Sulfanilamide

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)-[alpha] 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-[alpha] 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.

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

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Vitamin A and its active metabolites are important 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 a restitution process characterize the airway disease (4) 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 responses and late airway response in high prevalence, (6) showing high levels of specific IgE on active immunization (6) 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)-[alpha] and nitric oxide (NO) by alveolar macrophage and airways epithelial cells, and it can be inhibited by dexamethasone. (10) We hypothesized that 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 [+ or -] 2[degrees]C), relative humidity (65 [+ or -] 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 x [10.sup.9]/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 Hamehnann 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) x 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 liquid chromatography (12) 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 x [10.sup.6] cells/mL in Dulbecco's modified Eagle's medium including fetal calf serum and plated in 100 [micro]L/well plates. The cells were incubated for 2 h at 37[degrees]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-[micro]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-[alpha].

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-[alpha] Assay

Bioassay using indicator cells sensitive to TNF-[alpha] according to the method described by Flick and Gifford (13) was performed. Briefly, test supernatants were added to culture of HeLa cells. HeLa cells are sensitive to killing by TNF-[alpha], and death of these cells was determined by the release of neutral red dye. The concentration of TNF-[alpha] 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-[alpha]. In addition, the nature of the TNF-[alpha] 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-[alpha], percentage 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-[alpha], 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 liver supplies vitamin A to the serum various organs and maintains normal serum concentration as long as vitamin A is not 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 studies (15,16) 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 coworkers (17) 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 pneumocyte 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. (10) 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-[beta]-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 trials (22,23) 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 production (26) and decreases release of lysosomal enzymes. (27) Vitamin A deficiency increases circulating leukocytes (28) and exacerbates ozone-induced inflammation. (29) Vitamin A deficiency may occur as a result of the increased proliferation during tissue repair and accelerate the ongoing inflammation. Both TNF-[alpha] 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 studies (25-30) that have demonstrated an anti-inflammatory effect of vitamin A and the increase of the inflammatory process during vitamin A deficiency.

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 study (30) 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 FE[V.sub.1]. (31) Paiva and coworkers (32) 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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* 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: reifen@agri.huji.ac.il

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