Signalling and cellular specificity of airway nitric oxide production in pertussis

Signalling and cellular speci®city of airway nitric oxideproduction in pertussis

Tod A. Flak² and William E. Goldman*

Department of Molecular Microbiology, Washington

University School of Medicine, 660 South Euclid Avenue,

St. Louis, MO 63110, USA.

Summary

Bordetella pertussis, the aetiological agent of whoop-

ing cough (pertussis), causes selective destruction

of ciliated cells of the human airway mucosa. In a

hamster tracheal organ culture model, B. pertussis

causes identical cytopathology as does tracheal cyto-

toxin (TCT), a glycopeptide released by the bacter-

ium. The damage caused by B. pertussis or TCT has

been shown to be mediated via nitric oxide (NO?).

Using immuno¯uorescence detection of the cyto-

kine-inducible NO synthase (iNOS; NOS type II), we

determined that B. pertussis induced epithelial NO?

production exclusively within non-ciliated cells. This

epithelial iNOS activation could be reproduced by

the combination of TCT and endotoxin. However,

neither TCT alone nor endotoxin alone was capable

of inducing epithelial iNOS. This result mirrors the

synergistic activity of TCT and endotoxin exhibited

in monolayer cultures of tracheal epithelial cells.

Therefore, TCT and endotoxin are both important

virulence factors of B. pertussis, combining syner-

gistically to cause the speci®c epithelial pathology

of pertussis.

Introduction

The pertussis syndrome is the result of colonization of the

large airways by Bordetella pertussis. The primary epithe-

lial cytopathology associated with this colonization is the

selective destruction of the ciliated cell population. A

small peptidoglycan fragment produced by B. pertussis,

tracheal cytotoxin (TCT), can reproduce the ciliated cell

pathology in explanted tracheal tissue (Goldman et al.,

1982). We have previously shown that a key mediator of

this destruction is nitric oxide (NO?) (Heiss et al., 1994).

TCT activates cytokine-inducible NO? synthase (iNOS;

NOS type II) in tracheal tissue, and inhibition of iNOS by

selective inhibitors abrogates the damage caused by

TCT exposure. Thus, TCT-induced NO? production has

been proposed as the central mechanism in generating

the speci®c epithelial damage of B. pertussis infections

(Flak and Goldman, 1996).

An important unexplored issue is the identi®cation of the

precise source of NO? within respiratory tissue. Although

the ciliated cells are those most obviously affected, they

are not necessarily the source of NO? because it is a diffu-

sible gas. It is possible that the non-ciliated cells within the

epithelium, which include mucus-secreting cells and basal

cells, play some role in the disease. These non-ciliated

cells are not colonized by B. pertussis and appear to be

unaffected by either bacterial infection (Collier et al.,

1977) or by treatment with TCT (Goldman et al., 1982).

In fact, the function of mucus-secreting cells seems to be

unchanged, or possibly enhanced, and mucus accumula-

tion in the absence of ciliary clearance is a major problem

in clinical cases of pertussis.

The effects of TCT in an in vitro cell culture model have

also led us to question whether another bacterial product,

endotoxin, plays a role in the cytopathology of pertussis.

This in vitro system uses hamster trachea epithelial

(HTE) cells, a homogeneous epithelial cell population

derived from hamster tracheal tissue (Goldman and Base-

man, 1980). In HTE cells, DNA synthesis is inhibited in a

dose-dependent fashion by the combination of TCT and

endotoxin (Heiss, 1993; Heiss et al., 1993). This effect

occurs via an NO?-mediated mechanism, like the destruc-

tion of ciliated cells in tracheal rings. However, whereas

TCT alone is suf®cient to cause epithelial damage in

tracheal rings, in cultured epithelial cells both TCT and

endotoxin are necessary to elicit the NO? response. In

these HTE cells, neither TCT nor endotoxin has much effect

alone, but together they are highly synergistic (T.A. Flak,

L.N. Heiss, J.T. Engle and W.E. Goldman, submitted).

The apparent discrepancy between the epithelial cell

model system and the explanted tracheal tissue poses a

question of substantial signi®cance in the pathobiology of

pertussis: does endotoxin play a signi®cant role, as sug-

gested by the in vitro cell culture model, or is TCT alone

suf®cient to explain the epithelial cytopathology, as sug-

gested by the tracheal organ culture model?

In this paper, we use immunological probes to elucidate

the manner in which the toxins produced by B. pertussis

elicit the production of NO? and ultimately the destruction

Cellular Microbiology (1999) 1(1), 51±60

Q 1999 Blackwell Science Ltd

Received 18 March, 1999; revised 19 May, 1999; accepted 24 May,1999. ²Present address: Acacia Biosciences, 4136 Lakeside Drive,Richmond, CA 94806, USA. *For correspondence. E-mail [email protected]; Tel. (�1) 314 362 2742; Fax (�1) 314 362 4879.

of ciliated cells. We demonstrate that endotoxin indeed

plays an important, and previously unappreciated, syner-

gistic role in combination with TCT to induce the produc-

tion of NO? within airway epithelium. Furthermore,

whereas the NO? primarily affects the ciliated epithelial

cells, we show that the epithelial source of NO? is not

the ciliated cell themselves, but rather the neighbouring

secretory cells. This study illustrates how analysis of an

architecturally intact differentiated tissue was necessary

to truly penetrate the mechanism of pathogenesis, signi®-

cantly expanding our understanding beyond that possible

with an in vitro cell culture model.

Results

Synergistic induction of iNOS by TCT/endotoxin in

cultured trachea epithelial cells

We have shown in previous studies that TCT and endo-

toxin are highly synergistic in the NO?-mediated toxicity

observed in hamster trachea epithelial (HTE) cells (T.A.

Flak, L.N. Heiss, J.T. Engle and W.E. Goldman, submitted).

To evaluate whether the elevated NO? production by HTE

cells is due to an increase in iNOS protein level, we exam-

ined the production of iNOS by Western blotting. We used

a mouse monoclonal antibody directed against a C-termi-

nal fragment of mouse macrophage iNOS. This antibody is

speci®c for iNOS; it does not react with neuronal (type I) or

endothelial (type III) NOS. As shown in Fig. 1, TCT alone

or endotoxin (lipopolysaccharide, LPS) alone each

induces synthesis of a small amount of iNOS. However,

the two signals combined trigger a high level of iNOS pro-

duction, which is more than the sum of the individual

effects. This ®nding correlates well with the previously

observed synergy between TCT and endotoxin in NO?

production by HTE cells.

Also shown in Fig. 1 is the effect of exogenous interleu-

kin 1 (IL-1) on the induction of iNOS. As we have described

previously, IL-1 is a strong inducer of NO? production in

HTE cells (Heiss et al., 1994), as it is in many other sys-

tems (see Dinarello, 1991). Figure 1 shows the effect of

IL-1b only, but exogenous IL-1a was also capable of indu-

cing iNOS immunoreactivity in HTE cells (data not shown).

IL-1 was capable of inducing strong iNOS activity without

the addition of endotoxin; we also determined that the IL-

1b source was free of endotoxin contamination (data not

shown).

TCT and endotoxin induction of NO ? in tracheal tissue

As shown in Fig. 1, TCT and endotoxin are highly syner-

gistic in iNOS induction in cultured trachea epithelial

(HTE) cells. However, in explanted tracheal tissue, TCT

alone is suf®cient to induce a strong NO? response and

to cause epithelial cytopathology (Goldman et al., 1982;

Heiss et al., 1994). To begin to address this disparity

between the two model systems, we determined the NO?

production by tracheal organ cultures in response to com-

binations of TCT and endotoxin.

Fig. 2 presents a quantitative assessment of toxin

activity in tracheal rings (i.e. transverse segments of the

excised trachea). Production of NO? was assessed by

the accumulation of nitrite, a stable breakdown product

of NO?; this is the same assay system used to measure

NO? production by cultured HTE cells (Heiss et al.,

1994). As described previously, TCT by itself induced

NO? production by tracheal rings (Heiss et al., 1994). In

this experiment, however, we included samples exposed

to bacterial endotoxin. We observed that incubation with

endotoxin alone also resulted in NO? production. The com-

bination of TCT with endotoxin consistently induced higher

NO? production than either toxin alone, but the NO? pro-

duction was less than the sum of that produced by the

Q 1999 Blackwell Science Ltd, Cellular Microbiology, 1, 51±60

Fig. 1. Induction of iNOS protein by TCT/LPS. Growing HTE cellswere treated with TCT (3.2 mM), LPS (100 eU mlÿ1), or IL-1b(10 ng mlÿ1) for 4 h in serum-free medium; serum was added to15%, and cell lysates were prepared after an additional 22 hincubation. As a positive control for iNOS, RAW264.7 murinemacrophage-like cells were treated with LPS (100 eU mlÿ1) for 22 h.Western blot analysis was performed using an antimurine iNOSmonoclonal antibody.

Fig. 2. NO? production by hamster tracheal rings. Hamstertracheas were harvested and cut into ring segments, and wereexposed to TCT (10 mM) and/or endotoxin (E. coli LPS100 eU mlÿ1). Nitrite accumulation was assessed at 20 h. For eachbar, n� 7 or 8. Pairwise t-test analysis established that all meanswere signi®cantly different from each other (P < 0.005) except TCTcompared with LPS, which are not signi®cantly different from oneanother.

52 T. A. Flak and W. E. Goldman

two toxins individually. Thus, for whole tracheal rings,

some cooperation was observed between TCT and endo-

toxin, but the cooperativity was far less pronounced than

the strong synergy observed in HTE cells.

iNOS immunostaining in cultured trachea epithelial

cells

In order to determine the speci®c cellular source(s) of NO?

in tracheal tissue, we established an immuno¯uorescence

assay for iNOS. The initial development of the immuno-

¯uorescence detection method was accomplished using

HTE cells, utilizing the same antibody used for the iNOS

Western (Fig. 1). HTE cells on glass coverslips were

treated with TCT and endotoxin to trigger NO? production,

and after 16 h they were processed for iNOS immunoreac-

tivity. HTE cells not exposed to toxins showed no iNOS

staining (not shown), whereas those exposed to TCT/

LPS demonstrated strong ¯uorescent immunoreactivity

of iNOS, as shown in Fig. 3. The iNOS reactivity was dif-

fuse and, in general, was evenly distributed throughout the

cytoplasm but excluded from the nucleus. There was nota-

ble variation in reactivity between cells, with some cells not

staining at all.

Bordetella pertussis induction of iNOS in epithelium

and cartilage

To localize NO? production during a B. pertussis infection,

we used an established tracheal ring infection protocol

that has previously been used to characterize ciliated

cell-speci®c colonization and pathology (Collier et al.,

1977). When maintained in tissue culture medium, ham-

ster tracheal rings retain epithelial integrity and strong cili-

ary activity for weeks. For this experiment, we embedded

the tracheal tissue for sectioning 20±24 h after infection

with B. pertussis, a time at which little ciliated cell destruc-

tion was expected to have occurred. By this time point,

nitrite accumulation in the medium of infected tissue was

typically < 2±3 nmol per ring, whereas uninfected controls

produced less than 0.5 nmol per ring. Frozen sections

were prepared and stained for iNOS immunoreactivity.

The bulk of each tracheal ring consists of a C-shaped

hyaline cartilage region, with a thin epithelial layer

attached to the inner (luminal) surface of the cartilage

ring. The cartilage itself contains two major subsections:

most of the material is composed of chondrocytes, rather

quiescent cells which reside in a cavity (lacuna) within the

territorial matrix that they produce; surrounding the chon-

drocytes is the perichondrium, a layer of connective tissue

and less differentiated cartilage cells that serve as source

of new chondrocytes during development or after cartilage

damage (Kennedy et al., 1978; Ross et al., 1995). The

tracheal epithelium comprises three major cell types: the

columnar ciliated cells; columnar secretory cells, which

include mucus-secreting goblet cells; and basal cells,

which lie along the basement membrane and do not

reach the luminal surface of the epithelium (for a detailed

study of respiratory epithelial structure, see Becci et al.,

1978)

Fig. 4 depicts typical results that we observed for this

type of experiment. B. pertussis colonization of the ciliated

cells resulted in iNOS induction both within the epithelium

and, somewhat unexpectedly, within the cartilage. As

shown in Fig. 4A, iNOS immunoreactivity (red) is evident

along the apical surface of many of the epithelial cells, as

well as throughout the cartilage. Within the cartilage, there

was sometimes stronger staining in the perichondrial cells

than in the chondrocytes deeper in the tissue, as shown in

Fig. 4A. Uninfected rings resulted in images that were


Fig. 3. iNOS detection in HTE cells treated with TCT/LPS. HTE cells were exposed to a combination of TCT (3 mM) and LPS (100 eU mlÿ1).After 16 h, the cells were ®xed and permeabilized, and incubated with an anti-iNOS monoclonal primary antibody, followed by an anti-IgGsecondary antibody conjugated to Cy3. Samples were stained with Hoechst nuclear stain.A. A phase-contrast view plus ¯uorescence from the Hoechst nuclear stain.B. The same ®eld showing ¯uorescence of Cy3, demonstrating immunoreactivity for iNOS. Magni®cation 286 ´.

Toxin synergy eliciting NO production in epithelial cells 53

almost completely dark under ¯uorescence excitation,

indicating that there was no signi®cant activation of iNOS

in these control samples (not shown).

Fig. 4B presents a higher magni®cation bright®eld view

of the epithelium, with Hoechst-stained nuclei ¯uorescing

in blue. Two levels of blue nuclear staining are evident

within the epithelium, the lower level being the nuclei of

basal cells and the upper level that of columnar ciliated

and secretory cells. Although tissue processing some-

times did not preserve the integrity of the loose elastic

connective tissue that binds the epithelium to the underly-

ing cartilage, separation of the epithelial layer from the

cartilage did not interfere with interpretation of iNOS

immunoreactivity. We also observed nuclear staining

within this submucosal area, indicative of the many

types of cells typically found here, including lymphocytes,

eosinophils, and ®broblasts (Ross et al., 1995).

Within the infected epithelium, iNOS reactivity was

located exclusively in non-ciliated cells. In Fig. 4B, iNOS

immunoreactivity can be seen in cells immediately neigh-

bouring several ciliated cells. The iNOS reactivity was

generally concentrated near the apical end of the colum-

nar epithelial cells. We never observed iNOS reactivity

in ciliated cells or basal cells.

TCT induction of iNOS in cartilage

As TCT is able to reproduce the ciliated cell-speci®c

damage caused by B. pertussis infection, we wanted to

determine whether TCT could also replicate the pattern

of cellular iNOS induction we observed in B. pertussis-

infected tracheal rings. Therefore, we treated tracheal

organ cultures with TCT for 20±24 h and then embedded

them for sectioning. Previous studies have shown that

nitric oxide production by tracheal rings can be detected

as early as 12 h after TCT treatment, although epithelial

damage is typically not apparent until after 60 h (Heiss et

al., 1994). As expected, all TCT-treated rings exhibited

normal ciliary activity at the time of embedding. The

amount of nitrite that accumulated in the medium varied

between experiments, ranging from 1 to 3 nmol per ring,

but it was always signi®cantly more than untreated rings.

As shown in Fig. 5A, treatment of tracheal rings with

TCT resulted in strong induction of iNOS only within carti-

lage cells. The induction was generally uniform across the

entire cartilage layer, but in some cases there seemed to

be preferential induction in the perichondrium compared

with the central cartilage. There was no induction of

iNOS in any of the cells within the epithelium, in contrast

to the iNOS activation of non-ciliated epithelial cells

caused by B. pertussis infection.

TCT and endotoxin induction of epithelial iNOS

Because the combination of TCT and endotoxin is capable

of triggering more NO? production than either of the two

toxins alone (Fig. 2), we suspected that the epithelium

might be the source of this additional NO?. This would cor-

respond to the results with HTE cells, which are derived

from the epithelium of hamster trachea and which synthe-

size iNOS only when treated with both TCT and endotoxin

(Fig. 1).

Tracheal rings exposed to both TCT and endotoxin

exhibited strong iNOS induction within both the epithelium

and the cartilage, as shown in Fig. 5C. The epithelial

immunoreactivity was generally along the apical edge of

a subset of the epithelial cells and was virtually identical

to the reactivity seen in B. pertussis-infected samples.

No iNOS immunoreactivity was apparent in the basal

cells. The iNOS reactivity in the cartilage was generally

strong and uniform across the cartilage, in both the peri-

chondrocytes and chondrocytes, similar to the induction


Fig. 4. Induction of iNOS in hamster tracheal tissue by B. pertussis. Hamster tracheal rings were incubated in control medium (not shown), orinfected with B. pertussis (A and B), and embedded after 24 h. Red ¯uorescence denotes iNOS immunoreactivity; blue ¯uorescence is fromthe Hoechst nuclear stain; some ciliated cells are indicated (arrows). Yellow bars indicate the extent of the epithelial layer; green bars indicatethe cartilage. Untreated tissue had little staining (not shown), whereas infected tissue (A; 173 ´) shows iNOS induction in the cartilage andsome epithelial cells. A high magni®cation view (B; 522 ´) of infected tissue shows that the iNOS reactivity is along the apical edge of epithelialcells; superposition of the red ¯uorescence upon a Nomarski DIC image of the same ®eld demonstrates that iNOS reactivity is only in non-ciliated cells.


seen in rings treated with TCT alone. Exposure of the rings

to endotoxin alone resulted in iNOS reactivity only within

the cartilage, similar to that observed with TCT alone

(data not shown). Therefore, only the combination of

TCT and endotoxin could reproduce the complete pattern

of cellular iNOS activation observed during B. pertussis

infection of tracheal tissue (Fig. 4).

Determination of the epithelial cell type producing

iNOS

Identifying speci®c cell types in the epithelium was dif®cult

when relying only on morphological criteria, so we used

cell-speci®c markers to determine the identity of the sub-

set of epithelial cells that produced iNOS. First, we wanted

to con®rm our morphology-based conclusion that none of

the iNOS� epithelial cells were ciliated. In the bacterial

infection experiments, cilia were usually very easy to

see because they were colonized with bacteria, which

caused the cilia to remain extended during processing.

However, the cilia in toxin-treated samples could be

more dif®cult to visualize by the processing techniques

used. To be certain that we could identify every ciliated

cell, we used a monoclonal antibody against b-tubulin to

detect cilia. Tubulin has been shown to be present both

in the cilia themselves and in the basal bodies (Gordon

et al., 1977), and proved to be an effective means by

which to highlight ciliated cells. In Fig. 6, iNOS immunor-

eactivity is again shown in red, whereas b-tubulin immu-

nostaining is green. As seen in the micrograph, iNOS

reactivity was observed exclusively in non-ciliated epithe-

lial cells after treatment with TCT/endotoxin. In no case

did we detect iNOS immunoreactivity in ciliated cells (as

determined either by the presence of visible cilia or with

b-tubulin staining). This is in agreement with B. pertussis

infection, in which the epithelial cells showing iNOS activa-

tion were exclusively non-ciliated.

The majority of non-ciliated cells in the hamster tracheal

epithelium are secretory cells, but other non-ciliated cell

types have been described, such as brush cells, inter-

mediate cells, or unidenti®ed columnar cells (Kennedy et

al., 1978; McDowell et al., 1983; Shimizu et al., 1992).

To help identify the non-ciliated epithelial population

responding to TCT/endotoxin, we used the lectin UEA-1

(Ulex europaeus agglutinin I), which recognizes a-linked


Fig. 5. Induction of iNOS in tracheal tissue by TCT and endotoxin. Tracheal rings were treated with TCT alone (A; 10 mM TCT; 342 ´), TCTplus endotoxin (B; 10 mM TCT, 100 eU mlÿ1 LOS; 210 ´). Red ¯uorescence denotes iNOS immunoreactivity. Yellow bars indicate the extent ofthe epithelial layer; green bars indicate the cartilage. The red ¯uorescence image of (A) is overlaid on a dim bright®eld view of this ®eld todemonstrate the location of the epithelium, which lacked even background ¯uorescence. Untreated rings showed no iNOS reactivity (notshown).

Fig. 6. Double staining for iNOS and tubulin. Tracheal ringsexposed to TCT (10 mM) and endotoxin (100 eU mlÿ1) were stainedsimultaneously for b-tubulin (green), to highlight cilia, and iNOS(red). Fluorescence images are superimposed upon Nomarski DICviews. Magni®cation 167 ´.


fucose residues and which has been used to identify

secretory cells in other studies of respiratory epithelium

(Mariassy et al., 1988; Shimizu et al., 1991). The ¯uores-

cein-conjugated lectin was used simultaneously with

iNOS staining, as shown in Fig. 7. UEA-1 stained the gly-

coproteins within the secretory vesicles, effectively high-

lighting secretory cells interspersed among the ciliated

cells. Not all secretory cells will stain with UEA-1 because

the epithelium includes cells in various stages of mucus

accumulation or discharge. However, we observed a

strong correlation between UEA-1-stained epithelial cells

and those producing iNOS in rings treated with TCT/endo-

toxin (i.e. >50% of iNOS-positive cells were also UEA-1

positive). We obtained similar results using rings infected

with B. pertussis (not shown).

iNOS induction after luminal exposure to TCT/

endotoxin

The cartilage iNOS induced by TCT alone or endotoxin

alone is certainly substantial. In fact, because exposure

to TCT in the absence of endotoxin elicits iNOS exclusively

within the cartilage, chondrocytes and perichondrocytes

are likely to be the sole sources of the NO? that mediates

epithelial damage in rings treated with TCT alone. How-

ever, it is not clear whether this cartilage reactivity actually

plays a role in a natural B. pertussis infection. It seems

possible that this non-epithelial iNOS activation may be

an artefact of immersing a cut tracheal segment in a

bath of toxin-containing medium, thus unnaturally expos-

ing the cartilage to toxin.

To determine whether the production of NO? in cartilage

was physiologically relevant, we devised a system to

expose only the lumen of the trachea to toxins as a more

accurate model of the luminal surface colonization estab-

lished by B. pertussis. In this model, whole excised

tracheas were mounted onto hollow steel supports. Sterile

medium containing the desired samples was slowly

pumped through the tracheas, achieving continuous lumi-

nal oxygenation and exposure to the sample. The entire

system was placed in a bath of normal medium to maintain

hydration and provide nutrients to the cartilage. To check

the health of the trachea under these conditions, we cut

rings from tracheas that had been maintained in this fash-

ion for 24 h. They showed very strong ciliary activity, and

subsequent exposure of these rings to toxins resulted

in iNOS induction equivalent to that described above for

normal rings (data not shown).

Using this system, whole tracheas were exposed intra-

luminally to control medium or TCT/endotoxin for 22 h.

At this time, there was still very strong ciliary activity and

no indication of damage in the TCT/endotoxin-treated

trachea. Segments of the tracheas were then embedded,

sectioned, and stained as usual. In these experiments,


Fig. 7. Double staining for secretory cells andiNOS. Tracheal rings exposed to TCT (10 mM)and endotoxin (100 eU mlÿ1) were stainedsimultaneously for iNOS (red) and forsecretory cell glycoproteins with the lectinUEA-1 (green).A. The green UEA-1 ¯uorescencesuperimposed upon a Nomarski DIC image(509 ´) showed brightly stained secretorycells interspersed between ciliated cells.B. The red iNOS staining showed a similarset of cells.C. Simultaneous visualization of both the redand green ¯uorescence revealed iNOSreactivity within UEA-1 plus secretory cells;equal red and green staining appears asyellow in the combined image.


the cartilage was not activated by TCT/endotoxin to pro-

duce iNOS. As expected, TCT/endotoxin did induce

epithelial iNOS, exclusively in non-ciliated cells. Figure 8

shows a view of a TCT/LOS-treated trachea, in which

there is apical iNOS immunoreactivity in the epithelium

but almost no iNOS activation in the cartilage, which occu-

pies the dark bottom half of the image. Thus, in an intact

trachea, TCT and endotoxin do not seem to diffuse into

the cartilage to a signi®cant extent.

Discussion

The primary model systems described in this paper were

originally used to identify TCT as a potential virulence

factor, based on the ability to reproduce the respiratory

epithelial pathology caused by B. pertussis infection

(Goldman et al., 1982; Cookson et al., 1989). We have

expanded the analysis of these models with assays and

reagents to monitor production of NO?, a primary mediator

of this epithelial damage. This study has revealed that,

although ciliated epithelial cells exhibit the major airway

pathology of pertussis, they are not the source of the

NO? that leads to their destruction. B. pertussis coloniza-

tion of ciliated cells in hamster tracheal rings results in

the induction of epithelial iNOS by neighbouring secretory

cells. Identical iNOS activation in secretory cells is caused

by the synergistic combination of TCT and endotoxin.

Neither TCT alone nor endotoxin alone causes the induc-

tion of epithelial iNOS. Furthermore, this work is the ®rst

evidence that endotoxin plays an essential role in pertus-

sis pathology.

We also discovered that TCT and endotoxin, either indi-

vidually or in combination, are able to induce iNOS in

chondrocytes. Chondrocytes are known to express a

macrophage-like iNOS (Charles et al., 1993) in response

to cytokines, such as IL-1 and tumour necrosis factor a

(TNF-a), or LPS (Stadler et al., 1991). Previous reports

have suggested that peptidoglycan may also mediate

NO? production by chondrocytes. NO? has been impli-

cated as a mediator of in¯ammatory arthritis in a model

using injected streptococcal cell wall fragments (McCart-

ney-Francis et al., 1993). LPS is synergistic with muramyl

dipeptide, a synthetic peptidoglycan monomer analogue,

in the inhibition of proteoglycan synthesis in chondrocytes

(Ikebe et al., 1993), an effect which may be mediated by

NO? (Taskiran et al., 1994). Furthermore, a mixture of

anhydrous muramyl peptide monomers, mostly TCT and

the corresponding disaccharide±tripeptide derived from

Neisseria gonorrhoeae, causes arthritic effects in carti-

lage (Fleming et al., 1986). Despite these previous indica-

tions, this report provides the most direct evidence that a

peptidoglycan fragment can induce iNOS in chondrocytes.

Nevertheless, the induction of iNOS in cartilage is most

likely of little physiological relevance in pertussis, espe-

cially early in the infection while the epithelium is still intact.

Our studies suggest that the intact epithelium provides an

effective barrier to both toxins. Using whole tracheas can-

nulated to allow exposure of toxins only to the luminal

surface, we found that the iNOS production in the cartilage

was not activated by TCT and endotoxin. Therefore, it

seems likely that the induction of iNOS in cartilage is an

artefact of our tracheal ring model system, in which

the cartilage is arti®cially exposed to toxin-containing

medium on the cut ends of the ring segment as well as

on submucosal and serosal perichondrial surfaces via the

loose connective tissue in these areas. The iNOS induced

in cartilage is probably the source of NO? responsible for

epithelial damage in trachea rings treated with TCT

alone. Theoretical modelling of NO? diffusion indicates

that the epithelium is well within the range likely to be

affected by NO? produced in the cartilage (Lancaster,

1994).

It cannot be discounted that NO? production by carti-

lage could play a pathogenic role after epithelial disrup-

tion, when signals for chondrocyte iNOS activation

might penetrate the submucosa. However, our luminal

toxin exposure experiments imply that the initial destruc-

tion of ciliated cells almost certainly has its genesis in

epithelium-derived NO?. Furthermore, the loss of ciliated

cells in pertussis is followed by migration of the remain-

ing epithelial cells to ®ll the voids and keep the epithelial

layer essentially continuous (Goldman et al., 1982).

Therefore, it seems unlikely that cartilage iNOS produc-

tion becomes a signi®cant issue in the pathogenesis of

this particular disease.


Fig. 8. Whole tracheas exposed only on the luminal surface toTCT and endotoxin. Hamster tracheas were perfused with mediumcontaining TCT plus endotoxin for 22 h. Segments of the tracheawere then embedded, sectioned and stained for iNOS (red¯uorescence) as usual. Yellow bars indicate the extent of theepithelial layer; green bars indicate the cartilage. Signi®cant iNOSreactivity can be seen in the epithelium. In this image (183 ´), thered ¯uorescence has been superimposed upon a dim bright®eldimage to demonstrate the location of the cartilage, which wasalmost completely non-¯uorescent. Sections from untreatedtracheas had no iNOS immunoreactivity (not shown).


Pertussis is characterized by very prolonged coughing

(weeks), long after the bacteria have been cleared

(whether naturally or by antibiotic treatment). If the

tracheal epithelium is only damaged mechanically, the

restoration of a normal columnar ciliated epithelium

takes place over the course of several days (Keenan et

al., 1983). The long-term symptomatology and pathology

of pertussis suggests a defect in this epithelial regenera-

tive process. In fact, as secretory cells do not seem to

be damaged by B. pertussis infection (based on ultrastruc-

tural observations), they may continue to produce NO? for

an extended period of time and be at least partly respon-

sible for slow epithelial recovery.

Endotoxin is not actively secreted by B. pertussis, but it

has been previously established that there is a physiologi-

cally signi®cant amount of endotoxin present in normally

growing cultures. During exponential growth of B. pertussis,

endotoxin activity in the culture supernatant is greater than

105 eU mlÿ1. Upon entering stationary phase, endotoxin

accumulates rapidly in the culture supernatant, probably as

a result of outer membrane blebbing and/or some small

amount of bacterial lysis, reaching levels of < 5 ´ 106 eU

mlÿ1 (Heiss, 1993). Therefore, the level of soluble endotoxin

necessary to observe NO? production by tracheal tissue

(< 100 eU mlÿ1) is well within the range probably present

at the mucosal surface during a B. pertussis infection.

The possible importance of endotoxin in the pertussis

syndrome has been generally unappreciated, and there

have been no experimental data to challenge a 1988

authors' conclusion that `B. pertussis endotoxin appa-

rently does not play a crucial role, either in the pathogeni-

city of the bacteria in humans, or as a protective antigen in

immunity to whooping cough' (Chaby and Caroff, 1988).

However, the evidence we have presented indicates that

B. pertussis endotoxin plays an important role in the initial

ciliated cell pathology seen in pertussis, working synergis-

tically with TCT to induce a toxic level of NO? production

from secretory epithelial cells.

Experimental procedures

We used both Escherichia coli lipopolysaccharide (LPS) andB. pertussis lipooligosaccharide (LOS) as the source of endo-toxin activity. These molecules have similar biological activityand are biochemically very similar, differing primarily in thatB. pertussis LOS has a much shorter O-polysaccharidechain (Chaby and Caroff, 1988). Nearly all of the experimentswere performed with both B. pertussis LOS and E. coli LPS,and we detected no difference in activity between the two.Given the identical activity exhibited by these molecules, wehave used the term `endotoxin' throughout this paper to dis-cuss effects observed with B. pertussis LOS or E. coli LPS.Endotoxin activity was determined by the Limulus amoe-bocyte lysate assay (Whittaker Bioproducts), and isexpressed in endotoxin units (eU) per millilitre. Puri®ed B.pertussis lipooligosaccharide (LOS) (< 3 eU ngÿ1) was from

List Biological Laboratories; lipopolysaccharide (LPS) fromE. coli strain 026:B6 (< 1 eU ngÿ1) was from Sigma.

TCT was puri®ed from B. pertussis culture supernatant, aspreviously described (Cookson et al., 1989). The IgG2amonoclonal antibody against murine macrophage nitricoxide synthase (type II) was from Transduction Laboratories;it was used at a concentration of 0.5 mg mlÿ1. The IgM mono-clonal antibody to b-tubulin (C-terminal structural domain)was from Sanbio; it was used at a dilution of 1:49. The sec-ondary antibodies used were Cy3-conjugated goat anti-mouse IgG, Fcg fragment speci®c, at a concentration of2 mg mlÿ1; and ¯uorescein-conjugated goat anti-mouse IgM,m-chain speci®c, used at 15 mg mlÿ1; both were from JacksonImmunoResearch. Fluorescein-conjugated UEA-1 lectin wasobtained from Vector Laboratories and used at 0.2±1 mg mlÿ1. Tissue culture media were obtained from Life Tech-nologies. All other chemicals were from Sigma.

HTE cell monolayer experiments

Hamster trachea epithelial (HTE) cells were grown in F-12medium with 10% fetal bovine serum (FBS), as previouslydescribed (Heiss et al., 1994). Cells were seeded on acid-cleaned coverslips in 24-well plates in minimum essentialmedium (MEM) with 2.5% FBS at a density of 6 ´ 104 cellsper well. After 24 h, toxins were applied in serum-free MEM;after 4 h, FBS was added to a ®nal concentration of 15%.After another 12 h (16 h total exposure to toxins), the cover-slips were rinsed with PBS and processed for immuno¯uores-cent staining as described below for tissue sections.

iNOS Western

Treated HTE cell monolayers were rinsed and solubilizedin sodium dodecyl sulphate sample loading buffer (withoutreducing agent) (Ausubel et al., 1987). Protein concentrationswere determined using a bicinchoninic acid protein assay kit(Pierce), and then 2-mercaptoethanol was added to a concen-tration of 1%. Equivalent amounts of protein were electrophor-esed on a 10% discontinuous gel (Laemmli, 1970; Ausubel etal., 1987), and transferred to nitrocellulose. The blot was incu-bated with antibody against nitric oxide synthase (see above)at 0.5 mg mlÿ1, and the iNOS protein detected using anenhanced chemiluminescence kit (Amersham) according tothe manufacturer's directions.

Tracheal ring assays

Tracheal organ cultures were established as previouslydescribed (Goldman et al., 1982). Brie¯y, tracheas wereremoved from male Golden Syrian hamsters (Charles RiversLaboratories), at least 58 days old. The tracheas were sec-tioned into rings and placed into separate wells of a 48-welltissue culture plate in 250 ml of serum-free F-12 medium sup-plemented with antibiotics. Tracheal rings were maintained ina humidi®ed 95% air/5% CO2 atmosphere at 378C. Generally,rings were held in normal medium for at least 12 h beforemoving them into new wells containing the samples to betested, also in 250 ml of F-12 medium. Rings were visuallymonitored to assess ciliary activity. Nitrite accumulation in



the medium was assessed using the Griess reagent, asdescribed previously (Heiss et al., 1994). All procedures forobtaining hamster tracheal tissue were approved by theWashington University Animal Studies Committee.

Bordetella pertussis infection of tracheal rings

Virulent B. pertussis Tohama I was grown on agar platesmade with modi®ed Stainer±Scholte medium (SSM) (Stainerand Scholte, 1971; Hewlett and Wolff, 1976) and 10% de®bri-nated sheep blood (Colorado Serum Co.). Liquid cultureswere started in SSM supplemented with 0.15% casaminoacids (Difco Laboratories) and then subcultured in normalSSM. Actively growing bacterial cultures were used for allinfection experiments.

Infection of tracheal rings was performed essentially asdescribed previously (Collier et al., 1977). A B. pertussis culturewas diluted to an OD540�0.05 in medium consisting of a 1:1mix of SSM (titrated to pH 7.3 with HCl) and 2´ strength F-12(prepared from powder), without any added antibiotics. Tra-cheal rings, prepared as described above, were incubated in250 ml of this medium, with or without bacteria, for 3 h. Therings were rinsed three times in PBS to remove unattached bac-teria, blotting on sterile Kimwipes between each rinse. Incuba-tion was continued in a new well, in the same medium withoutbacteria.

Tissue processing and immuno¯uorescent staining

Toxin-treated rings and rings infected with bacteria were pro-cessed identically. They were blotted on a Kimwipe andplaced into a freezing mould containing OCT embedding com-pound (Miles Inc.); several rings were often embeddedtogether. The bottom of the mould was held in a dry ice/etha-nol bath until the compound was frozen, and then held atÿ708C until sectioning. Sections of 6 mm thickness were cutat ÿ208C and transferred to glass slides that had been treatedwith Vectabond reagent (Vector Laboratories). The slideswere stored at ÿ708C until use.

Slides were not air-dried before use because this greatlydiminished iNOS reactivity. Fixative solution was preparedby heating a 4% mixture of paraformaldehyde in PBS to608C and adding NaOH to 2±3 mM until clari®cation; thenHCl was added to neutralize the solution. Slides wereimmersed in freshly prepared ®xative for 20 min, then rinsedby immersion in PBS for 3 min. The tissue was permeabilizedby immersion in 0.2% Triton X-100 in PBS for 3 min, followedby four 3-min immersions in PBS. Approximately 50 ml block-ing solution (1% normal goat serum, 0.2% crystallized BSA)was applied to the sections for 20 min. Primary antibody in0.2% BSA in PBS was applied in a similar volume, andthe slides were incubated at room temperature for 3±5 h ina humidi®ed chamber. Unbound primary antibody wasremoved by four 3-min immersions in PBS, and secondaryantibodies were applied in the same BSA /PBS mixture.For UEA-1 staining, the ¯uorescein-conjugated UEA-1 lectinwas included with the secondary antibody. After 45 min,secondary antibody was rinsed away by a 3-min immersionin PBS, followed by one 10 min immersion in PBS containing< 0.1 mg mlÿ1 Hoechst stain no. 33258 (bis-benzimide), andtwo more 3 min PBS immersions. Excess liquid was wiped

away, mounting medium applied, and a coverslip was sealedin place with nail polish. Mounting medium consisted of 1:1mix of glycerol with PBS, pH 8.0, plus 0.5 mg mlÿ1 n-propylgallate to reduce ¯uorescence photobleaching.

Microscopy, photography and image processing

Samples were examined using epi¯uorescence excitation,Nomarski differential interference contrast (DIC), andphase-contrast light microscopy. Most samples were exam-ined using an Olympus BX60 microscope, equipped with ahigh-resolution low-light CCD camera (Optronics Engineer-ing). Images were captured on a Macintosh PowerPC compu-ter system using a video capture card (Truevision) and IPLabSpectrum software (Scanalytics). Some samples were photo-graphed using a Leitz Laborlux 12 microscope, and theimages were then transferred to the Macintosh using a slidescanner (Microtek International). Digital overlays and otherimage processing were carried out with ADOBE PHOTOSHOP

and IPLAB SPECTRUM, and matched samples in each experimentwere processed in an identical fashion. Images shown in thispaper were selected as representative of the results seen inexamining a minimum of three sections from duplicate or tripli-cate tracheal ring samples, and all reported results were repli-cated in at least three separate tracheal ring experiments.

Acknowledgements

This work was supported by Public Health Service grantsAI22243 and HL56419 (to W.E.G.) and AI07172 (to Washing-ton University).

References

Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seid-man, J.G., Smith, J.A., and Struhl, K. (eds) (1987) CurrentProtocols in Molecular Biology. New York: John Wiley &Sons.

Becci, P.J., McDowell, E.M., and Trump, B.F. (1978) Therespiratory epithelium. II. Hamster trachea, bronchus,and bronchioles. J Natl Cancer Inst 61: 551±561.

Chaby, R., and Caroff, M. (1988) Lipopolysaccharides of Bor-detella pertussis endotoxin. In Pathogenesis and Immunityin Pertussis. Chichester: John Wiley & Sons, pp. 247±271.

Charles, I.G., Palmer, R.M., Hickery, M.S., Bayliss, M.T.,Chubb, A.P., Hall, V.S., et al. (1993) Cloning, characteriz-ation, and expression of a cDNA encoding an induciblenitric oxide synthase from the human chondrocyte. ProcNatl Acad Sci USA 90: 11419±11423.

Collier, A.M., Peterson, L.P., and Baseman, J.B. (1977)Pathogenesis of infection with Bordetella pertussis inhamster tracheal organ culture. J Infect Dis 136: S196±S203.

Cookson, B.T., Cho, H.-L., Herwaldt, L.A., and Goldman,W.E. (1989) Biological activities and chemical compositionof puri®ed tracheal cytotoxin of Bordetella pertussis. InfectImmun 57: 2223±2229.

Dinarello, C.A. (1991) In¯ammatory cytokines: Interleukin-1and tumor necrosis factor as effector molecules in autoim-mune diseases. Curr Opin Immunol 3: 941±948.



Flak, T.A., and Goldman, W.E. (1996) Autotoxicity of nitricoxide in airway disease. Am J Respir Crit Care Med 154:S202±S206.

Fleming, T.J., Wallsmith, D.E., and Rosenthal, R.E. (1986)Arthropathic properties of gonococcal peptidoglycan frag-ments: Implications for the pathogenesis of disseminatedgonococcal disease. Infect Immun 52: 600±608.

Goldman, W.E., and Baseman, J.B. (1980) Selective isola-tion and culture of a proliferating epithelial cell populationfrom the hamster trachea. In Vitro 16: 313±319.

Goldman, W.E., Klapper, D.G., and Baseman, J.B. (1982)Detection, isolation, and analysis of a released Bordetellapertussis product toxic to cultured tracheal cells. InfectImmun 36: 782±794.

Gordon, R.E., Lane, B.P., and Miller, F. (1977) Electronmicroscope demonstration of tubulin in cilia and basalbodies of rat tracheal epithelium by the use of an antitubu-lin antibody. J Cell Biol 75: 586±592.

Heiss, L.N. (1993) Cellular Mediators of Bordetella pertussisTracheal Cytotoxin Damage to the Respiratory Epithelium.PhD Dissertation. Division of Biology and BiomedicalSciences, Washington University: St. Louis, MO.

Heiss, L.N., Moser, S.A., Unanue, E.R., and Goldman, W.E.(1993) Interleukin-1 is linked to the respiratory epithelialcytopathology of pertussis. Infect Immun 61: 3123±3128.

Heiss, L.N., Lancaster, Jr, J.R., Corbett, J.A., and Goldman,W.E. (1994) Epithelial autotoxicity of nitric oxide: Role inthe respiratory cytopathology of pertussis. Proc NatlAcad Sci USA 91: 267±270.

Hewlett, E., and Wolff, J. (1976) Soluble adenylate cyclasefrom the culture medium of Bordetella pertussis: Puri®ca-tion and characterization. J Bacteriol 127: 890±898.

Ikebe, T., Hirata, M., Yanaga, F., and Koga, T. (1993) Syner-gism between muramyl dipeptide and lipopolysaccharide inthe inhibition of glycosaminoglycan synthesis in cultured ratchondrocytes. Ann Rheum Dis 52: 32±36.

Keenan, K.P., Wilson, T.S., and McDowell, E.M. (1983)Regeneration of hamster tracheal epithelium after mechan-ical injury. IV. Histochemical, immunocytochemical andultrastructural studies. Virchows Archiv B 43: 213±240.

Kennedy, A.R., Desrosiers, A., Terzaghi, M., and Little, J.B.

(1978) Morphometric and histological analysis of thelungs of Syrian golden hamsters. J Anat 125: 527±553.

Laemmli, U.K. (1970) Cleavage of structural proteins duringthe assembly of the head of the bacteriophage T4. Nature227: 680±685.

Lancaster, Jr, J.R. (1994) Simulation of the diffusion andreaction of endogenously produced nitric oxide. Proc NatlAcad Sci USA 91: 8137±8141.

Mariassy, A.T., Plopper, C.G., St. George, J.A., and Wilson,D.W. (1988) Tracheobronchial epithelium of the sheep. IV.Lectin histochemical characterization of secretory epithe-lial cells. Anat Rec 222: 49±59.

McCartney-Francis, N., Allen, J.B., Mizel, D.E., Albina, J.E.,Xie, Q.-W., Nathan, C.F., and Wahl, S.M. (1993) Suppres-sion of arthritis by an inhibitor of nitric oxide synthase. JExp Med 178: 749±754.

McDowell, E.M., Combs, J.W., and Newkirk, C. (1983) Aquantitative light and electron microscopic study of ham-ster tracheal epithelium with special attention to so-calledintermediate cells. Exp Lung Res 4: 205±226.

Ross, M.H., Romrell, L.J., and Kaye, G.I. (1995) Histology: AText and Atlas. Baltimore, MD: Williams & Wilkins.

Shimizu, T., Nettesheim, P., Mahler, J.F., and Randell, S.H.(1991) Cell type-speci®c lectin staining of the tracheobron-chial epithelium of the rat: Quantitative studies with Griffoniasimplicifolia I isolectin B4. J Histochem Cytochem 39: 7±14.

Shimizu, T., Nettesheim, P., Ramaekers, F.C.S., and Ran-dell, S.H. (1992) Expression of `cell-type-speci®c' markersduring rat tracheal epithelial regeneration. Am J Respir CellMol Biol 7: 30±41.

Stadler, J., Stefanovic-Racic, M., Billiar, T.R., Curran, R.D.,McIntyre, L.A., Georgescu, H.I., et al. (1991) Articularchondrocytes synthesize nitric oxide in response to cyto-kines and lipopolysaccharide. J Immunol 147: 3915±3920.

Stainer, D.W., and Scholte, M.J. (1971) A simple chemicallyde®ned medium for the production of phase I Bordetellapertussis. J Gen Microbiol 63: 211±220.

Taskiran, D., Stefanovic-Racic, M., Georgescu, H., andEvans, C. (1994) Nitric oxide mediates suppression of car-tilage proteoglycan synthesis by interleukin-1. BiochemBiophys Res Commun 200: 142±148.



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Signalling and cellular specificity of airway nitric oxide production in pertussis