Repeated Allergen Challenge in Rats
Increases Vitamin A Consumption*
David Shoseyov, MD; Haim Bibi, MD; Hans Biesalski, MD; and
Ram Reifen, MD, MSc
Background: Vitamin A plays an important role in airways epithelial repair and differentiation.
Allergen challenge induces epithelial damage and shedding, which cause an increase in repair
activity.
Objective: To examine whether repeated allergen challenges could increase vitamin A consumption in a rat model.
Design: Allergic bronchitis was induced in 12 animals, and 12 rats remained naive. After 14 days,
repeated allergen inhalation challenges were performed in the sensitized rats for 2 weeks. On day
16, allergen challenge was performed and bronchoconstriction was measured in all 24 rats. On
day 30, all rats were killed. BAL was performed and ex vivo tumor necrosis factor (TNF)-␣ and
nitric oxide (NO) production was measured in the lavage cells. Liver, lung tissue, and serum were
collected for measurement of vitamin A concentration.
Results: The study rats showed severe bronchoconstriction after allergen challenge compared to
the naive rats, and ex vivo TNF-␣ and NO production was significantly higher in the sensitized
rats. Serum and lung concentrations of vitamin A were not different among the two groups.
However, the vitamin A liver concentration in the study rats was significantly lower compared to
the naive rats.
Conclusions: We conclude that vitamin A utilization is increased during repeated allergen
challenge and allergic bronchitis, most probably due to increased demand for epithelial repair.
(CHEST 2002; 122:1407–1411)
Key words: allergic bronchitis; animal model; vitamin A
Abbreviations: BN ⫽ Brown Norway; NO ⫽ nitric oxide; ODC ⫽ ornithine decarboxylase; Penh ⫽ enhanced pause;
RSV ⫽ respiratory syncytial virus; Tr ⫽ relaxation time; TNF ⫽ tumor necrosis factor
A and its active metabolites are important
V itamin
factors in promoting normal respiratory epithelial differentiation and growth.1,2 They exhibit a wide
spectrum of activities, including anti-inflammatory
properties. Children infected with respiratory syncytial virus (RSV) had a low serum concentration of
vitamin A during acute illness, and these low values
were associated with more severe illness. This phenomenon could be attributed to the increased rate of
vitamin A utilization by the tissue, damaged by the
virus.3 In asthma, continuous epithelial shedding and
*From the Pediatric Pulmonology Clinic (Dr. Shoseyov), Bikur
Cholim Hospital, affiliated with Haddasa Medical School Jerusalem, Israel; Pulmonology Clinic (Dr. Bibi), Barzilai Hospital,
Ashkelon, Israel; Institute of Biological Chemistry (Dr. Biesalski),
University of Hohenheim, Stuttgart, Germany; and School of
Nutritional Sciences (Dr. Reifen), The Hebrew University of
Jerusalem, Rehovot, Israel.
Manuscript received July 25, 2001; revision accepted March 18,
2002.
Correspondence to: Ram Reifen, MD, MSc, The School of Nutritional Sciences, The Hebrew University of Jerusalem, PO Box 12,
Rehovot, Israel 76100; e-mail: [email protected]
www.chestjournal.org
a restitution process characterize the airway disease4
and are responsible for the airway hyperresponsiveness. The degree of the hyperresponsiveness measured by methacholine challenge correlates with the
severity of asthma.
The Brown Norway (BN) rat model has been
extensively characterized and appears to be among
the best animal models thus far described.5 The
asthmatic rats have early airway response5 and late
airway response in high prevalence,6 showing high
levels of specific IgE on active immunization6 and
airway eosinophilia.7 Chronic asthma and airway
remodeling was reported earlier to have been induced with repeated allergen inhalation.8 Hyperresponsiveness to methacholine following allergen inhalation as well as presence of inflammatory changes
have previously also been described in this model.9
Allergen inhalation in sensitized BN rats causes an
increase in production of tumor necrosis factor
(TNF)-␣ and nitric oxide (NO) by alveolar macrophage and airways epithelial cells, and it can be
inhibited by dexamethasone.10 We hypothesized that
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allergen inhalation might interfere with cellular or
systemic vitamin A supply. To elucidate whether
allergic bronchitis interferes with vitamin A metabolism, we measured the vitamin A levels in the
serum, the lung, and the liver following repeated
allergen challenge in a rat model and compared
them to normal rats.
Materials and Methods
Animals
Highly inbred BN rats (n ⫽ 24) were obtained from Harlan.
The animals were housed in metal cages in a room with
controlled temperature (25 ⫾ 2°C), relative humidity (65 ⫾ 5%),
and light (8 am to 6 pm). Ethical permission was obtained for the
study, and all procedures were conducted in full compliance with
the strict guidelines of the Hebrew University Policy on Animal
Care and Use. The rats were weighed at the beginning and end
of the study, and their food intake was measured every other day.
Induction of Allergic Bronchitis
The rats were randomly assigned to either the test group
(n ⫽ 12) or the control group (n ⫽ 12). Allergic bronchitis was
induced in the test group rats at the beginning of the study
according to a protocol described by Du et al.5 Briefly, on day 0,
the rats received a single subcutaneous injection of 1 mg
ovalbumin plus aluminum-hydroxide (200 mg/mL in 0.9% NaCl)
and an intraperitoneal injection of 6 ⫻ 109/mL, heat-killed Bordetella pertussis bacteria (Pasteur Marieux; Lyon, France).
Twelve untreated rats were used as naive control rats.
Allergen Challenge
Fourteen days after the induction of allergic bronchitis, the test
animals were sensitized three times a week for 2 weeks (days 14,
16, 18, 21, 23, and 25). The unrestrained rats were placed in a
20-L box connected to an ultrasonic nebulizer (LS 230 System;
Villeneuve Sur Lot, France) and administered ovalbumin inhalation solution (1 mg/mL) for 5 min.
Bronchoconstriction was measured by barometric plethysmography (Buxco; Troy, NY) using a modification of the noninvasive
method by Hamelmann et al,11 and expressed as the enhanced
pause (Penh). Penh is a dimensionless value that reflects changes
in the waveform of the box-pressure signal. Using the waveform,
we measured for each breath the peak inspiratory pressure, peak
expiratory pressure, expiratory time, inspiratory time, and relaxation time (Tr), which is the time of pressure decay to 36% of
total box pressure during expiration. The Penh formula is as
follows: Penh ⫽ (peak expiratory pressure/peak inspiratory
pressure) ⫻ expiratory time ⫺ Tr)/Tr. Penh was calculated in
each measurement as the mean of the three breathing cycles with
the highest Penh measured from a 5-s period.
On day 16, Penh was measured before and 5 min after allergen
inhalation challenge using a 1 mg/mL ovalbumin solution. The
percent of increase in Penh compared to baseline was calculated
and used to compare the difference between the two groups.
Vitamin A Concentration Measurement
Vitamin A was measured in the serum, lung (whole lung
extracts), and liver samples by reversed-phase, high-pressure
1408
liquid chromatography12 on a C18 column using retinol acetate as
the internal standard and fluorescence detection.
BAL
On day 28, BAL was performed. The rats were anesthetized
with sodium thiopental intraperitoneal injection and killed by
bleeding from the abdominal aorta. The rats were than tracheotomized and cannulated through the trachea. BAL was performed with 50 mL of phosphate-buffered saline solution in
aliquots of 10 mL each time. The lavage fluid was collected in
50-mL tubes and placed in ice.
Primary Cells Culture
Cells extracted from the BAL were suspended at 1.3
⫻ 106 cells/mL in Dulbecco’s modified Eagle’s medium including fetal calf serum and plated in 100 ␮L/well plates. The cells
were incubated for 2 h at 37°C, and then the “nonadherent” cells
were washed off with phosphate-buffered saline solution. The
adherent cells (macrophages) were resuspended in Dulbecco’s
modified Eagle’s medium 10% fecal calf serum medium in 200␮L wells with or without lipopolysaccharide, 100 ng/mL, and
incubated for 48 h. Later, the supernatant was collected for assay
of NO and TNF-␣.
NO Assay
Griess reagent (naphthyl ethylenediamine dihydrochloride
[0.1% weight/volume], sulfanilamide [1% weight/volume], phosphoric acid [3%]) were mixed in equal volume with the culture
supernatant. After 10 min at room temperature, color absorption
was read at 550 nm against a standard curve prepared with
various concentrations of sodium nitrite.
TNF-␣ Assay
Bioassay using indicator cells sensitive to TNF-␣ according to
the method described by Flick and Gifford13 was performed.
Briefly, test supernatants were added to culture of HeLa cells.
HeLa cells are sensitive to killing by TNF-␣, and death of these
cells was determined by the release of neutral red dye. The
concentration of TNF-␣ in test medium was determined by
comparing the degree of killing of HeLa cells to that produced by
a titration curve obtained, using known amount of recombinant
TNF-␣. In addition, the nature of the TNF-␣ as the secreted
cytokine present in the experimental supernatant was also determined and confirmed using enzyme-linked immunosorbent assay
kits (Criston). Each measurement was done in triplets, and the
results are presented as mean of the three measurements.
Statistics
Results are expressed as means and SD. Statistical analysis was
performed using unpaired Student t test. p ⬍ 0.05 was considered significant.
Results
There was no difference in food consumption or
body weight at the beginning and end of the study
between the sensitized (test group) and the naive
(control group) rats. The mean NO, TNF-␣, perLaboratory and Animal Investigations
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centage of Penh, and concentration of vitamin A in
liver, lung, and plasma for the test group and control
group rats are shown in Table 1. Bronchoconstriction
5 min after allergen challenge was significantly
higher in the sensitized rats compared to the naive
rats (p ⬍ 0.001).
The plasma and lung vitamin A levels between the
two groups of rats were not significantly different
(p ⬎ 0.05). Nevertheless, the test rats had higher
levels of NO, TNF-␣, and percentage of Penh
(p ⬍ 0.05) compared with the control rats. The liver
vitamin A concentration of the sensitized rats was
significantly lower than that of the normal control
rats (p ⬍ 0.005).
Discussion
In this study, we demonstrated that recurrent
allergen challenges result in a decrease of hepatic
vitamin A stores in sensitized rats. Vitamin A intake
was not different between the two groups as was
measured by food intake and also expressed by body
weight. The liver supplies vitamin A to the serum
and the various organs and maintains normal serum
and tissue concentration as long as vitamin A is not
depleted.14 A decrease of hepatic vitamin A stores
might be a result of an increased demand of target
tissues or decreased intake. During acute and
chronic inflammation, the vitamin A demand of
tissues increases as a result of increased epithelialization.1,2
Several studies15,16 have shown the important role
of vitamin A in the respiratory and alveolar mucosa.
Respiratory mucosa requires vitamin A to ensure
mucous and ciliated cell proliferation and differentiation. Baybutt and coworkers17 showed that vitamin
A deficiency in rats leads to emphysematous lungs
and reduced content of lung elastin in areas of
interstitial pneumonitis, decreased type II pneumo-
Table 1—Results of Measurements in Sensitized and
Naive Rats*
Variables
Sensitized
Rats
Naive Rats
Liver vitamin A concentration, ␮g/g
236 ⫾ 41.5† 317 ⫾ 61.37
Lung vitamin A concentration, ␮g/g
3.6 ⫾ 2.3
3.47 ⫾ 2.1
Serum vitamin A concentration,
34.5 ⫾ 6.6
32 ⫾ 8.5
␮g/dL
Increase of Penh, %
370 ⫾ 38‡
56 ⫾ 7
NO
34.2 ⫾ 7.2‡
0.1 ⫾ 0.015
TNF-␣
183.78 ⫾ 5.9‡ 60.13 ⫾ 1.89
*Data are presented as mean ⫾ SD.
†p ⱕ 0.005 compared to the control group.
‡p ⱕ 0.001 compared to the control group.
www.chestjournal.org
cyte synthesis of surfactant, and decreased ornithine
decarboxylase (ODC) activity in pneumocytes. Reduction of ODC activity as a result of vitamin A
deficiency results in a decreased proliferation of type
II pneumocytes.18 Indeed, retinoic acid, which controls proliferation in a variety of cells, controls ODC
expression and activity.19 Vitamin A plays an important role in cellular growth and differentiation, and
exhibits a wide spectrum of activities, including
anti-inflammatory properties. Retinoids can inhibit
the respiratory burst and degranulation of stimulated
human polymorphonuclear leukocytes probably
through the mediation of lipoxygenase products,20 as
well as transforming growth factor-␤–induced differentiation of tracheal epithelial cells into squamous
cells.21 Low serum concentration of vitamin A is
reported during acute illness in children with RSV;
these low values are associated with more severe
illness and are attributed presumably to an increased
rate of utilization by the damaged bronchiolar epithelium.3 This may not be the case, as RSV bronchiolitis is too short to cause complete depletion of
vitamin A in the liver; rather, it may be due to
reduced levels of retinol-binding protein in the
serum. Further clinical trials22,23 of treatment RSV
bronchiolitis with high doses of vitamin A failed to
show any improvement. The importance of vitamin
A in measles (a Paramyxovirus like the RSV) is well
established, and it is currently recommended by the
American Pediatric Association as a treatment of
measles and its pulmonary complications.24 The
higher incidence of respiratory tract infections during measles may thus be attributed to the vitamin A
deficiency. Vitamin A supplementation may decrease
an inflammatory response when rats are administered monocrotaline, a proinflammatory pneumotoxin,16,24 1-nitronaphtalene,25 or bleomycin. In contrast, monocrotaline treatment of rats reduces lung
and liver vitamin A concentration.17 The role of
vitamin A in preventing inflammation is furthermore
related to its interaction with leukocytes particularly
with neutrophils. Vitamin A reduces neutrophil superoxide production26 and decreases release of lysosomal enzymes.27 Vitamin A deficiency increases
circulating leukocytes28 and exacerbates ozoneinduced inflammation.29 Vitamin A deficiency may
occur as a result of the increased proliferation during
tissue repair and accelerate the ongoing inflammation. Both TNF-␣ and NO were higher in the
vitamin A-deficient group (namely, the inflammatory
markers when an insufficient amount of vitamin A is
available). These data are in agreement with previous studies25–30 that have demonstrated an antiinflammatory effect of vitamin A and the increase of
the inflammatory process during vitamin A deficiency.
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1409
Continuous epithelial shedding and a restitution
process characterize the airway disease in asthma,
even in early stages of the disease.4 Treatment of
asthma with inhaled steroids and other anti-inflammatory medications promotes epithelial restitution
and decreases the hyperresponsiveness.4 Our data
are also consistent with a study30 in humans documenting an increased risk for COPD with decreased
vitamin A intake and an inverse relationship between
plasma retinol status and degree of airways obstruction assessed by FEV1.31 Paiva and coworkers32
demonstrated a lower plasma retinol level in patients
with moderate-to-severe COPD. In addition, it was
reported that high intake and high serum retinol
levels were associated with lower prevalence of
dyspnea in COPD patients.33
In conclusion, our study shows that sensitization in
a rat model, leading to bronchial constriction and
thus mimicking asthma, increases depletion of liver
vitamin A and inflammation. We postulate that
supplementation of vitamin A during airway bronchoconstriction may have some potential benefit by
accelerating bronchial epithelial repair following
asthmatic attacks, and consequently may reduce the
sensitivity of the respiratory mucosa against inflammatory attacks. Further studies are in progress to
elucidate the effect of retinoids on asthma in humans
and in an animal model.
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