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HOG BARN DUST EXTRACT INCREASES MACROMOLECULAR

EFFLUX FROM THE HAMSTER CHEEK POUCH

Israel Rubinstein1,2,3 and Susanna Von Essen4

Departments of Medicine1, and Biopharmaceutical Sciences2

Colleges of Medicine and Pharmacy, University of Illinois at Chicago,

and Jesse Brown VA Medical Center3, Chicago, Illinois 60612,

and Department of Internal Medicine4, University of Nebraska Medical Center,

Omaha, Nebraska 68198

Running Head: Hog barn dust and plasma exudation

Address for correspondence: Dr. Israel Rubinstein

Department of Medicine (M/C 719)

University of Illinois at Chicago

840 South Wood Street

Chicago, Illinois 60612-7323

Phone: (312) 996-8039

Fax: (312) 996-4665

E-mail: IRubinst@uic.edu

Page 1 of 31 Articles in PresS. J Appl Physiol (March 30, 2006). doi:10.1152/japplphysiol.01092.2005

Copyright © 2006 by the American Physiological Society.

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ABSTRACT

The purpose of this study was to determine whether short-term exposure to an

aqueous extract of hog barn dust increases macromolecular efflux from the intact

hamster cheek pouch and, if so, to begin to determine the mechanism(s) underlying this

response. By using intravital microscopy, we found that suffusion of hog barn dust

extract onto the intact hamster cheek pouch for 60 min elicited a significant,

concentration-dependent leaky site formation and increase in clearance of FITC-labeled

dextran (mol mass, 70 kDa). This response was significantly attenuated by suffusion of

catalase (60 U/ml), but not by heat-inactivated catalase, and by pretreatment with

dexamethasone (10 mg/kg, i.v.)(p<0.05). Catalase had no significant effects on

adenosine-induced increase in macromolecular efflux from the cheek pouch. Suffusion

of hog bran dust extract had no significant effects on arteriolar diameter in the cheek

pouch. Taken together, these data indicate that hog bran dust extract increases

macromolecular efflux from the in situ hamster cheek pouch, in part, through local

elaboration of reactive oxygen species that are inactivated by catalase. This response is

specific and attenuated by corticosteroids. We suggest that plasma exudation plays an

important role in the genesis of upper airway dysfunction evoked by short-term

exposure to hog barn dust.

Key Words: microcirculation; post-capillary venules; reactive oxygen species; catalase;

dexamethasone; plasma exudation; FITC-dextran; hamster

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INTRODUCTION

A growing body of clinical evidence indicates that short-term exposure of workers

and healthy volunteers to hog barn dust is associated with intense airway inflammation

manifested in the upper airway as marked nasal congestion (4, 16-19, 25, 33-36). This

response, in turn, compromises upper airway patency and may adversely affect work

performance of these individuals (12-14, 18). Hence, there is an ongoing need to

elucidate mechanisms underlying hog barn dust-induced upper airway inflammation so

that appropriate preventive and therapeutic interventions could be implemented

accordingly (4, 32).

A hallmark of the host inflammatory response to upper airway injury is plasma

exudation from post-capillary venules leading to interstitial edema and tissue

dysfunction (7, 15, 20, 26). To this end, Kolbeck et al (16) and Ek et al (4) showed that

short-term exposure of the nasal mucosa of healthy individuals to hog barn dust is

associated with an increase in albumin concentration in the nasal lavage. These data

implied that hog barn dust evoked plasma exudation from the nasal mucosa. Support

for this notion came from the study of Vesterberg et al (33) who showed that 3-h

exposure of healthy volunteers to swine dust is associated with an increase in the

concentration of α2-macroglobulin, a robust biomarker of plasma exudation, in the

bronchoalveolar lavage fluid of these individuals. However, the mechanism(s)

underlying the edemagenic effects of hog barn dust in the upper airway were not

investigated in these studies.

Hence, the purpose of this study was to begin to address this issue by

determining whether short-term exposure to an aqueous extract of hog barn dust

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increases macromolecular efflux from the intact hamster cheek pouch and, if so, to

determine the mechanism(s) underlying this response.

METHODS

General methods

Preparation of animals. Adult male golden Syrian hamsters weighing 120-130 g

were used in these studies as previously described in our laboratory and by other

investigators (1, 5, 6, 20-22, 28-30). Each animal was anesthetized with pentobarbital

sodium (6 mg/100 g body weight i.p.). A tracheostomy was performed to facilitate

spontaneous breathing. The left femoral vein was cannulated to inject the intravascular

tracer, fluorescein isothiocyanate-labeled dextran (FITC-dextran; mol mass, 70kDa) and

supplemental anesthesia (2-4 mg/100 g body weight/h). The left femoral artery was

cannulated to obtain arterial blood samples and to monitor systemic arterial pressure

and heart rate during the experiment. Body temperature was kept constant (37-38 °C)

during the experiment using a heating pad.

To visualize the microcirculation of the cheek pouch, we used a method

previously described in our laboratory and by other investigators (1, 5, 6, 20-22, 28-30).

Briefly, the left cheek pouch was spread gently over a small plastic base plate and an

incision was made in the skin to expose the cheek pouch membrane. The avascular

connective tissue layer was carefully removed and a plastic chamber was positioned

over the base plate and secured in place by suturing the skin around the upper

chamber. This chamber contained the suffusion fluid. This arrangement forms a triple-

layered complex: the base plate, the upper chamber and the cheek pouch membrane

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exposed between both plates. The hamster was then transferred to a heated

microscope stage. The chamber was connected through thermally insulated tubing to a

reservoir containing warmed (37-38 °C) bicarbonate buffer (composition, in mM: NaCl,

131.9; KCl 2.95; CaCl2, 1.48; MgCl2, 0.76; NaHCO3, 11.87) that enabled continuous

suffusion of the cheek pouch. The buffer was bubbled continuously with 95% N2-5%

CO2 (pH=7.4). The temperature of the suffusate in the chamber was checked

periodically using a thermistor and kept at 37°C throughout the experiment by adjusting

the buffer’s temperature in the reservoir accordingly. The chamber was also connected

via a three-way valve to an infusion pump (Sage Instruments, Cambridge, MA) that

allowed for constant administration of drugs into the suffusate.

Determination of clearance of macromolecules. The cheek pouch

microcirculation was visualized with an Olympus microscope (Olympus America Inc,

Melville, NY) coupled to a 100-W mercury light source at a magnification of x40.

Fluorescence microscopy was accomplished with the aid of filters that matched the

spectral characteristics of FITC-dextran as previously described (1, 6, 20-22, 28-30).

Macromolecular leakage was determined by extravasation of FITC-dextran, which

appeared as fluorescent “spots” or leaky sites around post-capillary venules (1, 6, 20-

22, 28-30). The number of leaky sites was determined by counting three random

microscopic fields every minute for the first 7 min and then at 5-min intervals for 30-60

min after each intervention (see below). The total number of leaky sites was averaged

and expressed as the number of leaky sites per 0.11 cm2 of cheek pouch, which

corresponds to an area of one microscopic field.

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In experiments in which clearance of FITC-dextran was calculated, the suffusate

was collected at 5-min intervals throughout the experiment by a fraction collector

(Microfractionator, Gilson Medical Electronics, Middleton, WI). Samples were collected

in glass tube tests and the concentration of FITC-dextran was determined in each tube.

Arterial blood samples were collected in heparinized capillary tubes (70-µl volume;

Scientific Products, McGaw Park, IL) beginning 5 min before and 5, 30, 60, 120, 180

and 240 min after intravenous injection of FITC-dextran. The concentration of FITC-

dextran was determined in all plasma samples as previously described in our laboratory

(1, 6, 20, 28-30).

To quantitate the concentration of FITC-dextran in the plasma and suffusate, a

standard curve for FITC-dextran concentrations versus percent emission was generated

on a spectrophotofluorometer (Perkin-Elmer, Norwalk, CT). The standard was FITC-

dextran prepared on a weight per volume basis. With bicarbonate buffer as background,

a standard curve was generated for each experiment and each curve was subjected to

linear regression analysis. The percent emission for unknown samples (plasma and

suffusate) was determined by the spectrophotofluorometer and the concentration of

FITC-dextran was then calculated from the standard curve. In preliminary experiments,

minimal fluorescence signal (<2% above background) was detected when drugs were

added to the buffer and when plasma and suffusate samples were examined before

adding FITC-dextran. Clearance of FITC-dextran was determined by calculating the

ratio of suffusate (ng/ml) to plasma (mg/ml) concentration of FITC-dextran and

multiplying this ratio by the suffusate flow rate (2 ml/min) as previously described (1, 5,

6, 20-22, 28-30).

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Preparation of hog barn dust extract. Settled surface dust from a large (~500

animals) hog confinement facility in Nebraska was collected in the winter as previously

described by one of us (19, 27). It was prepared in a manner similar to that for grain

sorghum dust extract as previously described in our laboratory (1, 6). Briefly, an

aqueous extract of hog barn dust was prepared by placing 1 g of dust in 10 ml of Hanks’

balanced salt solution without calcium. The suspension was vortexed and allowed to

settle for 1 h at room temperature. The suspension was then centrifuged at 1,800 rpm

for 5 min, the supernatant removed and centrifuged again. The resulting supernatant,

designated as 10% hog barn dust extract, was removed and filtered through a 0.22 µ

pore filter, diluted to the desired concentrations in Hanks’ balanced salt solution without

calcium and used immediately (see below). In preliminary studies we determined that

the concentration of endotoxin in 10% hog barn dust extract was 2.8 EU/ml (0.28 ng/ml;

Limulus Amebocyte Lysate Test, Associates of Cape Code, East Falmouth, MA).

Matsuda et al (20) and Gao et al (5) showed that this concentration of endotoxin has no

significant effects on macromolecular efflux and vasomotor tone in the intact hamster

cheek pouch.

Experimental protocols

Effects of hog barn dust extract on macromolecular efflux. The purpose of

these studies was to determine whether hog barn dust extract increases

macromolecular efflux from the intact hamster cheek pouch. After suffusing buffer for 30

min (equilibration period), FITC-dextran was injected intravenously and the number of

leaky sites and clearance of FITC-dextran were determined for 60 min. Then, increasing

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concentrations of hog barn dust extract (0.1 and 1.0%) were suffused onto the cheek

pouch in a non-systematic fashion. Each concentration was suffused for 60 min. The

number of leaky sites was determined before and every min for 7 min and at 5 min

intervals for 60 min thereafter. Clearance of FITC-dextran was determined before and

every 5 min thereafter for 60 min. The time interval between subsequent suffusions of

hog barn dust extract was at least 45 min (1, 5, 6, 20, 28-30). In preliminary studies, we

determined that repeated suffusions of hog barn dust extract (0.1 and 1.0%) were

associated with reproducible results. In addition, suffusion of saline (vehicle) for the

entire duration of the experiments was not associated with visible leaky site formation or

significant increase in clearance of FITC-dextran. The concentrations of hog barn dust

extract used in these experiments were based on preliminary studies.

Effects of catalase on hog barn dust extract-induced responses. The

purpose of these experiments was to determine whether reactive oxygen species

mediate, in part, hog barn dust extract-induced increase in macromolecular efflux from

the intact hamster cheek pouch (2, 3, 9, 15, 20-22). The experimental design was

similar to that outlined above except that catalase (60 U/ml) was now suffused onto the

cheek pouch 30 min before and during suffusion of hog barn duct extract (1%) for 60

min. The number of leaky sites and clearance of FITC-dextran were determined during

each intervention as outlined above. In preliminary studies, we determined that

suffusion of catalase (60 U/ml) for 90 min was not associated with visible leaky site

formation or significant increase in clearance of FITC-dextran (9). The concentrations of

catalase used in these experiments were based on preliminary studies.

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Specificity of catalase-induced responses. To determine the specificity of

catalase-induced responses, we utilized two experimental approaches. In the first series

of experiments, catalase (60 U/ml) was incubated in saline at 60 °C for 15 min before

being suffused onto the cheek pouch 30 min before and during suffusion of hog barn

duct extract (0.1%) for 60 min as outlined above. In the second series of experiments,

catalase (60 U/ml) was suffused 30 min before during and for 30 min after adenosine

(10 µM) was suffused for 10 min (1, 7, 10, 11, 24, 30). We chose adenosine because it

modulates microvascular responses through a reactive oxygen species-independent

mechanism(s)(24, 30, 38).

The number of leaky sites and clearance of FITC-dextran were determined

during each intervention as outlined above. In preliminary studies, we found that the

concentration of adenosine used in these studies evoked leaky site formation and

increased clearance of FITC-dextran from the cheek pouch to a similar extend to that

observed with suffusion of 0.1% hog barn dust extract (see below).

Effects of dexamethasone on hog barn dust extract-induced responses.

The purpose of these studies was to determine whether dexamethasone attenuates hog

barn dust extract-induced increase in macromolecular efflux from the intact cheek

pouch (1, 4, 32). The experimental design was similar to that outlined above except that

dexamethasone (10 mg/kg) was infused intravenously for 30 min before suffusing hog

barn dust extract (1.0 %) onto the cheek pouch for 60 min. The number of leaky sites

and clearance of FITC-dextran were determined during each intervention as outlined

above. In previous studies we found that intravenous administration of dexamethasone

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(10 mg/kg) alone for 30 min was not associated with a significant decrease in clearance

of FITC-dextran (1). In addition, intravenous administration of dexamethasone (10

mg/kg) alone for 30 min had no significant effects on arteriolar diameter (1). The

concentration of dexamethasone used in these experiments was based on a previous

study in our laboratory (1).

Effects of hog barn dust extract on arteriolar diameter. The purpose of these

studies was to determine whether suffusion of hog barn dust extract modulates

arteriolar diameter in the intact cheek pouch microcirculation. To accomplish this goal,

we utilized a technique previously described in our laboratory (1, 5, 10, 11). Briefly, the

cheek pouch microcirculation was visualized with an intravital microscope (Nikon,

Tokyo, Japan) coupled to a 100-W mercury light source at a magnification of 40X. The

microscope image was projected through a low-light television camera (Panasonic TR-

124 MA, Matsushita Communication Industrial, Yokohama, Japan) onto a video screen

(Panasonic). The inner diameter of second-order arterioles (baseline diameter 42-51 µ)

was determined during the experiment from the video display of the microscope image

using a videomicrometer (Model VIA 100, Boeckler Instruments, Tucson, AZ). In each

animal, the same arteriolar segment was used to measure vessel diameter during the

experiment.

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Drugs and chemicals

Fluorescein isothiocyanate-labeled dextran, bovine catalase and adenosine were

purchased from Sigma-Aldrich Co. (St Louis, MO). Dexamethasone was obtained from

American Regent Laboratories, Inc. (Shirley, NY). Hanks’ balanced salt solution without

calcium was purchased from Biosource International (Camarillo, CA). All drugs were

prepared and diluted in saline to the desired concentrations on the day of each

experiment.

Data and statistical analyses

Data are expressed as means±SEM. Because the number of leaky sites returned

to baseline (nil) between successive applications of test compounds, all vehicle (saline)

control data are expressed as a single value for each experimental condition. When a

test compound was suffused onto the cheek pouch, the maximal change in arteriolar

diameter was measured and compared to baseline diameter as previously described in

our laboratory (1, 5, 10, 11). Statistical analysis was performed on actual values using

repeated-measures analysis of variance with Neuman-Keuls multiple-range post hoc

test to detect values that were different from control values. A p<0.05 was considered

statistically significant. n is given as the number of experiments, with each experiment

representing a separate animal.

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RESULTS

Mean arterial pressure was 98±4 mmHg at the beginning and 95±3 at the

conclusion of the experiments (n=42 animals; p>0.5). Heart rate was 311±5 at the

beginning and 306±7 at the conclusion of the experiments (n=42 animals; p>0.5).

Effects of hog barn dust extract on macromolecular efflux. Suffusion of hog

barn dust extract induced a significant concentration-dependent increase in leaky site

formation and clearance of FITC-dextran (Figure 1; each group, n=4 animals; p<0.05).

The number of leaky sites increased significantly from nil during suffusion of saline

(vehicle) to 8±2/0.11 cm2 and 26±3/0.11 cm2 during suffusion of 0.1% and 1.0% hog

barn dust extract, respectively (Figure 1, upper panel; each group, n=4 animals;

p<0.05). Likewise, clearance of FITC-dextran increased significantly from 11±2 ml/min x

10-6 during suffusion of saline (vehicle) to 34±9 ml/min x 10-6 and 84±16 ml/min x 10-6

during suffusion of 0.1% and 1.0% hog barn dust extract, respectively (Figure 1, lower

panel; each group, n=4 animals; p<0.05). Leaky sites were visible within 15 min of

initiating hog barn dust extract suffusion, reached a maximum 35-40 min thereafter and

were no longer visible 15 min after suffusion was stopped.

Effects of catalase on hog barn dust extract-induced responses. Suffusion

of catalase (60 U/ml) significantly attenuated hog barn dust extract (1.0%)-induced

leaky site formation and increase in clearance of FITC-dextran from the cheek pouch

(Figure 2; each group, n=4 animals; p<0.05). The number of leaky sites decreased

significantly from 26±3/0. 11 cm2 during suffusion of hog barn dust extract (1%) to 9±1

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/0. 11 cm2 during suffusion of hog barn dust extract and catalase (Figure 2, upper panel;

each group, n=4 animals; p<0.05). Likewise, clearance of FITC-dextran decreased

significantly from 84±16 ml/min x 10-6 during suffusion of hog barn dust extract (1.0%) to

45±12 ml/min x 10-6 during suffusion of hog barn dust extract and catalase (Figure 2,

lower panel; each group, n=4 animals; p<0.05).

Specificity of catalase-induced responses. Suffusion of heated catalase (60

U/ml) had no significant effects of hog barn dust extract (0.1%)-induced leaky site

formation and increase in clearance of FITC-dextran from the cheek pouch (Figure 3;

each group, n=4 animals; p>0.5). Likewise, catalase (60 u/ml) had no significant effects

on adenosine (10 µM)-induced leaky site formation and increase in clearance of FITC-

dextran from the cheek pouch (Figure 4; each group, n=4 animals; p>0.5).

Effects of dexamethasone on hog barn dust extract-induced responses.

Pretreatment with dexamethasone (10 mg/kg; i.v.) significantly attenuated hog barn dust

extract (1.0%)-induced leaky site formation and increase in clearance of FITC-dextran

from the cheek pouch (Figure 5; each group, n=4 animals; p<0.05). The number of

leaky sites decreased significantly from 26±3/0. 11 cm2 during suffusion of hog barn

dust extract (1.0%) to 12±3 during suffusion of hog barn dust extract and

dexamethasone (Figure 5, upper panel; each group, n=4 animals; p<0.05). Likewise,

clearance of FITC-dextran decreased significantly from 84±16 ml/min x 10-6 during

suffusion of hog barn dust extract (1.0%) to 49±16 ml/min x 10-6 during suffusion of hog

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barn dust extract and dexamethasone (Figure 5, lower panel; each group, n=4 animals;

p<0.05).

Effects of hog barn dust extract on arteriolar diameter. Suffusion of hog barn

dust extract (1.0%) had no significant effects of arteriolar diameter throughout the 90-

min suffusion period (1±1% change from arteriolar diameter at baseline; n=4 animals;

p>0.5).

DISCUSSION

There are three new findings of this study. Firstly, we found that an aqueous

extract hog barn dust elicits a significant concentration-dependent increase in

macromolecular efflux from the intact hamster cheek pouch (Figure 1). These effects

were not related to non-specific damage to post-capillary venular endothelium because

FITC-dextran efflux returned to baseline once suffusion of hog barn dust extract was

stopped. Secondly, hog barn dust extract-induced increase in macromolecular efflux

was mediated, in part, by local elaboration of reactive oxygen species because

catalase, an enzyme that catalyzes conversion of hydrogen peroxide to water and

oxygen (2, 3, 9, 15, 20), significantly attenuated this response (Figure 2). The salutary

effects of catalase were specific because heat-inactivated catalase had no significant

effects on hog barn dust extract-induced responses, and because catalase had no

significant effects of adenosine-induced increase in macromolecular efflux from the

cheek pouch (Figures 3&4). We chose adenosine because it modulates microvascular

responses through a reactive oxygen species-independent mechanism(s)(7, 24, 30, 38).

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Lastly, dexamethasone, a potent anti-inflammatory drug (1, 4, 32), attenuated

hog barn dust extract-induced increase in macromolecular efflux (Figure 5). These data

are consistent with the study of Akhter and his colleagues (1) who showed that

dexamethasone attenuates grain sorghum dust extract-induced increase in

macromolecular efflux from the intact cheek pouch. In addition, they showed that

dexamethasone had no significant effects on adenosine-induced responses and on

arteriolar diameter in the cheek pouch (1). Collectively, these data indicate that an

aqueous extract of hog bran dust increases macromolecular efflux from the intact

hamster cheek pouch, in part, through local elaboration of reactive oxygen species in a

specific fashion and that this response is attenuated by corticosteroids. We suggest that

plasma exudation elicited by reactive oxygen species plays an important role in the

genesis of upper airway dysfunction evoked by short-term exposure to hog barn dust

(12-14, 17, 18, 25, 26, 35).

We have previously shown that grain sorghum dust extract elicits neurogenic

plasma exudation in the intact hamster cheek pouch and that dexamethasone

attenuates this response (6). These data coupled with the results of this study suggest

that different organic dusts present in the agricultural environment activate distinct

dexamethasone-responsive inflammatory pathways in the upper airway leading to

plasma exudation, interstitial edema and tissue dysfunction (1, 4, 6, 25, 32). The

mechanism(s) underlying the anti-edemagenic effects of dexamethasone during short-

term exposure to hog barn and grain sorghum dust extracts remains to be determined.

The results of this study support and extend those reported by Ek et al (4) and

Vesterberg et al (33). They showed that short-term exposure of healthy volunteers to

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swine dust is associated with plasma exudation in the upper and lower respiratory tract,

respectively. However, the mechanisms underlying this response were not elucidated in

these studies. We found that local elaboration of reactive oxygen species inactivated by

catalase mediate, in part, the edemagenic effects of hog barn dust extract in the intact

hamster cheek pouch. We did not attempt to quantify overall production of reactive

oxygen species during exposure to hog barn dust extract nor to identify their cellular

origin(s) in the cheek pouch (2, 3, 9, 20-22, 38). Rather, our goal was to probe their pro-

inflammatory effects in the microcirculation during this intervention. Whether reactive

oxygen species play a role in the host inflammatory response to hog barn dust in the

human upper airway and, if so, which reactive species are involved remains merits

further investigation.

The above notwithstanding, the salutary effects of dexamethasone on hog barn

dust extract-induced responses in the intact cheek pouch are consistent with those

reported by Ek et al (4) who showed that intranasal administration of fluticasone, a

potent corticosteroid, attenuates plasma exudation evoked by topical application of hog

barn dust to the nose of healthy individuals. Clearly, further studies to determine the

effects of corticosteroids on elaboration of reactive oxygen species in the upper airway

of humans during short-term exposure to hog barn dust are warranted.

The hamster cheek pouch is a well-established animal model used in our

laboratory and other investigators to study the effects of environmental toxicants and

inflammatory mediators, such as grain sorghum dust and reactive oxygen species, on

macromolecular efflux from the microcirculation in situ and mechanisms underlying

these phenomenae (1-3, 6-9, 20-22, 28-31). Solute efflux emanates from post-capillary

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venules and is determined by two reproducible parameters, leaky site formation and

clearance of FITC-dextran, thereby providing quantitative appraisal of macromolecular

transport across post-capillary venules in the cheek pouch during experimental

interventions. Importantly, successive suffusions of test compounds, such as aqueous

extracts of grain sorghum and hog barn dust (1, 6), at appropriate time intervals are

associated with reproducible formation of leaky sites and increases in clearance of

FITC-dextran in the absence of tachyphylaxis. Consequently, changes in

macromolecular efflux can be tested repeatedly in the same cheek pouch so that each

animal serves as its own control. This, in turn, reduces the overall number of animals

required to conduct the study and facilitates data analysis.

Conceivably, the increase in macromolecular efflux elicited by hog barn dust

extract may have been mediated, in part, by changes in vasomotor tone and/or increase

in venular driving pressure in the cheek pouch (1, 6, 7, 8, 20-23, 28-31, 37). However,

this possibility seems unlikely because we found that suffusion of the extract had no

significant effects of arteriolar diameter in the cheek pouch throughout the observation

period. In addition, other investigators showed that agonist-induced increases in

macromolecular efflux from post-capillary pressure in the hamster cheek pouch and

other microvascular beds are independent of changes in vasomotor tone and increase

in venular driving pressure (8, 23, 31, 37).

The component(s) in hog barn dust extract that stimulates resident and/or

migrant cells in the cheek pouch to elaborate reactive oxygen species was not identified

in this study. Dissecting this component(s) would require intensive analysis of this

complex organic material. Nonetheless, current concepts suggest that endotoxin is an

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important inflammatory stimulus in organic dusts, including hog barn dust (12-14, 16-18,

25, 35). To this end, Matsuda et al (20) showed that antioxidants attenuate endotoxin-

induced increase in macromolecular efflux from the intact hamster cheek pouch. In

addition, Gao et al (5) showed that allopurinol, a scavenger of reactive oxygen species,

attenuates vasodilation evoked by endotoxin in this preparation. However, the

concentration of endotoxin suffused onto the cheek pouch in these studies was in the

sub-milligram range whereas the concentration of endotoxin detected in hog barn dust

extract used in this study was very low, 0.28 ng/ml. The latter has no effects on

macromolecular efflux from the cheek pouch implying that endotoxin is an unlikely

candidate to mediate hog barn dust extract-induced increase in macromolecular efflux

in the upper airway mucosa. Separation of materials present in the aqueous extract of

hog barn by molecular weight distribution and testing them in the cheek pouch could

represent an attractive experimental approach to address this issue in future

experiments planned for this research project. Irrespective of the offending

component(s) in hog barn dust, these data suggest that antioxidants may be beneficial

in the treatment of plasma extravasation evoked by hog barn dust in the upper airway

mucosa. Additional studies are indicated to support or refute this hypothesis.

In summary, we found that hog bran dust extract increases macromolecular

efflux from the in situ hamster cheek pouch, in part, through local elaboration of reactive

oxygen species that are inactivated by catalase. This response is specific and

attenuated by corticosteroids. We suggest that plasma exudation plays an important

role in the genesis of upper airway dysfunction evoked by short-term exposure to hog

barn dust and that corticosteroids abates this process.

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ACKNOWLEDGMENTS

We thank Drs. Akhter and Ikezaki for technical assistance. This study was

supported, in part, by VA Merit Review and NIH grants RO1 AG024026 and RO1

HL72323.

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FIGURES

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FIGURE LEGENDS

Figure 1. Effects of suffusion of hog barn dust extract (HBDE) for 60 min on

leaky site formation (upper panel) and clearance of FITC-dextran (lower panel) from the

hamster cheek pouch. Data are means±SEM. Each group, n=4 animals; *p<0.05 in

comparison to saline; #p<0.05 in comparison to 0.1% hog barn dust extract.

Figure 2. Effects of suffusion of catalase (60 U/ml) on hog barn dust extract

(HBDE; 1.0%)-induced leaky site formation (upper panel) and increase in clearance of

FITC-dextran (lower panel) from the hamster cheek pouch. Data are means±SEM. Each

group, n=4 animals; *p<0.05 in comparison to saline; #p<0.05 in comparison to hog

barn dust extract (1.0%).

Figure 3. Effects of suffusion of heat-inactivated catalase (60 U/ml) on hog barn

dust extract (HBDE; 1.0%)-induced leaky site formation (upper panel) and increase in

clearance of FITC-dextran (lower panel) from the hamster cheek pouch. Data are

means±SEM. Each group, n=4 animals; *p<0.05 in comparison to saline.

Figure 4. Effects of suffusion of catalase (60 U/ml) on adenosine (10 µM)-

induced leaky site formation (upper panel) and increase in clearance of FITC-dextran

(lower panel) from the hamster cheek pouch. Data are means±SEM. Each group, n=4

animals; *p<0.05 in comparison to saline.

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Figure 5. Effects of dexamethasone (10 mg/g, i.v.) on hog barn dust extract

(HBDE; 1.0%)-induced leaky site formation (upper panel) and increase in clearance of

FITC-dextran (lower panel) from the hamster cheek pouch. Data are means±SEM. Each

group, n=4 animals; *p<0.05 in comparison to saline; #p<0.05 in comparison to hog

barn dust extract (1.0%).

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