Immunity, inflammation, and remodeling in the airway epithelial barrier: epithelial-viral-allergic...

Immunity, Inflammation, and Remodeling in the AirwayEpithelial Barrier: Epithelial-Viral-Allergic Paradigm

MICHAEL J. HOLTZMAN, JEFFREY D. MORTON, LAURIE P. SHORNICK, JEFFREY W. TYNER,MARY P. O’SULLIVAN, AURITA ANTAO, MINDY LO, MARIO CASTRO, AND MICHAEL J. WALTER

Departments of Medicine and Cell Biology/Physiology, Washington University School of Medicine, St. Louis, Missouri

I. Introduction: Traditional and Alternative Views of Airway Inflammatory Disease 19II. Molecular Basis of Airway Epithelial Immune Function From In Vitro Studies 21

A. Gene products for mediating transepithelial traffic of immune cells 21B. Cytokine-dependent gene network: transcriptional regulation 24C. Virus-dependent gene network: posttranscriptional regulation 27D. The final common pathway: epithelial cell death 28

III. Airway Immunity and Inflammation in a Mouse Model of Viral Infection 30A. Epithelial gene expression: interaction between virus and host cell 31B. Toward epithelial gene knockouts: defining an active role in innate immunity 31

IV. Airway Immunity and Inflammation in Human Subjects With Asthma 34A. Constitutive abnormalities in epithelial gene expression (Stat1 and IL-12) 34B. Glucocorticoid-sensitive abnormalities in epithelial gene expression (RANTES) 35C. A revised model for Th1/Th2 contributions to asthma 36

V. Long-Term Airway Hyperreactivity and Remodeling in Mice and Humans 37A. Segregating acute from chronic phenotypes 37B. Genetic and viral determinants for persistence 37

VI. Smart Strategies for Correcting Epithelial Inflammation and Remodeling 38A. Reversing viral mimicry using mutant E1A oncoprotein 39B. Modifying epithelial signaling with mutant Stat1 41C. Future considerations 41

VII. Summary 42

Holtzman, Michael J., Jeffrey D. Morton, Laurie P. Shornick, Jeffrey W. Tyner, Mary P. O’Sullivan, Aurita

Antao, Mindy Lo, Mario Castro, and Michael J. Walter. Immunity, Inflammation, and Remodeling in the AirwayEpithelial Barrier: Epithelial-Viral-Allergic Paradigm. Physiol Rev 82: 19–46, 2002; 10.1152/physrev.00020.2001.—Theconcept that airway inflammation leads to airway disease has led to a widening search for the types of cellular andmolecular interactions responsible for linking the initial stimulus to the final abnormality in airway function. It has not yetbeen possible to integrate all of this information into a single model for the development of airway inflammation andremodeling, but a useful framework has been based on the behavior of the adaptive immune system. In that paradigm,an exaggeration of T-helper type 2 (Th2) over Th1 responses to allergic and nonallergic stimuli leads to airwayinflammatory disease, especially asthma. In this review, we summarize alternative evidence that the innate immunesystem, typified by actions of airway epithelial cells and macrophages, may also be specially programmed for antiviraldefense and abnormally programmed in inflammatory disease. Furthermore, this abnormality may be inducible byparamyxoviral infection and, in the proper genetic background, may persist indefinitely. Taken together, we propose anew model that highlights specific interactions between epithelial, viral, and allergic components and so better explainsthe basis for airway immunity, inflammation, and remodeling in response to viral infection and the development oflong-term disease phenotypes typical of asthma and other hypersecretory airway diseases.

I. INTRODUCTION: TRADITIONAL AND

ALTERNATIVE VIEWS OF AIRWAY

INFLAMMATORY DISEASE

A critical step toward defining molecular mecha-nisms of airway disease came with formal recognition of

the role of immunity and inflammation. In the case ofasthma, evidence of immune abnormalities and excessiveairway inflammation (induced by allergic and nonallergicstimuli) has led to a widening search for the types ofinflammatory cells and mediators that might be responsi-ble for abnormal airway function. Cell types implicated in

Physiol Rev

82: 19–46, 2002; 10.1152/physrev.00020.2001.

www.prv.org 190031-9333/02 $15.00 Copyright © 2002 the American Physiological Society

the development of airway inflammation include immunecells as well as parenchymal cells. Cell-cell interactionsare attributed to classes of mediators that include lipids,proteases, peptides, glycoproteins, and cytokines. Theleading scheme for integrating this information has beenbased on the classification of the adaptive immune sys-tem, and especially the responses of T helper (Th) cells. Inthis scheme, CD41 T cell-dependent responses are clas-sified into T helper type 1 (Th1) or type 2 (Th2). Th1 cellscharacteristically mediate delayed-type hypersensitivityreactions and selectively produce interleukin (IL)-2 andinterferon (IFN)-g, whereas Th2 cells promote B cell-dependent humoral immunity and selectively produceIL-4, IL-5, and IL-13. Thus Th2 reactions may underlie theairway hyperreactivity and inflammation characteristic ofthe late response to allergen inhalation and so account forthe overproduction of Th2-derived cytokines that is char-acteristic of asthma (109, 151). How then can a Th2-polarized response account for asthma that is also trig-gered by exposure to nonallergic stimuli (especiallyrespiratory viruses) that would ordinarily trigger a Th1response? On the basis of the possibility that Th2-skewedresponses may develop in this setting as well (21, 62, 121),it is possible that a Th2-dominant response may mediateinflammation and hyperreactivity in response to nonaller-gic stimuli as well (as modeled in Fig. 1). However, thisdichotomous view of the immune response may not becompletely accurate, since there is often ambiguity in thetype of Th response triggered by most stimuli as well assignificant cross-regulation between the two types of re-sponses (57). In fact, recent studies in mice using adop-tive transfer with Th1 and Th2 cells have indicated thatTh1 cells may even be involved in initiating the allergicresponse (16, 110).

Because of these uncertainties, we have questionedwhether other aspects of immunity and inflammationmight also be critical for the pathogenesis of airway dis-ease. In particular, we aimed to develop a model thatbetter accounted for the dissociation between the devel-opment of allergy and asthma in many subjects and wasbased on a more precise appraisal of the Th1 system inthe airway. Furthermore, the “Th2 hypothesis” does notaccount for the possibility that the airway epithelial cellsmay act as active sentinels of innate immunity, and so,like other components of the innate immune system, mayprovide critical signals to the adaptive immune system.This review summarizes how our view of the barrierepithelial cell has evolved based on the identification ofits specialized programming for host defense and abnor-mal programming in disease.

The review is divided into six major sections. SectionII summarizes studies of isolated airway epithelial cells.The section proceeds from a relatively simple scheme forleukocyte recruitment (based on cell adhesion and che-moattraction) to one that depends on the coordinated

expression of a network of epithelial immune-responsegenes under distinct transcriptional and posttranscrip-tional controls. The transcriptional program depends pri-marily on interferon-Jak/Stat signaling while posttran-scriptional regulation uses a distinct RNA-proteininteraction that is responsive directly to viral replication.Section III summarizes studies that extend these samemolecular pathways to studies done in vivo using a mousemodel of viral bronchiolitis that takes advantage of agenetically defined host. Section IV summarizes studies ofhuman subjects with airway disease, focusing on howthese same epithelial gene networks behave in subjects

FIG. 1. Scheme for the role of the airway immune response in thedevelopment of asthmatic airway inflammation. A: illustration of howdecreases in virus-driven production of Th1 cytokines [e.g., interferon-g(IFN-g)] and increases in allergen-dependent production of Th2 cyto-kines [e.g., interleukin (IL)-4, IL-5, and IL-13] are characteristic ofasthma (designated as an “A”). This Th2-skewed setting thereby pro-vides for flares of the disease driven either by virus infection (with afunctional blockade in the normal Th1 response and a concomitant shiftto an increase in the Th2 response) or by allergen (with an increase inthe Th2 response). B: illustration of a modified scheme based on anincreased activity of both Th1 and Th2 responses. In this case, pathogen-activated Th1 cells in the airway tissue generate factors [e.g., tumornecrosis factor-a (TNF-a)] that mediate subsequent recruitment of Th2cells [e.g., via vascular cell adhesion molecule-1 (VCAM-1)] that providea setting for enhanced allergen responsiveness. [Modified from Castro etal. (16).]

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with stable or flared asthma in the presence or absence ofanti-inflammatory treatment with glucocorticoids. Thissection presents a model for how the epithelial genenetwork might interact with the Th1/Th2 balance to causeairway inflammation in asthma and introduces the possi-bility that this same gene network may also underliepersistent abnormalities in airway epithelial mucosal be-havior found in chronic airway disease. Section V summa-rizes studies of this possibility done in vitro and in vivo inmice and humans, highlighting the role of genetic suscep-tibility to virus-induced modification of epithelial pheno-type and consequent remodeling. Section VI, the final sec-tion, summarizes the review.

II. MOLECULAR BASIS OF AIRWAY

EPITHELIAL IMMUNE FUNCTION

FROM IN VITRO STUDIES

This section summarizes studies of isolated airwayepithelial cells, placing special emphasis on the use ofprimary-culture human cells that exhibit high-level fidelityto in vivo behavior. Section IIA begins with a model ofepithelial barrier function and so presents a workingscheme for epithelial-dependent traffic of immune cellsinto the airway. This model depends on at least one majorligand for cell adhesion, i.e., intercellular adhesion mole-cule-1 (ICAM-1) and another for chemotaxis, i.e., regu-lated upon activation, normal T cell expressed and se-creted (RANTES) chemokine, each of which depends onthe level of gene expression for function. Accordingly,section II, C and D, proceeds to summarize the basis forepithelial expression of these two corresponding genes.In doing so, these studies define two larger gene net-works. One is typified by ICAM-1 and is under the controlof IFN-driven Jak/Stat signal transduction and uses Stat1as a key intermediate. The other is typified by RANTESand relies on posttranscriptional regulation that is moredirectly inducible by viral replication. In this section, wealso introduce recent findings related to the IL-12 p40gene product that also appears chemotactic, but the reg-ulation of this pathway is not yet fully defined. Latersections will summarize evidence for the role of thesesame systems in mediating immunity and inflammation invivo in animal models and human subjects.

A. Gene Products for Mediating Transepithelial

Traffic of Immune Cells

The initial approach to defining epithelial control ofimmune cells and specific epithelial-leukocyte interac-tions relied on studies of immune cell interactions witheach other and with the endothelium. In these systems,the development of monoclonal antibodies against im-mune cell determinants indicated that a critical step in

leukocyte influx from the circulation is the coordinatedinteraction of specific cellular receptors on the migratingleukocytes with corresponding ligands on adjacent endo-thelial cells. A scheme for leukocyte recruitment wasdeveloped that revolved around the regulated expressionof distinct families of cell communication molecules, no-tably the selectins, the integrins, and the cell adhesionmolecule (CAM) members of the immunoglobulin (Ig)supergene family (12). In addition to these cell adhesionsystems for direct cell-cell contact, endothelial cells andimmune cells also appear capable of generating a series ofchemoattractants [e.g., platelet activating factor (PAF)]and chemoattractant cytokines (or chemokines) that mayact over a greater distance to direct immune cell move-ment and activation in tissue (85). These three systems, 1)selectin binding to carbohydrate mucinlike molecules, 2)cell adhesion molecules from the Ig and the extracellularmatrix protein families binding to integrins, and 3) che-moattractants and chemokines binding to G protein-cou-pled receptors, may act in a specific combination to dic-tate the type of immune cells that enter and get retainedin the tissue. Especially in the case of endothelial-leuko-cyte interaction, this information has enabled the devel-opment of a stepwise molecular scheme for leukocyteinflux into tissue from the circulation (120).

1. Cell adhesion molecules and the role of ICAM-1

in the epithelium

Our initial studies of epithelial-immune cell adhesionwere undertaken with airway epithelial cell monolayersestablished under conventional cell culture conditions,thereby exposing only the apical epithelial surface to theimmune cell (an approach that is more logically taken forstudies of endothelial cells). Improving on earlier meth-ods, however, we studied the basis for immune cell ad-hesion to airway epithelial and venular endothelial cellsusing a quantitative flow cytometry-based assay (98). Thistechnique avoids extensive leukocyte purification, cul-ture, or labeling steps that may alter leukocyte functionand by their nature may eliminate heterotypic cell-cellinteractions that may be important in vivo. The quantita-tive aspect of the flow cytometry methodology was alsocritical for examining the low levels of constitutive adher-ence that might be expected for resting T cells. Comparedwith standard cell-labeling methods, the flow cytometry-based assay yielded a lower level of constitutive T celladhesion despite a similar level of stimulated adhesion(after T cell activation with phorbol dibutyrate) usingendothelial or epithelial cell monolayers. Endothelial-Tcell adhesion was further increased by monolayer treat-ment with tumor necrosis factor-a (TNF-a) (less so withIL-1b and least with IFN-g), whereas epithelial-T cell ad-hesion was most sensitive to IFN-g. Cytokine stimulationof adhesion was invariably concentration dependent and

EPITHELIAL-VIRAL-ALLERGIC PARADIGM 21


closely matched to the cellular levels of ICAM-1 (79).Accordingly, T cell adhesion was markedly inhibited byanti-ICAM-1 or anti-b2-integrin antibody (95–97% inhibi-tion for epithelial cells and 57–67% inhibition for endo-thelial cells) directed against ICAM-1 interaction withleukocyte function-associated antigen 1 (LFA-1). Residualendothelial-T cell adhesion that correlated with endothe-lial vascular cell adhesion molecule-1 (VCAM-1) levelswas blocked by an anti-a4-integrin antibody directedagainst VCAM-1 interaction with very late-activation anti-gen 4 (VLA-4). The results suggest that 1) peripheralblood T cells without exogenous activation exhibit littleLFA-1- or VLA-4-dependent adherence except to endothe-lial or epithelial cells expressing high levels of ICAM-1and/or VCAM-1; and 2) differences in endothelial versusepithelial cell mechanisms to bind activated and unacti-vated T cells (e.g., dependence on a mixed- versus asingle-ligand system and distinct cytokine-responsivenessof ligand levels) may help to coordinate T cell traffic toepithelial barriers. These findings also support the viewthat T cell trafficking into inflamed tissues (especially inmucosal barriers) depends critically on local activationevents.

These initial studies highlight two distinct features ofairway epithelial cell behavior: relative hypersensitivity toIFN-g and a critical role for ICAM-1 in epithelial-immunecell adhesion. This pattern of cell adhesion molecule ex-pression and function on airway epithelial cells appearsdistinct from the one for venular endothelial cells. In thecase of endothelial cells, at least three interactions of celladhesion receptors have been implicated in the develop-ment of inflammation in epithelial barriers: 1) E-selectinbinding to its leukocyte sialyl-Lewis X carbohydrate li-gand (38, 49, 145); 2) VCAM-1 binding to the leukocyteb1-integrin VLA-4 (a4b1) (7, 97); and 3) ICAM-1 binding tothe leukocyte b2-integrins LFA-1 (aLb2) or Mac-1 (aMb2)(101, 146). In each case, there is evidence that the ligandmay be overexpressed and/or activated under basal orallergen-challenge conditions in asthmatic subjects andthat pretreatment with a blocking monoclonal antibodiescan inhibit immune cell influx in a model of asthmaticairway inflammation. The findings provide for two sys-tems (E-selectin and VCAM-1) capable of initiating leuko-cyte rolling and tethering to the vascular endothelium aswell as two that may mediate diapedesis through thevascular wall (VCAM-1 and ICAM-1). However, neitherisolated cells in culture nor endobronchial tissue fromnormal and asthmatic subjects indicated that airway epi-thelial cells (or fibroblasts or smooth muscle cells) ex-pressed significant levels of E-selectin or VCAM-1. Thesefindings are therefore consistent with the view that airwayepithelial cells present fewer adhesion ligands (than en-dothelial cells) for directing leukocyte movement. Thebiologic basis for this difference may reflect the capacityof endothelial cells to be armed with ligands that slow

down passing leukocytes (to allow tethering and trigger-ing) and that select a specific leukocyte subset (to allowadhesion and transmigration) from the diverse circulatingpool. This requires multiple molecular interactions withvarying specificities. In contrast, other parenchymal cellsmay come into contact with immune cells after somedegree of selection and activation so that they are re-quired only to facilitate further leukocyte migration andretention. ICAM-1 is well suited to mediate this processbecause nearly all immune cells constitutively express itsreceptors, i.e., the b2-integrins LFA-1 and Mac-1. In thecase of T cell traffic, the endothelial cells (and likelyepithelial cells) also may express a series of receptors(designated homing receptors or addressins) that serve todirect distinct subsets of lymphocytes to appropriate lo-cations of lymphoid tissue. This type of cell adhesion(exemplified by some of the b7-integrin interactions) isprobably most important in maintaining a resident popu-lation of immune cells, but whether this system is alsoregulated during airway inflammation remains uncertain.

Interestingly, a specific addressin for T cell localiza-tion to the lung has not yet been identified. We reasonedthat patterned expression of more common adhesion mol-ecules might also facilitate traffic of specific T cell subsetsto the airway mucosa. To define the relationship betweenT cell phenotype and adhesiveness, we examined T celladhesion to endothelial cell, fibroblast, and epithelial cellmonolayers as well as extracellular matrix proteins (col-lagen and fibronectin) using a three-color flow-cytometry-based adherence assay that minimizes basal adhesionlevels and facilitates quantitative lymphocyte subtyping(99). Regardless of monolayer type, monolayer stimula-tion conditions, or T cell activation status, we found thatthe gd-TCR bearing T cells adhered more efficiently thanab T cells. The difference was based predominantly onincreased levels of activatable LFA-1 (and to a lesserdegree VLA-4) because 1) it correlated precisely withinhibitability by anti-LFA-1 (and VLA-4) monoclonal anti-bodies and the levels of LFA-1 (and VLA-4) on the cellsurface; and 2) it persisted after maximal LFA-1 (andVLA-4) activation with phorbol dibutyrate. In contrast tomost cases of ab T cell behavior, gd T cell adhesion to cellmonolayers was not linked to memory status, i.e., therewas no difference between naive Vd11 and memory Vd21

populations in levels of LFA-1 (or VLA-4) expression orLFA-1- (or VLA-4-)dependent adhesion to cell monolayers.However, Vd11 cells exhibited higher levels of VLA-5 thatcorrelated with an increased adhesiveness to fibronectinand to a 120-kDa fibronectin fragment (FN-120) that con-tains only the VLA-5-binding domain but not to type Icollagen or to a fibronectin fragment (FN-40) that bindsonly VLA-4. Taken together, the results define a hierarchyfor integrin (LFA-1, VLA-4, and VLA-5) expression andconsequent adhesion among T cell subsets that is linkedto TCR gene usage (but not necessarily linked to memory

22 HOLTZMAN ET AL.


status) and may thereby help to explain the accumulationand retention of Vd11 gd T cells in epithelial and connec-tive tissues. The findings underscore the general conceptthat immune cells (like parenchymal cells) may regulatetheir expression of cell adhesion molecules to achievepreferential localization in tissues. The relationship ofregulation of more general cell adhesion receptors (suchas LFA-1) to more specific systems directed by addressinsor by specific antigens needs to be further defined. Atpresent, however, it appears that these more general re-ceptors may work in concert with more specific ones, aconcept that is illustrated during antigen presentationwhere cell adhesion molecules facilitate engagement andactivation of the T cell receptor for antigen in the appro-priate major histocompatibility complex (MHC) context.Recruitment of specific subsets of leukocytes may also bemediated by a diverse capacity for epithelial productionof chemokines as noted in section IIA2.

2. Chemokines and the role of RANTES

in the epithelium

As noted above, schemes for transendothelial movementof immune cells (extravasation) depend on the coordinatedexpression of cell adhesion molecules and chemoattractantsthat interact with corresponding receptors on the immune cellsurface (12, 85, 120). Thus it appeared reasonable to proposethat similar molecular mechanisms may regulate transepithelialmovement of immune cells. However, there are critical differ-ences between epithelial and endothelial cell adhesion andtransmigration, the most obvious of which may be that immunecell recruitment through endothelium and epithelium generallyoccur in opposite directions with respect to the luminal surfaceof cells. For endothelium, immune cells leave the circulation inan abluminal direction moving from apical-to-basal endothelialsurfaces, whereas for epithelium, cells move toward the lumentraversing a basal-to-apical direction with respect to epithelialcell surfaces. In the case of the endothelium, this directionalprocess may be coordinated by the actions of selectins, celladhesion molecules, and chemokines all acting as haptotaxins(90), but the driving force for immune cell movement acrossthe epithelial barrier (if it exists at all) was uncertain. Thusexpression of epithelial ICAM-1 and consequent interactionwith T cell LFA-1 appeared to be the major determinant of Tcell adhesion to the apical surface of the airway epithelial cellmonolayers (98, 99), but the extent to which ICAM-1/LFA-1interaction or other molecular interactions might regulate Tcell traffic through the epithelium and the directionality ofmovement was uncertain for airway and other epithelia.

Accordingly, we next developed a system for moni-toring immune cell adhesion and transmigration throughan epithelial model in apical-to-basal and basal-to-apicaldirections (129). Immune cell (in this case, T cell) behav-ior was again monitored by quantitative flow cytometry toavoid a need for extensive leukocyte purification, culture,

and labeling. Epithelial monolayers were established withprimary-culture human tracheobronchial epithelial (hTBE)cells that exhibit differentiated structural and functionalfeatures of polarized epithelial barriers found in situ (149). Inparticular, monolayers of hTBE cells emulate in vivo behav-ior with low basal levels of ICAM-1 and cytokine-dependentincreases in ICAM-1 expression (66, 79, 114). In this ex vivosystem, T cell adhesion and subsequent transmigration wereblocked in both directions by monoclonal antibodies againstLFA-1 or ICAM-1 (induced by IFN-g treatment of epithelialcells). The total number of adherent plus transmigrated Tcells was also similar in both directions, and this pattern fitwith uniform presentation of ICAM-1 along the apical andbasolateral cell surfaces. However, the relative number oftransmigrated to adherent T cells (i.e., the efficiency oftransmigration) was increased in the basal-to-apical relativeto the apical-to-basal direction, so an additional mechanismwas needed to mediate directional movement toward theapical surface. Screening for epithelial-derived T cell chemo-kines indicated that IFN-g treatment caused predominantexpression of RANTES (68). The functional significance ofRANTES production was then demonstrated by inhibition ofepithelial-T cell adhesion and transepithelial migration byanti-RANTES monoclonal antibody. In addition, we foundthat epithelial (but not endothelial) cells preferentially se-creted RANTES through the apical cell surface, therebyestablishing a chemical gradient for chemotaxis across theepithelium to a site where they may be retained by highlevels of RANTES and apical ICAM-1. In this system,RANTES did not account for all chemotactic activity, andsubsequent studies have indicated that other chemokines(and other chemotaxins such as IL-12 p80) provide for func-tional redundancy (see sect. III). Nonetheless, the resultsdefine how the patterns for cell-specific apical sorting ofRANTES serve to mediate the level and direction of T celltraffic and provide a basis for how this process is preciselycoordinated to route immune cells to the mucosal surfaceand maintain them there (Fig. 2). Potent effects of RANTESon T cells (4), eosinophils (2), and macrophages (as de-scribed below), distinct from effects on cell movement, in-dicate an additional role of epithelial cells in regulating theactivation status of local immune cells.

In sum, it appears that airway epithelial-T cell adhesionand transmigration depend on the cytokine-dependent expres-sion of ICAM-1 on the epithelial cell surface (98), concomitantlevels of expression and activation of LFA-1 on the T cellsurface (99), and polarized secretion of RANTES through theapical cell surface (129). For ICAM-1, distribution on both cellsurfaces mediates efficient cell adhesion at the basal cell sur-face (to aid in transmigration) and at the apical cell surface (forretention and movement along the airway). For RANTES, thepattern of preferential apical secretion provides for a solublechemical gradient for T cell movement from the subepithelium(where levels are low) to the mucosal surface (where levelsare higher). These patterns fit well with available data for



ICAM-1 and RANTES expression in airway epithelium invivo (see below), but we emphasize that this pattern of celladhesion molecule expression and chemokine secretions isdistinct. Other types of epithelial cells (notably colonocytesand pneumocytes) express ICAM-1 only along the apical cellsurface (11, 59, 106). Perhaps in this setting, ICAM-1 func-tions mostly in cell movement along that surface or acts inconcert with other cell adhesion receptors to aid in hostdefense (e.g., as opsonins) (102). Similarly, epithelial capac-ity for polarized secretion of RANTES may be distinct fromother cell types. In the case of endothelial cells, it appearsthat chemokines with immobilization domains (includingRANTES and IL-8) are anchored to the cell surface to act ashaptotaxins rather than freely secreted to act as chemotax-ins (41, 113, 131). Apparently in this haptotactic mode, thereis no mechanism for polarization because both luminal andabluminal surfaces of endothelial cells are coated with che-mokine (90). It is also possible that the cellular source ofendothelial-bound chemokine is the epithelial cell, since

transcytosis and subsequent presentation of exogenous che-mokine may occur in either direction. Taken together withcomparative data for endothelial cells, our studies of airwayepithelial cell-T cell interaction offer a means for progressivemovement of T cells from endothelium to the airway lumenthrough distinct cell-specific mechanisms for cell adhesionmolecule expression and chemokine secretion. As notedbelow in studies of human subjects, these same molecularsystems appear activated in asthma because there are in-creased numbers of activated T cells as well as increasedlevels of ICAM-1 and RANTES expression in the epitheliumof asthmatic subjects depending on the severity of diseaseand treatment conditions.

B. Cytokine-Dependent Gene Network:

Transcriptional Regulation

Section IIA concentrated on the topological basis forhow epithelial cells coordinate their expression of cell

FIG. 2. Molecular interactions that mediate immune cell adhesion to airway epithelial cells, transepithelial cellmigration, and retention at the airway lumen. Critical steps include the following: 1) initial integrin [e.g., leukocytefunction-associated antigen 1 (LFA-1)] binding to cell adhesion molecule [e.g., intercellular adhesion molecule-1(ICAM-1)] on the basolateral epithelial cell surface; 2) subsequent b-chemokine receptor (e.g., CCR5) binding tochemokine (e.g., RANTES) and migration along the chemical gradient for RANTES, which is secreted preferentially tothe apical epithelial cell surface; and 3) renewed LFA-1 binding to ICAM-1 for retention and/or migration along the apicalcell surface. Examples for cell adhesion molecules and chemokine and their receptors are most applicable to mononu-clear cell traffic. [Modified from Holtzman et al. (53).]

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adhesion molecules and chemoattractants with that in theunderlying vascular plexus so that leukocytes migrateefficiently through the tissue. Another challenging ques-tion is how this process is coordinated to be stimulusspecific. The first clue to this process was the finding thatvascular endothelial and airway epithelial cells have dis-tinct but complimentary profiles of cytokine responsive-ness for induction of ICAM-1 and ICAM-1-dependent celladhesion and transmigration. In particular, it appears thatairway epithelial cells exhibit a selective sensitivity toIFN-g (79), and this selectivity in cytokine responsivenessof ICAM-1 expression offered an opportunity to decipherthe genetic code of airway inflammation. In general, in-ducible gene expression may be mediated by signal trans-duction leading to regulation at transcriptional or post-transcriptional levels (controlled by DNA- or RNA-proteininteractions, respectively). In the case of the ICAM-1gene, nuclear run-off assays aimed at monitoring tran-scriptional initiation rate indicated that IFN-g regulationof epithelial ICAM-1 levels occurs at least in part at atranscriptional level (80). Because gene transcription isoften regulated by DNA-protein interactions in the pro-moter region, the basis for selective cytokine control ofICAM-1 expression could be investigated by structure-function analysis of the ICAM-1 gene promoter region.This section therefore summarizes findings on one aspectof this genetic code: the DNA-protein interactions in theICAM-1 gene promoter region that control IFN-g-induc-ible transcription of the ICAM-1 gene. As will be devel-oped, the molecular building blocks used to regulate theICAM-1 gene are also used to control an entire immune-response gene network in the epithelium.

Initial identification of DNA-protein interactions isoften undertaken with a functional analysis of promoter/reporter gene constructs and a concomitant analysis ofproteins that bind to any identified regulatory sites. Whenthis approach was used to analyze IFN-g-inducible ex-pression of the ICAM-1 gene in primary-culture airwayepithelial cells, a 37-bp region of the gene from nucleo-tides 2130 to 293 was found to be responsible for selec-tive IFN-g responsiveness of airway epithelial cell ICAM-1expression (80). This region (designated the IFN-g-re-sponse element or IRE) contained an 11-bp inverted re-peat (the gamma-activation site) that was necessary andsufficient for IFN-g-responsiveness of the ICAM-1 gene.This function was closely linked to the capacity of thissite to bind to Stat1 (the first member of the signal trans-duction and activation of transcription factor family). Be-cause vascular endothelial cells also appear capable ofStat1 activation and binding in response to IFN-g treat-ment, their failure to fully respond to IFN-g may be due tothe presence of a concomitant interaction of a repressorprotein that prevents the response. This possibility maybe reflected by restoration of IFN-g responsiveness inendothelial cells by protein synthesis inhibition with cy-

cloheximide treatment (D. C. Look and M. J. Holtzman,unpublished observations). The relatively small ICAM-1response to IFN-g in endothelial cells may be reconciledwith the fact that IFN-g-targeted viruses are not oftenencountered at the endothelial barrier. However, as dis-cussed next, the shared capacity of both endothelial andepithelial cells to utilize an alternative DNA element forIFN-g-activation of the MHC class I genes may provide acommon pathway for antigen recognition and processingthat is essential for immune defense against inhaled aswell as circulating agents.

Additional studies of airway epithelial cells in thecontext of work on other cell types by us and othersindicate that transcriptional regulation of the epithelialICAM-1 gene is typical of genes controlled by an IFN-g-responsive Janus family kinase (Jak)-Stat signaling path-way (summarized in Fig. 3). This pathway consists of theIFN-g-receptor, receptor-associated Jak1 and Jak2 ty-rosine kinases, the Stat1 transcription factor, and specificStat1/DNA and Stat1/protein interactions in the ICAM-1gene promoter region (79, 80, 141). Signaling depends onthe capacity of Stat1 to relay the signal from the cyto-plasm to the nucleus through its capacity to interact bothwith the IFN-g-receptor a-chain (46) as well as the GASsite in the gene promoter region (80). In addition toStat1/DNA interaction, it appears that Stat1 interactionwith the constitutively active transcription factor Sp1 isalso critical for full activation of the ICAM-1 gene (81). Atthe promoter, direct and indirect interactions with thecoactivators p300/CBP and p/CIP may further facilitateenhanceosome formation and more efficient gene tran-scription, and these components appear active in airwayepithelial cells as well (58, 82, 134, 153). In other cellsystems, an additional action of a serine/threonine mito-gen-activated protein kinase is needed to convey full ac-tivity, but this step appears less influential in airway epi-thelial cells (141).

In further work, it appears that these same molecularbuilding blocks serve to regulate IFN-g responsiveness ofother epithelial immune-response genes. Thus Stat1 bind-ing also confers IFN-g responsiveness of genes for trans-porter and antigen processor-1 (TAP-1), interferon regu-latory factor-1 (IRF-1), and Stat1 itself (80–82, 115, 141).As discussed further below, induction of Stat1 and con-sequent autoamplification of this pathway may serve toexaggerate the inflammatory response in experimentalmodels and in disease. Similarly, the IRF-1 gene product isalso a member of a transcription factor family and so mayamplify the pathway (in concert with other factors) byactivating the genes for inducible nitric oxide synthase(iNOS), MHC class I molecules, and IFN-a/b (35, 42, 65).In turn, IFN-a/b (generated as a result of these events ormore directly in response to replicating virus) may acti-vate the IFN-a receptor complex and overlapping signal-ing components to activate additional antiviral genes, in-



cluding IRF-1 and Stat1 (24; M. Lo and M. J. Holtzman,unpublished observations). Thus a cascade of common sig-naling components enables IFN-g to efficiently activate asubset of immune-response genes that are oriented towardseveral levels of antiviral defense. These include 1) antigenrecognition and T cell costimulation as well as immune cellrecruitment (ICAM-1); 2) antigen processing (TAP-1); and 3)amplification of the immune response (Stat1 and IRF-1) withadditional capacity for antigen recognition (MHC class I)and antiviral toxicity (iNOS and IFN-a/b).

Based on the apparent efficiency of epithelial im-mune-response genes for antiviral defense, one might ex-

pect little problem in coping with respiratory viruses.However, this is clearly not the case, and these failures ofthe immune response provide another informative strat-egy for unraveling the genetic code of epithelial-depen-dent immunity. Thus viruses have established an array ofstrategies for immune subversion (108). In the case ofriboviruses with relatively small genomes, molecular ad-aptation may take advantage of host cell machinery tosubvert the immune response. In fact, ICAM-1 itself wasdiscovered in part by its capacity to also serve as thereceptor for the major group of human rhinoviruses (47,123). Paramyxoviruses appear to block type I IFN signal-

FIG. 3. Signal transduction pathway from cell surface (left) to nucleus (right) for cytokine (IFN) activation of anairway epithelial immune-response gene network. IFN-g signaling begins when IFN-g binds to its heterodimeric receptor(IFN-gR) and triggers activation of a-chain-associated Jak1 and b-chain-associated Jak2 tyrosine kinases and consequenta-chain phosphorylation. This step enables a-chain recruitment of Stat1 via its SH2 domain and subsequent Stat1phosphorylation and release of the phosphorylated Stat1 homodimer. Activated Stat1 homodimer translocates to thenucleus where it binds to an inverted-repeated motif designated the gamma-activation site (GAS) and activates (inconcert with Sp1, p300/CBP, and p/CIP) transcription of a subset that includes the ICAM-1, TAP-1, IRF-1, and Stat1 genes.Increased levels of Stat1 may then act to autoamplify Stat1-dependent gene activation, while newly generated IRF-1 maybind to an IFN-response stimulation (IRS) site and activate (in concert with other factors) a second wave of transcrip-tion, including the HLA-B, inducible nitric oxide synthase (iNOS), and IFN-b genes. IFN-b driven gene expression isinitiated by activation of the IFN-a/b receptor (IFN-aR) and subsequent activation of IFN-aR1-associated Tyk2 andIFN-aR2-associated Jak1 with consequent IFN-aR1 phosphorylation and recruitment of Stat2. Phosphorylation of Stat2enables recruitment of Stat1 and release of the phosphorylated Stat1/Stat2-heterodimer. This heterodimer in concertwith IRF-9 (p48) forms the interferon-stimulated gene factor 3 (ISGF3) complex that binds to the interferon-stimulatedresponse element (ISRE) and increases transcription of a subset that includes the MxA, IRF-1, and Stat1 genes. For IFN-bsignaling, increased levels of Stat1 may autoamplify Stat1-dependent gene activation, but this pathway may also beeffectively downregulated by paramyxoviral (e.g., RSV) infection. However, as discussed in Fig. 4, paramyxoviruses mayactivate other epithelial immune-response pathways for host defense.

26 HOLTZMAN ET AL.


ing (Lo and Holtzman, unpublished observations). Otherviruses may encode proteins with the capacity for molec-ular mimicry that specially target IFN-g-dependent immu-nity by modifying host gene expression. In particular,adenoviral E1A oncoprotein may interfere with p300/CBPaction and may also directly target Stat1 (82). The molec-ular basis for this action and for how it can be used todevelop anti-inflammatory strategies is discussed furtherin the section on strategies for correcting abnormalities inepithelial signaling and remodeling (sect. VI).

C. Virus-Dependent Gene Network:

Posttranscriptional Regulation

Similar to the case for ICAM-1, only little was previ-ously known for mechanisms that regulate RANTES geneexpression (or b-chemokine gene expression in general) (6).On the basis of the standard approach noted above fordefining cis- and trans-acting controls for gene transcrip-tion, initial studies by others suggested that RANTES geneexpression depended on NF-kB sites in the RANTES pro-moter region (93, 100, 111). However, no attempt was madeto determine the functional importance of these sites bydirect determination of transcriptional initiation rate, and noinformation was available on any possible role of posttran-scriptional regulation of the RANTES gene. This sectionsummarizes our recent findings related to transcriptionaland posttranscriptional regulation of the RANTES gene inairway epithelial cells using systems for IFN-g- and virus-inducible expression in isolated cells. Later sections definehow this system behaves in vivo.

In initial experiments (as noted above), we determinedthe capacity of primary culture airway epithelial cells toproduce chemotaxins that could mediate T cell transmigra-tion (68, 129). In this setting, we found that IFN-g causedproduction of RANTES consistent with the IFN responsive-ness of this cell type. However, we were interested in deter-mining whether airway epithelial cells could respond di-rectly to viral infection without a requirement for signalsfrom immune cells. We were especially interested inwhether the types of viruses associated with asthma mightproduce distinct effects on epithelial immune-response geneexpression. The effects of paramyxoviruses appeared par-ticularly relevant, based on the epidemiological evidencethat members of the Paramyxoviradae family, especiallyrespiratory syncytial virus (RSV) and parainfluenza viruses,were closely associated with recurrent wheezing andasthma in young children (19, 26, 107, 119, 122, 124). In thatcontext, we were interested to find that RSV infection ofhuman airway epithelial cells (using an ex vivo system lack-ing IFN-g or other immune cell contribution) caused induc-tion of RANTES gene expression in marked excess of otherb-chemokine genes (69).

We next aimed to define the mechanism responsible

for virus-inducible RANTES production. Previous analysisof the host response to viral infection has generally fo-cused on the capacity of viruses to activate or represstranscription of cellular genes (52, 87), and this approachis also characteristic of work on riboviruses. In relatedexamples from the Paramyxoviruses, the effect on hostgenes is mediated by DNA regulatory elements that bindNF-kB, IRF-1, ATF-2/c-Jun, and high mobility group pro-tein HMG-I(Y) in the IFN-b gene or HMG-I(C) in theRANTES gene (78, 130, 132). Accordingly, we assumedthat NF-kB sites in the RANTES gene promoter (93, 100,111) might be responsible for virus induction of RANTESgene expression. Indeed, it initially appeared that RSV-driven expression of epithelial RANTES also depended oninducible gene transcription because expression was ac-companied by coordinate increases in transcriptional initia-tion rate and gene promoter activity. However, RSV-drivenincreases in RANTES gene transcription and promoter ac-tivity were small and transient relative to RANTES expres-sion, and they were no different in size and duration than forinactivated RSV that was incapable of inducing RANTESexpression. These findings suggested that the increase inRANTES gene transcription was required but not sufficientfor inducible expression and that critical regulatory effectsoccurred at a posttranscriptional level. This type of mecha-nism for virus-inducible expression of RANTES was estab-lished when we found that replicating RSV markedly in-creased RANTES mRNA half-life (69).

In contrast to what appear to be likely DNA-proteininteractions for mediating transcriptional activation ofhost genes by viruses and other stimuli, little is knownabout viral mechanisms for controlling mRNA stability. Inconcomitant work on cytokine responsiveness of epithe-lial immune-response genes (including ICAM-1, IRF-1, andRANTES), we have noted that IFN (like RSV) stimulatesRANTES production via mRNA stabilization (68). It istherefore possible that RSV-driven signals for alteringstability of RANTES mRNA may overlap with those forIFN-g signal transduction. However, the available exam-ples for IFN-g-dependent increases in mRNA stability ap-pear distinct from the characteristics of RANTES expres-sion. For example, IFN-g stabilization of ICAM-1 mRNA ismediated by a region of the translated sequence encodingthe ICAM-1 cytoplasmic domain (103), but the RANTESgene (encoding a secreted protein) does not contain thissequence. Furthermore, this instability mechanism (incontrast to the one for RANTES) is uninfluenced by acti-nomycin D treatment. Similarly, IFN-g upregulates ex-pression of the complement components C3 and C4 bystabilization of mRNA, but this system is also uninflu-enced by transcriptional inhibition (91). In addition, theRANTES mRNA does not contain consensus sites forpreviously identified mRNA turnover elements, includingAU-rich elements in other cytokine (as well as ICAM-1)mRNAs or UC-rich cleavage sites in gro a and 9E3 mRNAs



(13, 103, 126, 127). Thus the mechanism underlying basalinstability as well as RSV-dependent stabilization ofRANTES mRNA may be biochemically distinct from othergenes. Indeed, RNase protection assays of heterologous pro-moter/reporter plasmids indicate that basal instability ofRANTES mRNA is mediated at least in part by nucleotides11–389 of the RANTES gene, and this region is also thetarget for induction by virus (69). This region contains adistinct RNA turnover element in the 39-untranslated region(UTR) that forms a complex with a putative RANTES-bind-ing factor (RBF) under basal conditions but not during RSVinfection (A. Antao, W. Roswit, M. Pelletier, and M. J. Holtz-man, unpublished observations) (Fig. 4). The precise natureof this RNA-protein interaction and how it is regulated byviral replication still needs to be determined.

Even at this point, however, the findings provide thebasis for an alternative model for virus-dependent inductionof epithelial immune-response genes (depicted in Fig. 5). Inthis model, cytokine- (especially IFN-g)-dependent induc-tion of immune-response genes depends on transcriptionalactivation of gene expression, whereas direct viral inductionof expression may be regulated by transcriptional or post-transcriptional events. Transcription may depend on viral

interaction with Toll-like receptors (TLR) and activation ofNF-kB dependent pathways (71, 132, 133). This type ofregulation depends on viral surface proteins (e.g., RSV Fprotein) and may therefore be triggered by dead or livevirus. In addition, viruses may also act downstream at theposttranscriptional level. This action requires viral replica-tion and alters gene expression by stabilizing mRNA (whichis the case for RANTES) or improving protein translationand processing (which appears to be the case for otherepithelial immune-response genes). These two actions, acti-vation of gene transcription and stabilization of mRNA,would be highly synergistic for gene expression. In fact, ourexperience has been that viral induction of epithelial geneexpression is minimal in the absence of viral replication andconsequent action at a posttranscriptional level (69; A. An-tao, M. Pelletier, and M. J. Holtzman, unpublished observa-tions).

D. The Final Common Pathway:

Epithelial Cell Death

Each of the systems described above aims to explainhow epithelial cells communicate with the immune sys-

FIG. 4. Regulation of RANTES mRNA turnover under basal conditions and during viral infection. Under basalconditions, a trans-acting mRNA turnover factor (i.e., RBF) may be generated and activated and so be available to bindto a cis-acting mRNA turnover element in the 39-untranslated region (UTR). RBF-mRNA interaction alters the confor-mation of the mRNA to render it susceptible to degradation by active endoribonuclease (RNase) at the degradation targetsite. During viral infection (e.g., by RSV), the RBF is prevented from binding (by downregulating the level or activationstatus) and from mediating RNase action. This mRNA stabilization may synergize with transcription to mediate markedincreases in gene expression. [Modified from Holtzman et al. (53).]

28 HOLTZMAN ET AL.


tem. In the setting of viral infection, this communicationis generally designed to recruit immune cells that destroythe infected host (epithelial) cells and then clear thecellular debris from the site. In this setting, cytotoxic Tcells can mediate epithelial cell death via Fas-dependentand perforin/granzyme-dependent pathways (96), andmacrophages may clear the debris from the site. We willreturn to the specifics of this process in section III, whichdefines the host response to viral infection in vivo, but atthis point, we introduce the role of intrinsic pathways toregulate airway epithelial cell death and survival duringthe course of viral infection.

The approach to defining epithelial life and deathpathways that are targeted during acute respiratory viralinfection begins with the premise that the virus and thehost have opposing motives. The virus aims to first main-tain host viability (to allow for viral replication) and thentrigger cell death (to allow for lysis and viral spread toadjacent cells). In contrast, the epithelial host aims todestroy itself as rapidly as possible, presumably via apo-ptosis, to protect its neighbors from infection and further

inflammation. Thus self-programmed epithelial cell deathis an effective innate host defense mechanism. For exam-ple, activation of caspase-dependent epithelial death path-ways by the Fas death receptor may be critical for hostdefense against Pseudomonas aeruginosa infection inmice (45). However, we have found that primary culturehuman airway epithelial cells exhibit minimal Fas-depen-dent cell death unless the response is augmented by con-comitant treatment with cycloheximide or actinomycin D,and paramyxovirus-inducible cell death is not influencedby Fas blockade (105). Thus the relevance of this systemremains uncertain, especially in response to intracellularstimuli like respiratory viruses.

Recognizing that the pathways governing cell deathversus survival are complex, we have analyzed the behav-ior of airway epithelial cells during viral infection using anoligonucleotide microarray in conjunction with assays ofcell death parameters, e.g., caspase activation and mito-chondrial dysfunction. Our preliminary results indicatethat RSV infection causes mitochondrial dysfunction, butlethal effects appear to be delayed until viral replication is

FIG. 5. Regulation of epithelial immune-response gene expression by IFN-g or paramyxovirus (e.g., RSV). For IFN-gsignaling (1), sequential steps include IFN-gR activation/phosphorylation, Stat1 translocation and interaction at theinterferon regulatory element (IRE) in the gene promoter region, generation of pre-mRNA transcripts, and mRNAmaturation and release to ribosomal machinery for translation and then processing of protein for expression at theappropriate cellular site. The mRNA transcripts may alternatively interact with mRNA turnover factors such asRANTES-binding factor (RBF) that mediate rapid mRNA degradation by endoribonuclease (RNase). For paramyxovirussignaling (2), RSV may interact with Toll-like receptors (TLR) and so activate NF-kB and consequent NF-kB-dependentgene transcription. This type of transcriptional regulation depends on viral surface proteins and may therefore betriggered by dead or live virus. In addition, RSV may act downstream at the posttranscriptional level and alter geneexpression by stablizing mRNA or improving protein translation and processing. These posttranscriptional events requirelive, replicating virus.



well developed (M. O’Sullivan, K. Takami, and M. J. Holtz-man, unpublished observations). The initial phase mayrely on alterations in Bax-, TNF-, growth factor-, and cellcycle-dependent pathways that are each pushed towardgene expression that would favor cell survival during theacute phase of RSV infection (Fig. 6). Presumably,paramyxoviruses have developed a diverse program forpreserving host cell viability during this early phase ofinfection, because their survival depends on it. Concom-itant with these events, we also observe changes in Bcl-2-dependent pathways that would lead to mitochondrialdysfunction and cell death. Whether this change reflectshost defense or the initiation of cell lysis by the virus willrequire additional work. In either case, an alteration inthis step may provide a target for improving control ofparamyxoviral infection, even before immune cells arriveat the scene. We still know little about how these life and

death pathways may be altered in airway disease, but theindispensable role for them to govern cell growth anddifferentiation argues that they must be involved in patho-genesis. For example, preliminary evidence suggests thatthe Fas death pathway may be abnormally regulated inimmune cells in asthma (61). In section V, we presentexperimental evidence for virus-inducible alterations inthe epithelial repair process, and these alterations arealso linked to the controls for epithelial cell death versusgrowth and differentiation.

III. AIRWAY IMMUNITY AND INFLAMMATION

IN A MOUSE MODEL OF VIRAL INFECTION

Results from murine models of airway inflammationhave been reported on numerous occasions (21, 22, 36, 43,

FIG. 6. Schematic diagram for representative pathways that mediate epithelial cell death (left box) or survival (right

box) during acute viral infection. The death box features pathway initiation by TNFR superfamily members (e.g., Fas)that recruit death domain-containing adapter proteins (e.g., FADD and TRADD) and then activate caspases that mediatedegradation of structural proteins and apoptosis. Other signals trigger pro-apoptotic BCL-2 family members (e.g., BAX)that lead to mitochondrial damage and then to apopotsome formation and apoptosis or to necrosis. This pathwayappears to be the primary target for cell death induced by TNF-a plus IFN-g. The survival box features inhibition of thissame pathway by anti-apoptotic members of the BCL-2 protein family (e.g., BCL-2). Other survival pathways includeactivation of Rho family members, growth factor receptor tyrosine kinases (RTK), or TNFRII receptors, each leading toactivation of MAP kinases (MAPK) that may influence BCL-2 proteins and/or gene transcription that supports cellsurvival. In addition, inhibition of cell cycle proteins may be necessary to prevent inactivation retinoblastoma protein(pRb) and so allow for cell cycle progression to S phase. Preliminary data indicate that paramyxoviruses (PMV) mayinfluence each of these steps: 1) downregulation of BAX; 2) upregulation of BCL-2; 3) downregulation of cyclins; 4)upregulation of Rho; and 5) upregulation of TNFRII pathways to facilitate viral replication in the epithelial host cell.

30 HOLTZMAN ET AL.


83, 97, 116, 121) and reviewed by us and others (28,55–57). In the present context, we aimed to develop amouse model to better determine how the airway epithe-lial system operates in vivo and to subject the system togenetic modification. Because the epithelial system is pro-grammed for antiviral defense, we also aimed for a modelwith high fidelity to viral bronchiolitis in humans. Al-though RSV is often used for studies of human airwayepithelial cells and human subjects, based on its link tochildhood asthma, this virus does not cause a similar typeof bronchiolitis in the mouse. For that reason, we choseanother Paramyxoviridae family member, mouse parain-fluenza type I or Sendai virus based on its capacity, as anatural pathogen in rodents, to cause top-down infectionleading from the nose to the bronchi to the bronchioles tothe alveoli. By limiting the inoculum, infection is limitedto the airways and so resembles the pathology and patho-physiology of the human condition (136, 137, 139, 140).This pathogenesis is the same as observed in humansubjects with paramyxoviral infection and so offers anappropriate model for analysis. Sendai virus also infectsisolated human airway epithelial cells, presumably be-cause the allantoic fluid medium provides the Clara celltrytpase that is ordinarily provided by the rodent airway.Thus heterologous cell systems can be used to validatefidelity to RSV behavior when possible. This section sum-marizes our initial experience with this model for firstidentifying gene expression that is prominently induciblein airway epithelial cells during paramyxoviral infectionand then determining the response in same-strain micewith targeted mutagenesis of these genes.

A. Epithelial Gene Expression: Interaction

Between Virus and Host Cell

The initial step in defining epithelial cell-dependentimmunity came with viral induction of epithelial immune-response genes in vitro. The second step came with in-duction of the same genes in vivo. In particular, we havefound that paramyxoviral bronchiolitis causes inductionof epithelial ICAM-1, Stat1, and RANTES gene expressionin a pattern that is remarkably similar to the one observedin isolated airway epithelial cells. Accordingly, it appearsthat the mechanism for viral induction of epithelial im-mune-response genes in vivo may be similar to the post-transcriptional mechanism observed in vitro for RANTES.Thus, like RANTES, Stat1 gene expression is markedlyupregulated in the epithelial host cell by Sendai viralinfection in vivo (138), similar to the earlier experiencewith RSV infection of isolated human airway epithelialcells (T. Koga, D. Sampath, M. Lo, and M. J. Holtzman,unpublished observations). The sequence of the 59-regu-latory region of the mouse Stat1 gene shows a GAS con-sensus site for binding Stat1 homodimer in a location

comparable to the human gene promoter region. How-ever, this type of activation would require activation ofStat1 by IFN-g, and we have found that IFN-g-deficientmice exhibit the same virus-inducible expression of epi-thelial Stat1 in vivo. In contrast to human, there are noputative regulatory sites for binding ISGF3, NF-kB, orIRF-1 in the mouse Stat1 promoter. These findings suggestthat paramyxovirus may directly alter Stat1 gene expres-sion without a direct requirement for IFN- or NF-kB-dependent signaling. Consistent with these findings, ex-periments using isolated airway epithelial cells indicatethat RSV causes little change in transcription rate of theStat1 gene despite marked increases in gene expression(T. Koga, M. Pelletier, A. Antao, and M. J. Holtzman,unpublished observations). Additional definition of Stat1(and other epithelial) gene expression is needed, but eachof the available findings points to the possibility thatviruses trigger the network of epithelial immune-responsegenes at a posttranscriptional level using a mechanismthat is sensitive to viral replication.

Despite the uncertainty over the precise mechanismfor viral regulation of epithelial gene expression, it isevident that the site and the timing of viral replicationcorrelate closely with induction of expression. Thus bothevents are localized predominantly and coordinately toepithelial cells lining the airway. The entire profile ofepithelial gene expression in this setting is still beingdefined by oligonucleotide microarray, but initial resultscontinue to suggest close correlation between in vitro andin vivo changes in expression. As was the case in vitro,induction of epithelial gene expression in vivo also re-quires viral replication and is uninfluenced by prepara-tions of inactivated virus. Furthermore, epithelial geneexpression is followed closely by immune cell accumula-tion and activation at the site of viral infection. Each ofthese findings reinforces the proposal that the intrinsicresponse of the epithelial host cell to replicating virusmay be critical for innate immunity, and so begs thequestion to more precisely define the role of the barrierepithelial cell in this setting. The next section describesour current understanding of epithelial function for hostdefense in the short term, and subsequent sections willdefine the implications of epithelial-viral interaction for amore chronic process.

B. Toward Epithelial Gene Knockouts: Defining

an Active Role in Innate Immunity

This section summarizes the behavior of mice withtargeted null mutations in selected immune-responsegenes in the setting of paramyxoviral infection. Becausethe tools are still being developed to achieve specifictargeting, in airway epithelial cells and epithelial cell sub-sets (i.e., ciliated, Clara, goblet, or basal cells), the results



do not yet fully determine whether and which type ofepithelial cell may be critical for innate immunity to re-spiratory viruses. Nonetheless, even the current evidencefrom the mouse model strongly favors the hypothesis thatepithelial cells contain a specialized genetic program thatis critical for antiviral host defense as catalogued belowand summarized in Table 1.

1. IFN-g and IL-12

The possibility that epithelial barrier cells might con-tribute to host defense first came from experiments thatexamined the role of immune cells. Thus the Th1-depen-dent response has been traditionally designated as criticalfor defense against respiratory viruses, and essential reg-ulators of Th1 cell responses are IFN-g and IL-12 (94).Accordingly, these cytokines might be expected to bederived from immune cells and be essential for host de-fense in the setting of viral infection. However, IFN-gdeficiency caused no significant change in the response toparamyxoviral infection (139). In particular, induction ofepithelial immune-response genes proceeded at the usuallevel, indicating that viral effects on the epithelial cell didnot depend on IFN-g and instead might reflect directactions of replicating virus within the host cell (as notedabove for viral effects on epithelial cells in vitro). Inaddition, IFN-g-deficient mice exhibited no defect in re-covery from paramyxoviral infection, indicating that theepithelial program may be capable of directly helpingwith protection or for arming other aspects of the muco-sal immune response.

The response of IL-12-null mice to paramyxoviralinfection proceeded with a similar lack of immunocom-promise but was even more informative for the role of theepithelium. Thus IL-12 is a heterodimeric protein consist-ing of p35 and p40 protein subunits. Initial results withnull mutations targeted to the IL-12 p35, p40, or both IL-12subunit genes produced mice that exhibited no defect inclearance of Sendai viral infection, indicating that IL-12like IFN-g production was also not essential for antiviraldefense (137). Somewhat unexpectedly, however, micewith IL-12p35 deficiency exhibited increased airway in-

flammation (characterized by excessive macrophage ac-cumulation) and increased mortality during infection. Be-cause IL-12 is generally produced by antigen-presentingcells (i.e., macrophages, dendritic cells, B cells) (23, 84,125, 135), we next defined the site of IL-12 induction andsurprisingly found that expression was inducible by viralinfection and was predominantly localized to airway epi-thelial cells. Initial IL-12 induction was followed by exces-sive expression of IL-12 p40 (often as homodimer IL-12p80) that could be further enhanced in IL-12 p35-deficient mice. Others have provided evidence that IL-12p40 may function as an antagonist of IL-12 action (50, 67,76, 152), but in the present case, its production wasassociated with increased mortality and epithelial macro-phage accumulation. Although toxicity has been observedfor overproduction of IL-12 (37, 112), inflammation due toIL-12 p40 had not been observed. Thus the results placedepithelial cell overgeneration of IL-12 p40 as a key inter-mediate for virus-inducible inflammation and as a candi-date for epithelial immune-response genes that are abnor-mally programmed in inflammatory disease. As notedbelow, this possibility was further supported when weobserved increased expression of IL-12 p40 selectively inairway epithelial cells in subjects with asthma and con-comitant increases in airway levels of IL-12 p40 (as ho-modimer) and airway macrophages.

Taken together, these experiments with IL-12 suggesta novel role for epithelial-derived IL-12 p40 in modifyingthe level of airway inflammation during mucosal defenseand disease. The results also serve to introduce a themethat will develop for each of the epithelial immune-re-sponse genes, i.e., the usual level of epithelial gene ex-pression may aid in host defense, whereas excessive ex-pression may lead to inflammatory disease. In this case,epithelial IL-12 p80 production may help to mediate mac-rophage recruitment and/or activation, whereas higher orinappropriate levels of production may lead to macro-phage-dependent inflammation. This view favors cellularrecruitment as a feature of host defense, but from theviral perspective, recruitment may facilitate viral spread

TABLE 1. Influence of immune-response genes in paramyxoviral bronchitis/bronchiolitis and bronchopneumonia

based on behavior of inbred mice with targeted null mutations and SdV infection

Gene Target Viral Clearance Infiltrate Mortality Airway Reactivity

IFN-g* No change No change No change No changeIL-12 p40* No change No change No change No changeIL-12 p35 No change Increased (Mac) Increased No changeICAM-1* Decreased Decreased (PMN/L) Decreased DecreasedStat1* Decreased Increased (PMN) IncreasedRANTES* Decreased Increased (Mac) Increased

Mac, macrophage; PMN, polymorphonuclear leukocyte/neutrophil; L, lymphocyte; IFN-g, interferon-g; IL, interleukin; ICAM-1, intracellularadhesion molecule-1. * Genes that exhibit virus-inducible expression in airway epithelial cells during infection.

32 HOLTZMAN ET AL.


to additional cell populations, including neighboring epi-thelial cells that may also respond to chemotaxins.

2. ICAM-1

ICAM-1 is a predominant determinant of epithelial-im-mune cell adhesion, and transmigration in vitro and epithe-lial expression is prominently inducible by paramyxoviralinfection in vitro and in vivo. Thus mutagenesis of theICAM-1 gene was a natural target to influence airway inflam-mation in paramyxoviral bronchiolitis. As might be pre-dicted, ICAM-1-null mice are indeed protected from airwayinflammation (as assessed by accumulation of immune cellsin airway tissue) induced by paramyxoviral infection com-pared with same-strain controls (136). The blunted inflam-matory response in ICAM-1-null mice was accompanied byless efficient viral clearance but was still beneficial to thehost, since this cohort experienced less weight loss afterbronchiolitis and lower mortality rates after a larger inocu-lum that causes bronchopneumonia.

We have long proposed that airway inflammationmay lead to airway hyperreactivity (53, 54), but definitiveproof has been difficult to obtain through pharmacologi-cal and physiological approaches. Accordingly, we nextdetermined if ICAM-1-null mice that are relatively pro-tected from virus-induced airway inflammation are alsoprotected from postviral hyperreactivity. Indeed, usingwhole body barometric plethysmography to measure en-hanced pause (Penh) as an index of airway obstruction atbaseline and after methacholine challenge in wild-typeand ICAM-1-null mice, we found 1) slightly increasedbaseline and reactivity by 1 wk postinoculation that waspartially blocked in ICAM-1-null mice, and 2) normalbaseline but markedly increased reactivity by 3 wk afterinoculation that was completely blocked in ICAM-1-nullmice. As noted above, the predominant site of ICAM-1expression during paramyxoviral bronchiolitis is the air-way epithelial cells that are home to the virus, butwhether this cellular site is the one responsible for down-regulating inflammation and hyperreactivity will requiremore cell-specific targeting. As discussed further in sec-tion V, genetic susceptibility to virus-inducible hyperreac-tivity can also be defined in this system, since only certainstrains of inbred mice develop the acute inflammation/hyperreactivity phenotype as well as the subsequent andpersistent remodeling response.

3. RANTES

Experimental paramyxoviral bronchiolitis in micecauses marked induction of RANTES gene expression inthe lung, and expression is localized predominantly toairway epithelial cells and to adjacent tissue macrophages(N. Kajiwara, M. J. Walter, M. O’Sullivan, J. Tyner, O.Uchida, D. N. Cook, Y. Makino, T. M. Danoff, and M. J.Holtzman, unpublished observations). This pattern corre-

lates precisely with the location of viral replication and sofits closely with findings in humans with paramyxoviralinfection and in isolated cells, indicating that viral repli-cation is a potent and direct inducer of RANTES geneexpression (69). Additional experiments indicate thatmice with targeted disruption of the SCYa5/RANTES geneare immunocompromised to the point of overwhelmingviral infection and death (Kajiwara et al., unpublishedobservations). This defect appears to be manifest becauseRANTES is required to block apoptosis of virus-infectedmacrophages. The physiological source of RANTES invivo could be either the epithelial cell or the macrophage,so these experiments did not yet add to the primary roleof the airway epithelial cell in this setting. Nonetheless,this chemokine function is distinct from ones that havebeen previously identified in the setting of infection, suchas recruiting and activating immune cells, interfering withviral entry receptors, or triggering cell death receptors,and so establish a novel mechanism for host defensebased on preserving viability of the infected macrophagevia a distinct combination of antiapoptotic and antiviralactions of a chemokine. Thus defense against intracellularpathogens, particularly viruses, depends on programmeddeath of infected host cells and then clearance of thesecells by phagocytic macrophages. For effective clearanceto take place, the viability of macrophages must be main-tained in the face of infection, and these results suggest amolecular basis for preservation of virus-infected macro-phages. The precise molecular mechanism for RANTES toinfluence cell death pathways still needs to be deter-mined, but initial observations indicate that signaling tothe death pathway proceeds via specific chemokine re-ceptors that are also susceptible to regulation by viralreplication (J. Tyner, O. Uchida, and M. J. Holtzman,unpublished observations). However, the results withRANTES- and IL-12-deficient mice reinforce anothertheme of this system, i.e., both epithelial and macrophagecomponents of the innate immune response are criticaltargets for the virus and for effective antiviral defense.

4. Stat1

Stat1 mediates the expression of a subset of inter-feron-inducible genes (typified by ICAM-1) in airway epithe-lial cells, so Stat1 deficiency should have a major impact onepithelial function during viral infection. Indeed, Stat1-nullmice exhibit markedly increased weight loss and decreasedsurvival after Sendai viral infection (118). Thus intranasalinoculation with low levels of Sendai virus causes littleeffect in same-strain control mice but 100% mortality inStat1-null mice. These results are similar to reports of Stat1-null mice in other viral models, but the basis for the defectin host defense has not been determined (29). In the presentsetting, Stat1 deficiency is associated with marked increasesin viral replication rates and severe airway inflammation



with cellular infiltrate and debris in the lumen. This hostresponse may not simply reflect increased viral tissue load,since the response is not observed in wild-type mice even ata high inoculum that results in comparable viral load andlevels of mortality. The luminal infiltrate is comprised mainlyof neutrophils that contain virus and exhibit apoptosis and isfully manifest even at 8 days after infection. Because neu-trophils are ordinarily cleared by this time in wild-type mice(ordinarily the responsibility of activated macrophages), theresults suggest a possible delay in neutrophil clearance inStat1-null compared with wild-type control mice.

These findings suggested that Stat1 in epithelial cells(leading to enhanced viral replication) or macrophages(leading to decreased activation) might underlie the de-fect in antiviral defense. Additional experiments withbone marrow radiation chimeras were used to dissect therole of Stat1 in the radiation-resistant compartment (es-pecially the airway epithelium) versus radiation-sensitivehematopoietic cells (including macrophages). In thiscase, we found that lethally irradiated Stat1-deficient micereconstituted with wild-type bone marrow were still sus-ceptible to infection with Sendai virus, whereas wild-typemice that received Stat1-deficient bone marrow retainedresistance to virus (L. Shornick, D. Briner, M. Lo, and M. J.Holtzman, unpublished observations). The viral suscepti-bility of chimeras with Stat1-deficient epithelium exhib-ited the same pattern of persistent luminal inflammation.Taken together, the results suggest that epithelial Stat1may be critical for antiviral defense, perhaps by limitingviral replication and so aiding the removal of virus-in-fected, apoptotic neutrophils.

Because Stat1-deficient mice may have a selectiveand profound defect in interferon signaling (30, 88) andIFN-g-deficient mice exhibit no significant immunocom-promise with Sendai viral infection, the findings implicateStat1-dependent effects of IFN-a/b signaling as critical forantiviral defense against respiratory paramyxoviral infec-tion. However, related experiments in isolated airwayepithelial cells indicate that paramyxoviruses downregu-late IFN-a/b signaling as part of a strategy to establishinfection (M. Lo and M. J. Holtzman, unpublished obser-vations). Whether further decreases in this pathway or inother pathways lead to Stat1-deficient immunocompro-mise in vivo still needs to be determined. Defining themechanism for Stat1-dependent protection in paramyxo-viral bronchiolitis and the particular role of epithelial(versus immune cell) function of Stat1 may finally definea primary and distinct role of the epithelial barrier cell ininnate immunity.

IV. AIRWAY IMMUNITY AND INFLAMMATION

IN HUMAN SUBJECTS WITH ASTHMA

On the basis of the rationale that an abnormal im-mune response is part of inflammatory disease, it ap-

peared reasonable to next determine the behavior of ep-ithelial immune-response genes in asthma. Initial studiesindicate that airway epithelial cell expression of Stat1 andits target genes as well as IL-12 p40 and RANTES are allaltered in asthma, and this section summarizes thesefindings. Because each of these epithelial immune-re-sponse genes are highly responsive to paramyxoviral in-fection (with or without concomitant production of IFN-g), their activation in asthma brings into question thehypothesis that asthma develops due solely to decreasedTh1- and increased Th2-type T cell responses. This issueis also discussed in this section.

A. Constitutive Abnormalities in Epithelial Gene

Expression (Stat1 And IL-12)

Cytokine effects on immunity and inflammation oftendepend on STAT signaling pathways, so these are idealcandidates for influencing inflammatory disease. We rea-soned that selective interferon responsiveness of the firstSTAT family member (Stat1) and Stat1-dependent im-mune-response genes such as ICAM-1, IRF-1, and Stat1itself in airway epithelial cells provided a basis for detect-ing cytokine signaling abnormalities in inflammatoryairway disease. Based on nuclear localization and phos-phorylation, epithelial Stat1 (but not other control tran-scription factors such as Stat3, AP-1, and NF-kB) wasinvariably activated in asthmatic compared with normalcontrol or chronic bronchitis subjects (114). Stat1 activa-tion was relatively selective for epithelial cells, since ac-tivation was not detectable in neighboring macrophages.Furthermore, epithelial levels of activated Stat1 corre-lated with levels of expression for epithelial ICAM-1,IRF-1, and Stat1, and in turn, ICAM-1 level correlated withT cell accumulation in tissue. However, only low levels ofIFN-g or IFN-g-producing cells were detected in airwaytissue in all subjects. Since tissue levels of IFN-g are notincreased, asthma appears to be characterized by consti-tutive activity of Th1-like cytokine signaling even withoutevidence of stimuli (such as IFN-g or viral infection) (69,138) that normally drive this type of immune reaction. Theresults therefore provide initial evidence linking abnor-mal behavior of STAT pathways for cytokine signaling (asopposed to cytokine production) to the development ofan inflammatory disease. In that context, the results alsochange the current scheme for asthma pathogenesis toone that must include a localized gain in transcriptionalsignal ordinarily used for a Th1 cytokine (IFN-g) and so isdistinct from mechanisms that depend on allergy-drivenoverproduction of Th2 cytokines.

Localizing the abnormality in Stat1 activity to airwayepithelial cells is critical to the impact of these findings.Constitutive STAT activities in lymphocytes are associ-ated with malignant transformation, whereas excessive

34 HOLTZMAN ET AL.


Stat1 activity in chondrocytes driven by a mutant fibro-blast growth factor receptor may be associated withgrowth arrest (128). Thus depending on the programmedcytokine response of specific cell types in specific tissues,abnormal Jak-STAT activity may lead to diseases as di-verse as lymphoma or dwarfism. Consequently, analysisof cytokine-dependent responses in asthma using sitesdistant from the airways may not reflect behavior in air-way cells. For example, others have indicated that de-creased Th1 responses may predispose to asthma basedon an association with diminished delayed-type hypersen-sitivity reactions to tuberculin (117). However, this asso-ciation was based on immune responses in the skin and sodoes not reflect the specific activity of Th1-driven signal-ing pathways in airway epithelial cells.

To next determine whether the pattern of IL-12 up-regulation in murine viral bronchitis also develops inasthma, we measured levels of IL-12 expression in airwaytissue and bronchoalveolar lavage fluid obtained fromasthmatic subjects. In fact, subjects with asthma exhib-ited higher levels of IL-12 as well, with expression pre-dominantly in airway epithelial cells and in the form ofIL-12 p40 (and p80 homodimer). Upregulation of IL-12 p40expression also correlated with increased number of mac-rophages in the airway, so each of the features of IL-12behavior was similar to ones found in the mouse duringparamyxoviral infection. This finding offers two new pos-sibilities for a role of IL-12 p40 in asthma: 1) antagonismof endogenous IL-12, and so skewing the local cytokineenvironment toward a Th2 immune response, and/or 2)function as an agonist, e.g., as a macrophage chemotacticand activating factor, and so causing airway inflamma-tion. In fact, some (but not all) previous studies findsignificant macrophage accumulation in the submucosaland intraepithelial airway tissue of asthmatic comparedwith normal subjects (104, 109), whereas others provideevidence of macrophage activation in asthma (18) as wellas an increase in number and activation state of airwaymacrophages during allergen challenge in asthma (89). Ineither case, these results provide initial evidence thatasthma, often characterized as a condition that dependson overexpression of Th2 and underexpression of Th1cytokines by immune cells, does in fact also exhibit over-expression of IL-12 p40 that appears to be chiefly derivedfrom airway epithelial cells. In conjunction with previousobservations of constitutive activation of Stat1 and Stat1-dependent genes, the findings further support the possi-bility that pathways normally responsive to Th1 cytokinesare also dysregulated in airway inflammatory disease. Asdiscussed further below, upregulation of epithelial Stat1and IL-12 pathways in asthma is found in both allergic andnonallergic subjects and with or without treatment withglucocorticoids.

B. Glucocorticoid-Sensitive Abnormalities

in Epithelial Gene Expression (RANTES)

Recognizing that RANTES and Stat1-dependentgenes exhibit distinct but complementary function (che-motaxis versus cell adhesion) and regulation (posttran-scriptional versus transcriptional), we also determinedthe level of epithelial RANTES expression in asthma.Most previous reports indicated that RANTES is ex-pressed at similar basal levels in the airway epithelium ofnormal versus asthmatic subjects with mild disease (8, 33,60, 144). Similarly, we did not expect much change inepithelial RANTES levels in response to allergen, basedon our experience with segmental allergen challenge (M.Castro and M. J. Holtzman, unpublished observations).Accordingly, we sought an experimental protocol for en-dogenous exacerbation of asthma that might better reflectnatural flares of the disease and might better define themechanism of action of anti-inflammatory treatment.

In that context, we developed a protocol for con-trolled glucocorticoid withdrawal in stable asthmatic sub-jects that includes measurements of airway function andendobronchial biopsy and lavage during treatment andthen after withdrawal of inhaled glucocorticoids (15). Insome subjects, glucocorticoid withdrawal results inasthma exacerbation characterized by increases in airwayhyperreactivity and immune cell (predominantly T cell)infiltration. Interestingly, these disease flares are consis-tently associated with increased expression of RANTES(by in situ hybridization for RANTES mRNA) that appearslocalized predominantly to airway epithelial cells (129; M.Castro, S. Block, D. L. Hamilos, M. V. Jenkersen, Q. Li, T.Horiuchi, K. Schechtman, and M. J. Holtzman, unpub-lished observations). Concomitant work has indicatedthat there is no increase in IFN-g production or NF-kBactivation in this setting, similar to the findings in stableasthma. Thus epithelial RANTES production is inducibleduring asthma exacerbation even in the apparent absenceof viral infection or IFN-g production. Furthermore, in-duction is sensitive to glucocorticoid treatment but ap-pears to proceed via an NF-kB-independent pathway.These findings are analogous to those in paramyxoviralinfection, so it is possible that posttranscriptional eventsunderlying RANTES expression during paramyxoviral in-fection may also be relevant to expression in asthma. Asdiscussed in section IVC, we have proposed that the in-creased expression of each of these pathways, i.e., Stat1,IL-12 p40, and RANTES, may reflect a response to virus thatno longer causes signs of infection, but in the case ofRANTES, expression is unmasked by glucocorticoid with-drawal. Defining the basis for induction of RANTES geneexpression and its functional implications will requireadditional work on the molecular basis for RANTES ex-pression in isolated cell and animal model systems as wellas correlative protocols in human subjects. For example,



glucocorticoid effects on posttranscriptional regulation ofthe RANTES genes still need to be determined. Nonethe-less, the findings for Stat1, IL-12, and RANTES alreadysuggest that a propensity for epithelial gene expressionmay be conditioned by viral exposure and so underlie therelationship of childhood viral infection to ongoingasthma even later in life.

C. A Revised Model for Th1/Th2 Contributions

to Asthma

Asthmatic inflammation has been attributed to anabnormal sensitivity to inhaled allergens, and by exten-sion, to a skewed T helper cell response with increasedTh2 and decreased Th1 components compared with nor-mal. In some settings, Th1 cells (perhaps activated byviral infection) may assist Th2 cells in initiating an allergicresponse, but this scheme also relies on Th2-dependentproduction of cytokines, notably IL-4, IL-5, and IL-13 todrive asthmatic inflammation and consequent pathophys-iology (as summarized in Fig. 1). However, this pathogen-esis does not account for the discrepancy between aller-gic and asthmatic phenotypes and, as indicated in theprevious section, does not provide for the heightened

antiviral state of the airway epithelium in asthma. Inparticular, increased activity of innate antiviral program-ming in the epithelium appears to be a fundamental fea-ture of asthma. This alternative pathway is normally use-ful for airway barrier cells to defend against respiratoryviruses, but if overactive, could lead to inflammation, asappears to be the case in asthma. Some epithelial pro-grams appear insensitive to glucocorticoids and so arepresent constitutively even in stable disease (i.e., Stat1,IL-12 p40 networks), whereas others are sensitive to glu-cocorticoids and so inducible by glucocorticoid with-drawal and flares of the disease (i.e., RANTES). As per-haps expected for an antiviral system, this epithelialnetwork is generally oriented toward a Th1 response.

To better integrate these concepts and so better ex-plain the pathogenesis of airway disease, we have revisedthe model for the role of the airway immune response inasthma. The revised model accounts for how an alterna-tive Th1-oriented epithelial network may act in combina-tion with an enhanced Th2 response, and the combinationof epithelial, viral, and allergic components led to itsdesignation as an Epi-Vir-All paradigm (Fig. 7). In thisscheme, increases in epithelial antiviral signals (e.g., Stat1activation and IL-12 p40 expression) and allergen-driven

FIG. 7. Revised model for the role of the airway immune response in the development of airway inflammation andremodeling. A: illustration of how increases in epithelial antiviral signals (e.g., Stat1 activation and IL-12 p40 expression)and allergen-driven production of Th2 cytokines (e.g., IL-4, IL-5, and IL-13) are characteristic of subjects with asthma(designated as an “A”) studied under stable conditions during treatment with inhaled glucocorticoids. B: illustration ofhow further increases in epithelial signaling (driven by viral infection) or Th2 cytokine production (driven by furtherallergen exposure) may develop in subjects with asthma studied during a flare without glucocorticoid treatment. Theabnormality in epithelial cell behavior may depend on persistence of virus at low copy number and proper geneticbackground. The combination of epithelial, viral, and allergic components led to designation of this pathogenesis modelas an Epi-Vir-All paradigm.

36 HOLTZMAN ET AL.


production of Th2 cytokines (e.g., IL-4, IL-5, and IL-13) arecharacteristic of subjects with asthma under stable con-ditions during treatment with inhaled glucocorticoids.Further increases in epithelial signaling (driven by viralinfection) or Th2 cytokine production (driven by furtherallergen exposure) would develop in subjects withasthma during a flare of the disease. In addition, increasedlevels of Stat1 may mediate a hypersensitive Th1-typeresponse in the airway. At least in vitro, high levels ofStat1 are capable of priming the Stat1-dependent pathwayand so lead to exaggerated gene expression in response tonormal levels of stimulation (D. Sampath, Y. Zhang, andM. J. Holtzman, unpublished observations). In addition,flares of the disease are closely associated with inductionof RANTES gene expression. This system, which appearsto be regulated at the posttranscriptional level during viralinfection, may rely on a similar regulatory mechanism inasthmatic flares. Each of these possibilities begs the ques-tion as to how the abnormality in epithelial cell behaviororiginally develops. In section V, we summarize initialevidence that viral infection and the genetic backgroundof the host may interact to permanently reprogram thebehavior of airway epithelial cells.

V. LONG-TERM AIRWAY HYPERREACTIVITY

AND REMODELING IN MICE AND HUMANS

A relationship between viral infection and the devel-opment of chronic inflammatory disease has been pro-posed for diverse clinical syndromes, but the basis for thisrelationship is still uncertain. In the particular context ofasthma, paramyxoviral infections are the leading cause oflower respiratory tract illness in infants and young chil-dren (19, 26), and children with clinically significant viralbronchiolitis appear to be marked for the subsequentdevelopment of a chronic wheezing illness that is inde-pendent of allergy (107, 119, 124). Presumably paramyxo-viral infection triggers a switch to an abnormal host re-sponse, since paramyxoviruses (or other respiratory RNAviruses) are not thought to persist in airway tissue as acause of chronic respiratory disease (1). With or withoutviral persistence, however, the role of specific host fac-tors in the development of chronic wheezing or life-longasthma has not been determined. This section describesour initial results from studies of mice and human sub-jects in defining the linkage between respiratory viralinfection and persistent asthma and in so doing extendsthe concepts of the previous sections that were focusedpredominantly on short-term immunity and inflammation.

A. Segregating Acute From Chronic Phenotypes

To better define viral and host factors in the devel-opment of the asthma phenotype, we took further advan-

tage of the mouse model of paramyxoviral bronchiolitis.As noted above, inhibition of the acute inflammatory re-sponse could be achieved by disruption of epithelial im-mune-response genes. On the basis of the work in vitro,this epithelial gene network is directly inducible by viralreplication and is dominated by an array of interferon-responsive genes, but among candidates that might medi-ate immune cell traffic, ICAM-1 appears to be a predom-inant determinant for immune cell transmigration (69, 82,129). In fact, as noted above, we found that ICAM-1 isinducible primarily on host airway epithelial cells by viralinfection and is necessary for full development of acuteinflammation and concomitant postviral airway hyperre-activity that peaks at ;3 wk after infection in the mousemodel. Unexpectedly, however, as we followed wild-typeand ICAM-1-null mice in a homogeneous genetic back-ground, we also found that primary viral infection causedessentially permanent airway hyperreactivity and con-comitant epithelial remodeling that were manifest despiteICAM-1 deficiency and were not accompanied by low-level persistence of viral transcripts (136, 140; M. J.Walter, J. D. Morton, N. Kajiwara, E. Agapov, T. Horiuchi,M. Castro, and M. J. Holtzman, unpublished observa-tions). The predominant features of this remodeling phe-notype, i.e., goblet cell hyperplasia and mucin production,are also inducible in human subjects with asthma. Takentogether, the results indicate that paramyxoviral infectionmay cause both acute airway inflammation/hyperreactiv-ity and chronic airway remodeling/hyperreactivity pheno-types that can be segregated by their dependence onICAM-1 and time and so may depend on distinct geneticcontrols (as modeled in Fig. 8). In a more general context,the findings establish the capacity of a single paramyxo-viral infection to cause both acute and chronic manifes-tations of the phenotype for hypersecretory disease andthe relevance of specific host defense genes in moderatingthe acute but not necessarily the chronic phenotype.

B. Genetic and Viral Determinants for Persistence

The striking possibility that viral infection at an earlyage may lead to permanent alterations in airway epithelialbehavior and airway function requires significant furtherinvestigation. Initial results indicate that both viral andhost factors may determine the outcome of infection.Thus only specific strains of mice are susceptible to de-veloping the chronic phenotype, analogous but distinctfrom the diversity in resistance to acute infection (10;J. D. Morton and M. J. Holtzman, unpublished observa-tions). Mouse age also affects the severity of the pheno-type, indicating that developmental factors also influencethe outcome. Improvements in the technology for geneticmapping (using single-nucleotide polymorphisms as infor-mative markers) and gene expression (using oligonucle-



otide microarray) will allow more precise definition ofepithelial programming that is responsible for asthma andbronchitis phenotypes in the mouse model (39). Exten-sion of these results to analysis of candidate genes inhuman subjects using precise case-control studies maythen provide further insight into the genetic susceptibilityfor virus-inducible disease in humans.

Although genetic susceptibility for the host responsehas been the target of studies by our lab and others, itappears likely that viral genetics are also critical to thedevelopment of the chronic remodeling/hyperreactivityphenotype. Thus previous studies of Sendai virus andrelated paramyxoviruses indicated that viral particles arenormally eliminated from tissue after 10–12 days (25).However, these studies relied on relatively insensitivemethods that are responsive to replicating (nonmutant)forms of virus, and recent studies of other ribovirusesindicate that they may persist in the tissue as mutantquasi-species. In these cases, viral persistence may befacilitated by high mutation rates at critical epitopes andconsequent escape from immune surveillance (27), butwhether persistence is necessary or sufficient for chronicalterations in host cell behavior still needs to be deter-mined. Initial analysis of Sendai viral titers using kineticPCR suggests that viral persistence in host tissues at lowcopy number is not a common feature of paramyxoviralinfection (J. D. Morton, E. Agapov, D. Palamand, and M. J.

Holtzman, unpublished observations). Moreover, initialresults with the Sendai viral mouse model indicate thatviral persistence is not necessary or sufficient for thechronic phenotype, since sensitive and resistant strains ofmice exhibit similar effectiveness in viral clearance. Theresults suggest a hit-and-run hypothesis for the viral ef-fect, i.e., transient infection causes permanent alterationin host cell behavior. Even so, however, the viral geneproducts responsible for triggering the change in hostphenotype still need to be defined. New systems for re-covery of negative-strand RNA viruses entirely fromcDNA may allow better definition of these issues as wellas general understanding of replication and pathogenesisfor this order of viruses (20, 95).

VI. SMART STRATEGIES FOR CORRECTING

EPITHELIAL INFLAMMATION AND

REMODELING

The airway epithelium (by virtue of location) is di-rectly accessible to inhaled agents, so modifying epithelialfunction is a natural target for therapy of asthma and forcombating respiratory viral infection. In the case ofasthma, even current agents (such as glucocorticoids andsodium cromoglycate) may owe part of their efficacy tothe attenuation of cell adhesion molecule and chemoat-

FIG. 8. Segregation of virus-inducible phenotypes. Model for genetic- and time-dependent events in the developmentof the asthma phenotype induced by respiratory paramyxoviral infection. On the basis of studies of genetically definedmice, it appears that viral replication peaks on postinfection days 3–5, epithelial immune-response gene expressionpeaks on day 5, immune cell infiltration peaks on day 8, acute airway hyperreactivity (that depends on ICAM-1 geneexpression) peaks on approximately day 21, and chronic remodeling (e.g., goblet cell hyperplasia) and chronichyperreactivity are present thereafter.

38 HOLTZMAN ET AL.


tractant function (as noted above for RANTES) and socould target the epithelium to achieve therapeutic effects.Similarly, delivery of blocking antibodies, soluble ligands,or synthetic peptide analogs via the airway may indicatethe importance of local epithelial events if the reagentscannot effectively reach subepithelial sites (34, 49, 75, 86,101). Using the epithelial antiviral network as a model, wehave begun to develop more specific and perhaps smarterstrategies for modifying epithelial gene expression and soinfluencing epithelial function. The approach derives di-rectly from the molecular pathways defined in this reviewand is summarized in Figure 9. The overall aim is toimprove airway disease, with special reference to asthmaand related phenotypes in other forms of bronchitis/bron-chiolitis. In view of the proposed nature of epithelialprogramming against respiratory viruses, another naturaltherapeutic target is to improve antiviral host defense.

A. Reversing Viral Mimicry Using Mutant

E1A Oncoprotein

Immunity to viral pathogens depends on coordinatedcontrol of host cell genes, so viruses (including adenovi-ruses) have developed strategies to redirect the normalgenetic program (108). As one of the most informativeexamples, the adenoviral early region 1A (E1A) gene en-

codes for an oncoprotein that activates host cell cycle andso enables DNA synthesis needed for viral replication. Toaccomplish this, E1A oncoprotein presents two distinctbinding sites for competing with cell cycle regulators: onesite uses conserved regions 1 and 2 (CR1 and CR2) to binda family of antioncoproteins typified by retinoblastoma(Rb) protein; the other site uses NH2-terminal residuesand CR1 to bind a family of transcriptional coactivatorstypified by p300 (92, 148) (Fig. 10). The Rb protein andRb-related p107 and p130 proteins normally bind to theE2F transcription factor and silence genes needed forprogression through the cell cycle (147). The p300 protein(17) and the related cAMP response element binding pro-tein (CREB)-binding protein (CBP) (31) serve as adaptorproteins that link transcription factors such as CREB (3,72) as well as p53 antioncoprotein (48, 74) to the basaltranscription complex. In addition, p300/CBP proteins in-fluence gene transcription by intrinsic histone acetyl-transferase activity and by linking additional coactivators(such as p300/CBP/cointegrator-associated protein orp/CIP) to core histones (134). Thus, by sequestering (andperhaps linking) Rb- and p300/CBP-type proteins in com-petition with endogenous transcription factors and coac-tivators, three E1A domains (NH2 terminus, CR1, andCR2) are sufficient for mediating cellular proliferation(143).

FIG. 9. Strategies for modifying epithelial gene expression to preserve immunity but inhibit inflammation andremodeling. Anti-asthma strategies (top box) are aimed at downregulating the Stat1 pathway using mutant viral proteins(e.g., adenoviral E1A) or mutant Stat1 that each interrupts Stat1-dependent gene transcription. Anti-viral strategies(bottom box) include expression of mutant RBF or native RANTES to increase RANTES levels and so enhance innateantiviral defense.



In addition to altering control of the cell cycle, ad-enoviruses also aim to subvert host defense by evadingimmune detection. Early studies in cell lines suggestedthat E1A might inhibit activation of immune-responsegenes by disrupting events (such as protein phosphoryla-tion) that lead to protein-DNA interactions at interferon-responsive enhancers (64). However, subsequent studiesdemonstrated that E1A capacity for immune suppressionmight also depend on targeting p300/CBP. Thus p300/CBPcoactivator function was extended to c-fos and c-Juncomponents of activator protein-1 (3, 5), the p65 compo-nent of NF-kB (40), steroid and nuclear-hormone recep-tors (70, 150), and to the first two members of the STATfamily (9, 58, 153) that mediate interferon-driven geneactivation. In all cases, including Stat1 and Stat2, E1Ainfluenced host gene expression by competing with theseendogenous factors for binding to p300/CBP (5, 9, 40, 70,150, 153). Because Stat1 is critical for IFN-g-driven genetranscription, competition for p300/CBP by E1A may un-derlie its capacity to subvert IFN-g-stimulated immunity.

In reviewing this previous work, we questionedwhether E1A might also act directly on a specific DNA-binding transcription factor and thereby provide determi-nants for more precisely influencing gene expression. Prec-edent for this possibility may be found in interactions ofthe transactivating domain of E1A (conserved region 3)with transcription factors needed for viral gene expres-sion (77), but at least to date, E1A NH2-terminal interac-tions appear more highly restricted (92). Nonetheless,additional interactions might not be detected if E1A de-terminants for binding p300/CBP overlapped with thosefor binding more specific cellular activators. In addition,previous studies of adenoviral infection and host genebehavior often used transformed cell lines rather than thenatural adenoviral host cell and so could not excludeconfounding effects of other oncogenes (such as SV40large T antigen) on E1A action (32).

In that context, we have used primary culture hTBEcells (along with endobronchial tissue and mouse modelsof viral bronchitis) to define epithelial cell-dependent im-

FIG. 10. Reverse viral mimicry using adenoviral E1A oncoprotein to downregulate Stat1 function. A: diagram for E1A(13S and 12S splice variants) that includes the NH2-terminal (N-term), conserved region 1 (CR1), conserved region 2(CR2), conserved region 3 (CR3), and COOH-terminal (C-term) domains. B: proposed structure and the critical sites(including Arg2) for binding and sequestering p300/CBP and pRb. C: illustration of how E1A wild-type (E1A-WT) and E1Awith Arg to Gly mutation (E1A-RG2) may act to disrupt p300/CBP and Stat1 interaction and so inhibit transcription ofthe ICAM-1 gene. DNA/protein and protein/protein interactions that control transcription rate include the following: 1)Stat1/DNA interaction mediated by the GAS site at 2106 to 106; 2) Sp1/DNA interaction mediated by the GC box at 299to 294; 3) Stat1/Sp1 interaction; 4) Sp1 interaction with the Pol II-specific basal transcription complex that binds at theTATA box and initiates transcription at the transcription initiation site (TIS); 5) Stat1 interaction with p300; and 6) p300interaction with the basal transcription complex via p/CIP. [Modified from Holtzman et al. (53) and Moran (92).]

40 HOLTZMAN ET AL.


munity to respiratory pathogens (129, 138). Our work onisolated cells indicates that a subset of epithelial immune-response genes typified by ICAM-1 are controlled by anIFN-g-responsive Jak-STAT signaling pathway that relieson Stat1 to first be phosphorylated at the IFN-g receptorcomplex and then to bind to the ICAM-1 promoter region(80, 81, 141). At the promoter, Stat1 interacts with adja-cent transcription factors such as Sp1 and with p300/CBP(58, 81, 153) to facilitate enhanceosome formation andinduce gene transcription. As noted above, E1A-p300/CBPinteraction may therefore underlie its capacity to subvertIFN-g-driven gene activation, or alternatively, may ex-plain the antiviral effect of IFN-g (153).

Recently, however, we demonstrated that E1A canalso modify Stat1-dependent gene activation by directaction on Stat1 itself (82). Using mutant forms of E1A, wedefined an NH2-terminal determinant (Arg2) for bindingp300/CBP that is not needed for binding Stat1. Further-more, we showed that mutant E1A with no capacity forbinding p300/CBP or inhibiting p300/CBP-dependentevents (73, 143, 148) still binds Stat1 and inhibits IFN-g-induced, Stat1-dependent transactivation of exogenousand endogenous target genes early in the course of ad-enoviral infection. This effect is distinct from downregu-lation of Stat1 phosphorylation that occurs later in thecourse of infection. Taken together, these results providefor an overall scheme for adenoviral disruption of hostgene expression during early and late-phase infection, anovel action of the E1A NH2 terminus in mediating im-mune suppression, and a revised model of Stat1-depen-dent gene expression. The results also provide an appro-priate E1A-mediated strategy for more selectivelydownregulating Stat1-dependent events in the airway.

B. Modifying Epithelial Signaling

With Mutant Stat1

Transcription factors generally contain at least twoindependent domains for DNA binding versus activationof transcription (44). Removal of the transactivation do-main has been shown in many cases to result in aninactive factor that can bind to the DNA element anddisplace wild-type protein, thereby creating a dominant-negative action (51). Targeting such a dominant-negativeconstruct so that it is expressed in a specific tissue hasbeen useful in understanding the function of specific tran-scription factors in different tissues. Accordingly, thisstrategy offers an advantage over complete deletion of thefactor by homologous recombination with the endoge-nous gene if there is a goal of defining function in aspecific tissue or cell type. The creation of a dominant-negative mutation for Stat1 is more challenging than fortranscription factors with less complex function, but alsooffers an opportunity for dissecting the relative impor-tance of Stat1 modular function.

Accordingly, we aimed to selectively downregulatethe pathway using a dominant-negative strategy for inhi-bition of epithelial Stat1 in the primary culture airwayepithelial cell model (141). In initial experiments using aStat1-deficient cell line, we demonstrated that transfec-tion of wild-type Stat1 expression plasmid restored ap-propriate Stat1 expression and IFN-g-dependent phos-phorylation as well as consequent IFN-g activation ofcotransfected ICAM-1 promoter constructs and endoge-nous ICAM-1 gene expression. However, mutations ofStat1 at Tyr-701 (the Jak kinase phosphorylation site),Glu-428/429 (the putative DNA-binding site), His-713 (thesplice-site resulting in Stat1b formation), or Ser-727 (themitogen-activated protein kinase phosphorylation site) alldecreased Stat1 capacity to activate the ICAM-1 pro-moter. The Tyr-701 mutant (followed by the His-713 mu-tant) was most effective in disabling Stat1 function and inovercoming the activating effect of cotransfected wild-type Stat1 in this cell system, thereby highlighting theeffectiveness of blocking Stat1 homo- and heterodimer-ization. In experiments using primary culture airway epi-thelial cells and each of the four Stat1-mutant plasmids,transfection with the Tyr-701 and His-713 mutants againmost effectively inhibited IFN-g activation of an ICAM-1gene promoter construct. By then transfecting airwayepithelial cells with wild-type or mutant Stat1 tagged witha Flag-reporter sequence, we used dual immunofluores-cence to show that cells expressing the Tyr-701 or His-713mutants but not the two other Stat1 mutants or wild-typeStat1 were prevented from expressing endogenousICAM-1 in response to IFN-g treatment. The results pro-vide the initial indication that loss of function may corre-late with dominant-negative activity for Stat1 in a biolog-ically relevant human cell model. The capacity of specificStat1 mutations to exert a potent dominant-negative ef-fect on IFN-g signal transduction provides for furtherdefinition of Stat1 structure/function. The strategy alsoprovides a means for natural or engineered expression ofmutant or truncated Stat1 to selectively downregulateactivity of this pathway in a cell type- or tissue-specificmanner during immune and/or inflammatory responses invivo.

C. Future Considerations

The studies summarized above indicate that molecu-lar strategies can be devised that selectively target epithe-lial signaling pathways. Thus downregulation of epithelialStat1 signaling (to correct the abnormality in asthma) andpossibly upregulation of RANTES signaling (to aid inantiviral defense) may be devised from the regulatoryfeatures of these pathways, and these strategies appeareffective in vitro. Studies in animal models are poised todetermine whether modifying epithelial immune function



may be effective as a sole treatment for airway inflamma-tion or may render other anti-inflammatory drugs moreeffective in vivo. However, the efficacy of this approachwill depend on developing methods to achieve selectiveand efficient expression in the airway epithelium. Thusproof of concept may be feasible using transgenic animals(limited only by specificity of available gene promotersystems for epithelial expression), but vectors with repro-ducible, sustainable, and high-level expression in the air-way epithelium still need to be developed.

VII. SUMMARY

This review summarizes increasing evidence that air-way epithelial barrier cells actively mediate airway immu-nity and inflammation. In fact, the role of the epitheliumhas evolved from a relatively simple scheme for mediatingleukocyte recruitment to one that depends on the coor-dinated expression of a network of epithelial immune-response genes coordinated for host defense under dis-tinct transcriptional and posttranscriptional controls. Thetranscriptional program is typified by an interferon-drivenJak/Stat signaling pathway, while posttranscriptional reg-ulation uses RNA-protein interactions that are responsivedirectly to viral replication. Respiratory viruses ordinarilyinteract with this Th1-style gene network in a battle ofhost defense versus immune subversion, but the samenetwork is also activated in asthma even in the absence ofovert viral infection. Thus the barrier epithelial cell pop-ulation appears specially programmed for normal hostdefense but abnormally programmed in inflammatory air-way disease. Recent results indicate that airway epithelialcells may be reprogrammed for permanent proliferationand skewed mucus cell differentiation by asthmagenicRNA viruses that persist at low copy number. This alter-ation in the epithelial repair process exhibits genetic sus-ceptibility and so appears analogous to abnormal mucosalphenotypes in asthma and other hypersecretory diseases.These concepts can be integrated into a scheme thatincorporates epithelial, viral, and allergic components(designated an Epi-Vir-All paradigm) for a more completeexplanation of the pathogenesis of airway disease. Byextension, the same epithelial network is a target fortherapy in airway disease and for improving host defenseagainst respiratory viruses. Smart strategies have alreadybeen defined for reversing viral mimicry and engineeringdominant-negative mutations that alter epithelial behav-ior, but full utilization of these approaches will depend onalso achieving selective and high-level gene expression inthe epithelium. This goal depends in turn on determiningthe critical biology of the airway epithelium in antiviraldefense and airway disease and in particular on definingthe contributions of specific viral and host genetic com-ponents.

We acknowledge the contributions of our colleagues Eu-gene Agapov, Yael Alevy, Jan Bragdon, Steve Brody, DavidBriner, Naohiro Kajiwara, Edy Kim, Takeharu Koga, DwightLook, Shin Nakajima, Divya Palamand, Mark Pelletier, WilliamRoswit, Deepak Sampath, Massaguchi Taguchi, KazutakaTakami, and Yong Zhang.

The research underlying this review was supported bygrants from the National Heart, Lung, and Blood Institute; Mar-tin Schaefer Fund; and Alan A. and Edith L. Wolff CharitableTrust.

Address for reprint requests and other correspondence:M. J. Holtzman, Washington University School of Medicine,Campus Box 8052, 660 South Euclid Ave., St. Louis, MO 63110(E-mail: [email protected]).

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