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Repeated Allergen Challenge in Rats Increases Vitamin A Consumption
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 CHEST / 122 / 4 / OCTOBER, 2002
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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 200L 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
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. CHEST / 122 / 4 / OCTOBER, 2002
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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. References 1 Biesalski HK, Stoftt TK. Biochemical, morphological and functional aspects of systemic and local vitamin A deficiency in the respiratory tract. Ann N Y Acad Sci 1992; 559:325–331 2 Georgieff MK, Radmer WJ, Sowell AL, et al. The effect of glucocorticoids on serum, liver and lung vitamin A and retinyl ester concentration. J Pediatr Gastroenterol Nutr 1991; 13: 376 –382 3 Neuzil KM, Gruber WC, Chytil F, et al. Serum vitamin A level in respiratory syncytial virus infection. J Pediatr 1994; 124:433– 436 4 Erjefalt JS, Erjefalt I, Sundler F, et al. Effect of topical budesonide on epithelial restitution in vivo in guinea pig trachea. Thorax 1995; 50:785–792 5 Du T, Xu LJ, Lei M, et al. Morphometric changes during the early airways response to allergen challenge in rat. Am Rev Respir Dis 1992; 146:1037–1041 6 Eidlman DH, Bellofiore S, Martin JG. Late airway responses to antigen challenge in sensitized inbred rats. Am Rev Respir Dis 1988; 137:1033–1037 7 Pauwels R, Bazin H, Plateau B, et al. The influence of antigen dose on IgE production in different rat strains. Immunology 1979; 36:151–157 8 Shoseyov D, Bibi H, Ofer S, et al. Montelukast prevents airways remodeling in rats with chronic asthma [abstract]. Am J Respir Crit Care Med 2000; 161:3 9 Elwood W, Barnes PJ, Fan Chung K. Airway hyperresponsiveness is associated with inflammatory cell infiltration in Brown Norway rats. Int Arch Allergy Immunol 1992; 99: 91–97 10 Renzi PM, Olivenstein R, Martin JG. Effect of dexametha1410
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12 13 14 15
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17 18 19
20 21 22
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24 25 26
27 28 29 30
sone on airway inflammation and responsiveness after antigen challenge of the rat. Am Rev Respir Dis 1993; 148:932–939 Hamelmann E, Schwarze J, Takeda K, et al. Noninvasive measurement of airway responsiveness in allergic mice using barometric plethysmography. Am J Respir Crit Care Med 1997; 156:766 –775 Burri BJ, Jacob, RA. Vitamin A analogs as tests for liver vitamin A status in the rat. Am J Clin Nutr 1988; 47:458 – 462 Flick DA, Gifford GE. Comparison of in vitro cytotoxic assays for TNF-␣. J Immunol Methods 1984; 68:167–175 Reifen R, Zaiger G, Uni Z. Effect of vitamin A on small intestinal brush border enzymes in a rat. Int J Vitam Nutr Res 1998; 68:281–286 Manna B, Ashbaugh P, Bhattachryya SN. Retinoic acid regulated cellular differentiation and mucin gene expression in isolated rabbit tracheal-epithelial cells in culture. Inflammation 1995; 19:489 –502 Ann G, Luo G, Wu R. Expression of MUC 2 gene is down regulated by vitamin A at transcriptional level in vitro in tracheoepithelial cells. Am J Respir Cell Mo Biol 1994; 10:546 –551 Baybutt RC, Hu L, Molteni A. Vitamin A deficiency injures lung and liver parenchyma and impairs function of rat type II pneumocytes. J Nutr 2000; 130:1159 –1165 Nabeyrat E, Besnard V, Corroyer S, et al. Retinoic acid induced proliferation of lung alveolar epithelial cells: relation with the IGF system. Am J Physiol 1998; 275:L71–L79 Francz PI, Conrad J, Biesalski HK. Modulation of UVA induced lipid peroxidation and suppression of UVB-induced ornithine decarboxylase response by all trans retinoic acid inhuman skin fibroblasts in vitro. Biol Chem 1998; 379:1263– 1269 Fumaralo R, Conese M, Riccardi S, et al. Retinoids inhibit the respiratory burst and degranulation of stimulated human polymorphonuclear leukocytes. Agents Actions 1991; 34:339–344 Jetten AM, Shirley JE, Soner G. Regulation of proliferation and differentiation of respiratory tract epithelial cells by TGF-. Exp Cell Res 1986; 167:539 –549 Bresee JS, Fischer M, Dowell SF, et al. Vitamin A therapy for children with respiratory syncytial virus infection: a multicenter trial in the United States. Pediatr Infect Dis J 1996; 15:777–782 Dowell SF, Papic Z, Bresee JS, et al. Treatment of respiratory syncytial virus infection with vitamin A: a randomized, placebo-controlled trial in Santiago, Chile. Pediatr Infect Dis J 1996; 15:782–786 American Academy of Pediatrics Committee on Infectious Diseases. Vitamin A treatment of measles. Pediatrics 1993; 91:1014 –1015 Swamidas GP, Basaraba RJ, Baybutt RC. Dietary retinol inhibits inflammatory responses of rats treated with monocrotaline. J Nutr 1999; 129:1285–1290 Sauer JM, Hooser SB, Sipes IG. All-trans-retinol alteration of 1-nitronaphtalene-induced pulmonary and hepatic injury by modulation of associated inflammatory responses in the male Sprague-Dawley rat. Toxicol Appl Pharmacol 1995; 133:139 – 149 Sharma A, Lewandoski JR, Zimmerman JJ. Retinol inhibition of in vitro human neutrophil superoxide anion release. Pediatr Res 1990; 27:574 –579 Camisa C, Eisenstadt B, Ragaz A, et al. The effects of retinoids in vitro. J Am Acad Dermatol 1982; 6(4 suppl): 620 – 629 Wiedermann U, Chen XJ, Enerback L, et al. Vitamin A deficiency increases inflammatory responses. Scand J Immunol 1996; 44:578 –584 Paquette NC, Zhang LY, Ellis WA, et al. Vitamin A deficiency Laboratory and Animal Investigations
enhances ozone-induced lung injury. Am J Physiol 1996; 270:L475–L482 31 Morabia A, Menkes MJ, Comstock GW, et al. Serum retinol and airway obstruction. Am J Epidemiol 1990; 132:77– 82 32 Paiva SA, Godoy I, Vannuchi H, et al. Assessment of vitamin
A status in chronic obstructive pulmonary disease patients and healthy smokers Am J Clin Nutr 1996; 64:928 –934 33 Rautalhti M, Virtamo J, Haukka J, et al. The effect of alpha tocopherol and beta carotene supplementation on COPD symptoms. Am J Respir Crit Care Med 1997; 156:1447–1452
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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 CHEST / 122 / 4 / OCTOBER, 2002
1407
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 200L 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
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. CHEST / 122 / 4 / OCTOBER, 2002
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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. References 1 Biesalski HK, Stoftt TK. Biochemical, morphological and functional aspects of systemic and local vitamin A deficiency in the respiratory tract. Ann N Y Acad Sci 1992; 559:325–331 2 Georgieff MK, Radmer WJ, Sowell AL, et al. The effect of glucocorticoids on serum, liver and lung vitamin A and retinyl ester concentration. J Pediatr Gastroenterol Nutr 1991; 13: 376 –382 3 Neuzil KM, Gruber WC, Chytil F, et al. Serum vitamin A level in respiratory syncytial virus infection. J Pediatr 1994; 124:433– 436 4 Erjefalt JS, Erjefalt I, Sundler F, et al. Effect of topical budesonide on epithelial restitution in vivo in guinea pig trachea. Thorax 1995; 50:785–792 5 Du T, Xu LJ, Lei M, et al. Morphometric changes during the early airways response to allergen challenge in rat. Am Rev Respir Dis 1992; 146:1037–1041 6 Eidlman DH, Bellofiore S, Martin JG. Late airway responses to antigen challenge in sensitized inbred rats. Am Rev Respir Dis 1988; 137:1033–1037 7 Pauwels R, Bazin H, Plateau B, et al. The influence of antigen dose on IgE production in different rat strains. Immunology 1979; 36:151–157 8 Shoseyov D, Bibi H, Ofer S, et al. Montelukast prevents airways remodeling in rats with chronic asthma [abstract]. Am J Respir Crit Care Med 2000; 161:3 9 Elwood W, Barnes PJ, Fan Chung K. Airway hyperresponsiveness is associated with inflammatory cell infiltration in Brown Norway rats. Int Arch Allergy Immunol 1992; 99: 91–97 10 Renzi PM, Olivenstein R, Martin JG. Effect of dexametha1410
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sone on airway inflammation and responsiveness after antigen challenge of the rat. Am Rev Respir Dis 1993; 148:932–939 Hamelmann E, Schwarze J, Takeda K, et al. Noninvasive measurement of airway responsiveness in allergic mice using barometric plethysmography. Am J Respir Crit Care Med 1997; 156:766 –775 Burri BJ, Jacob, RA. Vitamin A analogs as tests for liver vitamin A status in the rat. Am J Clin Nutr 1988; 47:458 – 462 Flick DA, Gifford GE. Comparison of in vitro cytotoxic assays for TNF-␣. J Immunol Methods 1984; 68:167–175 Reifen R, Zaiger G, Uni Z. Effect of vitamin A on small intestinal brush border enzymes in a rat. Int J Vitam Nutr Res 1998; 68:281–286 Manna B, Ashbaugh P, Bhattachryya SN. Retinoic acid regulated cellular differentiation and mucin gene expression in isolated rabbit tracheal-epithelial cells in culture. Inflammation 1995; 19:489 –502 Ann G, Luo G, Wu R. Expression of MUC 2 gene is down regulated by vitamin A at transcriptional level in vitro in tracheoepithelial cells. Am J Respir Cell Mo Biol 1994; 10:546 –551 Baybutt RC, Hu L, Molteni A. Vitamin A deficiency injures lung and liver parenchyma and impairs function of rat type II pneumocytes. J Nutr 2000; 130:1159 –1165 Nabeyrat E, Besnard V, Corroyer S, et al. Retinoic acid induced proliferation of lung alveolar epithelial cells: relation with the IGF system. Am J Physiol 1998; 275:L71–L79 Francz PI, Conrad J, Biesalski HK. Modulation of UVA induced lipid peroxidation and suppression of UVB-induced ornithine decarboxylase response by all trans retinoic acid inhuman skin fibroblasts in vitro. Biol Chem 1998; 379:1263– 1269 Fumaralo R, Conese M, Riccardi S, et al. Retinoids inhibit the respiratory burst and degranulation of stimulated human polymorphonuclear leukocytes. Agents Actions 1991; 34:339–344 Jetten AM, Shirley JE, Soner G. Regulation of proliferation and differentiation of respiratory tract epithelial cells by TGF-. Exp Cell Res 1986; 167:539 –549 Bresee JS, Fischer M, Dowell SF, et al. Vitamin A therapy for children with respiratory syncytial virus infection: a multicenter trial in the United States. Pediatr Infect Dis J 1996; 15:777–782 Dowell SF, Papic Z, Bresee JS, et al. Treatment of respiratory syncytial virus infection with vitamin A: a randomized, placebo-controlled trial in Santiago, Chile. Pediatr Infect Dis J 1996; 15:782–786 American Academy of Pediatrics Committee on Infectious Diseases. Vitamin A treatment of measles. Pediatrics 1993; 91:1014 –1015 Swamidas GP, Basaraba RJ, Baybutt RC. Dietary retinol inhibits inflammatory responses of rats treated with monocrotaline. J Nutr 1999; 129:1285–1290 Sauer JM, Hooser SB, Sipes IG. All-trans-retinol alteration of 1-nitronaphtalene-induced pulmonary and hepatic injury by modulation of associated inflammatory responses in the male Sprague-Dawley rat. Toxicol Appl Pharmacol 1995; 133:139 – 149 Sharma A, Lewandoski JR, Zimmerman JJ. Retinol inhibition of in vitro human neutrophil superoxide anion release. Pediatr Res 1990; 27:574 –579 Camisa C, Eisenstadt B, Ragaz A, et al. The effects of retinoids in vitro. J Am Acad Dermatol 1982; 6(4 suppl): 620 – 629 Wiedermann U, Chen XJ, Enerback L, et al. Vitamin A deficiency increases inflammatory responses. Scand J Immunol 1996; 44:578 –584 Paquette NC, Zhang LY, Ellis WA, et al. Vitamin A deficiency Laboratory and Animal Investigations
enhances ozone-induced lung injury. Am J Physiol 1996; 270:L475–L482 31 Morabia A, Menkes MJ, Comstock GW, et al. Serum retinol and airway obstruction. Am J Epidemiol 1990; 132:77– 82 32 Paiva SA, Godoy I, Vannuchi H, et al. Assessment of vitamin
A status in chronic obstructive pulmonary disease patients and healthy smokers Am J Clin Nutr 1996; 64:928 –934 33 Rautalhti M, Virtamo J, Haukka J, et al. The effect of alpha tocopherol and beta carotene supplementation on COPD symptoms. Am J Respir Crit Care Med 1997; 156:1447–1452
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