Biochemical and Molecular Roles of nutrients

Vitamin A Deficiency Diminishes the Salivary Immunoglobulin A Response and Enhances the Serum Immunoglobulin G Response to Influenza A Virus Infection in BALB/c Mice1»2'3 CHARLES B. STEPHENSEN,4 ZINA MOLDOVEANÜ* AND NUPUR N. GANGOPADHYAY Department of International Health, School of Public Health, and *Department of Microbiology, School of Medicine, (Jniuersity of Alabama at Birmingham, Birmingham, AL 35294

1We wish to acknowledge the support of research grant ROÕ HD30293 from the National Institutes of Health. 2 Some of these data were presented at Experimental Biology 95, April 1995, Atlanta, GA [Stephensen, C., Moldoveanu, Z., Gangopadhyay, N. & Blount, S. (1995) Vitamin A deficiency impairs the secretory IgA response to influenza A virus infection in BALB/c mice. FASEB J. 9: A732 (abs.)]. 3 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734

INDEXING KEY WORDS: •mice

•vitamin A •IgA •IgG •influenza

solely to indicate this fact. 4 To whom correspondence

Vitamin A deficiency is associated with an increased risk of death from common childhood infections. This

and reprints requests should be ad

dressed.

0022-3166/96 S3.00 ©1996 American Institute of Nutrition. Manuscript received 19 June 1995. Initial review completed 11 August 1995. Revision accepted 5 October 1995. 94

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is particularly true for diarrheal diseases and, at least when associated with measles, lower respiratory tract infections (Glasciou and Mackerras 1993). This in creased risk of death typically is not associated with an overall increase in the incidence or prevalence of infectious episodes, although vitamin A deficiency does seem to increase the incidence of infectious ep isodes that become serious enough to require a clinic visit (Glasciou and Mackerras 1993, Ghana VAST Study Team 1993) and, under some circumstances, the incidences of diarrhea and respiratory infections may also be increased (Bloem et al. 1990, Biswas et al. 1994, Lie et al. 1993). There are at least two principal mech anisms by which vitamin A deficiency impairs host resistance to infection. Vitamin A deficiency can im pair regeneration of normal mucosal epithelium dam aged by infection or inflammation (Ahmed et al. 1990, Stephensen et al. 1993) and thus could increase the severity of an infectious episode and/or prolong re covery from that episode. Vitamin A deficiency also affects immune function, particularly the antibody re sponse to T cell-dependent antigens (Ross 1992). A diminished primary antibody response could also in crease the severity and/or duration of an episode of

ABSTRACT We examined the effect of vitamin A de ficiency on the secretory immunoglobulin (Ig) A and serum IgG response to influenza A virus infections in BALB/c mice. Mice fed a vitamin A-deficient (VAD mice) or a control diet were inoculated with influenza virus at 7 or 9 wk of age when serum retino! concen tration had dropped to 0.05). In a separate experiment, this salivary IgA response was significantly lower in the VAD mice (0.3 ±0.4% of total IgA) fol lowing a more severe infection (intranasal infection while under anesthesia) than it was in control mice (4.2 ±4.6% of total IgA, P < 0.0001). In contrast, the con centration of total salivary IgA was uniformly greater in the VAD mice than in the control mice during both the mild infection (VAD, 17 ±6 mg/L vs. control, 8 ± 11 mg/L at 3 wk, P < 0.0001) and the severe infection (VAD, 38 ±30 mg/L vs. control, 9 ±7 mg/L, P < 0.0001). Similarly, the influenza-specific serum IgG re sponse was also greater in the VAD mice than in the control mice during both the mild infection (VAD, 194 ±91 mg/L vs. control, 79 ±95 mg/L at 5 wk, P = 0.0002) and the severe infection [VAD median, 202 mg/L (25th, 75th percentiles, 153, 409 mg/L) vs. con trol, 123 mg/L (42, 165 mg/L), P = 0.0023]. Thus VAD significantly impairs the secretory IgA response to influenza infection but modestly increases the serum IgG response to the same infection. J. Nutr. 126: 94102, 1996.

VITAMIN

A AND ANTIBODY

RESPONSE TO INFLUENZA

infection, whereas a diminished secondary response could increase the risk of developing a second episode of infection. The secretory immunoglobulin (Ig)5A system is an important first line of defense against infections of mucosal surfaces (McGhee et al. 1992). Several studies in animal models have shown that the intestinal IgA response is impaired by vitamin A de ficiency (Davis and Sell 1989, Puengtomwatanakul and Sirisinha 1986, Rombout et al. 1992, Sirisinha et al. 1980, Wiedermann et al. 1993b). Our preliminary study suggested a similar pattern in the respiratory tract (Stephensen et al. 1993). In this study, we ex amine the effect in mice of vitamin A deficiency on the salivary IgA and serum IgG responses to a respi ratory tract infection with influenza A virus.

AND METHODS

Animals and diets. The animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Alabama at Bir mingham. Pregnant BALB/c mice (Charles River, Ral eigh, NC) were fed a vitamin A-deficient (VAD) or control diet as has been described (Smith and Hayes 1987, Stephensen et al. 1993). Pups were weaned to the same diets. The mild infection experiment in volved 26 animals (9 male, 17 female) fed the control diet and 18 animals (10 male, 8 female) fed the VAD diet. The severe infection experiment involved 23 an imals (9 male, 14 female) fed the control diet and 23 animals (5 male, 18 female) fed the VAD diet. Im munologie assays and total protein measurements were performed on all animals. Serum retino/. Serum retinol was measured by HPLC as has been described (Stacewicz-Sapuntzakis et al. 1987, Stephensen et al. 1993). Serum retinol was measured on five or six randomly selected animals from each diet group to confirm that animals were becoming vitamin A deficient. A group of animals was considered deficient when the group mean dropped to , interferon--y; plgR, polymeric immunoglobulin receptor, VAD, vitamin A-defi cient.

95

tial mortality occurs at higher doses. To initiate the mild infection, a much higher dose, IO35 plaqueforming units, was given to unanesthetized mice in the same volume. This route causes an initiation of infection in the nose with a progression down the res piratory tract into the trachea and lungs over a period of days (Novak et al. 1993). Antibody assays. Saliva for measuring IgA was collected after subcutaneous stimulation with 0.05 to 0.1 mL of 50 mg/L carbamylcholine chloride diluted in sterile phosphate buffered saline (carbachol, Sigma Chemical, St Louis, MO). Blood was collected during the study from the tail veins of unanesthetized animals using capillary micropipettes. Influenza A-specific IgA and IgG were titered in an ELISA assay as previously described (Stephensen et al. 1993). The concentrations of IgA, IgG, IgGl and IgG2a were determined using a standard curve in which serial dilutions of a reference serum of known antibody concentration (ICN ImmunoBiologicals, Costa Mesa, CA) were added to wells pre-coated with either 1 mg/L (IgA assay) or 0.5 mg/L (IgG assay) of the appropriate capture antibody (goat anti-mouse a-chain and -y-chain reagents were purchased from Kirkegaard and Perry, Gaithersburg, MD, and IgG subclass antibodies were purchased from Southern Biotechnology Associates, Birmingham, AL). Samples were assayed in duplicate or triplicate and were repeated when duplicate values disagreed by >0.1 absorbance units. Correlation coefficients for standard curves were always >0.98. Influenza-specific IgA val ues are expressed as a percentage of total salivary IgA to correct for differences in the rate of saliva secretion, which will vary within and between animals as a func tion of several factors, including method and site of saliva collection, the dose of carbachol and time after its administration when samples are collected, and the level of hydration of the animal. Total protein assay. Total protein was measured in saliva using the bicinchoninic acid reagent from Pierce Chemical (Rockford, IL). Statistical analysis. Statistical analysis was per formed with the SigmaStat program (Jandel Corpora tion, San Rafael, CA). Body weight, salivary IgA and total protein data, and serum IgG data were compared using two-way ANOVA (comparing sex and diet group) on each day. If data were not normally distrib uted or variances were not equal (at P < 0.05), then Iog10-transformed data were analyzed. If these data were not normally distributed or had unequal variance, then the nonparametric Mann-Whitney rank-sum test was used to compare groups (Snedecor and Cochran 1967). When body weights were found to differ by diet group, the dietary difference was examined in dependently in males and females using Student's t test. Serum retinol data were compared using Student's t test or, if a normality test failed, the Mann-Whitney test. For uniformity of presentation, data in line graphs are always presented as mean ±so of untransformed

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MATERIALS

INFECTION

96

STEPHENSEN

data even if these data were not normally distributed. The significance of changes in salivary antibody and total protein levels within diet groups over time was assessed using the paired Student's t test or the Wilcoxon signed-rank test (Snedecor and Cochran 1967).

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FIGURE Z Body weights [A] and serum retinol concen trations [B] of male and female (FEM) vitamin A-deficient (VAD; 5 male, 18 female) and control (CON; 9 male, 14 female) mice inoculated with influenza A virus using the severe infection protocol at 9 wk of age. Values shown are means ±1 so. Significant differences between diet groups are indicated as follows: a, P