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Vitamin A, immunocompetence, and infection
Ranjit K. Chandra and Devhuti Vyas
The interactions between nutrition, immunity, and infection have been the focus of much recent work. It has been observed that protein-energy malnutrition (PEM) is associated with impaired immunocompetence [1-3], including depressed cell-mediated immunity, phagocyte dysfunction, decreased levels of complement components, reduced mucosal secretory antibody responses, and lower antibody affinity. At the same time, the complexity of clinical malnutrition is recognized. This has led to the examination of immunological effects produced by single nutrient deficiencies.
Among other nutrients, vitamin A has a profound effect on immune responses in man and laboratory animals. In humans, vitamin-A deficiency seldom occurs in isolation; it is usually associated with varying degrees of PEM. In many countries there is a high prevalence of xerophthalmia and night blindness among infants and children suffering from PEM. However, the numerical association between PEM and xerophthalmia varies enormously in different geographical regions of the world, from 75% in Indonesia to 32% in India, 1%-6% in Latin America, and 1%-2% in Lebanon and Uganda. However, it is possible that patient selection and subjective diagnostic criteria have influenced the reported low prevalences. Besides low dietary intake, reduced absorption of vitamin A in PEM can further aggravate its deficiency. Oral water-miscible vitamin A is absorbed in an erratic fashion in children with kwashiorkar. A study carried out in South Carolina showed that 50% of underprivileged preschool children of a poor income group had either low or marginal levels of serum vitamin A . Thus, vitamin-A deficiency is not completely restricted to underprivileged or developing countries but has worldwide prevalence and is the third most frequent nutritional problem after
PEM and iron deficiency. It is the most frequent cause of preventable blindness. This has led to international efforts at prevention by periodic massive doses using oral or injectable vitamin A. The rapidity with which vitamin A is absorbed is one of the determinants of early recovery from night blindness and xerophthalmia.
Vitamin A and infection
High death rates among PEM children parallel their vitamin-A status: children dying of PEM have lower levels of vitamin A than the survivors . Most of the deaths are due to respiratory and gastrointestinal infections. It was observed in one study that the overall mortality rate in victims of PEM was 15%, whereas it was as high as 80% if malnutrition was associated with xerophthalmia . The association between hypovitaminosis-A and bacterial and viral infections in humans and various species of animals has been reviewed [7-9]. Many pathological mechanisms probably contribute to the increased risk of infection in vitamin-A deficiency, including tissue changes and altered specific and non-specific immunity. Furthermore, infection itself can aggravate vitamin-A deficiency.
Other than its well-recognized role in rhodopsin synthesis and vision, vitamin A also regulates differentiation of epithelial tissues and inhibits keratinization. In hypovitaminosis-A, the secretory epithelia of respiratory tract and salivary and prostate glands show keratinization; this change offers less resistance against penetration of infectious agents and makes the individual susceptible to infections. Our recent studies indicate that bacteria bind to respiratory epithelial cells in greater numbers in vitamin-A-deficient subjects than in healthy controls (table 1) . Increased mortality has been noted in children with mild vitamin-A deficiency . Vitamin-A deficiency and infections aggravate each other, as the deficiency predisposes the host to infection, which in turn decreases the intestinal absorption of the vitamin . Infection can even precipitate the symptoms of deficiency in an individual with marginal levels of the vitamin. In partially vitamin-A-deficient rats, even a subclinical load of malarial parasites has been shown to precipitate deficiency symptoms at a faster rate and resulted in more severe parasitaemia . Vitamin-A-deficient patients sometimes develop xerophthalmia in only one eye, possibly because of the presence of infection in one site. Nasal mucosa of vitamin-A-deficient chicks keratinized only in areas of virus infection . From these observations, one can deduce that the effects of deficiency are compounded if there is an accompanying infection. In germ-free environments, vitamin-A-deficient rats live for a relatively long time; it is believed that the vitamin requirement is low in the absence of stress of infection .
TABLE 1. Clinical data and bacterial binding to epithelial cells
|Group||Number||Age (months)||Serum retinol (m mol/L)|
|I||14||8||6||22 ± 3||2.234 ± 0.28a||1.413-3.351|
|II||10||6||4||24 ± 4||1.117 ± 0 14b||0.733-1.397|
|III||12||7||5||20 ± 3||0.419 ± 0.10c||0.139-0.698|
|Group||Dietary Vitamin A (retinol equivalents)||Ocular signs (number of subjects)||Weight for height (% of standard)||Bacteria per epithelial cell|
|Xerophthalmia||Bitot's spots||Corneal opacity|
Data are given as mean ± SE.
Values in the same column designated by different superscript letters differ significantly from each other at a probability level of .01 or less.
Secretory antibody response, mucosal cell-mediated immunity, and non-specific barrier functions are important host defences. The disastrous effect of infection in vitamin-A-deficiency state may be due to the increased pathogenicity of infectious agents and/ or reduced immunocompetence of the host. Hypovitaminosis impairs tissue integrity by permitting keratinization of mucociliary linings. In the intestine, a reduction in the number of goblet cells and in mucus production disrupts non-specific defence mechanisms of the gastrointestinal tract. Impaired barrier function increases the systemic spread of infectious agents. By exposing vitamin-A-deficient rats to the nematode Angiostrongylus cantonensis, it was shown that the penetration power of the larvae increased and the animals had a higher worm burden and shorter survival period . The observed effect was not due to inanition, as the animals were tube fed with a vitamin-A-deficient diet. Vitamin-A-deficient chicks after Newcastle disease virus infection had 100 times more virus in the throat swabs than normal non-deficient chicks . The high frequencies of respiratory and gastrointestinal tract infections in PEM may in part be due to tissue changes resulting from associated vitamin-A deficiency.
Reduced levels of secretory IgA (slgA) have been observed in the saliva, nasopharyngeal secretions, duodenal fluid, and tears in malnourished children [18-21]. slgA antibody response to polio virus and measles vaccines is impaired . Intraepithelial Iymphocytes and slgA-secreting plasma cells have been shown to be numerically lower in malnourished patients . Also, levels of slgA in the intestinal fluid of rats on vitamin-A-deficient diets were reported to be very low compared with those in normal animals: rats given 5 mg retinoate per gram of diet showed a maximum of 180.2 mg of slgA per millilitre of intestinal fluid, and withdrawal of retinoic acid resulted in a low level of 46.0 mg of slgA per millilitre . Moreover, it was also shown that supplementation to the deficient animals of 500 mg of retinyl palmitate per day, for just two days, increased the levels of slgA to 148.5 mg per millilitre of intestinal fluid. Intestinal cells of the deficient animals did not show any decrease in immunofluorescence for secretory component in the initial period of deficiency, but, after eight days and more, decreased intensity of fluorescence staining was observed. In addition, vitamin-A-deficient animals showed poor anti-dinitrophenyl response to 2,4-dinitrophenylated bovine gamma globulin (50 mol DNP per mole BOG) fed through tap water after initial priming with an intraperitoneal injection. In fact, only one of seven deficient rats showed a detectable anti-DNP activity, but three of four control animals responded to the antigen. The only deficient rat that responded to the antigen had significantly low levels of antibody (7.4 pmol antibody combining site per millilitre of intestinal fluid) compared with that of controls (18.9 pmol antibody combining site per millilitre).
There are few data on cell traffic in vitamin-A deficiency. Mesenteric Iymph node lymphocytes (MLNL) are precursors of intestinal IgA-secreting plasma cells and have a tendency to localize selectively in the gut mucosa. Protein-deprived rodents show reduced localization of MLNL in the gut. McDermott et al.  have shown that PEM and vitamin-A deficiency alter the capacity of MLNL to localize in the gut. Weanling rats were fed a low-vitamin-A diet until growth ceased, but there was no evidence of xerophthalmia or alteration in intestinal mucosa. Labelled Iymph node cells of normal or vitamin-A-deficient animals were injected into normal or vitamin-A-deficient recipient animals. Localization to the gut was decreased irrespective of the nutritional status of the recipients. However, the separate effects of vitamin A and PEM were not clearly dissected out in this study . Reduced homing of MLNL in PEM and vitamin-A deficiency might be one of the reasons for reduced sIgA levels in this nutrient deficiency.
Increased keratinization, low levels of mucus secretion, and decreased numbers of goblet cells in the intestine in vitamin-A deficiency, together with reduced slgA secretion and reduced local antibody response, act synergistically to make the deficient individual vulnerable to a variety of gastrointestinal and respiratory infections. In addition, because of compromised systemic immunocompetence, such an individual may succumb to repeated and chronic infections [26; 27].
Once the infectious agent crosses the anatomic barriers of skin and mucous membrane and enters the body, phagocytes deal in a non-specific way with certain bacteria and fungi before antigen-specific cellular and humoral immune mechanisms come into play. Large doses of vitamin A given to normal mice have been shown to offer non-specific resistance to grampositive and gram-negative bacterial and fungal infection . A decreased mortality rate and low levels of bacteraemia were reported after challenge with Pseudomonas aeruginosa, Listeria monocytogenes, and Candida albicans. It was concluded that this increase in resistance is non-specific and might result from an augmented capacity of phagocytes (macrophages) to deal with these agents. However, there was no change in the uptake of colloidal carbon or aggregated human serum albumin by the reticuloendothelial system. The effect could not be due to changes in the organisms themselves, since vitamin A did not alter in vitro growth rates of the three microorganisms.
Hof and Emmerling  also observed a 100-fold increase in resistance to infection with L. monocytogenes. This was seen during the early stage of infection and was therefore attributed to increased functional capacity of the phagocytic system in rats treated with vitamin A. However, it should be noted that very large toxic doses (4 x 15,000 IU) of retinoic acid had to be used ro achieve such protective effects. Vitamin-A-deficient and PEM rats were shown to have a reduced number of glass-adhering cells (macrophages), and, moreover, these phagocytes had decreased ability to clear infection with a malarial parasite, Plasmodium berghei. Oral supplementation of vitamin A to deficient rats after infection has been shown to augment the recovery process . Survival time after infection was shorter in vitamin-A-deficient animals than in pair-fed animals, but in control animals no death occurred during the experimental period of five weeks.
An impaired immune system is one of the factors that add to the vulnerability of vitamin-A-deficient individuals to infection. Usually vitamin-A deficiency occurs concomitantly with PEM, and in experimental animals vitamin-A deficiency causes inanition. There is ample evidence of reduced immunocompetence in PEM [1-3]. If PEM is accompanied by vitamin-A deficiency, the deficiency adds significantly to susceptibility to infection because of its additional detrimental effects on Iymphoid tissues and organs [30-35].
A diet deficient in vitamin A has been shown to cause atrophic changes in the thymus and spleen in rats . Pair-fed rats that developed PEM also showed atrophic changes in bath tissues, but the magnitude of change was much greater in the vitamin-A-deficient group. The cortical region of the thymus of vitamin-A-deficient animals becomes devoid of Iymphocytes. The spleen also shows atrophy of germinal centers. However, Chandra and Au  observed only a slight difference in weight of the thymus and spleen from vitamin-A-deficient and pair-fed control animals, though the weights were significantly different Mom those of control animals fed ad libitum (table 2). These results suggest that the observed effect is mainly due to anorexia and malabsorption associated with vitamin-A deficiency. Similarly, Nauss et al.  also observed that weights of the spleen and thymus of vitamin-A-deficient and pair-fed controls were comparable but were lower than those from controls fed ad libitum.
TABLE 2. Organ weights
|Vitamin-A-deficient||6||181 ± 39||317 ± 41|
|Pair-fed control||6||208 ± 28||339 ± 47|
|Ad libitum control||6||281 ± 40||387 ± 59|
Source: Ref. 31.
Involution of the thymus and bursa of Fabricius have been observed in vitamin-A-deficient chicks . Chickens on a vitamin-A-deficient diet from the time of hatching showed atrophic changes in Iymphoid tissues. After 30 days on a vitamin-A-deficient diet, the epithelium of the bursa of Fabricius became pseudo-stratified, or cystic, and was disorganized compared with normal tissue. Follicles also displayed irregularity and fibrosis. The medulla showed invasion by heterophil cells, which replaced Iymphocytes. Panda and Combs  also observed a decrease in weight of bursae in vitamin-A-deficient chicks. Infection of deficient chicks by Newcastle disease virus caused further regression of bursae, interfollicular fibrosis, epithelial metaplasia, and keratinization. Follicles showed loss of lymphocytes and invasion of polymorphonuclear leukocytes and eosinophils five days after infection, and the medulla showed debris of disintegrating cell nuclei. These atrophic changes were progressive, and after nine days of infection, bursae of vitamin-A-deficient chicks were significantly different from those of vitamin-A-deficient non-infected controls. The thymus of vitamin-A-deprived chicks showed invasion of polymorphonuclear cells and loss of cortical width. After one day of virus infection, thymic corpuscles increased in size and number, and the cortex decreased in size and showed cell debris. These changes were progressive: Three days after infection a complete loss of cortex became evident, as shown by a reticular epithelial mesh, and thymic corpuscles showed dilation and degeneration. After seven days the organ appeared devoid of thymocytes and showed thymic corpuscles, degenerating cells, thick-walled vessels, and vacuoles containing polymorphonuclear cells. Atrophic changes were modest in the noninfective vitamin-deficient state . Thus, vitamin deficiency and infection had synergistic effects on the Iymphoid tissue, thereby increasing the severity of the course of infection.
It is evident that vitamin-A deficiency causes atrophic changes in Iymphoid tissue. At the same time, a high dose of retinoic acid (1,000 fig per mouse per day for seven days) also resulted in reduction in body weight and spleen weight; it affected cell populations in the spleen and thymus, but the bone marrow was more or less resistant to this toxic effect. The thymus and spleen showed 95% and 50% reduction in cellularity respectively when high doses of retinoic acid were used .
In clinical practice, it is difficult to sort out the individual effects of vitamin-A deficiency from those of other nutrient deficiencies. The frequent presence of PEM confounds the picture. Thus, the reported reduction in the number of T cells  may well be due to concomitant PEM rather than to vitamin-A deficiency itself. In rodents fed a low-vitamin-A diet, the level of serum thymic factor is unaltered . A decreased total leucocyte number has been observed in vitamin-A deficiency; the differential count revealed a relative increase in neutrophils and a decrease in the number of Iymphocytes. The mitogenic response of splenic Iymphocytes of vitamin-A-deficient rats to phytohaemagglutinin, concavalin A, and Escherichia coli lipopolysaccharide is reported to be less than one-third that of control and pair-fed animals [31; 36], although the mitogenic response of thymic Iymphocytes to concavalin A was comparable in vitamin-A-deficient, pair-fed, and control animals studied by 3H incorporation. Vitamin-A supplementation for three days was shown to increase circulating Iymphocytes and to restore the mitogenic response of splenic Iymphocytes to normal levels . Decreased 3H incorporation by thymacytes and splenocytes was also reported in vitamin-A-deficient and PEM rats .
Decreased nucleic acid synthesis and mitogenic response (table 3) may be due to defective synthesis of membrane receptors. Marked changes in glycoprotein synthesis have been observed in vitamin-A deficiency [38; 39], and similar changes in membrane glycoproteins of lymphocytes (R. K. Chandra and V. Kumar, unpublished data) may contribute to impaired cell-mediated immunity seen in vitamin-A deficiency. Alternatively, if changes in T cells in vitamin-A deficiency are accompanied by increased suppressor T cells, then decreased mitogenic response would also result.
TABLE 3. Mitogen stimulation response
rats (N = 6)
|Pair-fed controls(N = 6)||P value|
|0.2||20.7 ± 8.8||87.8 ± 39.5||<.05|
|0.5||37.5 ± 19.0||104.8 ±36.4||< .01|
|2.0||38.7 ± 31.9||121.8 ±57.5||<.01|
Source: Ref. 31. a. Amount of phytohaemagglutinin (PHA) in each well of microtitre plate.
In human volunteers, a modest supplement of beta-carotene is associated with increase in the number and function of CD4+ helper T cells. If this translates into enhanced resistance, it would have immense clinical significance. Vitamin-A supplementation has been shown to augment cell-mediated immune response, and injections of 150 IU per gram per day for five days accelerated the onset and decreased the duration of skin-graft rejection in mice . Mice treated with 150 mg of vitamin A and sheep red-blood cells (SRBC) simultaneously and challenged 3-21 days later with the same antigen by subcutaneous injection into the pinna showed increased cell-mediated immune response measured as an increase in the thickness of the ear . Conversely, it has been reported  that mice treated with 25 and 50 mg of retinoic acid, primed with SRBC, and then challenged later with SRBC into the footpad did not show any increase in levels of cell-mediated immune response measured as an increase in footpad diameter. Moreover, 100 and 300 mg of retinoic acid slightly suppressed the delayed hypersensitivity reaction. The discrepancy in the results of the two groups of investigators might be due to differences in the design of the experiment, particularly the dosage of retinoic acid and the time of its administration. In one study , vitamin A and SRBC were injected simultaneously, and, in the other , retinoic acid was injected for 1-6 days and the animals were primed with SRBC on day 10. Retinoic acid was shown to have no stimulatory effect on the mixed lymphocyte reaction in mice .
Vitamin A also modified the humoral response, especially to T-dependent antigens. The number of plaque-forming cells in vitamin-A-deficient rats was lower than in pair-fed animals (table 4) . Haemagglutinin titre against diphtheria and tetanus antigens were found to be reduced in vitamin-A-deficient rats as compared with pair-fed controls . Another study showed increased haemagglutinin titre to SRBC in mice after treatment with 600 IU of vitamin per gram of body weight per day for five days, either before or shortly after sensitization with antigen . The response to vitamin-A treatment was highly significant: the relative reciprocal titre of haemagglutininin the controls was 64 and in the vitamin-A-treated animals was in the range 1,024-4,096.
TABLE 4. Plaque-forming cell response
|Direct PFC per spleen (10 -3)||Vitamin-A- deficient||Pair-fed controls||P value|
|rats (N = 6)||(N = 6)|
|Background||0.39 ± 0.08||0.24 ± 0.09||<.05|
|Day 5 after||17.5 ± 4.8||56.4 ± 9.0||<.01|
Source: Ref. 31.
Cohen and Cohen  have also reported increased plaque-forming cells to SRBC in vitamin-A-treated mice. They showed that intraperitoneal administration of 1,000 IU of vitamin A per day for four days increased plaque-forming cells. The maximum effect (a fivefold increase) was observed with a dose of 300 IU per day for four days; the toxic dose of 9,000 IU per day did not increase antibody-forming cells in the spleen of mice. Increased antibody production to 2,4-dinitrophenyl conjugate of ovalbumin after two subcutaneous injections of either 2,000 or 5,000 IU of vitamin A was also observed in mice .
Partial vitamin-A deficiency in chicks reduces agglutinin titre against Salmonella pullorum antigen . Uhr et al.  have reported an immunosuppressive effect of vitamin A in guinea pigs. They did not find any increase either in the clearance of bacteriophage ox174 from the circulation or in the production of antibodies to it. However, they observed prolonged production of 19S antibodies if vitamin A was injected simultaneously with antigen . Dennert and Lotan , in studies in vitro, observed 80% reduction in plaque-forming cell response of spleen cells from retinoic-acid-treated mice, and 10-5-10-9 M concentration of retinoic acid during sensitization of spleen cells to SRBC was shown to completely suppress the induction of plaque-forming cells. In viva studies showed no effect of 100, 300, and 1,000 fig of retinoic acid for five days and sensitization with SRBC on day 4 on the number of plaque-forming cells in the spleen after eight days.
In mice, the simultaneous administration of vitamin and tetanus toxoid resulted in an enhanced antitoxin response. The effect of three different doses of vitamin A (3,000, 25,000, and 30,000 IU) in mice on the antitoxin response to tetanus toxoid indicated a direct relation of the response to the dose of vitamin . Comparable doses of vitamin A given to children would have had undesirable side effects. At the dose level of 30,000 IU, mice showed signs of toxicity. However, these investigators did not observe any significant effect of 200,000 IU of vitamin A on the production of antitoxin to tetanus toxoid in a field trial involving Bangladeshi children.
In PEM, in addition to a decrease in cell-mediated and humoral immune responses, haemolytic complement activity of the serum is depressed. Vitamin-A deficiency has been shown to aggravate the disastrous effects of PEM on immunocompetence. However, in rats, vitamin-A deficiency did not have any additive effect on serum complement levels in PEM. Contrary to the case with PEM, in which complement levels decrease [1-31, vitamin-A deficiency has been shown to enhance complement levels . In addition to this, Azar and Good  have shown suppression of serum complement haemolytic activity 24 hours after a large dose of vitamin A is administered.
Vitamin A acts as an adjuvant at non-toxic doses and enhances cell-mediated and humoral immune responses. Injections of vitamin A increased the cellularity of redonal Iymph nodes.. The vitamin has also been shown to stimulate antibody production to bovine gamma globulin, which would otherwise have resulted in immunological paralysis . The adjuvant property of vitamin A was thought to be due to its membrane-labilizing effect on Iysosomes. Lysosomal membrane labilization can induce Iymphoid cell proliferation . Vitamin A has been shown to enhance cell-mediated immune response when administered simultaneously with or close to the antigen challenge. Moreover, injection of vitamin A at a site remote from that of the antigen was shown to be ineffective in enhancing cellular immunity [40; 41], suggesting that the draining Iymph node might be the site of adjuvant action of vitamin A. Taub et al.  reported on the adjuvanticity of vitamin A. Two days after injection of 0.5 mg of vitamin A in liquid paraffin in the right footpad, the popliteal Iymph node showed enlargement and hypercellularity of paracortical areas. After six days, the germinal centres were observed in the cortex, and the medullary region showed slight expansion and a few place cells. Vitamin A was shown to work as a classic adjuvant without any antigenicity. The formation of germinal centres after vitamin-A administration was not as pronounced as with other adjuvants, such as alum-precipitated bovine gamma globulin, Freund's complete adjuvant, and pertussis coccobacillus. Enlargement of the Iymph node and increased Iymphocyte traffic after vitamin-A treatment close to the time of antigen treatment may help to augment immune response by increasing contact between lymphocytes and antigens. Allison and Davies  have also shown cellular proliferation and blast transformation in thymus-dependent areas of a draining Iymph node after vitamin-A administration.
The world-wide prevalence of isolated vitamin-A deficiency and its occurrence in association with other nutritional deficiencies, especially PEM, and with infection has stimulated considerable recent work on the role of vitamin A in host resistence. Vitamin A subserves a number of important physiological functions that contribute to effective immunocompetence. Moderately large doses of beta-carotene have an immunostimulatory effect and can reverse the suppression produced by pharmacological agents such as cortisone. It acts as an adjuvant and influences both cell-membrane and intracellular composition and function. This applies to Iymphocytes and monocytes. Thus, it is reasonable to expect that vitamin-A deficiency is associated with an increased incidence of infection and that its prevention or treatment will result in decreased illness. This may be due to epithelial changes and improved cell-mediated and mucosal immunity. Recent work [26; 27; 52-55] has affirmed the critical role of beta-carotene and vitamin A in optimum immunocompetence. At the same time, massive doses of vitamin A if given for prolonged periods may have a deleterious effect.
Our work has been supported by Health and Welfare Canada, the Medical Research Council, Ross Laboratories, Sandoz Nutrition, and the Carnation Company.
The present article is based on an earlier review article l27].
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