The innate immune response of the respiratory epithelium

27

Immunological Reviews 2000Vol. 173: 27–38Printed in Denmark. All rights reserved

Copyright © Munksgaard 2000

Immunological ReviewsISSN 0105-2896

Gill DiamondDiana LegardaLisa K. Ryan

The innate immune response of the respiratory epithelium

Authors’ addresses

Gill Diamond1, Diana Legarda1,2, Lisa K. Ryan3,1Department of Anatomy, Cell Biology and Injury Sciences, UMDNJ-New Jersey Medical School. 2Graduate School of Biomedical Sciences, Newark, New Jersey, USA.3Immunotoxicology Branch, U. S. EPA, NHEERL, MD-92, Research Triangle Park, North California, USA.

Correspondence to:

Gill DiamondDepartment of Anatomy, Cell Biology and Injury SciencesUMDNJ-New Jersey Medical School185 South Orange Ave.Newark NJ 07103USAFax: 1 973 972 7489e-mail: [email protected]

Acknowledgements

This research was supported by grants to GD from the NIH (HL53400), the USDA (9504034) and the Cystic Fibrosis Foundation (DIAMON97Z0). The authors wish to thank Drs Charles Bevins and Gary Hatch for critical reading of the manuscript, and Dr Scott Randell for providing the photomicrograph of the airway epithelium.Disclaimer: This chapter has been reviewed by the National Health and Environmental Effects Research Laboratory, U. S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

Summary: The respiratory epithelium maintains an effective antimicrobialenvironment to prevent colonization by microorganisms in inspired air. Inaddition to constitutively present host defenses which include antimicro-bial peptides and proteins, the epithelial cells respond to the presence ofmicrobes by the induction two complementary parts of an innate immuneresponse. The first response is the increased production of antimicrobialagents, and the second is the induction of a signal network to recruitphagocytic cells to contain the infection. Inflammatory mediators releasedby the recruited cells as well as from the epithelium itself further inducethe expression of the antimicrobial agents. The result is an effective pre-vention of microbial colonization. The epithelial cells recognize the patho-gen-associated patterns on microbes by surface receptors such as CD14and Toll-like receptors. Subsequent signal transduction pathways havebeen identified which result in the increased transcription of host defenseresponse genes. Diseases such as cystic fibrosis, or environmental expo-sures such as the inhalation of air pollution particles, may create an envi-ronment that impairs the expression or activity of the host defenses in theairway. This can lead to increased susceptibility to airway infections.

Introduction

The airway epithelium represents a primary site for the intro-

duction and deposition of potentially pathogenic microorgan-

isms into the body, mainly through inspired air. Yet only in

highly unusual cases does colonization of this tissue actually

take place. It has become evident that the ciliated epithelium

lining the airway prevents colonization by inhaled bacteria in

three general ways: 1) the physical removal by ciliary clearance

and cough; 2) the presence of broad-spectrum antimicrobial

agents in the mucus; and 3) the recruitment of phagocytic cells

and an inflammatory response. Moreover, the epithelial cells

that line the airway play a responsive role in the innate immune

defense against microorganisms by mechanisms that are pre-

dicted to mimic those found inside phagocytic cells. In this

review we will concentrate on the response of the airway epi-

thelia to potential pathogens and its role in preventing infec-

tions. We will also examine situations in which the response is

inhibited, leading to increased susceptibility to infection.

28 Immunological Reviews 173/2000

Diamond et al · Innate immunity in the airway

The airway is defined anatomically as the upper respiratory

tract, which includes the nasal sinuses and the nasopharynx,

and the lower respiratory tract, which begins at the larynx and

continues to the trachea before dividing into the smaller air-

ways until they reach the alveoli. The luminal surface of the air-

way is lined by a layer of epithelial cells. In the conducting air-

way these cells are pseudostratified columnar epithelial cells,

which become simple cuboidal epithelium as the branches

extend to the alveoli. While both alveolar epithelium and tra-

cheobronchial epithelium exhibit numerous host defense

mechanisms, we will concentrate here on the innate immune

response of the epithelium in the conducting airways.

The initial line of defense for the airway is the barrier func-

tion of the epithelium, which effectively separates the luminal

surface from the basolateral surface. The columnar epithelial

cells are also ciliated, and the co-ordinated beating of the cilia

provides clearance of much of the inhaled material. Constitu-

tively expressed chemical defenses provide the airway with a

constantly present antimicrobial milieu. Increasing evidence

has appeared that the cells lining the airway are responsive to

the presence of pathogens to provide an inducible response

(Table 1). Disease states and environmental exposures can

inhibit these defenses and responses, thus allowing unimpeded

growth of pathogenic microorganisms.

Constitutive chemical defenses

An examination of the antimicrobial agents found in the mucus

layer covering the airway epithelia has identified a wide range

of peptides, proteins, and organic molecules. These are gener-

ally derived from secretory cells found in the epithelia, includ-

ing mucous and serous cells in submucosal glands, as well as

goblet and Clara cells in the epithelial layer (Fig. 1). Pseudostrat-

ified columnar ciliated epithelial cells have also been implicated

in the secretion of antimicrobial factors into this airway surface

fluid (ASF), which consists of an underlying periciliary serous

layer and an outer viscous gel mucus layer (1). Beginning with

the discovery by Fleming that human nasal secretions contain

antimicrobial activity, lysozyme has been considered among

the most important of the host defense molecules. Indeed, it is

found at concentrations that vary from 1 to 10 µg/ml in airway

lavage fluid (2) to 500 µg/ml in nasal secretions (3), and up to

1 mg/ml in sputum (4). At similar concentrations is lactofer-

rin, another large, broad-spectrum antibiotic protein. Other

potential antimicrobial agents include secretory leukoprotease

inhibitor, uric acid, peroxidase (5), aminopeptidase, statherin,

secretory phospholipase A2 (3), and �-defensins (6). An active

antimicrobial reactive nitrogen species, nitric oxide (NO) (7)

is present in the airway, synthesized by both constitutive and

inducible nitric oxide synthetase (iNOS) (8).

Fig. 1. The respiratory epithelium. A. Cultured human bronchial epithelial cells grown on T-COL membranes. Well-differentiated cells (>21 days after seeding) were fixed with OsO4-perfluorocarbon and stained with Richardson’s stain. Courtesy of Dr Scott Randell, UNC-School of Medicine. B. A schematic view showing the basal cells, pseudostratified ciliated cells and secretory goblet cells.

A

Immunological Reviews 173/2000 29


The major protein component of the ASF are large mucin

glycoproteins which provide viscoelastic properties to the fluid

(9). While the mucins themselves do not exhibit antimicrobial

activity in vitro (2), they can contribute to the natural host

defense of the airway by physical protection of the airway tissue

and segregation of inhaled particles and microorganisms (9).

The salt concentration of the ASF is apparently tightly con-

trolled and may play a role in the defense of the airway. Some

studies have shown that high salt concentration inhibits the

activity of some antimicrobial peptides and proteins (10). It has

been proposed that such aberrant salt concentrations may occur

in cystic fibrosis (CF), a lethal recessive disorder caused by a

mutation in a chloride channel expressed in the airway epithe-

lium, which is characterized by recurrent airway infections.

Indeed, the airway antimicrobial factors from CF patients could

kill most CF-related pathogens, and the activity is inhibited by

high salt concentrations (2, 11). Alternately, it has been reported

that the defect lies in absorption of the liquid in the ASF, which

may result in other effects on chemical defenses (12).

Antimicrobial peptides are a recently described component

of host defense in the airway and throughout the body (13). A

major family of these peptides are the defensins, which can be

further divided into two classes, �- and �-defensins, based on

structural characteristics (14). The defensins are 29–40 amino

acid cationic peptides containing six disulfide-linked cysteines.

The cysteines in each class are invariantly conserved in a con-

sensus sequence which differs between the two classes.

Defensins exhibit strong microbicidal activity against Gram-

positive and Gram-negative bacteria, fungi, mycobacteria, and

viruses. Defensins are found in a variety of tissues and cell

types. They are highly abundant in phagocytic cells, where they

participate in the oxygen-independent killing of ingested

microorganisms. In epithelial cells, such as the small intestinal

crypts (15), female reproductive tract (16), and trachea (17),

they have been predicted to provide a first line of host defense

by acting in the luminal contents as a component of the innate

immune response.

We first described the presence of a 38-amino acid peptide

with broad-spectrum antimicrobial activity in the tracheal

mucosa of the cow, called tracheal antimicrobial peptide (TAP)

(17). The TAP gene is expressed at high levels in vivo in the ciliated

airway epithelium (18). TAP is the initial member of the �-

defensins (for review see (6)). Other �-defensins are highly

abundant in bovine neutrophils (19) and alveolar macrophages

(20) as well as in epithelial cells in the cow (21, 22). �-defensins

have also been identified in humans (23, 24), mouse (25–29),

sheep (30), pigs (31), chickens, and turkeys (32, 33).

In humans, an abundant �-defensin (human �-defensin

(hBD)1) was discovered in the kidney (24), with lower, consti-

tutive levels in numerous other tissues, including the trachea

(23). Inhibition of hBD1 gene expression with antisense oligo-

nucleotides correlates with the inability to prevent colonization

Table 1. Innate immune response in respiratory epithelial cells. Shown are: 1) infectious and inflammatory stimuli observed to cause a host response in AEC; 2) receptors expressed on AEC; 3) signal transduction mechanisms known to occur in these cells; 4) host defense genes known to be upregulated by the stimuli; 5) cytokines released from AEC in response to the stimuli.

1. Stimuli Selected references

Lipopolysaccharide 54, 136

Muramyl dipeptide 43

Lipoteichoic acid 43, 137

Respiratory syncytial virus 71

Rhinovirus 69, 95

TNF-� 42, 73

IL-1� 42, 46

PMA 41, 88

Interferon-� 72, 138

IL-4 72, 138

2. Receptors

CD14 41, 74

Toll-like receptors M. Becker, G. Diamond, M. Verghese, S. H. Randell, submitted

TNF receptors 76

IL-1 receptors 78

ICAM 67

3. Signal transduction events

P38 and MAP kinase cascades 97, 106

STAT1, STAT6 139

Activation of NF-�B 90, 97, 106

Activation of NF IL-6, AP-1 91

4. Host defense genes upregulated

TAP, LAP (cow) 22, 41

hBD2 (human) 46, M. Becker, G. Diamond, M. Verghese, S. H. Randell, submitted

LL37/CAP18 36

MUC2, MUC5C 50, 140

5. Cytokines released

IL-1 59

IL-5 60

IL-6 59

IL-8 55

RANTES 69

Endothelin 61

GM-CSF 62

TGF-B 63

Fibronectin 64

IP-10 65

Mig 65

I-TAC 65



of pathogenic bacteria in a tracheal xenograft model (34). In

this model system, human tracheal epithelial cells (TEC) are

seeded into a rat trachea whose own epithelium has been

denuded. This trachea is implanted into a nude mouse, and pro-

vides a unique system for examination of airway host defense.

Another antimicrobial peptide found constitutively in the

human airway is a member of the cathelicidin family, LL-37/

CAP-18 (35). This is a broad-spectrum antimicrobial peptide

found in the ASF (35), bronchoalveolar lavage fluid, bronchial

epithelial cells, bronchial epithelial glands, and alveolar mac-

rophages (35, 36). When epithelial cells from CF patients were

grown in the xenograft model, Bals et al. was able to show that

overexpression of exogenous LL-37/CAP-18 by gene transfer

could restore bactericidal activity (37). Together, these results

suggest that antimicrobial peptides are part of the innate

immune system in the conducting airway.

Inducible chemical defenses

In order to study the response of the airway to infection, a

number of organ and cell culture systems have been developed.

These systems, which include explant organ culture (38) and

primary cultures of epithelial cells (39) from several different

species, allow assessment of defense responses. Furthermore,

immortalized airway epithelial cell (AEC) lines have been

developed (40). Using these culture systems, two complemen-

tary inducible defense mechanisms have been observed. The

first mechanism is the increased production of antimicrobial

agents, and the second mechanism is the induction of a signal-

ing network to recruit phagocytic cells to contain the infection.

In order to characterize the role of antimicrobial peptides

in innate immune response in the airway, we examined the

expression of the TAP gene. Using primary cultures of bovine

TEC, we observed a 15-fold increase in the steady-state levels of

mRNA encoding TAP upon incubation with 100 ng/ml E. coli

lipopolysaccharide (LPS) (Fig. 2) (41). This suggested that the

AEC could respond to pathogens by the production of antimi-

crobial agents. Subsequently we discovered that these cells will

upregulate the expression of TAP in response to numerous

infectious and inflammatory agents, including phorbol 12-

myristate 13-actetate (PMA) (41), tumor necrosis factor

(TNF)-� (42), interleukin (IL)-1 �, muramyl dipeptide, lipote-

ichoic acid (43), and interferon (IFN)-� (J. Russell, C. Bevins,

G. Diamond, unpublished results). In addition, the homolo-

gous �-defensin, lingual antimicrobial peptide (LAP), origi-

nally described as a peptide whose expression in the tongue

epithelium was upregulated at sites of inflammation (22),

underwent an increase in expression in the airway in a co-ordi-

nated fashion with TAP (42). Together, these results indicated

that the airway was capable of responding directly to pathogens

by the production of antimicrobial agents.

In humans, �-defensin 2 (hBD2) was initially discovered

expressed in psoriatic skin (44). This peptide was also expressed

in other inflamed tissues, including the airway (45). There it

was observed that primary AEC upregulated hBD2 mRNA in

response to IL-1� (46). We also observed a similar induction in

an air–liquid interface culture of human AEC in response to

Pseudomonas aeruginosa LPS, as well as TNF (M. Becker, G. Diamond,

M. Verghese, S. H. Randell, submitted). Mouse �-defensin 2

(mBD2) also undergoes upregulation in response to LPS (27).

In vivo studies support this hypothesis. By intratracheal

instillation of Pasteurella haemolytica into a single lobe of a cow

lung, an increase in �-defensin expression in the airway epithe-

lium was observed to be correlated with the infection (47). In

the same study, cows testing positive for infection with Mycobac-

terium paratuberculosis exhibited increased expression of �-defen-

sins in the intestine. Similarly with the mouse homologs,

intratracheal instillation of P. aeruginosa was sufficient to increase

expression of mouse �-defensin 3 (mBD3) in the tracheal epi-

thelium as well as the intestinal tract (29). Similar system-wide

induction of hBD2 was seen in humans with bacterial pneumo-

nia (48) and of LL-37/CAP-18 in patients with sarcoidosis

(36). Thus, it is increasingly evident that colonization of the

airway by pathogenic bacteria may induce the production of

antimicrobial peptides in order to contain the infection.

Other inducible host defense molecules include mucins

and reactive nitrogen species such as NO (49). Mucin concen-

tration in the bronchoalveolar lavage is increased in response to

Fig. 2. LPS-mediated upregulation of TAP gene expression in bovine tracheal epithelial cells. Primary cultures of bovine tracheal epithelial cells were grown on a collagen matrix in serum-free medium, and treated with LPS from P. aeruginosa (Sigma) at the indicated concentrations. Cells were lysed and total mRNA was isolated. Northern blots were hybridized to a 48-base oligonucleotide corresponding to a portion of the TAP sequence. The blots were also hybridized to �-tubulin probe to control for loading.



LPS, and MUC2 and MUC5A gene expression is upregulated by

both LPS and Gram-positive and Gram-negative bacteria (50).

NO is primarily produced by iNOS, which is expressed in AEC

(51). The expression of the iNOS gene is modulated in response

to a variety of stimulants, including LPS (51, 52), resulting in

the increased production of NO in the airway. Together these

agents may contribute to an antimicrobial environment that

will prevent further colonization.

In concert with the upregulation of a direct antimicrobial

defense by microbial invasion, an inflammatory response is ini-

tiated (reviewed in (53)). When primary cultures of bovine

AEC were stimulated with LPS from E. coli, the production of

neutrophil chemotactic activity was observed in the superna-

tants (54). This activity appeared to be due to a combination of

inflammatory mediators, including arachidonic acid metabo-

lites, TNF (54), and IL-8 (55).

In addition to neutrophils, other inflammatory cells

recruited to the airway include lymphocytes (56), monocytes

(57), and eosinophils (58). The chemotactic agents for these

cells are induced in response to a variety of stimuli (53). Cyto-

kines released by AEC include IL-1 (59), IL-5 (60), IL-6 (59),

IL-8 (55), endothelin (61), granulocyte/macrophage colony-

stimulating factor (GM-CSF) (62), transforming growth factor-

� (TGF-�) (63), fibronectin (64) as well as the T-cell-specific

CXC chemokines IFN-induced protein of 10 kDa (IP-10),

monokine induced by IFN-� (Mig), and IFN-inducible T-cell

�-chemoattractant (I-TAC) (65). As part of the inflammatory

response in the airway, AEC will express adhesion molecules

such as intercellular adhesion molecule (ICAM)-1 in order to

allow the adhesion of recruited neutrophils. These molecules

are induced in response to TNF in human bronchial cell line

cultures (66) and IL-4 (67). TNF also stimulates rat tracheal

epithelial cells to secrete arachidonic acid metabolites (68).

The respiratory epithelium is able to induce an innate

immune response to viral infections. Rhinovirus replication in

primary bronchial epithelial cells was coupled with an increase

in secretion of IL-8, RANTES, a chemoattractant for CD4+

T cells, and GM-CSF, an activator of eosinophil survival and

adhesion molecule expression (69). An increase in CD15,

CD14, and CD18 expression was also observed Hep-2 cells (a

human epithelial cell line) infected with respiratory syncytial

virus (RSV) (70). As a result of the enhanced CD14 and CD18

expression, there was an increase in bacterial binding to these

cells. This indicates that virus infections alter both the surface

expression of molecules and the secretion of molecules in epi-

thelial cell surfaces, the first barrier pathogens encounter upon

infection. This, in turn, results in the recruitment of neutro-

phils, macrophages, and eosinophils, as well as T and B-lym-

phocytes. In some viral infections, such as RSV, eosinophils are

also recruited. A study by Olszewska-Pazdrak et al. (71) indi-

cated that respiratory epithelial cells recruit eosinophils in

response to RSV infection and induce eosinophil degranulation

and subsequent release of eosinophil cationic protein. RSV also

induced the upregulation of the �2 integrin CD11b on the eosi-

nophil membrane, and of ICAM-1 on AEC surfaces (71).

The response to both infectious agents and inflammatory

mediators suggests two strategies for antimicrobial defense of

the airway epithelium. One strategy is the direct induction of

antimicrobial factors in response to the pathogen; the second is

the initiation of an inflammatory response, beginning with the

secretion of cytokines for recruitment of inflammatory cells. In

addition to their role in recruitment, these cytokines can have

an autocrine and paracrine inductive effect on gene expression

of antimicrobial agents. As described above, we observed the

upregulation of TAP mRNA in response to TNF, IL-1�, and

IFN-� in cultured bovine cells. Human �-defensins are also

upregulated in response to the same mediators. Stimulation of

iNOS gene expression was observed in culture with IFN-�

(which was potentiated by IL-4) (72), and mucins are upreg-

ulated by TNF (73). Thus, the increase in cytokine-secreting

inflammatory cells has the added effect on the airway by the

maintenance of high levels of the peptides.

Mechanisms of induction

AEC express a variety of cell surface receptors to mediate their

response to infection and inflammatory agents. The primary

receptor for LPS is CD14. Originally characterized on the sur-

face of phagocytic cells, this receptor is a glycosylphosphati-

dylinositol-linked protein with no cytoplasmic domain. CD14

binds LPS as a membrane protein or as a soluble form. The

binding of LPS to CD14 is potentiated by a serum protein, LPS-

binding protein (LBP). Fearns et al. first identified the expres-

sion of CD14 in epithelial cells, as well as numerous other tis-

sues in the mouse, indicating that an endogenously expressed

membrane-bound CD14 could be involved in the airway’s rec-

ognition of pathogens (74). The expression of CD14 in the

mouse was upregulated by LPS, and required TNF and IL-1�

(75). We showed that bovine AEC expressed the CD14 gene,

and interference with its binding activity using specific mono-

clonal antibodies was able to inhibit the LPS-mediated upregu-

lation of the TAP gene (41). Similar results were seen with LPS

upregulation of the hBD2 gene in human AEC (M. Becker, G.

Diamond, M. Verghese, S. H. Randell, submitted), indicating

that CD14 plays a role in the recognition of bacteria as part of

the innate immune response of the airway. In cows, but not



human cells, this LPS-mediated upregulation occurred in the

absence of serum, indicating that LBP was not required for the

stimulation (41), although the response is potentiated by the

addition of serum (Fig. 2).

As CD14 has no cytoplasmic domain, other membrane-

linked co-receptors must be involved in the recognition of

pathogen-associated molecular patterns. The Toll-like receptors

(TLRs) have been shown to bind LPS as well as components of

Gram-positive bacteria to participate in the transduction of

these signals in concert with CD14. We have shown that TLR2

is highly expressed in human AEC, and may be involved in the

airway’s response to LPS by upregulating the expression of the

hBD2 gene (M. Becker, G. Diamond, M. Verghese, S. H. Randell,

submitted). Cultured bovine AEC express TLR3, and possibly

other homologs (D. Legarda, G. Diamond, unpublished data).

The role of the TLRs is actively being examined in phagocytic

cells, and they may share a similar function in AEC.

As part of the response of AEC to TNF, the cells have been

shown to express two types of TNF receptors, RI and RII (76).

Incubation of cells with TNF causes downregulation of RII

mRNA and shedding of the receptor (76). This shedding was

also observed with IL-1� and protein kinase C (77). This

appears to be a mechanism by which the airway cells control

the cytokine levels in their microenvironment. Human bron-

chial epithelial cultures were also shown to express IL-1 recep-

tor I, but not the inhibitory type II receptor, suggesting that the

airway is particularly sensitive to IL-1�, and is unable to

decrease its activity in the ASF (78).

Signal transduction

In response to LPS or proinflammatory cytokines, a typical gen-

eral result is the gene induction mediated by the activation of

transcription factors. An examination of the 5'-flanking region

of the TAP gene revealed consensus binding sites for both

nuclear factor (NF)-�B and NF IL-6 transcription factors. Mem-

bers of the NF-�B family mediate the induction of genes

involved in the immune and inflammatory response (79),

including the induction of antimicrobial peptides in insects

(80–82). The transcription factor NF IL-6 has been shown to

participate in activation of numerous innate immune

responses, often through interactions with NF-�B (83–87).

Transient transfection of bovine TEC with reporter constructs

containing these sites indicated that both NF-�B and NF IL-6

sites were necessary for the expression and upregulation of the

TAP gene. Electrophoretic mobility shift assays indicated that

the p50/p65 heterodimer of NF-�B was activated upon stimu-

lation with LPS, and that NF IL-6 was constitutively found in

the nucleus. The result was an increase in the rate of transcrip-

tion of the TAP gene as determined by nuclear run-on assays

(43). Immunohistochemical analysis with antibodies to NF-�B

showed translocation into the nuclei of human AEC after incu-

bation with LPS or IL-1� (M. Becker, G. Diamond, M. Verghese,

S. H. Randell, submitted), supporting the notion that airway

cells are subject to a similar innate immune response as circu-

lating myeloid cells. The results also strengthen the argument

that the innate immune response is highly conserved through-

out evolution, as we see that the induction of the expression of

antimicrobial peptide genes occurs through similar pathways

in both insects and mammals.

Other factors which have been shown to activate NF-�B in

airway cells include cytokines (IL-1, TNF, PMA (88)) and

inhaled particulates, including diesel exhaust (89), asbestos

(90), ozone (91) and air pollution particles (92). Activation of

NF-�B was also observed in asthmatic airways (93). The adher-

ence of Pseudomonas to the epithelial cells stimulates the activa-

tion of the p50/p65 dimer of NF-�B (94), as do viruses (95,

96). Genes which are induced by activation of NF-�B include

mucins (97), RANTES (96), GM-CSF (88), IL-6 (92) and IL-8

(98). Together with our data, these results lead to the hypo-

thesis that a co-ordinated response is mediated in AEC upon

stimulation by infectious agents, inflammatory mediators or

oxidants. The stimulation with these agents results in the

expression of innate immunity genes.

The intermediate events between LPS binding to CD14 and

NF-�B activation are incompletely defined and may involve

numerous signaling pathways. Shortly after treatment with LPS,

macrophages are known to activate these signal transduction

pathways by a number of kinase cascades. Among the first pro-

teins phosphorylated are those of the Src family, although they

are not necessary for LPS-mediated induction (99), suggesting

other potential initial events. Further in the pathway, three

structurally related families of mitogen-activated protein

kinases (MAPK) appear to mediate the signal transduction lead-

ing to the expression of host defense genes. These families are

the extracellular signal-regulated kinase (ERK), c-Jun NH2-ter-

minal kinase (JNK) and the p38 MAPK (100). In macrophages

it has been shown that LPS and other bacterial components can

induce the activation of all three pathways (101). The p38 and

ERK pathways are also activated by LPS in human umbilical vein

endothelial cells in the presence of soluble CD14 (102). Both

of these pathways are also activated by LPS in macrophages to

stimulate the expression of the cyclooxygenase gene (103).

In human airway epithelial cells, one pathway has been

defined for the bacterial induction of the MUC2 gene. Using a

MUC2-expressing AEC line, Li et al. showed that activation of



NF-�B by P. aeruginosa occurs via a Src-dependent pathway

involving the phosphorylation of ERK1/2 but not the JNK

pathway. The kinase cascade in this system was defined as: Src�

Ras � Raf1 � MAP/ERK kinase 1/2 � ERK1/2 � pp90rsk

(97). The ERK pathway was also observed in TEC stimulated by

neutrophil elastase to induce morphological changes (104).

Activation by TNF may occur through a distinct pathway

which differs from the LPS-induced pathway early in the cas-

cade (105). Indeed, Matsumoto et al. have shown that induc-

tion of IL-8 gene expression by TNF in human AEC occurs

through a p38 MAPK pathway (106). This pathway is activated

by other proinflammatory cytokines, as well, suggesting a

common pathway of response by the epithelial cell to infec-

tious agents and inflammatory mediators.

Other stimuli can induce tyrosine kinase pathways in AEC.

Samet et al. have shown that sublethal levels of combustion-

derived metals such as arsenic, copper, vanadium, and zinc can

rapidly induce activation of all three MAPK pathways (107).

Further research showed that activation of the ERK pathway

proceeded through the epidermal growth factor-receptor

tyrosine kinase (108), although the specific subsequent enzy-

matic steps involved differ between the metals (109). Both ERK

and JNK pathways are also activated in alveolar epithelial cells

stimulated by reactive oxygen species and TNF (110). Surpris-

ingly, the ERK pathway seems to inhibit activation of NF-�B in

these cells, suggesting that some cell types may regulate gene

activation through these pathways differently (110).

An important step in the NF-�B signaling pathway is the

inactivation of I�B by phosphorylation and subsequent degra-

dation. In bacterially induced AEC, this occurs by a pathway

involving pp90rsk (97), which leads to the degradation of I�B�

(111). A similar role of I�B� is observed with AEC stimulated

with RSV, where overexpression of I�B� inhibits the virus-

mediated upregulation of RANTES (96).

Inhibition of airway epithelial cell-mediated innate immunity

The host defense of the airway can be inhibited via several

mechanisms. Air pollutants such as ozone and nitrogen diox-

ides inhibit the phagocytic capacity of alveolar macrophages

(112). Air pollutants inhibit the action of cilia without being

cytotoxic to the airway epithelium (113). Certain pollutants

influence the secretion of mucus by goblet cells, which ulti-

mately affect airway host defense. If an air pollutant mimics a

cholinergic agent or it disrupts the integrity of the goblet cell,

the end result is increased mucus secretion (114).

Pollutants induce a variety of cytokines and mediators in the

airway epithelium. IL-8 release from AEC is stimulated by ozone

(115), particulate matter of 10 �m or less (PM10) (116), ciga-

rette smoke (117), and, to a lesser extent, asbestos (118). IL-6

and TNF release are also stimulated by PM10 and ozone in AEC

(115, 116). In addition to releasing IL-6 and IL-8, epithelial

cells were shown to release the CXC chemokines growth-related

protein-� (115) macrophage inflammatory protein-2, and

RANTES (119) in response to ozone exposure. Ozone induces

the release of cyclooxygenase and lipooxygenase products from

AEC (119). These products have been shown to play a role in

inhibiting alveolar macrophage phagocytosis of bacteria (120,

121). Epithelial cells may also influence the development of

T cells to reflect a T-helper type 2 (Th2) response and a suppres-

sion of a T-helper type 1 (Th1) response following ozone expo-

sure (122). Ozone inhibits IL-2 production (123) and IFN pro-

duction (124) in TEC; this inhibition may be mediated by pros-

taglandin E2 (PGE2). PGE2 inhibits the production of Th1 lym-

phokines, IL-2, and IFN-�, but does not affect the production of

Th2 lymphokines, IL-4, and IL-5 (125–127). IL-6 production

stimulates IL-4 production, and NO, released from alveolar type

II epithelial cells (128), inhibits the development of Th1 cells

but not Th2 cells (129). In addition, the responsiveness of alve-

olar macrophages to IFN-� may be compromised, affecting the

ability of alveolar macrophages to be activated to phagocytize

and kill bacterial pathogens (130). Thus, air pollutants can

interfere with host defense mechanisms by acting on AEC, influ-

encing both innate and acquired immune mechanisms for bac-

terial clearance in the lung.

The effect of air pollutant particles on TAP gene expression

Very little research has been carried out the effect of air pollut-

ants on antimicrobial activity in AEC. We tested the hypothesis

that air pollutant particles inhibit TAP gene expression by LPS. As

an air pollution agent, we chose residual oil fly ash (ROFA), a

combustion emission source ambient air particulate, kindly

donated by Dr Kevin Dreher of the U. S. Environmental Protec-

tion Agency, Research Triangle Park, NC. ROFA was collected on

a Teflon-coated fiber glass filter downstream from the cyclone

(“scrubber”) of an oil-burning power plant in Florida. The

physicochemical properties and composition have been

described (131), but it is important to note that ROFA was 90%

water soluble and was high in ionizable metal content, espe-

cially vanadium, nickel, and iron, and had negligible endotoxin

content (131, 132). ROFA (100 �g intratracheally instilled) has

been shown to increase mortality to Streptococcus zooepidemicus

(Group C) infection in a mouse animal model by approximately

50% (132, 133). Silica (SiO2, Sigma Chemical Co., St Louis,

MO) was used as a control in our study, as a similar dose of silica



had little effect on host defense in the mouse, increasing suscep-

tibility to pulmonary bacterial infection by only 17% (133).

The silica was baked at 190°C for 3 h to render it LPS free.

Bovine AEC were maintained in air–liquid interface cul-

tures in which the cells are grown on a collagen layer on a filter,

fed from the basolateral surface, with no media on the apical

surface. Cells grown in this manner more closely resemble the

actual airway, and respond to the administration of LPS to the

basolateral media similarly to the submerged cultures by

upregulating the expression of the TAP gene. Culture medium

containing ROFA or SiO2 at increasing concentrations (0–100

�g/ml) was applied to the apical surface for 18 h. The pH of the

medium remained neutral. The apical medium was aspirated,

and cells were grown for a further 24 h, with LPS (100 ng/ml)

added for the final 18 h. Cells were harvested and mRNA was

isolated. Northern blot analysis was performed, using the �-

defensin antisense oligonucleotide TAP48a as a probe, and nor-

malized to hybridization with a bovine �-tubulin probe as

described previously (41). Blots were quantitated by phosphor-

image analysis. The results shown in Fig. 3 indicate that ROFA

inhibited the LPS-mediated upregulation of TAP gene expres-

sion in a dose-dependent manner. As little as 5 �g/cm2 inhib-

ited TAP gene expression below constitutive levels. Similar incu-

bation with SiO2 showed only a moderate decrease of LPS-

induced TAP mRNA levels. The concentrations of ROFA used

here did not significantly increase cell death as determined by

trypan blue exclusion, or by the presence of tubulin mRNA.

Neither ROFA or SiO2 altered constitutive TAP gene expression

in the absence of LPS.

These results suggest that some aspect of the ROFA parti-

cles may be interfering with the induction of TAP gene expres-

sion by bacterial products in amounts that represent realistic

exposure levels that are not cytotoxic to the cells. Interestingly,

in the Hatch et al. study, the cytotoxic potential of various types

of particles on alveolar macrophages did not correlate well with

the ability to increase susceptibility to infection, suggesting

that an acellular mechanism may account for the decreased host

defense seen with inhalation of various types of pollutant par-

ticles (133). In our study the level of inhibition of LPS-induced

�-defensin gene expression by ROFA and silica correlated with the

inhibition of host defense in the mouse model described by

Hatch et al. (133). Other aspects of airway host defense may be

affected differently, however. One study showed that similar

amounts of ROFA increased the release of lactoferrin (134) and

the number of lactoferrin receptors on AEC following exposure

of these cells in vitro (135). Further investigation is needed to

examine the inhibitory effect of particulates on antimicrobial

peptides and antimicrobial activity of the airway epithelium.

Conclusions

The respiratory epithelium is an important interface with the

environment, and represents a dynamic system for innate host

defense. The cells lining the airway respond to the presence of

microorganisms by producing natural antimicrobial factors and

mounting an inflammatory response. Diseases such as CF, as well

as inhaled air pollutants, can change this environment to inhibit

the natural activity of the innate immune system in the airway.

Fig. 3. Effect of ROFA and silica on LPS-mediated upregulation of TAP gene expression. Cultures of bovine AEC were exposed to particles as described above, followed by stimulation by 100 ng/ml LPS. SiO2 (closed circles) was baked at 190°C to render it LPS free; ROFA (open circles) contained negligible (2.5 pg/mg) LPS (131). Effect is expressed as levels of TAP mRNA relative to unstimulated cultures with no particles. Particle concentration is expressed µg/ml of media. 100 µg/ml particle suspension equals 21.4 µg/cm2 over cell surface.



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The innate immune response of the respiratory epithelium