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The Journal of Nutrition Vol. 127 No. 4 April 1997, pp. 558-565
Copyright ©1997 by the American Society for Nutritional Sciences

Vitamin A Deficiency Alters Rat Neutrophil Function1,2

Sally S. Twining*, dagger , 3, David P. Schulte*, Patricia M. Wilson*, Brian L. FishDagger , and John E. MoulderDagger

* Departments of Biochemistry, dagger  Ophthalmology, and Dagger  Radiation Oncology, Medical College of Wisconsin, Milwaukee, WI 53226

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
LITERATURE CITED

ABSTRACT

Previous studies showed a higher percentage of neutrophils from vitamin A deficient rats are hypersegmented and contain lower levels of cathepsin G than the neutrophils from control rats. In this study chemotaxis, phagocytosis and oxidant generation were studied using either isolated neutrophils or neutrophils in whole blood from four dietary groups of rats: 1) vitamin A deficient rats; 2) vitamin A deficient rats that received vitamin A for 16, 8, 4 or 2 d prior to killing; 3) weight-matched rats pair-fed a vitamin A-complete diet; and 4) rats fed nonrestricted, vitamin A complete diet. Chemotaxis towards P. aeruginosa conditioned medium and formylated methinyl leucinyl phenylalanine was significantly lower for neutrophils from vitamin A-deficient rats than for neutrophils from weight-matched pair-fed rats, nonrestricted vitamin A sufficient rats and vitamin A deficient rats that received vitamin A for 16 d prior to killing. No differences in chemotaxis towards activated rat serum were noted among the neutrophils from the four groups of rats. Adhesion of P. aeruginosa organisms, phagocytosis of these organisms and generation of active oxidative molecules were significantly lower in the neutrophils from the vitamin A-deficient rats relative to these functions in the neutrophils from the vitamin A deficient rats that received vitamin A for 16 d, weight-matched rats pair-fed a vitamin A complete diet; and rats fed nonrestricted, vitamin A-complete diet. Eight days after vitamin A administration to vitamin A deficient rats, the ability of the neutrophils to phagocytose P. aeruginosa organisms and to generate active oxidant molecules was restored to the levels observed for weight-matched, pair-fed rats and rats fed nonrestricted, vitamin A complete diet. The elucidated alterations in neutrophil function in vitamin A deficient rats probably contribute to the altered ability of vitamin A deficient rats to fight infections.

Key words: vitamin A deficiency, neutrophils, phagocytosis, chemotaxis, rats, oxidant generation.

INTRODUCTION

Vitamin A deficient people and rats are prone to more severe infections and have a higher mortality than vitamin A sufficient people and rats (Beaton et al. 1992, Fawzi et al. 1994, Twining et al. 1996b). This increase is observed even in the early stages of the deficiency (Semba et al. 1993, Twining et al. 1996b). Numbers of Pseudomonas aeruginosa organisms that are subinfectious in corneas of normal and pair-fed, weight matched rats are capable of inducing keratitis in corneas of vitamin A deficient rats (Twining et al. 1996b).

The first line of defense against infections, including corneal infections, is the neutrophil (Smith 1994). This cell is the first to respond to infections and targets bacteria (Ferrante et al. 1993, Twining et al. 1996b), fungi, (Roilides et al. 1993), protozoa (Ferrante et al. 1989), viruses (Klebanoff and Coombs 1992, Ratcliffe et al. 1988), virus-infected cells (Ratcliffe et al. 1988) and tumor cells (Weitzman and Gordon 1990). The neutrophil phagocytoses the target organism or cell and then uses an arsenal of active oxygen species and other microcidal molecules to kill its target (Rosen et al. 1995, Smith 1994). The importance of the neutrophil is observed in animals with neutropenia or a deficiency of one or more key neutrophil enzymes (Edwards 1994). In these animals, infections which normally are mild can be life threatening.

The neutrophil differentiates and matures in the bone marrow. Differentiation from the promyelocyte to the mature neutrophil requires the oxidized form of retinol, retinoic acid (Robertson et al. 1992, Tsai and Collins 1993). Recently, we showed that in severely vitamin A deficient rats, the concentration of retinol is not less in the bone marrow as we hypothesized, but is elevated (Twining et al. 1996a). This sequestration of retinol indicates the importance of bone marrow cells for the survival of the animals.

Vitamin A deficiency does not alter the distribution of myeloid derived cells in the bone marrow with the exception of a significantly greater occurrence of hypersegmented neutrophils (six or more lobes) in vitamin A deficient rats (2.1%) relative to the control rats (<=  0.1%). The blood of the vitamin A deficient rats contained significantly higher numbers (P < 0.01) of hypersegmented neutrophils (67%) relative to those in the control rats (2-7%). The hypersegmentation of the neutrophils in vitamin A deficient rats was not due to a concurrent deficiency of vitamin B-12 or folate (Twining et al. 1996a).

Incubation of neutrophils with retinoids can influence their function. At a concentration of 25 µmol/L, all trans-retinal and retinol can stimulate the release of superoxide (Badwey et al. 1989). Retinoic acid in combination with other neutrophil activators such as phorbol esters or formylated methinyl leucinyl phenylalanine (f-Met-Leu-Phe),4 inhibit superoxide production (Fumarulo et al. 1991, Sharma et al. 1990, Varani et al. 1991), chemiluminescence (Fumarulo et al. 1991), degranulation (Fumarulo et al. 1991, Perkins et al. 1991) and endothelial cell injury (Varani et al. 1991).

Very little is known about polymorphonuclear leukocyte function in vitamin A deficient animals. Using a rat model, Ongsakul et al. (1985) reported fewer numbers of active phagocytic polymorphonuclear leukocytes in deficient animals. The number of bacteria ingested per active phagocytic cell was similar. The purpose of this study was to determine the effects of vitamin A deficiency on the neutrophil functions: chemotaxis, adhesion of organisms, phagocytosis and killing.

MATERIALS AND METHODS

Materials. An autoclavable vitamin A deficient diet and a control diet with retinyl palmitate added back (15 mg/kg) were purchased from Harlan-Sprague Dawley-Teklad (Madison, WI).5 Heparin was obtained from Elkins-Sinn, Inc. (Cherry Hill, NJ). Retinol, ascorbic acid and HPLC chromatography solvents were from J. T. Baker Chemical Co. (Phillipsburg, NJ). Hexane was purchased from Sigma-Aldrich Chemical Co. (Milwaukee, WI). Dextran T500 was obtained from Pharmacia Fine Chemicals AB (Uppsala, Sweden). Staphylococcus A antibody, zymosan, pyrogallol, retinyl palmitate, retinyl acetate, Histopaque, o-dianisidine, hydrogen peroxide, f-Met-Leu-Phe and other compounds used for buffers were also purchased from Sigma Chemical, (St. Louis, MO). Pentobarbital was purchased from Abbott Laboratories (North Chicago, IL). LeukoStat staining kit was from Fisher Diagnostics (Orangeburg, NY). 2',7'Dichlorofluorescein diacetate (DCFH-DA) and hydroethidine were from Molecular Probes (Eugene, OR). The Coulter Diagnostics Clone Immuno-Lyse and Clone Fixer (Hialeah, FL) were used. Unless otherwise stated chemicals and enzymes were obtained from Sigma Chemical Company (St. Louis, MO). Myeloperoxidase was from Athens Research Technologies (Athens, GA). Polycarbonate chemotaxis membranes were obtained from Neuropore (Cabin John, MD). RPMI medium was purchased from GIBCO-BRL (Grand Island, NY). A FITC-labeled rabbit anti-rat PMN antibody was obtained from Accurate (Westbury, NY).
Fig. 2. Effect of vitamin A-deficiency on rat neutrophil function shown by representative flow cytometry plots of phagocytosis of FITC labeled P. aeruginosa (LFL1) and superoxide generation (LFL3) by neutrophils from individual -A (vitamin A deficient), +A (weight-matched pair-fed), R (vitamin A deficient rats refed vitamin A for 16 d) and N (nonrestricted, vitamin A complete diet) rats with time of incubation. Whole blood, pretreated with hydroethidine, was incubated with FITC-labeled killed P. aeruginosa, strain 107, at 37°C for increasing times. The reaction was stopped at 2, 7, 15 and 30 min by the addition of Immuno-Lyse followed by Clone Fixer. Flow cytometry was used to quantify phagocytosis and superoxide generation by measuring the increase in florescence at 510-530 nm due to the association of FITC P. aeruginosa organisms with the cells (LFL1) and the increase in fluorescence at 600 nm from the oxidation of hydroethidine by superoxide (LFL3), respectively. The neutrophils were selected by electronic gating. Fluorescence values are given as log fluorescence (LFL) in these representative plots.
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Vitamin A deficient and control rats. WAG/Rij/MCW rats were derived from animals established in 1970 at Yale University from rats supplied by H. R. Reinhold (Radiological Institute, TNO, Rijswyk, The Netherlands). These rats were raised in a defined microflora environment, and vitamin A deficient and control rats were prepared as previously reported (Twining et al. 1996a). Briefly, nursing females were fed an autoclavable casein-based vitamin A deficient diet5 beginning 16 d postpartum. The pups were weaned on d 21. The male pups were divided into four dietary groups: 1) Sixteen rats (-A) were fed a casein-based retinoid deficient diet. 2) Twenty-six rats (R) were fed the retinoid deficient diet. On d 74 (16 d before killing), 14 rats were fed a bolus of 500 µg retinyl palmitate in 200 µL safflower oil and then given free access for 16 d to the retinyl palmitate-supplemented control diet (vitamin A deficient diet supplemented with retinyl palmitate at 15 mg/kg). On d 82, 86 and 88 (8, 4 and 2 d before killing), groups of four vitamin A deficient rats were fed a bolus of retinyl palmitate (500 µg retinyl palmitate in 200 µL safflower oil). These rats were then given free access to the retinyl palmitate-supplemented control diet. 3) Fourteen rats (+A) were restricted in their intake of the retinyl palmitate-supplemented diet so that their weight gain matched that of the -A rats. 4) Fourteen nonrestricted (N) rats were given free access to the retinyl palmitate-supplemented diet. Because P. aeruginosa was not present in the known microflora environment, the rats were immunized with killed P. aeruginosa, strain 107, on d 42, 49 and 56 (Twining et al. 1996b). Because the rats used in these experiments were from multiple litters born on different days, the experiments were staggered. Each of the experiments described was carried out as 3 or 4 independent trials.

On the last day of the experiment (90 days of age), each rat was anesthetized with methoxyflurane and bled by heart puncture using heparin as an anticoagulant. The blood was used for neutrophil function assays and the determination of plasma retinol concentration. The rats were then killed with an overdose of pentobarbital. Livers were removed and stored at -80°C until extracted for retinoids. All procedures were carried out under yellow lights to preserve the retinoids. Samples were stored in either foil or yellow tubes for protection from light.

The rat protocols used in these experiments were approved by the Animal Care Committee of the Medical College of Wisconsin and comply with the Guide for the Care and Use of Laboratory Animals (NRC 1985).

Liver and retinoid concentration determination. Plasma retinoids were extracted using hexane in the presence of 5.7 mmol ascorbic acid/L (Driskell et al. 1985). The entire liver was weighed, homogenized, saponified and extracted using hexane in the presence of 80 mmol pyrogallol/L (Nauss et al. 1985, Thompson et al. 1971). Retinyl acetate was added as an internal standard to all samples prior to extraction. The samples were solubilized in ethanol and separated by HPLC (Dionex, Sunnyvale, CA) on a Whatman (Hillsboro, OR) EQC 125A C18 column (4.6 mm i.d. × 108 mm) using a linear gradient of methanol/H2O (90:10, v/v) and ethyl acetate/isopropanol (90:10, v/v) at a flow rate of 1.0 mL/min (Rudy et al. 1992).

Neutrophil chemotaxis. Neutrophils were isolated from heparinized blood using dextran sedimentation followed by separation on a Histopaque density gradient (Twining et al. 1996). Preparations containing at least 95% neutrophils were used for chemotaxis experiments. Activated rat serum was prepared by incubating plasma from N rats with zymosan for 1 h at 37°C followed by heat inactivation at 56°C for 30 min (Metcalf et al. 1986). RPMI medium was conditioned by Pseudomonas aeruginosa strain 107 for 48 h at 37°C. The organisms were removed by centrifugation followed by filtration through a 0.2 µm filter.

Chemotaxis was carried out by the modified Boyden chamber method (Twining et al. 1986) using Neuro Probe 48-well micro chemotaxis chamber (Neuro Probe Inc., Cabin John, MD) and gelatin coated 5 µm pore membranes. Neutrophils (2 × 107), in RPMI medium, were placed in the bottom wells, and the chemoattractants, 10% activated rat serum, f-Met-Leu-Phe (10-10 to 10-5 mol/L) or 0-16 mL/100 mL P. aeruginosa conditioned medium were placed in the top wells. For 4 × 4 checkerboard assays (to test for chemotaxis rather than chemokinesis), chemoattractants were placed in both the upper and lower wells at graded concentrations (0, 4, 8 and 12 mL/100 mL for the P. aeruginosa conditioned medium and 10-10 to 10-6 mol/L for f-Met-Leu Phe). The chamber was inverted and incubated at 37°C in a humidified 5% CO2 environment for 1 h. The contents of the upper wells (containing the cells which chemotaxed) were mixed with hexadecyltrimethylammonium bromide to a final concentration of 5 g/L. Chemotaxis was quantified by determination of the myeloperoxidase content of these samples relative to the myeloperoxidase content of isolated neutrophils (Williams et al. 1983).

Neutrophil phagocytosis and generation of active oxidants. P. aeruginosa was killed by treatment with 10% formalin (Kessler 1976) and labeled with fluorescein isothiocyanate (Twining 1984). Whole blood was incubated with hydroethidine (added in dimethylformamide) at a final concentration of 5 µmol/L for 10 min at 37°C prior to initiation of experiments. Hydroethidine is permeable to membranes. Inside neutrophils, hydroethidine is oxidized to the membrane impermeable molecule, ethidium, mainly by superoxide during the respiratory burst (Rothe and Valet 1990). Upon oxidation, the fluorescence of this molecule undergoes a red shift. In some experiments, DCFH-DA (in ethanol) was also added at a final concentration of 5 µmol/L (Rothe and Valet 1990). DCFH-DA is a nonfluorescent, membrane permeable compound which upon oxidation becomes fluorescent and membrane impermeable (Rothe and Valet 1990). This reagent follows mainly the myeloperoxidase catalyzed formation of hypohalous compounds. The specificities of the oxidants for hydroethidine and DCFH-DA, however, is not absolute.

Table 1. Final weights, plasma retinol and total liver retinol concentrations of vitamin A-deficient and control rats1

[View Table]

Experiments were initiated by the addition of FITC-labeled or unlabeled phenol-killed P. aeruginosa organisms (when DCFH-DA was used) at either a ratio of 1:10 or 1:100 (neutrophils:organisms) to blood prewarmed at 37°C with or without hydroethidine pretreatment. Incubation was continued at 37°C until termination of the reactions at 2, 7, 15, 30 or 60 min by the addition of Coulter Clone Immuno-Lyse at room temperature, to lyse red cells, followed by Coulter Clone Fixative according to the suppliers recommended protocol. The cells were washed with phosphate buffered saline, pH 7.2 and resuspended in the same buffer at the same volume as the original blood sample. The fluorescence of the cells was quantified (Rothe and Valet 1990) using a Coulter Epics V Flow Cytometer. A 488 nm argon laser with a 470-500 nm bandpass filter was used for excitation. A 510-530 band pass filter was used for emission wavelength selection when either FITC-labeled P. aeruginosa organisms or DCFH-DA was used. When hydroethidine was used, a 600 nm longpass filter was used for emission wavelength selection. For each sample, the fluorescence of 5000 to 10,000 cells was measured. The neutrophils were gated using the forward angle scattering vs. 90° light scattering. The position of the neutrophils was determined using cells labeled with an FITC-labeled antibody specific for rat neutrophils. The data were analyzed using a Cytometer Elite software package (Coulter Diagnostics, Hialeah, FL).

Neutrophil adhesion. Hydroethidine (5 µmol/L final concentration) was added to whole blood and then placed at 4°C for 10 min. FITC-labeled P. aeruginosa organisms were added and samples were removed at 2, 15 and 30 min. The red cells were lysed using Coulter Clone Immuno-Lyse and the remaining cells were fixed using Coulter Clone Fixative at 4°C. In contrast to carrying out the lysis and fixing steps at room temperature, organisms were associated with the neutrophil membranes as determined microscopically. The fluorescence was quantified in the same manner as given for the phagocytosis and killing assays.

Statistical analysis. Statistical analysis of the data for overall differences was carried out using SigmaStat Statistical Software (Jandel Scientific Software, San Rafael, CA). Either the one-way ANOVA or the Kruskal-Wallis ANOVA (Kruskal and Wallis 1952) was used to determine whether an overall difference existed among the -A, R, +A and N groups of data. The Kruskal-Wallis analysis of variance was used when variances were unequal between groups or the data did not pass the normality test. Multiple comparisons were carried out using Tukey's Comparison Method (Tukey 1949) except where the data did not pass the normality test. In this later case, Dunn's multiple comparison of ranks (Dunn 1961) was used.

RESULTS

Vitamin A deficient rats used for these experiments were in the midweight plateau stage (25 ± 3 d after onset of weight plateau) of the deficiency, and 90 days of age. Final average weights of the -A and +A rats were significantly different from those of the R16 and N rats (P < 0.05; Table 1). No significant differences in the final average weights were observed among the -A, +A, R2, R4 and R8 groups. The plasma and liver retinol concentrations at the time of killing confirmed that the -A rats were vitamin A deficient and that the R rats had received vitamin A (Table 1). The plasma retinol concentrations of the -A rats were significantly different from those of the +A, R2, R4, R8, R16 and N rats (P < 0.05). The liver retinol concentrations of the -A rats were significantly different from those of the +A, R16 and N rats (P < 0.05) but not those of the R2, R4 or R8 rats (P < 0.05).
Fig. 1. Comparison of chemotaxis towards P. aeruginosa conditioned medium and f-Met-Leu-Phe using neutrophils from -A (Vitamin A deficient), +A (Weight-matched pair-fed), R16 (Vitamin A deficient rats refed vitamin A for 16 days) and N (Nonrestricted, vitamin A complete diet) rats. Isolated neutrophils from vitamin -A deficient and control rats were placed in modified Boydon Chambers and allowed to migrate towards 10% activated rat serum, 12% P. aeruginosa strain 107 conditioned RPMI medium and 1 nmol f-Met-Leu-Phe/L for one hour under 5% CO2 and a humidified atmosphere. Chemotaxis was quantified by assaying the cells that migrated through the filter into the well containing the chemoattractant for myeloperoxidase. Values were corrected for the myeloperoxidase content of the neutrophils isolated from the same rat. Chemotaxis of the cells toward activated rat serum was used as the control. Values are given as percent migration of the neutrophils towards either P. aeruginosa, strain 107 conditioned medium or f-Met-Leu-Phe relative to the migration of the neutrophils towards 10% activated rat serum (ARS). The bars represent the means, and the error bars represent the SD, n = 10. An overall significant difference in chemotaxis was determined by ANOVA among the neutrophils from -A, R, +A and N rats. P < 0.05 for a vs. b and c vs. d based on Tukey's Comparison Method (Tukey 1949).
[View Larger Version of this Image (35K GIF file)]

Chemotaxis of neutrophils isolated from the blood of the -A, R, +A and N rats was studied using activated rat serum (ARS, control), P. aeruginosa conditioned medium and f-Met-Leu-Phe. In dose response experiments, maximal chemotaxis towards the P. aeruginosa conditioned medium was observed at concentrations of 14 to 16 mL/100 mL and towards f-Met-Leu-Phe at concentrations of 1 to 10 nmol/L for the -A, R16, +A and N rat neutrophils. Checkerboard analyses (4 × 4) for both P. aeruginosa conditioned medium and f-Met-Leu-Phe revealed migration in the direction of the gradient and confirmed the assays measured chemotaxis and not chemokinesis. Chemotaxis towards ARS varied among experiments. However, within a given experiment, there were no significant differences in the chemotaxis activity of -A PMN relative to +A, R16 and N PMN towards this chemoattractant. Chemotaxis towards P. aeruginosa conditioned medium and 10 nmol f-Met-Leu-Phe/L relative to ARS was significantly less (P < 0.05) for the neutrophils from the -A rats when compared to the neutrophils from the +A, R and N rats (Fig. 1).


Fig. 3. Effect of vitamin A deficiency and refeeding of vitamin A to vitamin A deficient rats on neutrophil phagocytosis (A, C) and superoxide generation (B, D). Neutrophils in whole rat blood from vitamin A deficient rats (-A), vitamin A deficient rats refed vitamin A for 2, 4 or 8 d (R2, R4, R8) and non-restricted, vitamin A complete diet rats (N) were assayed as given in Figure 2. A: Comparison of FITC-P. aeruginosa fluorescence associated with neutrophils from -A, +A, and N rats. n = 8 per group. B: Comparison of superoxide oxidation of hydroethidine by neutrophils from -A, +A, and N rats. n = 8 per group. C: Effect of refeeding vitamin A to vitamin A deficient rats for 2, 4 or 8 d on FITC-P. aeruginosa fluorescence associated with neutrophils. n = 4 per group. D: Effect of refeeding vitamin A to vitamin A deficient rats for 2, 4 or 8 d on superoxide oxidation of hydroethidine by neutrophils. n = 4 per group. The points represent the average of the mean fluorescence channel, and the error bars represent the SD. An overall significant difference in phagocytosis for the data in A plus C and in killing for the data in B plus D was determined by ANOVA among the neutrophils from -A, +A, R2, R4, R8 and N rats at 7, 15 and 30 min for both phagocytosis and killing and, in addition, at 2 min for phagocytosis. P < 0.01 for a vs. b in A, and c vs. d in B; P < 0.05 for a (in A) vs. e (in C) and for c (in B) vs. f (in D) based on Tukey's Comparison Method (Tukey 1949).
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Neutrophil phagocytosis and killing were studied by incubation of fluorescein-labeled, dead P. aeruginosa organisms with whole blood containing hydroethidine (Fig. 2). Two populations of neutrophils were noted with increasing time; one population with low red fluorescence and one with higher levels of the red fluorescence (LFL3). The red fluorescence was due to oxidation of hydroethidine mainly by superoxide. With time, the percentage of cells with the higher red fluorescence increased in all groups of cells. The green fluorescence was due to the association of the FITC-labeled organisms with the neutrophils. The distribution of green fluorescence in the two populations overlapped. Cells containing hydroethidine but not exposed to P. aeruginosa organisms had red fluorescence values of < 0.2 fluorescence units compared to the observed 0.2 to 1 fluorescence units observed at 2 and 7 min for cells exposed to P. aeruginosa. Thus, in the population of cells with low red fluorescence, some oxidation of hydroethidine had occurred. The percentage of cells in the high red fluorescence population was significantly less (P < 0.01) (Turkey 1949) in the -A samples (32 ± 3%) at 7 min relative to the R16 (61 ± 5%), +A (58 ± 3%) and N (56 ± 4%) samples (n = 8 for each group). By 15 and 30 min, the differences in distribution of cells among the groups were insignificant. The average of the mean cell fluorescence channel for both green fluorescence, due to FITC-labeled P. aeruginosa organisms (Fig. 3A), and red fluorescence, due to hydroethidine oxidation (Fig. 3B), was significantly (P < 0.01) lower for the total -A neutrophil population than the total +A and the total N neutrophil populations at 7, 15 and 30 min. The number of organisms phagocytosed by the neutrophils reached a plateau by 10 min (Fig. 3A), however, the oxidation of hydroethidine was still linearly increasing at 30 min (Fig. 3B). In neutrophils obtained from rats refed vitamin A two (R2) d and four (R4) days before killing, the averages of the fluorescence channel means representing the phagocytosis of FITC-P. aeruginosa organisms (Fig. 3C) and the oxidation of hydroethidine (Fig. 3D) were higher at all time points. No significant differences were observed between the values for neutrophils from the R2 d and R4 d rats and those for the neutrophils from the -A rats. Neutrophils, obtained from vitamin A deficient rats 8 d after vitamin A was given, had significantly greater numbers of phagocytosed FITC-organisms (Fig. 3C) and oxidized hydroethidine (Fig. 3D) (P < 0.05) than that for the neutrophils from -A rats. Similar values were observed at 16 days (not shown). No significant differences in the amounts of phagocytosed FITC-organisms and oxidized hydroethidine were observed among the neutrophils from the vitamin A-deficient rats refed vitamin A for 8 and 16 d, the +A and N rats.

Unlabeled P. aeruginosa organisms were incubated with hydroethidine to follow mainly superoxide generation and with DCFH-DA to follow mainly myeloperoxidase catalyzed formation of hypohalous compounds. Similar results for superoxide generation were obtained as given in Fig. 3 for the -A, R, +A and N rats. The generation of hypochlorous acid was significantly less (P < 0.05), as quantified by the amount of DCFH-DA oxidized at 2, 7 and 15 min, in the neutrophils from the -A rats in comparison to the neutrophils from the R16, +A and N rats (Fig. 4). By 30 min, no significant differences were noted among the neutrophils from the -A, R, +A and R rats.

Fig. 4. The effect of vitamin A-deficiency on myeloperoxidase oxidation of 2'7'-dichlorofluorescein diacetate (DCFH-DA) determined by comparing activated neutrophils from -A (vitamin A deficient), +A (weight-matched pair-fed), R16 (vitamin A deficient rats refed vitamin A for 16 d) and N (nonrestricted, vitamin A complete diet) rats. The experiment was carried out in the same manner as given in Figure 2 with the exceptions that unlabeled P. aeruginosa and DCFH-DA were used. n = 8 for -A; n = 4 for +A, R16 and N. Values are the average of the mean fluorescence channel and the error bars represent SD. An overall significant difference in DCFH oxidation was determined by ANOVA among the neutrophils from -A, +A, R16 and N rats at 2, 7 and 15 min. P < 0.05 for a vs. b at each time point based on Tukey's Comparison Method (Tukey 1949).
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Adhesion was measured by incubation of hydroethidine loaded neutrophils with FITC-labeled P. aeruginosa organisms at 4°C for 2, 15 and 30 min. The hydroethidine was used to assure that the cells did not undergo a respiratory burst indicative of phagocytosis. The number of organisms associated with the neutrophils from the -A rats at 2 and 15 min was significantly less (P < 0.05) than that seen with the neutrophils from the R, +A or N rats (Fig. 5). At 30 min, only the -A and R neutrophils had significantly different numbers of organisms associated. No significant differences were observed among the neutrophils from the -A, +A and N rats.

Fig. 5. Comparison of adhesion of P. aeruginosa to neutrophils from -A (vitamin A deficient), +A (weight-matched pair-fed), and R16 (vitamin A deficient rats refed vitamin A for 16 d) rats and N (nonrestricted, vitamin A complete diet) rats. The experiment was carried out in the same manner as given in Figure 2 with the exception that the reaction was carried out at 4°C. n = 8 for -A, n = 4 for +A, R and N. Values are the average of the mean fluorescence channel and the error bars represent the SD. An overall significant difference in adhesion of P. aeruginosa was determined by ANOVA among the neutrophils from -A, +A, R16 and N rats at 2, 15 and 30 min. P < 0.05 for a vs. b at each time point based on Tukey's Comparison Method (Tukey 1949).
[View Larger Version of this Image (22K GIF file)]

DISCUSSION

This study revealed vitamin A deficiency alters neutrophil functions. Chemotaxis of neutrophils towards P. aeruginosa conditioned medium and f-Met-Leu-Phe is less for neutrophils from -A rats relative to neutrophils from +A, R and N rats. Chemotaxis of the neutrophils from the -A rats towards activated rat serum was not altered by the deficiency. In vivo, the differences in neutrophil chemotaxis towards bacterial products did not affect the total number of neutrophils that responded within 24 h to a corneal P. aeruginosa infection at high numbers of organisms topically applied to scratched corneas (10-7-10-8) (Twining et al. 1996b). This may be due to the fact that, in vivo, multiple chemotactic factors are generated by the host in response to the infective organism.

The rate of oxidant generation and association of P. aeruginosa organisms with the neutrophils from -A rats was lower than that of the neutrophils from the R, +A and N rats. This is probably related to a lower initial rate of adhesion of the organisms to the -A neutrophils. The initial number of cells which have undergone the respiratory burst (indicated by a large increase in red fluorescence) was less in the neutrophil population of -A rats. This may also reflect differences among the cells in the rate of adhesion of the organisms or it may also be related to a lower rate of the respiratory burst. The differences in kinetics in the generation of oxidized hydroethidine and DCFH-DA may reflect the partitioning of superoxide for the oxidation of hydroethidine and the generation of hydrogen peroxidase. Hydrogen peroxide is needed as a substrate for myeloperoxidase and subsequent oxidation of DCFH-DA. A difference in the amount of DCFH-DA oxidized, but not in the amount of hydroethidine oxidized, was noted by Rothe and Valet (1990) when the two fluorescent molecules were present during phagocytosis of bacteria.

The alterations in neutrophil function due to vitamin A deficiency could be due either to exposure to higher than normal retinol concentrations in the bone marrow during maturation of the neutrophils (Twining et al. 1996a) or to very low retinol concentrations in blood. The bone marrow of the vitamin A deficient rats sequesters retinol with fourfold higher levels than in control rats (Twining et al. 1996a). The low retinol levels in the blood could play a role in the adhesion, phagocytosis and killing experiments reported here because they were carried out using whole blood from the respective rats.

The differences observed in chemotaxis, adhesion, phagocytosis and killing probably are important in fighting infections initially and in determining whether insults by low numbers of organisms are infective. When low numbers of P. aeruginosa organisms are applied topically to scratched corneas on -A, +A, R and N rats, only the -A rat corneas became infected within 24 h (Twining et al. 1996b). These differences in neutrophil functions probably contribute to the lessened ability of vitamin A deficient animals to fight infections of various types.

Vitamin A status affects the ability of the individual to fight infections (Bendich 1992). The severity of infections is affected more than the incidence of infections by the vitamin A status. Because neutrophils are the first cell that responds to an infection, they play a role in limiting the scope of the infection by reducing the number of organisms present (Ferrante et al. 1993, Ratcliffe et al. 1988, Twining et al. 1996b). The reduced ability of the neutrophils from the vitamin A deficient rats to chemotax, phagocytose and kill P. aeruginosa probably is related to the increased severity of infections in vitamin A deficient animals.

Previously identified deficits associated with vitamin A deficiency include the following: an impaired ability to synthesized specific antibodies (Kinoshita and Ross 1993, Pastatiempo et al. 1990, and 1994, Rothe and Valet 1990), lower numbers of natural killer cells (Zhao et al. 1994), abnormal T-cell proliferation (Cantorna et al. 1994, Friedman and Sklan 1989), and increased interferon-gamma secretion by T-cells (Carman and Hayes 1991). Thus, changes in neutrophil, T and B cell and natural killer cell function probably contribute to the decreased ability of vitamin A deficient animals to fight infections.

FOOTNOTES

1   Supported in part by grants RO1EY-08388 and P30EY-01931 from the National Eye Institute-National Institute of Health and a grant from the Thrasher Foundation.
2   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 solely to indicate this fact.
3   To whom correspondence and reprint requests should be addressed. email: stwining@post.its.mcw.edu
4   Abbreviations used: -A, vitamin A deficient rats; +A, weight-matched pair-fed control rats; DCFH-DA, 2'7'-dichlorofluorescein diacetate; FITC, fluorescein isothiocyanate; f-Met-Leu-Phe, formylated methinyl-leucinyl-phenylalanine; LFL, log fluorescence; N, nonrestricted vitamin A-complete diet rats; PMN, polymorphonuclear neutrophil; R, vitamin A-deficient rats subsequently receiving vitamin A for 2, 4, 8 or 16 d (R2, R4, R8, R16).
5   Components of autoclavable casein-based vitamin A deficient diet were the following (g/kg diet): AIN-76 mineral mix, 35.0; calcium carbonate, 4.0; cellulose, 50.0; cornstarch, 649.3; cottonseed oil, 50.0; ethoxyquin (antioxidant), 0.01; DL-methionine, 4.0; vitamin-free test casein, 200.0; vitamin mixture, 7.7. The vitamin A complete diet contained all of the components of the vitamin A deficient diet plus retinyl palmitate, 0.015 g/kg diet. The AIN-76 mineral mix contained the following (g/kg diet): calcium phosphate dibasic, 17.5; chromium-potassium sulfate·12 H2O, 0.019; cupric carbonate, 0.011; ferric citrate, 0.21; magnesium oxide, 0.84; manganous carbonate, 0.12; potassium citrate monohydrate, 7.7; potassium iodate. 0.00035; potassium sulfate, 1.8; sodium chloride, 2.6; sodium selenite pentahydrate, 0.00035; zinc carbonate, 0.056. The vitamin mixture contained the following (mg/kg diet): p-aminobenzoic acid, 165; ascorbic acid, 1487; biotin, 0.66; cholecalciferol, 0.0825; choline, 2150; folic acid, 10; menadione, 75; niacin, 149; pantothenate, 91; pyridoxine, 27; riboflavin, 33; thiamine HCl, 83; all-rac-alpha -tocopherol, 160; vitamin B-12, 45.

Manuscript received 29 August 1996. Initial reviews completed 28 October 1996. Revision accepted 20 December 1996.

LITERATURE CITED

0022-3166/97 $3.00 ©1997 American Society for Nutritional Sciences



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