human nasal epithelium: characterization and effects of in vitro exposure to sulfur dioxide

Human Nasal Epithelium: Characterization and Effects of In Vitro Exposure to Sulfur Dioxide

Michael S. McManus, Leonard C. Altman, Jane Q. Koenig, Daniel L. Luchtel, David S. Covert, Frank S. Virant, and Coralie Baker

ABSTRACT: Human nasal turbinate tissue fiom surgical specimens was dissected fiee of connective t ime, and primary epithelial cultures were established by explant techniques. Transmission electron microscopy revealed that cultured cells retained homogeneous cytoplasmic granules, tonofilaments, and desmosomes and formed a homogeneous mono- laym f i e epithelial cells stained positively witb cytokeratin antibodies AE1, AE3, and 35BHll but failed to stain with two other cytokeratin antibodies, AEZ and 34BElZ. Staining was also positive with anti-desmoplakin I and 11 but negative with anti- vimentin (43BE8), anti-desmin, and anti-human factor v711 antibodies. Cultured cells were exposed to jltered air or sulfur dioxide at 1-5 ppm for 30-60 min. Although there was no increase in cell lysis as measured by chromium-51 release, SO, exposure sign$- cantly inhibited [3H]leucine incorporation compared to air exposure. This effect was dependent on both SO, concentration and exposure duration. Control experiments revealed that these SO, efects were not caused by the [H'] load produced by SO, exposure. Electron microscopy of cells exposed to air or SO, did not show any signtficclnt morphological differences.

INTRODUCTION

Sulfur dioxide is a common ambient and occupational pollutant. Prior studies have shown that with nasal breathing greater than 99.9% of this irritant gas is absorbed in the upper airways [l, 21. Exposures of animals and humans to high concentrations of SO, have been shown to cause airway epithelial cell

From the Departments of Environmental Health, Medicine, and Division of Allergy and Infectious Dis-

Address all correspondence to Michael S. McManus, M.D., Department of Medkine, Pulmonary Division,

Received 12 September 1988; accepted 6 May 1989.

use, University of Wiington, h t t l e , Wiington.

37-131, UCLA Center for the Health Sciences, Los Angeks, CA 90024-1690.

Experimental Lung Research 152349-865 (1989) Copyright 0 1989 by Hemisphere Publishing Corporation 849

Exp

Lun

g R

es D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

B H

eide

lber

g on

11/

15/1

4Fo

r pe

rson

al u

se o

nly.

850 M. S . McManus et al.

death [3, 41. In addition, in vivo and in vitro animals studies have demonstrated that chronic exposure to SO, causes morphological changes in airway mucosa, an increase in the relative density of secretory cells, impaired mucociliary transport, and increased epithelial permeability [S-131. In vivo exposures of human subjects to SO, have repeated demonstrated that relatively low concentrations of this gas are capable of producing significant decreases in both upper and lower airway function. Andersen et al. [14] demonstrated that exposure of normal subjects to SO, at 1, 5, and 25 ppm for up to 6 h increased nasal airflow resistance and impaired pulmonary function in a fashion that was both exposure duration and SO, concentration dependent. Simi- larly, other investigations have documented impaired upper and lower airway function after SO, inhalation [15-191. Although the upper airways effectively remove SO, from inspired air, nasal inhalation affords only partial protection from SO,-induced bronchoconstriction [20, 2 11. Sulfur dioxide may mediate airway reactivity by neuroreflex mechanisms triggered in the proximal por- tion of the respiratory tract [22]. In addition, direct lung epithelial damage by SO,, causing exposure of sensory nerve endings, loss of barrier function, and loss of epithelial relaxant factor production, could explain the increased bronchial sensitivity of asthmatic subjects to SO, [23]. Laitinen and co-workers [24] have shown that microscopic evidence of airway epithelial damage and increased exposure of sensory nerves is associated with bronchial hyperre- sponsiveness in asthmatic subjects. The demonstrated ability of SO, to damage the airway epithelium and current evidence for the role of the airway epithelial cell in mediating and modulating the pulmonary response to vari- ous irritating stimuli prompted the present investigation [25].

We examined the effect of low-level short-term SO, exposure on cultured human nasal epithelial cells. A range was selected of SO, exposure levels that have been shown to cause significant physiologic effects in both

normal (5 ppm) [26, 271 and atopic (I ppm) [18, 191 subjects. This study demonstrates a methodology for characterizing and examining the effects of gases and airborne toxins in cultured human airway epithelium and demonstrates that low-level SO, exposure can adversely affect airway epithelial cells.

METHODS

Materials

Base media (Dulbecco’s modified Eagle’s medium, Ham’s nutrient medium F- 12 with L-glutamine, and Ham’s minimal essential medium), phosphate- buffered saline (PBS), and HEWS buffer were purchased from M. A. Whitta- ker Bioproducts (Wilkerville, MD) and Joklik’s calcium-free minimal essential medium from Gibco-Life Technologies (Grand Island, NY). Fungizone and antibiotics were from Calbiochem-Behring (La Jolla, CA) and Nu-serum from

Exp

Lun

g R

es D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

B H

eide

lber

g on

11/

15/1

4Fo

r pe

rson

al u

se o

nly.

In Vitro Exposure to Sulfur Dioxide 85 1

Collaborative Research (Lexington, MA). All radiolabeled compounds were acquired from Dupont-New England Nuclear (Boston, MA). Primary monoclonal antibodies AE1, AE2, and AE3 were the kind gifts of Dr. T.-T. Sun of New York University, and antibodies 34BE12, 35BH11, and anti-vimentin (43BES) were the gifts of Drs. Gown and Vogal of the University of Washing- ton. Anti-human factor VIII related antigen was purchased from ATAB Atlan- tic Antibodies-Charles River (Scarborough, ME), antidesmoplakin I & II and anti-desmin from Boehringer Mannheim (West Germany), fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG F(ab ’ ), from Cooper Biomedical (Malvern, PA), and FITC conjugated goat anti-rabbit IgG F(ab ‘)2

from Miles-Yeda LTD Research Products (Israel).

Specimens

Nasal mucosal specimens were obtained from patients undergoing excision of inferior and middle turbinate tissue primarily for upper airway obstruction. Specimens from patients with tumors, polyps, chronic rhinitis or sinusitis, cystic fibrosis, or tissue that did not appear normal by light microscopy were not used. Specimens were obtained from 47 patients, 24 male and 23 female, with a mean age of 36 years ranging from 17 to 56 years. All patients had given informed consent, and the study was approved by the University Hu- man Subjects Committee.

Tissue Preparation and Culture

Human nasal epithelium was cultured by an explant technique similar to that described by others [28-311. Tissue specimens were rinsed 3 times in Joklik‘s calcium-free minimal essential medium supplemented with 60 IU/ml penicillin, 60 pg/ml streptomycin sulfate, and 50 pg/ml gentamicin sulfate. After rinsing, and under sterile conditions, the epithelium was dissected free of all visible underlying connective tissue until only a thin membrane of translu- cent pink epithelium remained. The dissection was performed under a dissect- ing microscope, first teasing away the bulk of the connective tissue and then using a combination of blunt and sharp dissection. The epithelium was rinsed 3 times in fresh medium, diced into small slices of approximately 1 mm2, and placed into 35-mm plastic culture wells at 10-12 explants per well. Explants were covered with a minimal amount of medium, a 1 : 1 mixture of Ham’s F- 12 : DMEM supplemented with 10% Nu-serum, 250 pg/ml L-glutamine, 60 IU/ml penicillin, 60 pg/ml streptomycin sulfate, %% Fungizone, and 20 mmol/ml HEPES buffer, and incubated in humidified 95% air-5% CO, at 37°C for 24 h to maximize explant adherence. After 24 h, 1.5 cm’ of culture medium was added to each well and subsequently changed every 2-3 days.

Exp

Lun

g R

es D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

B H

eide

lber

g on

11/

15/1

4Fo

r pe

rson

al u

se o

nly.

852 M. S. McManus et 4.

Human nasal epithelium cultured by this method reached near confluence at approximately 14 days, at which time the specimens were inspected by inverted phase-contrast microscopy and, if free of fibroblast contamination, were treated with 0.25% trypsin and 0.02% EDTA for 3-5 min at 37OC. Suspended cells were counted with a hemocytometer and passaged into 24- well Falcon microtiter plates (Becton Dickson, Oxnard, CA) at 6 x lo4 cells per well. The dissociated cells used for subculture were greater than 95% viable by trypan blue dye exclusion, and their plating efficiency at 24 h was 79.2% (SEM = 1.1, n = 10). The cells used in each exposure experiment were derived from a single donor.

Characterization of Cultured Human Nasal Epithelium

Phase-contrast microscopy was performed using an inverted phase-contrast microscope (Nikon, Tokyo, Japan), and photographs of the cultures were made with a vertically mounted 35-mm camera and Kodak Tri-X film. For scanning electron microscopy, cell cultures were grown or passaged as described above on glass cover slips. Cultures were fixed by immersion of the coverslips in a modified Karnovsky's fixative (1% formaldehyde, 2% glutar- aldehyde in 0.1 M sodium cacodylate buffer, pH 7.4) for 1-2 h at room temperature, rinsed in 0.16 M cacodylate buffer, and postfixed in 1.0% OSO, in 0.15 M cacodylate buffer for 1 h. Specimens were then dehydrated and critical-point dried, sputtercoated with gold palladium, and examined in a JEOL JSM-35U scanning electron microscope at 15 kV.

For transmission electron microscopy, cells grown in plastic culture wells were fixed as described above. The cultures were dehydrated in ethanol and embedded in Epon. Sections were cut parallel and, after reembedding, per- pendicular to the culture monolayers and examined with a JEOL 100-S transmission electron microscope.

The epithelial nature and purity of primary and passaged cell cultures were verified by indirect immunofluorescence using a panel of monoclonal and polyclonal antibodies as described below [32-351. Cell cultures were hydrated in PBS and fixed in 100% methanol at 20°C for 10 min and then washed with PBS. Fixed cells were extracted with 0.5% Triton X-100 for 15 min at 2OoC and then incubated with the primary antibody for 1 h at room temperature followed by 3 washes with PBS of 15 min each. The specimens were incubated for 30 min at room temperature in a dark humid chamber with the second antibody, FITC-conjugated IgG F(ab ')z fragment specific goat anti- mouse or goat anti-rabbit. The stained specimens were again washed 3 times with PBS and preserved with a 1 : 1 glycerol : PBS mounting medium on glass coverslips and stored at 4OC. Stained and mounted specimens were viewed with a Zeiss photomicroscope with an epifluorescene illuminator Ill RS and a high-intensity halogen illuminator.

Exp

Lun

g R

es D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

B H

eide

lber

g on

11/

15/1

4Fo

r pe

rson

al u

se o

nly.

In Vitro Exposure to Sulfur Dioxide 853

Environmental Exposures

An environmental exposure system was constructed that allowed simulta- neous exposure of cells to either filtered air alone or filtered air with SO, added at controlled concentrations (Fig. 1). For each experiment, sets of cultured cells used for control and SO, exposure were derived from the same surgical specimen, that is, the same patient. Pairs of 24-well culture plates containing cultured human nasal epithelial cells were simultaneously exposed in identical 0.5-ft’ exposure chambers on platforms tilted 15 degrees and rotat- ing at 1.0 rpm above a heated water bath. The exposure gas flowed into the chamber at 300 standard ft’/h (SCFH) and out via negative-pressure exhaust ports adjusted to maintain chamber pressure at 1 atm. The temperature inside the chambers was monitored continuously and maintained at 37OC. The humidity of the input gas was maintained at 80% with an H,O humidifier and continuously monitored with a dew-point hygrometer (General Eastern, Sys- tem 1lOODC Watertown, MA). The humidity within the exposure chambers under these conditions was greater than 95% as measured with dry- and wet- bulb thermometers. Sulfur dioxide was added from a compressed gas source. The flow of SO, was adjusted and continuously monitored by a pulsed fluo- rescent SO, analyzer (Thermo Electron, Series 43, Hopkinton, MA). Both chambers received CO, maintained at 5% of total input continuously monitored with a medical gas analyzer (Beckman, model LB-2, Irvine, CA).

Measurement of Cell Lysis

Cell lysis was assayed by 5’Cr release 136, 371. In brief, nasal epithelial cells were plated in microtiter plates at 6 x lo4 cells per well in 1 ml of medium and incubated for 24 h with sodium [51Cr]chromate, 1 Ci/ml in PBS, at a final concentration of 20 pCi/ml or 1.76 x lo7 cpm/ml. After incubation, the cells were gently washed 4 times and then 150 p1 of Ham’s F-12 with 0.05% gel and HEPES at 20 mmol/ml was added to each well. Paired plates were then exposed to filtered air alone or to filtered air, with 5 ppm SO, added for 60 min. Release of soluble 51Cr, a measure of cell lysis, was determined by aspirating 50 pl of supernatant from each well, transferring aliquots to glass tubes, and counting the gamma emissions in a gamma counter (Pack- ard Gamma Counter, 5000 Series). The cells were washed, lysed by treatment with 1.0% Triton X-100, and the gamma emissions in the lysate were counted. Percent lysis was calculated as [cpm supernatant/(cpm cells + cprn supernatant)] x 100.

Measurement of [3HlLeucine Incorporation

Cellular leucine incorporation was determined by adding 150 p1 of 1 pCi/ml (1.35 x lo5 cpm) [‘H]leucine to cells in microtiter wells, which were then

Exp

Lun

g R

es D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

B H

eide

lber

g on

11/

15/1

4Fo

r pe

rson

al u

se o

nly.

854 M. S . McManus et al.

thermometer motor

mlcrolitef platform wlth ceile

plate

heating unit heating unit

Figure 1 Schematic of cell exposure system.

incubated for 1 h at 37°C in 95% air-5Oh CO, [38]. After this, paired culture plates were exposed to filtered air alone or SO, at 1,3, or 5 ppm for 30 or 60 rnin. Next, the cells were incubated at 37°C for 24 h and then collected and washed on glass filters using an automatic cell harvester. Incorporation of ['Hlleucine was measured in a liquid scintillation counter (Beckman model 3801, Irvine, CA). Percent inhibition of cellular [3H]leucine incorporation was calculated as [(cpm air-exposed cells - cpm SO,-exposed cells)/(cpm air- exposed cells)] x 100.

Determination and Effect of Hydrogen Ion Load

To determine the cellular effect of the change in pH or acid load caused by exposure to SO,, cells were prepared in an identical fashion as for the [3H]leucine incorporation assays and exposed to air or SO, at 5 ppm for 60 min. Afterward, the medium from six wells of each plate was pooled and the pH of the pooled mecha was determined with a pH/ion meter (Corning model 150, Corning, NY). Determinations of pH were made preexposure (baseline), immediately postexposure and after incubation at 37°C in 5% CO, for 1 h. To examine the effect of acid load on ['Hlleucine incorporation, cells were prepared and incubated with ['Hlleucine as described above, after which 0.01 N HCI or 0.02 N acetic acid was added over 60 min. The volume of acid added was that predetermined to cause a fall in pH equal to that produced by exposure to 5 pprn SO, for 60 min. As controls, HEPES buffered Ham's F-12

Exp

Lun

g R

es D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

B H

eide

lber

g on

11/

15/1

4Fo

r pe

rson

al u

se o

nly.


or PBS was added to cells at the same volume as acid or no solution was added. After addition of acid or buffer, cells were reincubated for 24 h and [3HJleucine incorporation was assayed as described above.

Statistical Methods

Data were analyzed by analysis of variance (ANOVA) [39]. Before statistical analysis, percentage data were normalized by the angle transformation. Two- way analysis of variance was performed with exposure gas (air or SO,) and SO, concentration as grouping or between-factor variables and exposure duration as a within-factor variable. If ANOVA revealed a significant effect, multi- ple comparisons were made using the paired- or two-sample t-test. The BMDP statistical software package was used for all computer computations 1401.

RESULTS

Characterization of Cell Cultures

Inverted-phase contrast microscopy revealed a uniform monolayer of polygonal cells that were homogeneous in shape and density (Fig. 2). Scanning electron microscopy of primary explant cultures also showed a morphologically homogeneous monolayer of tightly packed polygonal cells that were epithe-

Figure2 Phase-contact micrograph of human nasal epithelium in primary culture at day 11. Note formation of a uniform monolayer of polygonal cells with sparse cilia. x 250.

Exp

Lun

g R

es D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

B H

eide

lber

g on

11/

15/1

4Fo

r pe

rson

al u

se o

nly.

856 M. S. McManus et al.

lial in nature but only sparsely ciliated. Transmission electron microscopy revealed uniform cellular ultrastructure with welldeveloped desmosomes and large numbers of mitochondria, tonofilaments, and homogeneous cytoplasmic granules (Fig. 3). Nasal epithelium cultured by the methodology described was usually free from fibroblast contamination. The occasional culture contaminated with fibroblasts was discarded.

Indirect immunofluorescence studies confirmed the epithelial nature and purity of cell cultures. Both primary and passaged cell cultures stained identi- cally with the panel of monoclonal and polyclonal antibodies. The antibodies, their specificities, and the results of immunofluorescence staining for both primary and passaged cells are summarized in Table 1.

Cell Lysis

There was no difference in lysis of cells exposed to 5 ppm SO, for 60 min compared to air exposed cells. For cells exposed to air, mean cell lysis was 15.3% (SEM = 0.Q and for cells exposed to SO,, lysis was 15.3% (SEM =

l.O), n = 20.

['HILeucine Incorporation

Exposure of cells to SO, at 5 pprn for 60 min produced a highly significant inhibition of ['Hlleucine incorporation. Mean uptake of [3H]leucine was 22.7% (SEM = 1.1) of maximum following air exposure and 9.9% (SEM = 0.3) following SO, exposure, n = 9. Expressed in another way, these data reveal that exposure to 5 ppm SO, for 60 min produced a 62.2% (SEM = 4.1, n = 9, p < .OOI) inhibition of [3H]leucine incorporation relative to air exposure. Further, this effect was significant at an SO, concentration as low as 1 ppm: 21.1% inhibition (SEM = 6.4, n = 6, p < .05). The effect of SO, was both concentration and exposure time dependent: time effect p value - 0.017, SO, concentration effect p value < .001 (Table 2).

Acid Load

The initial mean pH of the cell containing medium was 7.51 (SEM = 0.07, n = 10). After exposure to 5 pprn SO, for 1 h the mean p H decreased to 7.42 (SEM = 0.10, n = lo), while air exposure produced no reduction in the pH of the medium; mean pH = 7.53 (SEM = 0.09, n = 10). Thus, exposure to 5 ppm SO, for 60 min produced a mean decrease in p H of 0.095 (SEM = 0.030, n = 15, p < .Ol) compared to air-exposed cells. To determine if SO, produced inhibition of [3H]leucine incorporation indirectly due to pH change or directly as an effect on cellular function, a series of acid load studies were performed. As shown in Table 3, the addition of acetic and hydrochloric acids, although producing a comparable pH change as SO, exposure, did not

Exp

Lun

g R

es D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

B H

eide

lber

g on

11/

15/1

4Fo

r pe

rson

al u

se o

nly.

Figu

re 3

Tr

ansm

issio

n el

ectr

on m

icro

grap

hs o

f hu

man

nasal e

pith

eliu

m. (

A) N

ote

unifo

rm c

ell m

orph

olog

y, x

2300

. (B)

Cel

ls s

how

den

se m

itoch

ondr

ia, t

onof

ilam

ents

and

hom

ogen

e-

ous

cyto

plas

mic

gra

nule

s, x

6000

.

Exp

Lun

g R

es D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

B H

eide

lber

g on

11/

15/1

4Fo

r pe

rson

al u

se o

nly.


Figure 3 Transmission electron micrographs of human nasal epithelium in culture (Continued). (C) Prominent desmosomes adjoining cells, x 20,ooO.

inhibit [3H]leucine incorporation. These data indicate that the inhibitory effect of SO, exposure on ['Hlleucine incorporation was not the result of SO,- induced pH changes. However, the addition of lefold the volume of acetic acid required to mimic the change in pH produced by SO, exposure caused a small but statistically significant inhibition of ['Hlleucine incorporation (19.3% uptake, SEM - 0.4, n - 6, p < .OOS).

Morphological Effects

Cells exposed to air or SO, at 5 ppm for 60 min were examined for morphological changes. Neither scanning or transmission electron microscopy showed significant differences in cell appearance.

Exp

Lun

g R

es D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

B H

eide

lber

g on

11/

15/1

4Fo

r pe

rson

al u

se o

nly.


Table 1 Results of Indirect Immunofluorescence Studies on Human Nasal Epithelial Cells'*b

Monoclonal antibodies, anti-cytokerat ins Specificity Results

AE1 AE2 AE3 34BE12

35BHll

Acidic keratins + 56.5 kDa and 65-67 kDa keratins Basic keratins + 66-kDa and 57-kDa keratins, -

-

less reactive with 51-kDa and 49-kDa keratins, stains full thickness of squamous epithelium

nonsquamous epithelium 54-kDa keratins, stains +

Others

Anti-desmoplakin I and 11 Desmosomal plaque proteins + Anti-vimentin Cells of mesenchymal origin - Antidesmin Muscle cells - Anti-factor VIII Endothelial cells -

'IgG, nonimmune mouse globulin, and FITC controls all negative. %econd Ab: FITC conjugated goat-anti-mouse, or goat-anti-rabbit IgG, F(ab'), fragment specific.

Table 2 Inhibition of [3H]Leucine Incorporation in Nasal Epithelial Cells Exposed to SO,"

Exposure duration

SO, Conc. (min) Mean SEM n P

1 PPm 30 7.6% 10.8 6 > .10

3 PPm 30 24.9% 6.2 7 < .02

5 PPm 30 43.1% 5.1 7 c .002

60 21.1% 6.4 6 < .05

60 34.6% 7.5 7 < .02

60 62.2% 4.1 9 c .001

'Cultured human nasal epithelial cells were labeled with 150 pl of 1 pCi/ml [3H]leucine for 1 h at 37OC. Cells then were exposed to 1,3, or 5 ppm SO, for either 30 or 60 min. Cellular incorporation of ['Hlleucine was measured in a liquid scintillation counter, and the results expressed as mean * SEM percent incorporation.

Exp

Lun

g R

es D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

B H

eide

lber

g on

11/

15/1

4Fo

r pe

rson

al u

se o

nly.


Table 3 Effect of pH on ['HILeucine Uptake"

['HI Leucine Additions to cells uptake SEM n

None PBS Ham's F12 + HEPES HC1 Acetic acid Acetic acid (10 X ) SO,, 60 min Air, 60 min

23 .o% 23.7% 22.2% 25.0% 22.2% 19.3% 9.9%

22.7%

0.5 0.3 0.6 0.3 0.5 0.4 0.3 1.1

6 12 6

6 6' 9' 9

"The effect of acid load on ['Hlleucine uptake was examined after 0.01 N HCI or 0.02 N acetic acid was added for 60 min. The volume of acid added was that predetermined to cause a fall in p H equal to that produced by exposure to 5 ppm SO, for 60 min. The determination of ['H]leucine incorporation is as described in Table 2. h Significant increase of uptake, p < .01. 'Significant inhibition of uptake, p < .W1.

DISCUSSION

This study presents methods for culturing and characterizing human nasal epithelial cells and for studying the effects of toxic gases on these cells in vitro. Small, freshly excised surgical specimens of human nasal epithelium were grown in primary culture by an explant technique. This method allowed us to grow 2-3 x 106 cells from a single 0.8-1.2 cmz surgical specimen taken from a living donor of defined health status. Further, this approach permitted us to expose cells derived from the same donor to test or control atmospheres under identical and narrowly controlled environmental conditions. Human nasal epithelial cells grown under these conditions retained characteristics of differentiated epithelium, including tonofilaments, homogeneous cytoplasmic granules, desmosomes, and occasional cells with motile cilia [41]. In addition, immunofluorescence staining with specific antibodies showed that the cultured cells retained a characteristic epithelial staining profile. Specifically, the cells stained positively with cytokeratin antibodies AEI, AE3, and 35BHll and also with antidesmoplakin antibodies I and II but failed to stain with two other cytokeratin antibodies, AE2 and 34BE12, or with anti-vimentin (43BE8), antidesmin, and anti-human factor VIZI antibodies.

These findings indicate that cultured nasal epithelial cells retained their ability to express acidic and basic cytokeratins (AE1 and AE3) and a cytokeratin specific for nonsquamous epithelium (35BH11). In contrast, the cells did not stain with AE2 or 34BE12, which indicates the absence of terminal keratinization or epidermal type differentiation [42, 431. Finally, the cells did not dedifferentiate and express markers characteristic for cells of mesenchy-

Exp

Lun

g R

es D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

B H

eide

lber

g on

11/

15/1

4Fo

r pe

rson

al u

se o

nly.


mal origin (anti-vimentin), endothelial origin (anti-factor VIII), or muscle (anti-desmin).

Short-term exposure of cultured nasal epithelial cells to SO,, at levels known to produce significant physiologic effects in normal subjects, caused a highly significant impairment of ['Hlleucine incorporation. This impairment was dependent on both exposure duration and SO, concentration. In contrast, SO, exposures caused no apparent cytotoxic effect to these cells.

Current evidence implicates bisulfite ion and not H' or sulfite as the SO, product primarily responsible for the bronchoconstriction induced by SO, inhalation [44]. However, others have found that pathological changes in rat lungs, as a result of high-level SO, exposure, are secondary to the associated accumulation of H' ion [45]. We found that exposure to SO, at 5 ppm for 60 min significantly impaired [SHIleucine incorporation, while addition of an [H'] load did not. This effect of SO, on [3H]leucine incorporation may represent impaired cellular uptake of leucine or a defect in de novo protein synthesis.

Sulfur dioxide is a highly water-soluble gas that goes rapidly into solution as sulfurous acid (H2S03) [46, 471. Sulfurous acid then gives off H' and forms bisulfite (HSO,), which, with loss of a second H', forms sulfite [SO;]. Un- der physiologic conditions SO, in solution exists predominantly as a mixture of bisulfite, sulfite salts, and H' ions. Bisulfite is a highly reactive nucleophil or reducing agent that has been shown to combine with a variety of biologi- cally important macromolecules and to be capable of disrupting cell function [48, 491. Bisulfite anion may inhibit [3H]leucine incorporation by a variety of mechanisms. Bisulfite is capable of reducing disulfide bonds to form S- substituted thiosulfates and a thiol, interrupting the steriochemistry and function of globular proteins and inhibiting enzyme function [50, 511. Bisulfite also has been shown to react with both cytosine and uracil and to inhibit the coupling of transfer RNA with ribosomes and impair protein synthesis at the level of translation [52-541. Also, bisulfite and sulfite have been shown to impair cellular energy metabolism in mammalian tissues. Bisulfite has been shown to decrease cellular stores of ATP, possibly as a result of binding and disrupting the function of pyridine and flavin nucleotides [55, 561. This effect is inhibited by the enzyme sulfite oxidase. Studies on the organ and species variability in activity of sulfite oxidase and capacity to detoxify bisulfite suggest that human lung tissue may be particularly susceptible to this agent [56- 581. The inhibition of fH]leucine incorporation documented in the current study may be a secondary effect of depleted cellular energy stores.

In summary, our study presents a model for studying the effect of gases on human nasal epithelium in vitro and demonstrates that low-level exposure to SO, causes significant concentration- and exposureduration-dependent non- cytolytic inhibition of human upper airway epithelial cell ['HJleucine incorporation. This effect of SO, appears to be independent of the [H'] load accumulated with the SO, exposure. These findings suggest that the adverse

Exp

Lun

g R

es D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

B H

eide

lber

g on

11/

15/1

4Fo

r pe

rson

al u

se o

nly.


effects induced by nasal inhalation of SO, may be in part mediated by altered epithelial functioning. Further, these results indicate that the role of the upper airways in protecting the lungs against SO, is not without inherent risk of altered upper airway function.

Supported by Environmental Pathology/Toxicology training grant ES 07032, grant 5 R 0 1 ES 02366 from the National Institute of Environmental Health Sciences, grant DE 08229 from the National Institutes of Dental Research, and the University of Washington Graduate School Research Fund. The authors would like to express their appreciation to Leslie J. Becker, F. R. Sennewald, M. D., D. M. Madigan, M.D., C. M. Furuya, M.D., and S. K. Clark, M.D., for their assistance in providing tissue specimens and Irma G. McManus for manuscript preparation.

REFERENCES

1. Speizer FE, Frank NR: The uptake and release of SO, by the human nose. Arch Environ Health 12:725-728, 1966.

2. Frank NR, Yoder RE, Brain JD, Yokoyama E: SO, (35S labeled) absorption by the nose and mouth under conditions of varying concentration and flow. Arch Envi- ron Health 18:315-322, 1969.

3. Charan NB, Myers CG, Lakshminarayan S, Spencer TM: Pulmonary injuries associated with acute sulfur dioxide inhalation. Am Rev Respir Dis 119555-560, 1979.

4. Man SFP, Hulberg WC, Mok K, Ryan T, Thomson ABR: Effects of sulfur dioxide on pore populations of canine tracheal epithelium. J Appl Physiol60:416-426, 1986.

5. Hirsch JA, Swenson EW, Wanner A: Tracheal mucous transport in beagles after long-term exposure to 1 ppm sulfur dioxide. Arch Environ Health 30249-253, 1975.

6. Islam MS, Oellig WP, Weller W: Respiratory damage caused by long-term inhalation of high concentration of sulfurdioxide in dogs. Res Exp Med 171:211-218, 1977.

7. Wakabayashi M, Bang BG, Bang FB: Mucociliary transport in chickens infected with Newcastle disease virus and exposed to sulfur dioxide. Arch Environ Health

8. Vai F, Fournier MF, Lafuma JC, Touaty E, Pariente R SO,-induced bronchopathy in the rat: Abnormal permeability of the bronchial epithelium in vivo and in vitro after anatomic recovery. Am Rev Respir Dis 121:851-858, 1980.

9. Majima Y, Swif t DL, Bang BG, Bang FB: Mechanisms of slowing of mucociliary transport induced by SO, exposure. Ann Biomed Eng 13:515-530, 1985.

10. Majima Y, Okuyama H, Bang BG: Effect of SO, on nasal secretory cells. Acta Otolaryngol (Stockh) 102:302-307, 1986.

11. Spicer SS, Chakrin LW, Wardell JR: Effect of sulfur dioxide inhalation on the carbohydrate histochemistry and histology of the canine respiratory tract. Am Rev Respir Dis 110:13-24, 1974.

12. Greene SA, Wolff RK, Hahn EF, Henderson RF, Mauderly JL, Lundgren DL: Sulfur dioxide-induced chronic bronchitis in beagle dogs. J Toxic01 Environ Health 13:945-958, 1984.

32:lOl-107, 1977. Exp

Lun

g R

es D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

B H

eide

lber

g on

11/

15/1

4Fo

r pe

rson

al u

se o

nly.


13. Clark JN, Dalbey WE, Stephenson KB: Effects of sulfur dioxide on the morphology and mucin biosynthesis by the rat trachea. J Environ Pathol Toxic01 4:197- 207, 1980.

14. Andersen I, Lundqvist GR, Jensen PLY Proctor DF: Human response to controlled levels of sulfur dioxide. Arch Environ Health 28:31-39, 1974.

15. Frank R: The effects of inhaled pollutants on nasal and pulmonary flow resistance. Ann Otol Rhino Laryngol 79540-546, 1970.

16. Koenig JQ, Morgan MS, Horike My Pierson WE: The effects of sulfur dioxide on nasal and lung function in adolescents with extrinsic asthma. J Allergy Clin Im- munol 76:813-818, 1985.

17. Newhouse MT, Dolovich My Obminski G, Wolff RK: Effect of TLV levels of SO, and H,SO, on bronchial clearance in exercising man. Arch Environ Health 33:24-32, 1978.

18. Koenig JQ, Pierson WE, Frank R Acute effects of inhaled SO, plus NaCl droplet aerosol on pulmonary function in asthmatic adolescents. Environ Res 22: 145-153, 1980.

19. Sheppard D, Wong WS, Uehara CF, Nadel JAY Boushey HA: Lower threshold and greater bronchomotor responsiveness of asthmatic subjects to sulfur dioxide. Am Rev Respir Dis 122973-878, 1980.

20. Kirkpatrick MB, Sheppard D, Nadel JAY Boushey HA: Effects of the oronasal breathing route on sulfur dioxide induced bronchoconstriction in exercising asthmatic subjects. Am Rev Respir Dis 125:627-631, 1982.

21. Linn WS, Shamoo DA, Spier CE, Valencia LM, Anzar UT, Venet TG, Hackney JD: Respiratory effects of 0.75 ppm sulfur dioxide in exercising asthmatics: Influ- ence of upper-respiratory defenses. Environ Res 30:340-348, 1983.

22. Nadel JA, Salem H, Tamplin B, Tokiwa G: Mechanisms of bronchoconstriction during inhalation of sulfur dioxide. J Appl Physiol20:164-167, 1965.

23. Cuss FM, Barnes PJ: Epithelial mediators. Am Rev Respir Dis (Suppl) 135532- 534, 1987.

24. Laitinen LA, Heino M, Laitinen A, Kava T, Haahtela T: Damage of the airway epithelium and bronchial reactivity in patients with asthma. Am Rev Respir Dis 131599-606, 1985.

25. Hogg JC: Is asthma an epithelial disease?. Am Rev Respir Dis 129:207-208, 1984. 26. Frank DR, Amdur MO, Worcester J, Whittenberger JL: Effects of acute con-

trolled exposure to SO, on respiratory mechanics in healthy male adults. J Appl Physiol 17:252-258, 1962.

27. Snell RE, Luchsinger PC: Effects of sulfur dioxide on expiratory flow rates and total respiratory resistance in normal human subjects. Arch Environ Health

28. Heckman CAY Marchok AC, Nettesheim P: Respiratory tract epithelium in primary culture: Concurrent growth and differentiation during establishment. J Cell Sci 32:269-291, 1978.

29. Lechner JF, Haugen A, McClendon IA, Pettis EW: Clonal growth of normal adult human bronchial epithelial cells in a serum-free medium. In Vitro 18:633- 642, 1982.

30. Wiesel JM, Gamiel H, Vlodavsky I, Gay I, Ben-Basset H: Cell attachment, growth

18:693-698, 1969.

Exp

Lun

g R

es D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

B H

eide

lber

g on

11/

15/1

4Fo

r pe

rson

al u

se o

nly.


characteristics and surface morphology of human upper-respiratory tract epithelium cultured on extracellular matrix. Eur J Clin Invest 1357-63, 1983.

31. Van Scott MR, Yankaskas JR, Boucher RC: Culture of airway epithelial cells: Research techniques. Exp Lung Res 11:75-94, 1986.

32. Woodcock-Mitchell J, Eichner R, Nelson WG, Sun T-T: Immunolocalization of keratin polypeptides in human epidermis using monoclonal antibodies. J Cell Biol 95580-588, 1982.

33. Gown AM, Vogel AM: Monoclonal antibodies to intermediate filament proteins of human cells: Unique and cross-reacting antibodies. J Cell Biol 95414-424, 1982.

34. Wu Y-U, Parker LM, Binder NE, Beckett MA, Sinard JH, Griffiths CT, Rheinwald JG: The mesothelial keratins: A new family of cytoskeletal proteins identified in cultured mesothelial cells and non-keratinizing epithelia. Cell 3 1:693- 703, 1982.

35. Wu R, Yankaskas J, Cheng E, Knoweles MR, Boucher RC: Growth and differentiation of human nasal epithelial cells in culture. Serum-free, hormone- supplemented medium and proteoglycan synthesis. Am Rev Respir Dis 132:311- 320, 1985.

36. Spangberg L: Kinetic and quantitative evaluation of material cytotoxicity in vitro. Oral Surg 35389-401, 1973.

37. Magoffin DA, Kushell DL, Schlesinger RJ: The chromiumdl release cytotoxicity test: Comparison to the agar overlay and cell-growth inhibition tests. In: Brown SA, ed., Cell-Culture Test Methods. Philadelphia, American Society for Testing and Materials, 1983, pp. 94-101.

38. Massaro D, Weiss H, Simon MR: Protein synthesis and secretion by the lung. Am Rev Respir Dis 101:198-206, 1970.

39. Steel RGD, Torrie JH: Principles and Procedures of Statistics: A Biometrical Approach. New York, McGraw-Hill, 1985.

40. Dixon WJ, ed.: BMDP Statistical Software Manual. Los Angeles, University of California Press, 1983 (1985 printing).

41. Fey EG, Wan KM, Penman S: Epithelial cytoskeletal framework and nuclear matrix-inter mehate filament scaffold: Three-dimensional organization and protein composition. J Cell Biol 98:1973-1984, 1984.

42. Gown AM, Vogel AM: Monoclonal antibodies to human intermediate filament proteins: II. Distribution of filament proteins in normal human tissues. Am J Pathol 114:309-321, 1984.

43. Sum T-T, Tseng CG, Huang AJ-W, Lynch MH, Weiss R, Eichner R: Monoclonal antibody studies of mammalian epithelial keratins: A review. Ann NY Acad Sci

44. Fine J, Gordon T, Sheppard D: The roles of pH and ionic species in sulfur dioxide- and sulfite-induced bronchoconstriaion. Am Rev Respir Dis 136~122- 1126, 1987.

45. Gunnison AF, Sellakumar A, Currie D, Snyder EA: Distribution, metabolism and toxicity of inhaled sulfur dioxide and endogenously generated sulfite in the respiratory tract of normal and sulfite oxiddeficient rats. J Toxic01 Environ Health 21:141-162, 1987.

455:307-329, 1985.

Exp

Lun

g R

es D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

B H

eide

lber

g on

11/

15/1

4Fo

r pe

rson

al u

se o

nly.


46. Gunnison AF: Sulfite toxicity: A critical review of in vitro and in vivo data. Food

47. Gunnison AF, Jacobsen DW: Sulfite hypersensitivity. A critical review. CRC Crit

48. Petering DH, Shih NT: Biochemistry of bisulfite-sulfur dioxide. Environ Res

49. Gunnison AF, Benton AW: Sulfur dioxide: Sulfite, interaction with mammalian serum and plasma. Arch Environ Health 22:381-388, 1971.

50. Steinacker A: Presynaptic effects of sodium bisulfite at the frog neuromuscular junction. J Neurosci Res 7:313-319, 1982.

51. Mallon RG, Rossman TG: Effects of bisulfite (sulfur dioxide) on DNA synthesis and fidelity by DNA polymerase I. Chem-Biol Interact 46:lOl-108, 1983.

52. Braverman B, Shapiro R, Szer W: Modification of E. coli ribosomes and coliphage MS2 RNA by bisulfite: Effects on ribosomal binding and protein synthesis. Nu- cleic Acid Res 2501-507, 1975.

53. Shapiro R, Gazit A: Crosslinking of nucleic acids and proteins by bisulfite. Adv Exp Med Biol 86A:633-640, 1977.

54. Walter P, Ibrahim I, Blobel G: Translocation of proteins across the endoplasmic reticulum 1. Signal recognition protein (SW) binds to in-vitro-assembled poly- somes synthesizing secretory protein. J Cell Biol 91545-550, 198 1.

55. Oshino N, Chance B: The properties of sulfite oxidation in the perfused rat liver; Interaction of sulfite oxidase with the mitochondrial respiratory chain. Arch Bio- chem Biophys 170:514-528, 1975.

56. Beck-Speier I, Hinze H, Holzer H: Effect of sulfite on the energy metabolism of mammalian tissues in correlation to sulfite oxidase activity. Biochim Biophys Acta

57. Gunnison AF, Bresnahan CA, Palmes ED: Comparative sulfite metabolism in the

58. Tejnorova I: Sulfite oxidase activity in liver and kidney tissue in five laboratory

Cosmet Toxicol 19:667-682, 1981.

Rev Toxicol 12185-214, 1987.

955-65, 1975.

841 :8 1-89, 1985.

rat, rabbit and rhesus monkey. Toxicol Appl Pharmacol 42:99-109, 1977.

animal species. Toxicol Appl Pharmacol 44:251-256, 1978.

Exp

Lun

g R

es D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

B H

eide

lber

g on

11/

15/1

4Fo

r pe

rson

al u

se o

nly.

human nasal epithelium: characterization and effects of in vitro exposure to sulfur dioxide

Documents