Inflammatory Cells of the Lung: Polymorphonuclear Leukocytes

8.06 Inflammatory Cells of the Lung: Polymorphonuclear Leukocytes D M Hyde and S I Simon, University of California, Davis, CA, USA ª 2010 Elsevier Ltd. All rights reserved.

8.06.1 8.06.2 8.06.2.1 8.06.2.2 8.06.3 8.06.3.1 8.06.3.2 8.06.3.3 8.06.3.4 8.06.4 8.06.5 8.06.6 8.06.7 8.06.8 8.06.9 8.06.10 References

Introduction Normal Structure and Function in the Respiratory Tract PMNs Pulmonary PMNs Cell Kinetics and Migration Recruitment Migration Feedback Mechanisms Controlling Recruitment and Migration Calcium As a Central Regulator of Integrins and the Cytoskeleton Methods for Evaluating Structure and Function Markers of Injury Role in Pulmonary Injury Acute and Subchronic Responses to Toxic Exposure Chronic Responses to Toxic Exposure Human versus Animal Toxicology In Vitro Systems

Abbreviations BALF CRAC FMLP FNLP G-CSF IL-8 LFA-1

bronchoalveolar lavage fluid calcium release-activated calcium channel F-Met-Leu-Phe formyl Norleu-Leu-Phe granulocyte colony-stimulating factor interleukin-8 leukocyte function-associated antigen-1

8.06.1 Introduction Polymorphonuclear leukocytes (PMNs, or neutrophils), which form the majority of circulating leukocytes, are the first line of defense against microbial infections and play a significant role in bacterial phagocytosis and removal or debridement of injured epithelial cells in the respiratory tract. PMNs are positioned in the microvasculature of the lung to respond immediately to inflammatory stimuli. They sequester in the lung capillaries through unique hemodynamic and geometric properties of the

MCP-1 MIP NF-B PLC PMA PMN RANTES ROS SER

115 116 116 116 118 118 119 120 121 121 121 122 122 123 124 125 126

monocyte chemoattractant protein-1 macrophage inflammatory protein nuclear factor-B phospholipase C phorbol myristate acetate polymorphonuclear leukocyte regulated on activation, normal T-cell expressed and secreted reactive oxygen species smooth endoplasmic reticulum

pulmonary microvasculature as well as changes in their ligand-specific functions and biophysical properties during activation. PMNs can cause extensive damage to the lung in a variety of infectious and toxicological inflammatory disorders. This is accomplished by their production of reactive oxygen and nitrogen species and release of granule components upon activation. However, PMNs and the inflammatory process exist because they are beneficial to the host throughout the animal kingdom. Thus they are beneficial rather than injurious to host tissue in the majority of circumstances. 115

116 Inflammatory Cells of the Lung: Polymorphonuclear Leukocytes

8.06.2 Normal Structure and Function in the Respiratory Tract 8.06.2.1

PMNs

PMNs belong to the innate immune system (see Chapter 5.01). They comprise about half of the circulating leukocytes in most species and are marked by a multilobed nucleus and distinct cytoplasmic granules (Figure 1). The cytoplasmic granules include primary (azurophilic), secondary (specific), and tertiary granules that contain an arsenal of proteins, enzymes, and glycosaminoglycans believed to participate in various cell functions (Faurschou and Borregaard 2003). PMNs form the first line of defense against microbial infections and play a significant role in bacterial phagocytosis in the respiratory tract. PMNs also remove injured tissue and modulate pulmonary inflammation. To accomplish their role, PMNs must respond to chemotactic signals from the affected tissue, interact with and penetrate the wall of the microcirculation, migrate to the site of the chemotactic stimuli, and recognize, phagocytose, and kill microbes or injured cells. All of these stages represent the basic elements of PMN function. PMNs are principally present in three separate pools within the body. These include the bone marrow pool, the circulating pool in the systemic vasculature, and a localized sequestered pool in tissues such as the lung, kidneys, and gastrointestinal tract. The large marginated (or sequestered) pool of PMNs is confined to the microvasculature, which has been estimated to contain about two-thirds of the

Azurophid granule

Specific granule Neutrophil Figure 1 A transmission electron micrograph of a normal neutrophil in tissue showing a multisegmented nucleus, azurophilic granules, and specific granules.

total number of circulating PMNs (Doerschuk et al. 1987; Hogg 1987; MacNee and Selby 1993). It has been shown that rapid, dynamic shifts occur between the marginating and circulating pool of PMNs, in response to a variety of endogenous and exogenous inflammatory stimuli, such as granulocyte colony-stimulating factor (G-CSF) (Mukae et al. 2000), CXCR4/SDF-1 (Suratt et al. 2004) and CCL23 (Shih et al. 2005), or endotoxin (e.g., lipopolysaccharide). 8.06.2.2

Pulmonary PMNs

It is well documented that PMNs are retained in the lung in both physiological and pathological states. The strategic location of this intrapulmonary reservoir of PMNs, in close proximity to the gas exchange area, represents a first, rapid line of defense against pathogens from the external environment. There are significant differences between the interactions of circulating PMNs with the pulmonary and systemic microvasculature, with respect to the site of PMN sequestration in the lung and their interaction with the endothelium. In vivo microscopic studies in the systemic circulation have shown that PMNs slowly roll along the walls or margins (marginated pool) of ¨ nbein et al. 1980). small venules (Schmid-SchU However, PMNs do not normally adhere to vascular endothelium. PMN adherence to endothelium following an inflammatory insult can be induced within minutes by thrombin- or histamine-activated upregulation of endothelial P-selectin, and within 4–6 h via cytokine-mediated transcriptional activation and membrane upregulation of E-selectin. If PMNs rolling along the endothelium come into contact with an inflammatory mediator, then their CD11/CD18 integrins will be activated and bind to intercellular adhesion molecule-1 (ICAM-1) and other endothelial ligands that result in a firmer adhesion and initiate transmigration (Lawrence and Springer 1991). Although this phenomenon has also been suggested to occur in the lung (Kuebler et al. 1994), it is known that resting, unstimulated PMNs entering the pulmonary microvascular bed form the marginating pool that is located almost exclusively within lung capillaries (Doerschuk et al. 1987, 1994; Downey et al. 1993; Hogg 1987; Lien et al. 1987, 1991). It has also been shown that the PMNs are sequestered and retained in predisposed, discrete sites during their transit through the pulmonary capillary bed. The nature of this differential retention is uncertain, but it might either reflect differences in the local

Inflammatory Cells of the Lung: Polymorphonuclear Leukocytes

expression of endothelial adhesion molecules or represent unique hemodynamic or geometric properties of the pulmonary microvasculature. It is well documented that retention of PMNs in the lung is modulated by at least three major factors: (1) unique hemodynamic properties of the pulmonary microvasculature (Doerschuk et al. 1987; Hogg 1987), such as perfusion under low pressures (Nagasaka et al. 1984) and pulsatile flow (Wasserman et al. 1966), which slow the PMN transit through the lung and reduce the shear forces imposed on PMNs as they move through the lung; (2) discrepancy in size between circulating PMNs and pulmonary capillary diameter. It has been shown that the average pulmonary capillary diameter is smaller than the average PMN diameter (Doerschuk et al. 1993; Downey and Worthen 1988), which results in restriction of the transit of the PMN through the capillary bed of the lung; and (3) biophysical properties of the PMN, such as deformability and cell size.

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Unstimulated circulating PMNs within the vasculature are fairly deformable (Downey and Worthen 1988) but tend to become more rigid due to cytoskeletal changes and increase in volume in response to stimulation by inflammatory mediators and migration through the endothelium (Figure 2) (Tsai et al. 1994; Worthen et al. 1994). The importance of the cytoskeletal changes in PMNs in the pulmonary vasculature was shown by CD18 blockage 15 min before blood samples were obtained in Escherichia coli-induced pneumonia with more PMNs entering the lung containing F-actin rims than PMNs exiting, which in turn accounted for the sequestered PMNs (Yoshida et al. 2006). These PMNs with cytoskeletal rearrangements preferentially sequester within the lungs during pneumonia, but this sequestration was not due to CD11/CD18-mediated adhesion, L-selectin expression, or platelet adhesion to PMNs. Thus, the events that determine leukocyte sequestration appear to be unique to the lung and may reflect a

AL

N

Figure 2 An example of polymorphonuclear leukocyte (PMN) emigration into the injured epithelium of an airway. The transmission electron micrograph is representative of monkey tracheal epithelium 12 h after injury induced by inhalation of 1 ppm of ozone for 8 h. Note the necrotic ciliated cell (N) and the numerous PMNs () migrating in the epithelium. Arrowheads mark the epithelial basal lamina, while AL marks the tracheal airway lumen. Scale ¼ 5 mm.

118 Inflammatory Cells of the Lung: Polymorphonuclear Leukocytes

combination of a decrease in PMN deformability due to increased viscosity (Simon et al. 2007) as well as very site-specific, local hydrodynamic and geometric conditions within the pulmonary microvasculature. The exact molecular mechanisms of this CD11/CD18-independent PMN emigration in the lung that occurs with selected stimuli are still under investigation.

8.06.3 Cell Kinetics and Migration 8.06.3.1

Recruitment

A crucial event in the process of acute inflammation in the lung is the rapid recruitment and accumulation of neutrophils in airways and alveoli in response to inflammatory stimuli such as C5a (complement fragment), FMLP (F-Met-Leu-Phe) (bacterial product), or platelet-activating factor (phospholipid mediator). Multiple studies tried to elucidate the various mechanisms by which neutrophils accumulate in the lung, as well as the migratory process and sequestration of neutrophils that allow the rapid generation of alveolitis and inflammation of peripheral airways. A major factor in the development of an inflammatory response is the degree of local blood flow. The nature of the acute pulmonary inflammatory response is, at least in part, dependent upon the regulation of local blood flow to the area of

inflammation (Downey et al. 1988; Fisher and Wood 1980). In a variety of animal models, diversion of blood flow away from the inflamed area occurs via vasoconstriction. This phenomenon limits the migration of neutrophils to the inflamed area and might represent a protective mechanism because decreased blood flow to the inflamed region would minimize ventilation–perfusion mismatching and thereby prevent hypoxemia. The reduction in blood flow in acute pulmonary inflammation contrasts with blood flow patterns during systemic inflammation, where a dramatic increase in blood flow, due to sympathetic adrenergic control of small arteries and arterioles, is the first response to an inflammatory stimulus (Issekutz 1981). To successfully navigate to inflamed areas, leukocytes undergo a multistep process of recruitment (Figure 3). Following an inflammatory stimulus, the inflamed endothelium begins to secrete chemokines and expresses adhesion molecules such as P- and E-selectins and the cell adhesion molecules ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1). L-selectin on leukocytes and P- and E-selectins on inflamed endothelium form transient yet strong adhesive bonds lasting from milliseconds to seconds by recognizing glycoproteins expressed on the adjacent cell surface. The selectins serve to capture the leukocyte from circulation and instigate cellular rolling over the endothelium. All selectins

CD11/CD18 activation Intermediate

High affinity F-actin MAPK PI3K PLC

Low

PSGL-1

GPCR L-selectin

P/E-selectin

Tethering and rolling

ICAM-1

Arrest

Polarization and migration

Transmigration

Figure 3 Cooperativity between selectins, 2 integrin, and chemokine signaling of polymorphonuclear leukocyte (PMN) arrest and migration. 2 integrins in a resting PMN are expressed in a low-affinity conformation. Rolling via selectins begins to trigger a transition through intermediate- to high-affinity integrins that strengthen adhesion by binding to dimerized intercellular adhesion molecule-1 (ICAM-1). G-protein receptor-coupled chemokine receptors signal intracellular activation of kinases that are amplified by store release of calcium, which leads to F-actin formation. This in turn leads to cell polarization, migration, and diapedesis into the inflamed parenchyma. GPCR, G-protein-coupled receptor; MAPK, mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase; PLC, phospholipase C; PSGL-1, P-selectin glycoprotein ligand-1.

Inflammatory Cells of the Lung: Polymorphonuclear Leukocytes

contain a lectin domain, which allows them to recognize sialylated and fucosylated sugars that decorate membrane glycoprotein receptors. The bond between a selectin and its ligand functions as a catch bond that can provide tethering strength only within a specific regime of shear stress (Zhu and McEver 2005). Establishment of rolling adhesion is thought to provide sufficient contact area and duration for leukocytes to recognize chemokines on the endothelial surface and effect activation of integrin receptors to a higher affinity binding state for their respective CAM ligands, VCAM-1, ICAM-1, and ICAM-2. Thus, at relatively low shear stresses below 0.2 dyn cm2, selectins do not form strong bonds with glycosylated ligands such as sLex, and at high shear stress (i.e., 5 dyn cm2) distractive forces either dissociate selectin bonds or prevent them from forming. This creates a situation in the lung wherein PMN recruitment would be selectin-independent. Such a situation is observed under conditions of Gram-negative sepsis, which amplifies lung PMN sequestration and migration independent of adhesion mediated by CD18/ICAM-1. However, PMN activation and increased pulmonary microvascular permeability induced by E. coli are solely dependent on engagement of CD18 integrins, even when PMN accumulation and migration responses are independent of CD1 (Gao et al. 2001).

8.06.3.2

Migration

To transition from tethering via selectin glycoprotein and rolling along the inflamed vessel wall requires that chemokines signal cell arrest of granulocytes through activation of the 1 and 2 integrins

(Burke-Gaffney and Hellewell 1996; Simon et al. 2007; Walsh et al. 1991). The 2 integrins are heterodimeric adhesion receptors that consist of four CD11 subunits, which are noncovalently linked to the common 2 subunit CD18. The 2 integrins are not constitutively expressed in a conformation with high affinity for their ligands, but exhibit a rapid and inducible increase in affinity and adhesive avidity upon leukocyte activation with chemokines such as interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), or macrophage inflammatory protein (MIP)-1 . Data from both human in vitro endothelial cell monolayers and intravital mouse models of inflammation suggest that expression of CXC and CC chemokines on the glycocalyx that lines the vessel lumen may serve to present and coordinate leukocyte recruitment. In particular, IL-8, eotaxin-1/2, and MCP-1 are chemokines that specifically activate and serve as chemoattractants of neutrophils, eosinophils, and monocytes, through binding to various CC and CXC G-protein-coupled receptors (Ponath et al. 1996; Ravensberg et al. 2005). For instance, eotaxin-1 may be expressed very early in asthma, providing a primary activator of 1 integrinmediated arrest and transmigration of eosinophils. The roles of a number of chemokine receptors in airway inflammatory responses have been studied by using mice deficient in respective receptors, as listed in Table 1. Another significant difference between the systemic and pulmonary circulation is the site of PMN vascular emigration in response to an inflammatory stimulus. In the systemic circulation, neutrophils marginate and migrate across postcapillary venules. If neutrophils employed arteriolar or venular

Table 1 Assessment of defects in PMN function in vivo PMN function

References

Identification of subpopulations, especially immature forms, of PMNs Markers for PMN turnover Lysozyme Lactoferrin Alkaline phosphatase deficiency Leukocyte adhesion deficiency

Gallin (1984)

Demonstration of PMN functional defects by cytochrome b559 (chronic granulomatous disease) Myeloperoxidase deficiency Chediak–Higashi syndrome Pulmonary alveolar proteinosis (GM-CSF autoantibodies)

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Hansen (1973) Birgens (1985) Anderson et al. (1985) Anderson et al. (1985) and Anderson and Springer (1987) Segal (1989) Malech and Gallin (1987) Rausch et al. (1978) Uchida et al. (2007)

PMN, polymorphonuclear leukocyte; GM-CSF, granulocyte–macrophage colony-stimulating factor.

120 Inflammatory Cells of the Lung: Polymorphonuclear Leukocytes

emigration routes in the lung to gain access to the alveolus, they would be forced to migrate over long distances through pulmonary interstitial spaces. Rather, it has been suggested that in the lung the interaction and emigration of the circulating PMN into adjacent alveolar spaces take place in the pulmonary capillary bed (Downey et al. 1993). In principle, the sequence of events in the migratory process of extravasation from the pulmonary capillary bed into the alveolar space is divided into the following steps: (1) recruitment and accumulation of neutrophils from the circulating pool into the vascular bed of the lung; (2) margination (pavementing) and sequestration within the microvasculature of the lung; (3) adhesion to the microvascular endothelial cells; and (4) transmigration across the endothelium, basement membrane, and epithelial cells in the alveolar spaces. Molecular events attendant during inflammatory responses include cytokines and chemokines that trigger integrin-mediated adhesion of leukocytes to the vascular endothelium. The principal function of the cytokines and chemokines is to (1) induce expression of adhesion molecules on the luminal surface of vascular endothelium and upshift the affinity of integrins on the leukocyte, (2) signal increased release of granulocytes from the bone marrow, and (3) function as chemoattractants of circulating eosinophils and neutrophils to the inflamed airways. Activated T cells and macrophages resident in inflamed lung can produce Th2 cytokines that combine to elicit numerous responses from adjacent epithelial cells, including release of chemokines that function as stop signals and chemoattractants for neutrophils and eosinophils. The most abundant chemoattractants are IL-8 (or the murine homologue KC), RANTES (regulated on activation, normal T-cell expressed and secreted), MCP-1, MIP-1, and eotaxin (Erger and Casale 1995; Kameyoshi et al. 1992; Li et al. 1999; Ponath et al. 1996). These chemokines can mediate granulocyte adhesion to inflamed airway endothelium and epithelium (Gonzalo et al. 1996; Holgate et al. 1997; Lamkhioued et al. 1997; McNulty et al. 1999). Neutrophils are activated by chemokines, which transduce signals via sensory G-protein-coupled receptors on their membranes, which can detect minute concentration gradients of chemokines. A rapid functional response upon binding is the activation of adhesion receptors that enable neutrophils to gain a foothold at vascular sites of inflammation. Adhesion receptors enable neutrophils to migrate through the

endothelium to the site of inflammation, where they engulf and destroy microbes and clean up senescent cellular debris. At each step of the recruitment process, neutrophils demonstrate a high level of active motility. Active force generation is achieved through the local cytoplasmic conversion of G- to F-actin at the cell front where pseudopod formation occurs and contraction at the rear or uropod where traction forces are also generated. 8.06.3.3 Feedback Mechanisms Controlling Recruitment and Migration The mechanism by which neutrophils (PMNs) become activated in the free stream or while tethered to the endothelial wall is an area of active research. In PMN adhesion, CD11a/CD18 or leukocyte functionassociated antigen-1 (LFA-1) is instrumental in transitioning the cell from rolling to firm adhesion by undergoing conformational shifts on a subsecond time frame upon PMN activation via ligation of E-selectin and subsequently chemokine receptor (Shamri et al. 2005; Tessier et al. 1997). Site-directed mutagenesis of the LFA-1/ICAM-1 binding ‘I domain’ has led to the identification of stable conformations corresponding to low-, intermediate-, and high-affinity states for binding ICAM-1 of LFA-1 (Green et al. 2006). One mechanism by which the efficiency of PMN recruitment is regulated is through membrane redistribution of LFA-1 into dense high-affinity clusters that act to strengthen adhesion. When distributed in dense clusters, LFA-1 can mediate outside-in signals upon engagement with ICAM-1 (Takada et al. 2007). For instance, highaffinity LFA-1 ligation of ICAM-1 is necessary for F-actin-supported cell polarization and ensuing transmigration. The cytoskeleton can regulate avidity by increasing LFA-1 mobility during activation and directing movement of microclusters following engagement of ligand. On the endothelial side, binding of oligomeric ICAM-1 can induce microclustering of LFA-1 (Sampath et al. 1998). Spontaneous dimerization of ICAM-1 on the endothelial membrane facilitates the high-avidity binding through LFA-1 and an 10-fold slower rate of dissociation of LFA-1/ICAM-1 bonds (Sarantos et al. 2005; Yang et al. 2004). During PMN activation, the small Ras-related guanosine triphosphatase (GTPase) Rap1 induces association of its effector molecule, RapL, with the cytoplasmic domain of leukocyte integrins, regulating integrin affinity (Katagiri et al. 2003). The RapL–LFA-1 complex moves to polarize large caps of high-affinity LFA-1 on the activated cell membrane during adhesion to vascular

Inflammatory Cells of the Lung: Polymorphonuclear Leukocytes

endothelium. CD18 integrins on neutrophils reorganize from a uniform surface distribution to form both small punctuate clusters (<1 mm2) and large caps (3 mm2). Within 2 min of activation, over 60% of the active LFA-1 is colocalized into clusters with other active CD18 (Katagiri et al. 2003). Thus, both a conformational shift and multivalent binding, at least at the level of LFA-1/ICAM-1 dimers, are critical for adhesion strengthening and transitioning the cell from rolling to arrest.

8.06.3.4 Calcium As a Central Regulator of Integrins and the Cytoskeleton Shear stress of flowing blood promotes rolling and translation of neutrophils over the endothelial surface, increasing the opportunity to engage apical chemokines (Zhang and Neelamegham 2002). The adhesive and chemotactic signaling processes converge through secondary signaling pathways, notably through calcium flux (Schaff et al. 2007). Calcium flux, a sharp increase in intracellular calcium within the leukocyte, occurs through release of intracellular stores and subsequent opening of plasma membrane calcium channels (Tintinger et al. 2005). Calcium flux is initially generated from phospholipase C (PLC), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol-3-phosphate (IP3), which in turn causes emptying of intracellular stores such as the endoplasmic reticulum (ER) and calciosomes. Influx of calcium through the plasma membrane occurs concurrently with PMN arrest and upregulates high-affinity 2 integrin resulting

in arrest stabilization in microvessels of the lung (Lee et al. 2005). Thus, calcium release-activated calcium channel (CRAC) flux may play a critical role in coordinating recruitment of neutrophils and other leukocytes at precise locations of integrin ligand presentation on inflamed endothelium.

8.06.4 Methods for Evaluating Structure and Function Normal life requires the full repertoire of PMN functions to maintain homeostasis. Serious clinical conditions become apparent in patients suffering from disorders characterized by reduced PMN function. Therefore, it is essential to be able to assess PMN function under in vitro and in vivo conditions. Tables 1 and 2 provide an overview of a variety of approaches to evaluate PMN function in vivo and in vitro. Table 1 shows defects in PMN function in vivo, highlighting the importance of PMN function as a whole cell and subcellular components as contributors to effector functions. Table 2 shows an assessment of PMN function in vitro where alterations in receptors and other PMN components can be quantitated.

8.06.5 Markers of Injury PMNs are the most important and most numerous white blood cells in combating nonviral infections. During activation, PMNs emigrate from the vessels into the tissues. During this process, PMNs generate

Table 2 Assessment of defects in PMN function in vitro PMN function

References

Alkaline phosphatase activation Quantitation of stimuli-induced PMN activity Phagocytosis assay

Borregaard et al. (1987)

Collagenase activity Plasma membrane markers (evaluated quantitatively by flow cytometry) HLA1 C5a receptor Leukotriene B4 receptor Fc-receptor CD11/CD18 integrin complex Other PMN functions evaluated by flow cytometry include calcium mobilization, F-actin assembly, adhesion and aggregation, degranulation, PMN ROS generation, PMN priming and apoptosis PMN, polymorphonuclear leukocyte; ROS, reactive oxygen species.

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Metcalf et al. (1986) and Van Eeden et al. (1999) Weiss (1989) Williams and Barkeley (1988) Boulay et al. (1991) Omann et al. (1987) Akerley et al. (1991) Albelda and Buck (1990) Van Eeden et al. (1999)

122 Inflammatory Cells of the Lung: Polymorphonuclear Leukocytes

reactive oxygen species (ROS) (respiratory burst) and mobilize intracellular compartments (degranulation). During the process of degranulation, PMNs influence the local tissue by extracellular release of intragranular secretory products into the local environment. Plasma membrane-related processes take place simultaneously, such as chemotaxis and respiratory burst, as well as translocation of membrane-bound proteins to the surface (i.e., upregulation of adhesion molecules). It is evident that PMNs are endowed with a complex, highly integrated, sophisticated, and functional repertoire that enables cells to play a major part in the first line of host defense against infectious agents. The ultimate killing of microorganisms is executed by oxidizing radicals, oxidizing halogens, bactericidal proteins, or proteo- and hydrolytic enzymes. The oxygen-dependent and oxygen-independent systems can act separately, but the effect is amplified when they occur simultaneously (Gallin 1988; Spitznagel 1990; Weiss 1989). A major drawback of exuberant PMN activity is that they are nonselective in their targets. Instead, PMNs guided by antibody presenting immune complexes, chemokines, cytokines, and complement components operate to induce injury and necrosis to the host tissue via their repertoire of hydrolytic enzymes and oxygen radicals. Armed with this potent repertoire of enzymes, peptides, and proteins, they are able to cleave tissue constituents, basement membranes, and tissue matrix (Huber and Weiss 1989). These products are powerful mediators of endothelial injury and tissue damage and amplify the effects of the initial inflammatory stimulus. Thus, in a variety of chronic lung diseases, the PMN infiltrate itself becomes the offender in the pathogenesis of the disease.

8.06.6 Role in Pulmonary Injury Studies using the isolated perfused rat lung exposed to ozone showed that PMNs have a synergistic influence on epithelial injury, especially in the central acinus (Joad et al. 1993). PMN-mediated pulmonary tissue injury is well characterized in vivo. In vivo evidence of oxidant generation and proteolytic enzyme release from stimulated PMNs has been demonstrated in experimental lung injury induced by the administration of formyl Norleu-Leu-Phe (FNLP) intrabronchially and phorbol myristate acetate (PMA) intravenously to rabbits (Schraufstatter

et al. 1984). Oxidants and PMN elastase have also been demonstrated in bronchoalveolar lavage fluid (BALF) from patients with the adult respiratory distress syndrome (Cochrane et al. 1983). Human PMN defensins account for most (60%) of the cytotoxicity of PMN granule extracts to lung epithelial cells (Okrent et al. 1990). Defensins (HNP-1)single-strand DNA breaks in K562 and Raji target cells after 6–8 h of incubation (Gera and Lichtenstein 1991). Inhibitors of poly(ADP-ribose) polymerase increased defensin-induced DNA injury and enhanced cytolysis. Thus PMNs can act as the ‘villain’ in the pathogenesis of inflammatory disorders, carcinogenesis, autoimmune-mediated disorders, immunemediated vasculitis, asthma, chronic obstructive pulmonary disease, and adult respiratory distress syndrome (Table 3) (Borregaard 1988; Cross et al. 1987; Weiss and LoBuglio 1987).

8.06.7 Acute and Subchronic Responses to Toxic Exposure A wide variety of inhaled pollutants and ingested organic compounds (especially found in food and drinking water) commonly occurring in the environment produce lesions in bronchiolar epithelium of centriacinar regions. The organic compounds can be classified into seven different groups: furans, indoles, chlorinated hydrocarbons, aromatic hydrocarbons, Nnitroso compounds, trialkyl phophorothioates, and paraquat. The primary centriacinar response to a single injection of these organic compounds, and their subsequent delivery to the bronchiolar epithelium by the bloodstream, is bronchiolitis (Plopper and Dungworth 1987). The early phase (4–72 h) is nonciliated bronchiolar necrosis. Dilation of the smooth endoplasmic reticulum (SER) and nuclear envelope reaches a maximum between 24 and 72 h after injection. Higher doses of these organic compounds increase the number of cells showing a dilatation response, the degree of dilatation per cell, and the number of necrotic cells. Little is known of the role of the PMN in these organic compound-induced lung injuries. Of the inhaled environmental pollutants, ozone and nitrogen dioxide alone (see Chapter 8.14), or with respirable-sized aerosols, represent some of the most important potential threats to human and animal health. These pollutants arise primarily from oxidation of atmospheric nitrogen to NO and NO2 during high-temperature combustion of fossil fuels. Concentrations of O3 and NO2 attained in photochemical smogs are interdependent, with NO2, a

Inflammatory Cells of the Lung: Polymorphonuclear Leukocytes

precursor of most of the O3, formed via a complex series of free-radical reactions occurring in the polluted atmosphere. NO2 has been studied much less extensively than O3, but in general NO2, which is less soluble in water than O3, seems to require higher concentrations in air than O3 to elicit equivalent biologic and/or toxicological effects. Using an elevation of lung collagen synthesis rate after 7 days of exposure to oxidant gases as an index of lung injury, O3 was estimated to be about 18 times more toxic to rat lungs than NO2 at a given exposure (Last et al. 1983). Short-term inhalation of ozone causes damage to pulmonary epithelial cells in the anterior nasal cavity, trachea, and central acinus. Acute injury results in necrosis of ciliated cells, deciliation, and degranulation of secretory cells in conducting airways, and necrosis of type I cells and ciliated cells in proximal acini. Maximum epithelial necrosis occurs during the first 24 h after the initiation of exposure in the rat, and necrosis during this period is accompanied by a significant influx of PMNs (Castleman et al. 1980; Hotchkiss et al. 1989; Pino et al. 1992; Stephens et al. 1974). Epithelial necrosis in respiratory bronchioles of monkeys occurs as early as 4 h and is maximal between 12 and 24 h after the initiation of ozone exposure, and there is a strong relationship between epithelial necrosis and the emigration and retention of PMNs at all levels of the tracheobronchial tree by 1 h postexposure (Castleman et al. 1980; Hyde et al. 1992). However, experiments designed to evaluate the contribution of PMNs to O3-induced epithelial necrosis in monkeys showed a beneficial role for PMNs in removing O3-injured epithelial cells and enhancing the repair and differentiation of the bronchiolar epithelial cells (Hyde et al. 1999). In these blocking antibody studies combined with complement C5a instillation in nonhuman primates exposed to ozone, it became evident that neutrophils respond to chemokines produced by injury to epithelial cells and/or alveolar macrophages and play a key ‘beneficial role’ in epithelial repair via removal of necrotic epithelial cells (Hyde et al. 1999).

8.06.8 Chronic Responses to Toxic Exposure Upon repeated exposure to organic compounds, the bronchiolar epithelium develops the ability to avoid cellular damage from subsequent doses that are cytotoxic when they are administered in the absence of such pretreatment (O’Brien et al. 1989) (Figure 4). The tolerance to further injury is dose- and time-dependent

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over a 7-day period. Little is known about the role of PMNs in repeated exposure to organic compounds. In long-term ozone inhalation in rats and monkeys, the central acinus shows a dose-dependent response of increased numbers of cuboidal ozone-resistant epithelial cells, accumulation of alveolar macrophages, and interstitial thickening characterized by accumulation of smooth muscle cells, fibroblasts, interstitial macrophages, and mast cells (Barr et al. 1988; Fujinaka et al. 1985; Moffatt et al. 1987). Exposure of rhesus monkeys (8 h day1) for 6 or 9 days to 0.15 or 0.30 ppm ozone resulted in ciliated cell necrosis, shortened cilia, and secretory cell hyperplasia with less stored glycoconjugates in the nasal region. Respiratory bronchiolitis was also observed in these monkeys at 6 days and this persisted to 90 days of exposure (Harkema et al. 1993). Even at the lower concentration of 0.15 ppm ozone, nonciliated bronchiolar cells appeared hypertrophied and increased in abundance in respiratory bronchioles (Harkema et al. 1993). The principal lesion in monkeys in response to 0.25 ppm ozone (8 h day1) daily or episodes (nine cycles of 1 month of ozone followed by 1 month of filtered air) for 18 months was respiratory bronchiolitis (Tyler et al. 1988). Conclusions from these long-term monkey studies are as follows: (1) there is persistent epithelial injury in the anterior nasal cavity and respiratory bronchiole produced by exposure levels as low as 0.15 ppm ozone; (2) there is a worsening of the respiratory bronchiolar lesion in monkeys during the postexposure period; and (3) episodic exposures cause more severe injury than daily exposures. It is difficult to provide a simplistic outline of the inflammatory process in the lung with long-term daily or episodic exposure because of the lack of experimental data. Furthermore, it is perplexing that when the epithelium of the central acinus is adaptive and resistant to long-term ozone exposure, the interstitium is reactive and marked by progressive inflammation and fibrosis (see Chapter 8.04). To investigate how episodic ozone exposure can be a bridge between the acute and chronic ozoneinduced lesion, rats were exposed to four 2-week cycles of 5 days of ozone (8 h day1) followed by 9 days of filtered air, evaluating airway inflammation and epithelial injury and repair at numerous times during the experiment (Schelegle et al. 2003). After four episodes, the investigators observed a depressed epithelial cell proliferative response to ozoneinduced injury, which may in part be the result of a decreased PMN inflammation and/or release of mitogenic neuropeptides in response to ozoneinduced injury.

124 Inflammatory Cells of the Lung: Polymorphonuclear Leukocytes

(a)

(b)

Figure 4 A transmission electron micrograph of Clara cells from carrier-treated mice (a) or mice treated with naphthalene (200 mg kg1) for 21 days (b). The epithelium in (b) is representative of an epithelium that is tolerant to injurious doses of naphthalene and shows an increase in overall cell size, and increased smooth endoplasmic reticulum (SER), round endoplasmic reticulum (RER), and small mitochondria with cristae. Scale ¼ 1 mm. Micrographs courtesy of Dr. Charles Plopper.

8.06.9 Human versus Animal Toxicology In a comparative review of the greater sensitivity of the primate (macaque monkey) lung compared with the rodent (Sprague–Dawley rat) lung to ozone exposure, Plopper et al. (1991) showed that collagen metabolism after high levels of ozone exposure (1.5 ppm, 23 h day1, 7 days) was increased by an average of 200% above control rats, but in monkeys there was an 800% increase. Exposure of rats to 0.25 ppm ozone (8 h day1, 42 days) produced less than a 10% increase in epithelial thickness or the number of cells per Square millimeter of epithelial basal lamina. By comparison, exposure of monkeys

to 0.15 ppm ozone (8 h day1, for 6 or 90 days) produced a 150% increase in thickness and a 700% increase in the number of cells of the epithelium (Harkema et al. 1993). These data suggest that at environmentally relevant concentrations, ozone is much more toxic to the primate lung than to the rodent lung. In studies in which humans were exposed to 0.4 ppm O3 for 2 h with heavy exercise (60 l min1), BALF was collected at 1 and 18 h postexposure and showed that the inflammatory response (PMNs and protein accumulation in BALF) was quickly initiated and comparable to rhesus monkeys exposed at rest to higher levels of O3 for a longer time (Hyde et al. 1992). One could infer from these results that macaque monkey toxicology is particularly pertinent to predicting human toxicology.

Inflammatory Cells of the Lung: Polymorphonuclear Leukocytes

8.06.10 In Vitro Systems Isolated rat alveolar epithelial cells are capable of forming tight, polarized, sodium-absorbing monolayers when cultured on porous Transwell inserts in the presence of serum (Cheek et al. 1989). Serumfree medium enhances alveolar epithelial barrier function in vitro as evidenced by elevated peak bioelectric properties: transepithelial resistance (Rt), which provides an indication of epithelial paracellular junctional integrity, and short-circuit current (Ieq), which is the sum of net active ion fluxes across the monolayer. The use of serum-free medium also promotes the retention of type II cell phenotypic markers, and the primary secretory product of type II cells, surfactant, is detected solely in monolayers cultured in serum-free medium. The dose–response relationships and interactive effects of ozone injury and PMNs on alveolar epithelial barrier function in vitro have been investigated using this model system (Cheek et al. 1995). Quiescent PMNs modulate the response of alveolar epithelium to acute ozone exposure, as reflected by altered bioelectric properties and differences in the number of injured epithelial cells in monolayers treated with PMNs after ozone exposure. Both the direction and the magnitude of PMN effects on oxidant-induced impairment of alveolar epithelial barrier function were dependent on ozone dose. Monolayers exposed to either filtered air or 0.1 ppm ozone, and subsequently administered PMNs onto their apical surfaces, exhibited greater Rt values at 24 h postexposure relative to monolayers not subjected to PMNs. However, this trend was reversed with increasing ozone concentration: by 48 h postexposure, monolayers exposed to 0.2 or 0.5 ppm ozone followed by PMN treatment had significantly lower Rt values relative to monolayers exposed to the same concentration of ozone only. A significant loss of total epithelial cells was noted in monolayers exposed to 0.5 ppm ozone; this exfoliation at the high ozone concentration was independent of PMN treatment. With respect to injured cells, PMN-treated monolayers exposed to 0.1 ppm ozone had significantly fewer injured epithelial cells per unit area (as identified by uptake of vital dye) relative to monolayers exposed to the low level of ozone only. The bioelectric data suggest two roles for PMNs in the modulation of ozone impairment of epithelial barrier function in vitro: (1) at high levels of ozone, PMNs may exacerbate injury to oxidant-injured epithelial cells and (2) conversely, the presence of PMNs after

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exposure to lower levels of ozone may enhance recovery of epithelial barrier function. Concurrently, observations of monolayer cell viability and organization 5 h postexposure suggest that the addition of PMNs after low-level ozone injury to alveolar epithelium in vitro facilitated the removal of dead and/or injured cells. It is known that reactions in the epithelial lining fluid with O3 are followed by reactions with antioxidants, which can lead to secondary ROS production, which in turn creates oxidant stress in susceptible adjacent cells (epithelial cells and alveolar macrophages) (Ballinger et al. 2005; Postlethwait et al. 1998; Pryor 1986; Pulfer et al. 2005). Oxidant stress in epithelial cells initiates a multinavigational chemokine network that aids in recruitment of inflammatory cells that target the oxidant stress cells (Oslund et al. 2004). Oxidant-stressed cells upregulate thioredoxin (produced predominantly apically), which upregulates IL-8 (secreted predominantly basolaterally) in neighboring healthy epithelial cells via nuclear factor-B (NF-B) (Harper et al. 2001). Thus the PMNs are recruited to the epithelial basal lateral surface by IL-8 secretion from the majority of healthy epithelial cells where they encounter a higher thioredoxin gradient in the airspace compartment, which drives their migration to the apical surface of the epithelial cells (Miller et al. 2000). Once on the apical surface of the epithelium, they preferentially migrate to and become adherent to oxidant-stressed epithelial cells (Oslund et al. 2004). The mechanism of oxidantstressed epithelial cell removal still remains to be elucidated. Pulmonary epithelial cell injury in vitro from toxicants such as quartz and asbestos dusts combined with activated PMNs appears to be dependent primarily on PMN proteases (Donaldson et al. 1988; Kamp et al. 1993). These observations are in good agreement with studies showing that simple PMN emigration through pulmonary type II cells does not increase epithelial permeability, but prolonged PMN–epithelial contact or PMN stimulation with FMLP results in increased epithelial permeability (Bhalla et al. 1993; Parsons et al. 1987). PMN migration across monolayers of a colonic epithelial cell line formed clusters that were associated with focal epithelial cell loss and disruption of epithelial junctional complexes in adjacent epithelial cells (Ginzberg et al. 2001). The loss of colonic epithelial cells was attributed in part to PMN-derived proteases. Hence, PMNs have the potential to cause

126 Inflammatory Cells of the Lung: Polymorphonuclear Leukocytes

substantial epithelial injury during migration, but their usual function is to play a ‘beneficial’ role by directly targeting injured airway epithelial cells and sparing the healthy airway epithelial cells.

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