noted previously, however, the upper respiratory tract may be a source of infection when aspiration of small amounts of upper airway secretions leads to compromise of the host pulmonary system. Microbial colonization is normal, and although potential pathogens can be isolated from the nasopharynx of healthy persons, under normal conditions, resident flora competitively inhibit the growth of pathogens.

Cough/Sneeze

Mechanical or chemical stimulation of receptors in the nose, larynx, or trachea or elsewhere in the respiratory tree may produce bronchoconstriction to prevent deeper penetration of irritants and also may trigger the cough or sneeze reflex to expel particles deposited in the airways (Table 22-3). The cough reflex aids mucociliary transport to remove trapped particles. Mucus usually is conveyed to the carina by the cilia and then expelled by coughing from this location. Disruption of the cough reflex (e.g., in smokers or patients with vocal cord palsy or stroke) results in a predisposition to pneumonias.

Table 22-3 Phases of Cough/Sneeze

Component	Event(s)
1. Inspiratory phase	Deep inspiration, usually 1 to 2 times tidal volume
2. Compression phase	Begins with closure of the glottis and contraction of respiratory muscles, resulting in the generation of high intrathoracic pressure (up to 100 to 200 cm H₂O in adults)
3. Expressive phase	Glottal opening, with airflow at rates as high as 25,000 cm/sec (partly helped by compression of airway cross section)
4. Relaxation phase	Relaxation of respiratory muscles with temporary bronchodilatation

Glottis

The digestive tract and the respiratory tract share both an embryologic origin and an opening to the external world (the mouth). The glottis protects the lungs from potential contamination of the airways with digestive tract material. Nevertheless, small amounts of aspiration do occur from the nasopharynx and oropharynx or esophagus in healthy people, usually without pathologic consequences. Of importance, frequent aspiration or episodes of aspiration of large amounts of material, as is seen in persons with glottic dysfunction, can lead to bacterial pneumonia and chemical pneumonitis and possibly contribute to pulmonary fibrosis.

Respiratory Epithelium

The respiratory epithelium is a highly effective barrier to microbes. Conducting airways are lined with pseudostratified columnar epithelial cells that become cuboidal as the branches extend to the alveoli. Specialized structures termed tight junctions separate the epithelial monolayer into apical (luminal) and basolateral components that form an important barrier to the passive passage of molecules and microbes from the airway lumen or alveolar space.

In addition to this important physical barrier function, epithelial cells, including ciliated cells, goblet cells, serous cells, basal cells, and Clara cells, are integral to the normal function of the mucociliary escalator, the production of a variety of antimicrobial molecules, and the initiation and regulation of inflammatory responses (see Figure 22-4).

Mucociliary Escalator

The airway epithelium is lined from the trachea to the respiratory bronchioles by the airway surface liquid (ASL), a 5- to 25-µm-thick surface film the primary function of which is to trap and facilitate the physical removal of foreign particles, as well as to provide an environment conducive to the activity of antimicrobial molecules (Table 22-4).

Table 22-4 Properties of the Mucociliary Escalator

Structure	Function(s)	Dysfunction in Disease
• Present from trachea down to level of respiratory bronchioles • 200 cilia per ciliated cell • Each cilia: 6 µm long and 0.2 µm in diameter • Thickness of layer: 5-25 µm • Inner layer: low-viscosity, periciliary solutes • Outer layer: viscous mucous blanket

• Traps foreign particles and transports them up and out of respiratory tract

Transport rate: small airways: 0.5-1 mm/min; large airways and nose: 5-20 mm/min

• Production of antimicrobial compounds

• Smokers: decreased number of ciliated cells, depressed ciliary functions

• Bronchiectasis

• Cystic fibrosis

• Primary ciliary dyskinesia

• Stents

ASL is a critical component of the mucociliary escalator and is the result of secretion by glands and serous cells and of plasma transudation (Table 22-5). The inner low-viscosity periciliary sol facilitates the coordinated beating action of the cilia that propels the outer viscous mucous blanket toward the glottis, thereby facilitating the removal of trapped pathogens or particles by expectoration or ingestion. The viscous mucus is composed of mucopolysaccharides, produced predominantly by submucous glands in the larger airways, with increasing contributions from goblet cells and Clara cells with successively larger airway generations.

Table 22-5 Properties of Airway Surface Liquid (ASL)

Composition	Function	Dysfunction in Disease
Secretions from glands and goblet cells Plasma transudation	Antimicrobial properties due to low pH and secreted antimicrobial compounds	Cystic fibrosis Asthma COPD

COPD, chronic obstructive pulmonary disease.

Dysfunction of the ASL is seen in conditions such as asthma and chronic bronchitis, in which excessive mucus is produced, resulting in airway obstruction. In cystic fibrosis, defects in ion transport are thought to reduce ASL volume, thus increasing viscosity of the mucus. This altered environment impairs mucociliary clearance and predisposes the lungs to bacterial colonization, highlighting the importance of the antimicrobial function of normal ASL.

The cilia are the second key component of the mucociliary escalator and are present from the upper airways down to the terminal bronchioles. There are approximately 200 cilia per ciliated cell. Each of these protuberances is approximately 6 µm long and 0.2 µm in diameter. The cilia are arranged longitudinally to coordinate a beating activity of 500 to 1500 beats/minute, which serves to efficiently propel the mucous layer. Abnormal ciliary function may be related to primary ciliary dyskinesia (most frequently consequent to defects in the microtubule structure of the cilia) and predisposes the lungs in affected persons to development of pneumonia and bronchiectasis. In turn, bronchiectasis, whether associated with genetic causes such as cystic fibrosis or arising as an acquired condition such as sequelae of pulmonary infections, including pulmonary tuberculosis, can lead to localized ciliary dysfunction.

Although these structural components of the respiratory system are not formally considered to be part of the immune system, together they nonetheless constitute a vital and early component of the microbial defense systems of the lung.

Specific Cell Responses

Diverse cell populations contribute to the host defense system of the lung. In addition to the cells of the respiratory epithelium, these include other structural cells of the lung, the pulmonary vascular endothelium and fibroblasts, resident leukocytes, and, at later stages of immune responses, recruited immune cells (Table 22-6), as reviewed next.

Table 22-6 Cells Mediating Pulmonary Host Defense and the Inflammatory Response

Timing	Immune Cells	Nonimmune Cells
Early phase	Monocyte-macrophages Eosinophils Mast cells Neutrophils Natural killer cells

Late phase

Epithelial Cells

The contribution of the respiratory epithelium is not limited to its roles as a structural barrier and facilitator of mucociliary clearance (Box 22-1). Respiratory epithelial cells actively participate in the regulation of inflammation and are capable of mounting an immune response by internalization of organisms and secretion of cytotoxic and antimicrobial peptides. Inhaled microbial pathogens including bacteria and viruses and other antigens can trigger activation of pathogen recognition receptors (such as Toll-like receptors [TLRs], discussed later in some detail under “Innate Immune Receptors”) expressed by epithelial cells. Epithelial cells are induced by bacterial components, such as LPS, and by cytokines such as tumor necrosis factor (TNF)-α and interleukin-1β (IL-1β) to express various gene products (by the NFκB signaling pathway, discussed later on) that modulate the inflammatory response (Figure 22-4). Such inducers include the following:

• Cytokines such as TNF-α, IL-1β, and thymic stromal lymphopoietin (TSLP)

• Chemokines that include macrophage inflammatory protein (MIP)-2, CXC chemokines, monocyte chemoattractant protein (MCP)-1, IL-7, IL-8, IL-15

• Nitric oxide (NO) and reactive nitrogen species (ONOO⁻)

• Adhesion molecules such as β-integrins and intercellular adhesion molecule-1 (ICAM-1)

• TLRs such as TLR-2 and TLR-4

• TNF-α receptors, TNFR1 and TNFR2

• Growth factor receptors such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR)

• Plasminogen activator receptors

Figure 22-4 Epithelial cell defense mechanisms. The pulmonary epithelial monolayer functions as a highly effective defense system. The different mechanisms are illustrated, including the airway surface liquid that contains potent antimicrobial compounds, the mucous layer, junctions between the epithelial cells, and surface receptors expressed by the epithelial cells. ICAM, intracellular adhesion molecule; RNS, reactive nitrogen species; ROS, reactive oxygen species; SP-A, surfactant-associated protein A; SP-D, surfactant-associated protein D.

TSLP, also produced by fibroblasts, is thought to aid in the maturation of dendritic cells. Consequently, epithelial products lead to innate cell (neutrophil) recruitment to limit local infection. Epithelial products can promote dendritic cell maturation and thus induce an adaptive immune response (including memory T cells and neutralizing antibodies). In addition to these molecules, pulmonary epithelial cells also express a number of antimicrobial mediators that are unique to the lung. These include the surfactant proteins SP-A and SP-D (members of the collectin family) and the β-defensins, potent antimicrobial peptides (discussed later on).

Endothelial Cells

Endothelial cells also play a pivotal role in the regulation of host defense and propagation of the inflammatory response. Like epithelial cells, endothelial cells also form tight junctions that separate the endothelial monolayer into apical and basal surfaces and prevent passive movement of particles and molecules. In addition to this physical barrier, activated endothelial cells modulate the expression of numerous proteins involved in different pathways that contribute to host defense. These include the following:

• Reactive nitrogen and oxygen species, including nitric oxide (NO^•), peroxynitrite (ONOO⁻), superoxide (O₂⁻), and hydrogen peroxide (H₂O₂), which are cytotoxic to microorganisms and cells

• Inflammatory cytokines such as TNF-α, IL-1β, and IL-6, as well as chemokines such as IL-8, RANTES (regulated on activation, normal T cell–expressed and secreted), and MIP-1, which activate and recruit leukocytes

• Cytokine and chemokine receptors such as TNFR1 and IL-1R

• Adhesion molecules, including ICAM-1, ICAM-2, platelet endothelial cell adhesion molecule (PECAM), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and P-selectin, which have differential binding specificities for specific leukocyte populations

• Toll-like receptors (TLRs)

• Procoagulants and protease-activated receptors

• Proteases

• Leukotrienes and prostaglandins

• Growth factors such as vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-β

• Alterations in the surface adhesion molecules, cytoskeleton, and intercellular (junctional) proteins to allow for leukocyte adhesion transmigration and to regulate changes in vascular permeability

Fibroblasts

Although fibroblasts are classically viewed as structural cells and passive responders to exogenous influences, they are actually integral to the regulation of host defense. Fibroblasts synthesize extracellular matrix proteins, such as collagen and fibronectin, which are required to maintain the structural integrity of the lung during normal tissue turnover and tissue repair after infection and injury. They also express and secrete matrix-degrading proteases such as matrix metalloproteinases and cytokines such as IL-1, TNF, and TSLP, which play important roles in leukocyte recruitment and activation.

Immune Cells

Several leukocyte populations play distinct and vital roles in host defense in the lung and can be broadly classified as those that are normal residents of the lung (monocyte-macrophages, mast cells, dendritic cells) and those recruited in response to infection in injury (neutrophils and lymphocytes) (Table 22-7). Studies in animal models and in humans have demonstrated the increased risk of lung infection associated with defects in or absence of these cell types.

Table 22-7 Leukocyte Defense Mechanisms

Immune Cell	Primary Role(s)	Primary or Unique Inflammatory Mediators
Neutrophils	Killing and elimination of invading organisms

Reactive oxygen and nitrogen species

Proteolytic enzymes and cationic proteins

TNF-α, IL-1β, IL-6

Macrophages

Immune surveillance

Killing and containment of invading microorganisms

Removal of particulate matter

Reactive oxygen and nitrogen species

Mast cells

“Antennae” of immune response

Granule release (mediated via Fcε receptors)

TLRs

PAF, leukotrienes, and prostaglandins

IL-1, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IL-16, TNF-α, VEGF, TGF-β, MIP-1α, MCP-1

Dendritic cells

Eosinophils

Eosinophil-specific granules

Cationic proteins

Major basic protein

Eosinophil peroxidase

Eosinophil-derived neurotoxin

Lipid mediators—leukotriene C₄ and PAF

ICAM-1, intercellular adhesion molecule-1; IL-1, interleukin-1 [etc.]; MCP-1, monocyte chemoattractant protein-1; MIP-1, macrophage inflammatory protein-1; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α; PAF, platelet-activating factor; TLRs, Toll-like receptors; VEGF, vascular endothelial growth factor.

Eosinophils

Eosinophils (Figure 22-5) are considered to be effector cells of allergic responses and of parasite elimination. These bone marrow–derived cells contain four distinct granule cationic proteins: (1) major basic protein; (2) eosinophil peroxidase; (3) eosinophil cationic protein; and (4) eosinophil-derived neurotoxin.

Figure 22-5 Defense mechanisms mediated by eosinophils and basophils. Immunoglobulin E (IgE)-mediated degranulation induces release of four distinct granule cationic proteins from eosinophils that can modulate the inflammatory response and display antihelminthic properties. Eosinophils and basophils also are capable of releasing cytokines and chemokines in a stimulus-specific manner.

During allergic inflammation, eosinophils release granule contents, as well as inflammatory mediators including lipid mediators such as leukotriene C₄ and platelet-activating factor, which may cause dysfunction and destruction of other cells.

Polymorphonuclear Neutrophils

The primary function of neutrophils in the innate immune response is to contain and kill invading microbial pathogens. Neutrophils merit particular mention in pulmonary host defense as the pulmonary microvascular endothelium preferentially recruits this leukocyte population in response to infection and inflammation (see Figure 22-6) by the expression of adhesion molecules and chemokines that target neutrophils. Recently it has been recognized that the T_H17 pathway involving the production of IL-17 and IL-23 can lead to neutrophil recruitment. Different stimuli including allergen sensitization, mast cell activation, various infections and TNF production, can promote a T_H17 response and thereby recruit neutrophils to the lung.

Figure 22-6 Leukocyte recruitment in airways and alveolar spaces. Contact with noxious agents or pathogens induces activation and enhanced expression of adhesion molecules on the apical surface of vascular endothelial cells (1) that facilitate leukocyte adhesion, recruitment, and activation; leukocytes then transmigrate across the endothelium, through the interstitial tissues, and across the epithelium and into the air space (2); leukocytes and the epithelial cells may become activated during these processes (3), leading to enhanced inflammatory cytokine and chemokine production (4).

Neutrophils achieve their antimicrobial function through a series of rapid and coordinated responses that culminate in phagocytosis and destruction of the pathogens (Figure 22-7). Neutrophils have a potent antimicrobial arsenal that includes the following roles:

• Oxidants

• Powerful proteolytic enzymes (proteinases)

• Cationic peptides

Figure 22-7 Neutrophil-mediated defense mechanisms. Neutrophils play a central role in host defense of the lung through a variety of mechanisms (1 to 6), including their ability to phagocytose pathogens and, by oxidative and proteolytic mechanisms, kill the invading bacteria.

Oxidants such as O₂⁻ and H₂O₂ are produced by a multicomponent enzyme termed the phagocyte NADPH oxidase (NOX2). Granules within the cytoplasm of polymorphonuclear neutrophils (i.e., leukocytes) (PMNs) contain potent proteinases and cationic proteins that can digest a variety of microbial substrates. These compounds are released directly into the phagosome, compartmentalizing both the pathogen and the cytotoxic products (Figure 22-8). Conversely, neutrophil serine proteinases can be externalized in a weblike fashion together with nucleosomes to function as a trap for pathogens. These neutrophil extracellular traps (NETs) have been described in the vasculature and in the airways. NETs have an important antimicrobial function independent of phagocytosis by bringing and immobilizing microbial pathogens in close proximity to proteinases. NETs also provide a physical barrier to microbial spread. Of interest, some evidence suggests that eosinophils also produce NET-like structures in the lung.

Figure 22-8 Neutrophil-derived antimicrobial products. O₂⁻ and H₂O₂ are produced by a multicomponent enzyme, termed the phagocyte NADPH oxidase, which is expressed by neutrophils (PMNs) and macrophages. Leukocytes also contain unique secretory granules, the primary and secondary granules, which release potent proteolytic enzymes and cationic proteins that can digest a variety of microbial substrates. fMLP-R, fMet-Leu-Phe receptor; hCAP-18, human cathelicidin antimicrobial protein-18; NADPH, nicotinamide adenine dinucleotide phosphate (reduced); PMNs, polymorphonuclear neutrophils [leukocytes].

Antimicrobial Molecules

Antimicrobial molecules (Table 22-8) are expressed by multiple cell types and play important roles in destruction and removal of pathogens. An overview of the important factors that have been shown to impede microbial growth and infection in the context of host defense of the lung is presented next.

Table 22-8 Antimicrobial Factors in Airway Surface Liquid

Factor	Role(s)/Function(s)
Lactoferrin	Binds iron Inhibits bacterial growth Enhances PMN function Modulates LPS activity

Lysozyme

Hydrolyzes peptidoglycan

Fibronectin

Complement

Immunoglobulins IgA and IgG

Opsoninization

Complement activation

Defensins

Pore formation in microbial membranes

Chemotactic

Promotes cytokine production

Stimulates dendritic cells

Cathelicidins

Modulates LPS

Chemotactic

Stimulates dendritic cells

Collectins

Binds carbohydrate domains of organisms, with reduced virulence and increased phagocytosis

LPS, lipopolysaccharide; PMN, polymorphonuclear neutrophil [leukocyte].

Antimicrobial Components in the Airway Surface Liquid

The acidic pH (6.4 to 7.3) of the ASL is inhibitory to microbial proliferation, as well as providing an optimal environment for the activity of the antimicrobial molecules found in the ASL. Important among these are lactoferrin and lysozymes.

Lactoferrin

Lactoferrin is an iron-binding protein that is secreted by serous epithelial cells and neutrophils that competes with bacteria for iron, thereby inhibiting bacterial growth. Lactoferrin also possesses other antimicrobial properties, including the following:

• Enhancement of neutrophil functions—motility, adherence, and superoxide production

• Inhibition of biofilm formation by Pseudomonas aeruginosa

• The ability to injure bacterial membranes with resultant release of LPS

• Modulation of LPS activity by competitively binding LPS, thus preventing LPS-binding protein from binding and promoting an LPS-CD14 interaction, which would result in cell activation

Some evidence also suggests that lactoferrin can upregulate expression of various cytokine and growth factor genes such as IL-12 and IFN-β.

Lysozyme

Lysozyme is an enzyme produced by serous cells, macrophages, and PMNs that can hydrolyze peptidoglycan, a major component of gram-positive bacterial cell membranes, thus providing defense against infection by this type of bacteria. Lysozyme also aids in the defense against gram-negative bacteria and acts synergistically with lactoferrin, secretory leukoprotease inhibitor (SLPI), and LL-37 (an antimicrobial peptide) to provide multiple mechanisms for bacterial destruction and removal.

Complement

Complement proteins are sequentially activated in a cascade encompassing three distinct pathways: (1) the classic antibody-antigen complex–dependent pathway, (2) the alternate pathway that is initiated by foreign or microbial products, and (3) a more recently identified pathway that is initiated by mannose-binding lectin (MBL), a member of the collectin family, and a related family of proteins called ficolins that are present in the lung. Complement proteins exude from plasma into airways in response to inflammation and are also produced by macrophages, type II pneumocytes, and fibroblasts. Specific complement components have important roles in host defense:

C5a: potent anaphylatoxin and chemoattractant that recruits neutrophils into the lung and enhances neutrophil-mediated microbial killing

C3a: another potent anaphylatoxin that also stimulates mucus production from goblet cells and enhanced LPS-induced synthesis of TNF and IL-1 by adherent phagocytes while decreasing synthesis in nonadherent cells

C5a-C9 complex: also known as the membrane attack complex (MAC), which creates pores in cell membranes, effectively killing bacteria

Multiple complement components can function as opsonins aiding phagocytosis of foreign particles (Figure 22-9). Of importance, hereditary deficiency of specific components of the complement pathway results in recurrent respiratory tract infections.

Figure 22-9 Immune defense mechanisms mediated by receptor-mediated phagocytosis and signaling. Internalization of pathogens or particulate matter induces multiple host defense mechanisms, including degradation of the offending agent (1), induction of the NADPH oxidase (neutrophils and macrophages) or iNOS (2), induction of chemokine and cytokine expression (3), and antigen presentation (particularly in dendritic cells and macrophages) (4). iNOS, inducible nitric oxide synthase; NADPH, nicotinamide adenine dinucleotide phosphate (reduced); NO, nitric oxide; ROS, reactive oxygen species.

Antimicrobial Peptides

A number of antimicrobial peptides are present in the airways. These can be classified conveniently into four groups on the basis of structural motifs: defensins, cathelicidins, histatins, and collectins.

Defensins

Defensins are single-chain strongly cationic peptides that have a broad spectrum of antimicrobial activity against gram-positive bacteria, gram-negative bacteria, fungi, and viruses. They work synergistically with other host defense molecules such as lysozyme and lactoferrin. The antimicrobial activities of defensins include the ability to form pores in target membranes, to interfere with protein synthesis, and to directly damage DNA. Defensin activity is influenced by the salt concentration of the ASL, with higher concentrations decreasing their antimicrobial activity.

Defensins are defined by the presence of six cysteines and three intramolecular disulfide bridges and are classified into three different subgroups: α, β, and θ defensins. Only α and β defensins are of relevance in humans. The first four human α defensins (HD 1 to HD 4) are produced by neutrophils and are found in the airways, and the other two (HD 5 and HD 6) are found in the small intestine and female urogenital tract. Of the 28 human β defensins identified, 6 (HBDs 1 to 6) are expressed mainly by epithelial cells. HBDs 2 to 4 are inducible in response to a variety of stimuli, including bacterial and viral infection, IL-1, TNF, and LPS. The levels of HDs 1 to 4 are increased in inflammatory processes as a result of release by activated neutrophils.

In addition to their direct antimicrobial properties, defensins also contribute to host defense in other ways:

• Enhanced bacterial binding and killing and cytokine production by airway epithelial cells

• Chemotaxis for monocytes and immature dendritic cells

• Direct binding to CCR6 receptors on dendritic cells, thus linking the innate immune system to adaptive immunity (β-defensins)

• Promotion of phagocytosis in macrophages and neutrophils

• Downregulation of the inflammatory response by increasing SLPI release from airway epithelial cells, inhibiting the complement cascade, and reducing macrophage cytokine release and neutrophil oxidant production

Cathelicidins

The only human cathelicidin is LL-37, a peptide with a broad spectrum of antimicrobial activity that acts synergistically with lysozyme and lactoferrin. LL-37 is stored as a precursor, hCAP-18, in specific granules and also is secreted by mast cells and the respiratory epithelium. Other host defense activities of LL-37 include the following:

• Neutralization of LPS

• Chemotactic for neutrophils, eosinophils, mast cells, and T lymphocytes

• Direct interaction with dendritic cells to form T_H1 cells

• Mast cell degranulation

• Induction of cytokine and chemokine expression by epithelial cells and monocytes

• Induction of angiogenesis

• Induction of epithelial wound healing

Histatins

Histatins are a family of cationic histidine-rich peptides that are strongly antifungal and may have LPS-neutralizing properties. Histatins are present in saliva and play an important role in preventing oral infections. They have not been isolated from airway secretions.

Collectins

The collectins, or collagenous C-type lectins, are a family of polypeptides that bind collagenous carbohydrates. Collectin proteins that play a role in pulmonary host defense include MBL and the surfactant proteins SP-A and SP-D. These molecules are secreted by the respiratory epithelium and are present in the airways. The collectins have broad antimicrobial activity against viruses, bacteria, and fungi by binding to conserved carbohydrate domains. In addition, collectins can bind organic antigens, apoptotic cells, nucleic acids, and host proteins. As discussed previously, MBL can activate the complement cascade and also has been shown to induce inflammatory mediator production from monocytes. The importance of these molecules is illustrated by the susceptibility of SP-A– and SP-D–deficient mice to various lung infections. In addition, some clinical studies suggest a link between severe pneumonia and reduced lung levels of SP-A or SP-D.

Together, these groups of antimicrobial molecules in the ASL function to destroy or inhibit microbes before they have a chance to proliferate and compromise the health of the host. If pathogens evade structural defenses and then survive the antimicrobial properties of the ASL, they can come into contact with host cell membranes, which allows for interaction with Toll-like receptors.

Innate Immune Receptors

As highlighted earlier, the respiratory epithelium and other structural cells of the lung express innate immune receptors. These receptors function primarily as pattern recognition receptors to recognize conserved molecular patterns in microbial pathogens, called pathogen-associated molecular patterns (PAMPs), which are not normally found in the host (Table 22-9). This “pre-recognition” allows the host to respond to specific microbial products in the absence of an adaptive response; thus, the response does not require priming and is always present. Innate immune receptors include the membrane bound Toll-like receptors (TLRs) and additional cytosolic receptors. The 10 human TLRs share a protein structure characterized by an extracellular domain with a number of leucine-rich repeats (LRRs) and a cytoplasmic domain containing a Toll/IL1 receptor homology domain (TIR). They are highly expressed by leukocytes and in tissues in contact with the external environment, such as the lung. Cellular expression of TLRs is modulated by microbial stimuli. TLRs are found as homodimers with the exception of TLR-1 and TLR-6, which form heterodimers with TLR-2. TLR activation after binding of its cognate ligand triggers signal transduction pathways that ultimately lead to altered gene expression. The signaling pathway after recruitment of MyD88 is conserved for all TLRs (see Figure 22-10) with the exception of TLR-3, which utilizes TRIF. In addition, specific LPS chemotypes can activate TLR-4 in an MyD88-independent manner. Binding of MyD88 to the activated TLR leads to recruitment of IL-1 receptor–associated kinase (IRAK) and IRAK autophosphorylation. TNF receptor–associated factor-6 (TRAF-6) links with this complex and activates IκB kinase (IKK). IKK phosphorylates IκB, leading to its dissociation from nuclear factor κB (NFκB). NFκB then translocates to the nucleus to activate gene transcription. Alternatively, TRIF leads to activation of IRF3 (interferon regulatory factor 3) that can translocate to the nucleus and lead to the transcription of interferon-α and -β. This pathway also activates NFκB.

Table 22-9 Toll-Like Receptors (TLRs) and Their Ligands

Receptor	Reported Ligands
TLR-1	Heterodimerization with TLR-2, lipopeptides

TLR-2

Peptidoglycan and lipoteichoic acid (components of gram-positive bacteria)

LPS

Bacterial lipoproteins (components of Borrelia burgdorferi)

Components of mycobacterial cell walls

Mannuronic acid polymers (components of Pseudomonas aeruginosa)

Glycosylphosphatidylinositol lipid (components of Trypanosoma cruzi)

Phenol-soluble modulin (from Staphylococcus epidermidis)

Soluble tuberculosis factor (19-kDa lipoprotein secreted by Mycobacterium tuberculosis)

Whole bacteria (heat-killed Listeria monocytogenes)

TLR-3

dsRNA, polyinosinic : polycytidylic acid (poly I : C)

TLR-4

LPS

Mannuronic acid polymers (components of Pseudomonas aeruginosa)

RSV fusion protein

Endogenous inflammation-related products (heat shock proteins HSP-60 and HSP90, fibronectin)

TLR-5

Flagellin

TLR-6

Heterodimerization with TLR-2, lipopeptides

TLR-7

ssRNA, antiviral compounds

TLR-8

ssRNA, antiviral compounds

TLR-9

Unmethylated CpG, bacterial DNA

TLR-10

CpG, cytosine-phosphate-guanine [DNA sequence]; dsRNA, double-stranded RNA; LPS, lipopolysaccharide; RSV, respiratory syncytial virus; ssRNA, single-stranded RNA.

Figure 22-10 Toll-like receptor (TLR) signaling. The classical signaling pathway triggered by TLRs involves MyD88/IRAK (interleukin-1 receptor–associated kinase) and TRAF-6 (tumor necrosis factor [TNF] receptor–associated factor-6), leading to transcriptional regulation of immune and inflammatory genes. IκB, inhibitor of nuclear factor κB; IKK, IκB kinase; LBP, lipopolysaccharide-binding protein; LPS, lipopolysaccharide; NFκB, nuclear factor κB.

Additional nonmembrane receptors enable cytosolic detection of microbial products. NOD1 and NOD2 (nucleotide-binding oligomerization domain proteins 1 and 2) are members of the NLR family of proteins (nucleotide-binding domain, leucine-rich repeat–containing) that recognize bacterial products and eventually lead to NFκB activation. NOD2 also may bind to single-stranded RNA and thus sense viruses. RNA viruses are recognized in the cytoplasm by retinoic acid–inducible gene (RIG)-I–like receptors (RLRs). These include RIG-1, MDA-5 (melanoma differentiation-associated gene-5), and LGP2 (laboratory of genetics and physiology-2). These proteins have a helicase domain and induce interferon production when activated. Double-stranded RNA (dsRNA) also can activate protein kinase R (PKR). PKR has an amino-terminal (N-terminal) RNA-binding domain and a carboxyl-terminal (C-terminal) kinase domain that is proapoptotic. PKR also can induce activation of other important molecules, including NFκB.

These innate immune receptors, whether on the membrane or in the cytosol, are critical for initiating an appropriate innate immune response to a microbial threat. In addition, strong evidence indicates that in the absence of appropriate TLR signaling, the adaptive immune response is impaired. The TLR response represents a “danger signal” and marks an associated antigen for an immune response. This signaling may be particularly relevant for the lung, which is constantly exposed to antigens yet must maintain a noninflamed environment in order to maximize gas exchange.

Toll-Like Receptor-2

Toll-like receptor-2 (TLR-2) is expressed on the surface of numerous cells and has a large number of reported ligands. These include components of gram-positive and gram-negative bacteria, mycobacteria, the protozoan parasite Trypanosoma cruzi, and zymosan, a yeast wall component. Additional ligands include secreted factors from Staphylococcus epidermidis and Mycobacterium tuberculosis, as well as whole bacteria (Listeria monocytogenes). TLR-2 does not recognize these PAMPs in isolation but rather forms heterodimers with TLR-6 and TLR-1, thus contributing to the diversity and specificity of TLR-2–mediated immune response.

Toll-Like Receptor-3

Toll-like receptor-3 (TLR-3) is activated by dsRNA), a marker of viral infection, and, through the activation of NF-κB, triggers the production of type I interferons. TLR-3 is found on plasma membranes, where it is believed to contact dsRNA after the lysis of infected cells and release of dsRNA into the extracellular milieu. Of note, additional evidence suggests that TLR-3 is found inside cells in vesicles associated with the endocytic pathway. TLR-3 is thought to have a more restricted pattern of expression but is found in dendritic cell populations.

Toll-Like Receptor-4

Toll-like receptor-4 (TLR-4) is the prototypical and most extensively studied of the TLRs. It recognizes lipopolysaccharide (LPS) (i.e., endotoxin), mannuronic acid polymers (components of Pseudomonas aeruginosa), and the F protein of the respiratory syncytial virus (RSV). Unlike the other TLRs, TLR4 also recognizes several endogenously derived molecules, including heat shock proteins HSP60 and HSP70 and fibronectin.

Toll-Like Receptor-5

Toll-like receptor-5 (TLR-5) recognizes a highly conserved cluster of 13 amino acid residues in flagellin, the structural component of flagella found in many pathogenic bacteria. This interaction triggers an innate immune response, resulting in cytokine production from leukocytes and dendritic cell activation and maturation.

Toll-Like Receptor-9

Toll-like receptor-9 (TLR-9) binds to unmethylated CpG (i.e., cytosine-phosphate-guanine sequence) elements in DNA and triggers a predominantly T_H1-weighted immune response. Unmethylated CpG dinucleotides are present at high frequencies in most bacterial genomes, whereas in the human genome, these dinucleotides are suppressed, methylated, and flanked by bases that are immune-neutralizing. TLR-9 is believed to be localized within endosomal compartments, thus facilitating its interaction with bacterial DNA.

Other Toll-Like Receptors

TLR-1 and TLR-6, as mentioned previously, interact with TLR-2 and function as a complex that recognizes a broad range of antigens. TLR-10 is related to TLR-1 and TLR-6, but its role remains to be clarified. TLR-7 and TLR-8 recognize single-stranded RNA.

Inflammatory Mediators

Cytokines

Cytokines (Table 22-10) are soluble, low-molecular-weight proteins that play important roles in host defense by regulating the inflammatory response and are expressed not only by leukocytes but also by endothelial cells, epithelial cells, and fibroblasts. Expression of cytokines is transcriptionally regulated, and secretion can be quickly enhanced after cell stimulation.

Table 22-10 Cytokines and Chemokines and Their Functions

Cytokine	Function	Other Clinical Roles
TNF-α	Proinflammatory Neutrophil activation in ARDS

Proximate cytokine released in response to inflammatory stimulus IL-1β

Proinflammatory

Neutrophil activation in ARDS

Upregulation of adhesion molecules on leukocytes, endothelium, and airway epithelium

One of first cytokines to be released in response to inflammatory stimulus IL-6

Proinflammatory

Leukocyte activation

Promotes proliferation of myeloid progenitor cells

Induces pyrexia

Acute-phase reactant

Circulating levels are a marker of severity of ARDS of different causes IL-10

Antiinflammatory

Inhibits release of TNF-α, IL-1β, and IL-6 from monocyte-macrophages

Stimulates the production of IL-1 receptor antagonist (IL-1RA) and soluble p75 TNF receptor

GM-CSF

Alveolar macrophage function

Lung host defense

Surfactant homeostasis

Low circulating levels associated with poor prognosis in sepsis PAF

Acts by way of receptors on platelets, leukocytes, and endothelial cells

Increases vascular permeability

Leukocyte recruitment

Primes and triggers leukocyte secretion

ICAM-1

Leukocyte recruitment and retention

Increased in inflammation C5a

Product of classical and alternate complement cascade

Potent anaphylatoxin and chemoattractant

Acts via C5aR

Can be both pro- and antiinflammatory

Substance P

Neuropeptide that acts through its receptor NK1R

Proinflammatory and associated with development of lung injury

ARDS, acute respiratory distress syndrome; GM-CSF, granulocyte-macrophage colony-stimulating factor; ICAM-1, intercellular adhesion factor-1; IL, interleukin; NK1R, neurokinin 1 receptor; TNF, tumor necrosis factor; PAF, platelet-activating factor.

Signaling through cognate receptors, cytokines exert distinct responses in specific cell populations, stimulating some populations to activate, proliferate, and differentiate while having an inhibitory effect on other cell types. In this way, cytokines play a major role in regulating the intensity and duration of the inflammatory response. The cytokines that play important roles in inflammation, particularly in the early proinflammatory phase, include TNF-α, IL-1β, IL-6, and IL-10. Significant elevations of these cytokines are observed in generalized inflammatory states and particularly in gram-negative sepsis.

Chemokines

Chemokines (Table 22-11) are 8- to 10-kDa glycoproteins that, although structurally related to cytokines, are distinct from them in their ability to bind and signal via G protein–coupled receptors. Chemokines are both chemotactic and cellular activating factors for leukocytes and can be classified into four groups on the basis of their amino acid structure. The two primary groups that play an important role in host defense are the CC chemokines (e.g., MCP-1, MIP-1α, and RANTES), which are chemotactic for monocytes, lymphocytes, basophils, and eosinophils and the CXC chemokines (e.g., IL-8, GRO-α [growth-related oncogene α], and ENA-78 [epithelial cell–derived neutrophil-activating peptide-78]), which act primarily on neutrophils.

Table 22-11 Chemokine Families

Family	Member(s)	Function(s)
CXC	IL-8 MIP-2 GRO-α ENA-78 NAP-2

Chemotactic for and stimulatory to neutrophils

Chemotactic for and activation of:

Attracts T cells CXXXC

Fractalkine

Attracts T cells and monocytes
Promotes adhesion

ENA-78, epithelial cell–derived neutrophil-activating peptide-78; GRO-α, growth-related oncogene; IL-8, interleukin-8; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; NAP-2, neutrophil-activating protein-2.

The chemokine receptors are structurally related seven transmembrane–spanning proteins that transmit their signals through heterotrimeric G proteins. As with cytokines, the effect of chemokine activation results in diverse physiologic responses that are cell- and stimulus-specific. The binding specificity of individual chemokine receptors is determined by a region in the amino terminus of the protein. Some receptors are highly specific, whereas others bind multiple chemokines of both CC and CXC families. Differential regulation and expression of the chemokine receptor in different cell types play an important role in determining the biologic result of chemokine activation.

Summary

It is apparent that the lung has multiple lines of defense against microbial pathogens and noxious environmental agents. These include physical barriers such as mucus and soluble factors such as antimicrobial peptides and proteins produced by epithelial cells, as well as leukocytes of the innate and adaptive immune systems. It is this combination of diversity and redundancy that enables the lung to thwart infection under most circumstances.

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