PART VII. IMMUNOLOGY, CANCER, AND VITAMIN B,
Regulation of Immune Responses by Vitamin B, R A N J I T KUMAR CHANDRA A N D LEELA SUDHAKARAN
Department of Pediatrics Memorial University of Newfoundland, St. John’s, Newfoundland, Canada
INTRODUCTION It has always been acknowledged that there is a close relationship between malnutrition and infection, the one almost invariably being accompanied by and aggravating the other.’ The underlying mechanisms among underprivileged populations are many. These include poor sanitation and personal hygiene, overcrowding, and lack of health education. Recent studies have highlighted the role played by a depressed immune system in these individuals.233This is not surprising considering the fact that clinical manifestations of infection in malnutrition resemble those of infection seen in primary immunodeficiency syndromes, such as thymic defects, neutrophil dysfunctions and others. The main defects in immune responses in protein energy malnutrition are shown in FIGURE1. The question to be answered, then, is just what is it that causes a disordered immune system in malnourished individuals? Clinical malnutrition is a complex syndrome with a broad spectrum of manifestations ranging from life-threatening kwashiorkor and marasmus complicated by infection, to subclinical deficiencies of selected nutrients. It is a composite of deficiencies of calories and many nutrients including proteins, fatty acids, minerals, vitamins, and trace elements. Of these, some, like zinc and vitamin A, have been found to be associated with characteristic immunological changes that have responded to specific nutritional rehabilitati~n.~ Because vitamin B, plays such a key role in the metabolism of nucleic acids and protein synthesis, a deficiency of this nutrient could have a profound effect on the immune response in an i n d i v ~ d a lAlthough .~ isolated, vitamin B, deficiency is rare in humans, in association with other deficiencies, a reduced intake and body stores of vitamin B, may be an important contributory cause of the disordered immunocompetence observed in the malnourished. This is the focus of our review.
T H E IMMUNE SYSTEM The immune system is an important link in the chain of host defense mechanisms that are brought into action when the body faces external (e.g., bacteria, viruses, parasites) or internal (e.g., tumor cells, “forbidden clones” of antibody-producing cells) invaders. Primary immune deficiency syndromes are well recognized but rarely encountered in clinical practice. However, malnutrition is the commonest cause of secondary immunodeficiency worldwide. An optimum immune response requires the balanced interaction of thymic404
CHANDRA & SUDHAKARAN IMMUNE RESPONSES
dependent T lymphocytes, helper and suppressor subsets, antibody-producing B lymphocytes, macrophages, and both enhancing and inhibiting soluble factors.’ Lymphoid organs (thymus, spleen, lymph nodes), the cells comprising these organs, and a multitude of molecules participate in immune responses. These two components of antigen-specific immune protection, humoral immunity and cellmediated immune responses, interact with several nonspecific factors of host resistance such as complement or phagocytes to resist the invasion and multiplication of microorganisms and/or to eliminate them (TABLE1). The relative importance of various components of host defence depends on the nature, dose, and route of antigen exposure. Specific antibodies recognize and react with antigens and the complement system. B lymphocytes, which mature in bone
impaired immunity Cell-mediatedimmunity Microbicidal activity of phagocytes Complement system Secretory antibodies Antibody affinity
Vomiting & rnalabsorption Nutrient losses in urine & faeces Sequestration of nutrients Increased catabolism Diversion of nutrients
FIGURE 1. Interactions between undernutrition and infection.
marrow, respond to appropriate antigen stimulation by proliferation and differentiation into plasma cells. These in turn synthesize and secrete antibodies into lymph and serum. Antibodies are immunoglobulins and have a wide range of specificity for different antigens. In humans, there are five major classes of immunoglobulins, IgG, IgA, IgM, IgD, and IgE. Their basic structure is the same, but they differ in the type of amino acid heavy chain, in molecular weight, and in function. A deficiency or defect of B
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lymphocytes is associated with bacterial and viral infections. Cell-mediated immunity relies primarily on cells such as thymus-dependent T lymphocytes rather than molecules like immunoglobulins in humoral immunity. When T lymphocytes are exposed to antigens, usually by accessor cells such as phagocytic macrophages, metabolic changes occur at the cell surface as well as inside the cell. In addition to cell division or transformation, clones of cells are formed that carry receptors specific to the sensitizing agent. These are the long-lived memory cells. Upon re-exposure to the same antigen, T lymphocytes bearing the appropriate antigen receptor proliferate rapidly and release soluble lymphokines. These, with the aid of other cells, (e.g. macrophages) can destroy the antigen. Delayed cutaneous hypersensitivity testing is a useful tool for assessing cell-mediated immunity in viva Defective cell-mediated immunity is associated with infectious diseases caused by certain pathogenic bacteria, mycobacteria, viruses, fungi, and parasites. This has been observed in both man and laboratory animals. Immunoregulatory processes are very complex. T lymphocytes are a heterogeneous group of cells that mediate cellular immune responses, such as delayed hypersensitivity, allograft and xenograft rejection, and destruction of virus- and bacteria-infected cells and of cancer cells. T lymphocytes also regulate responses of other immunocompe-
TABLE 1. Host Protective Factors
Skin cell mediated Mucous membranes Cilia Mucus Phagocytes Complement system Lysozyme
tent cells. The different subsets of T cells are recognized by the presence of “differentiation antigens” with the help of monoclonal antibodies. Helper-inducer T cells facilitate antibody production by plasma cells and modulate interactions between lymphocytes and accessor cells through the release of lymphokines. Cytotoxic suppressor T cells destroy target cells or provide negative feedback to inhibit antibody response or downregulate inflammatory response. Antigenic nonspecific factors (TABLE1) provide anatomic barriers as well as the front-line phagocytosis, a nonspecific process of ingesting foreign material. In humans there are both circulatory phagocytes, which include monocytes and granulocytes (neutrophils, eosinophils), and “fixed” phagocytes, primary macrophages or larger scavenger cells. The latter are important in the initial processing of antigen for its recognition by T and B lymphocytes. Phagocytosis is facilitated by opsonins that coat particles before ingestion. Opsonization depends both on antibodies and the complement system, which is a complex set of proteins and is the primary humoral mediator and amplifier. Antibody and other molecules can activate the cascade of complement enzymes. Activation of complement promotes varied functions such as phagocytosis, viral neutralization, and lysis of virus-infected cells. Defects in the complement system
CHANDRA & SUDHAKARAN IMMUNE RESPONSES
are associated with increased susceptibility to bacterial infections. Lysozyme, interferon, and other factors provide additional avenues of host defense.
MALNUTRITION AND THE IMMUNE SYSTEM Cellular Response Clinical The cell-mediated immune response which is mediated through the thymusdependent lymphocyte ( T cell) plays a major role in host defence against most viruses, fungi, and mycobacteria. It has been seen that persons who are malnourished have a greater susceptibility to tuberculosis, that the mortality rate from measles is four times greater than that of well-nourished children, and that there is a greater incidence of hepatitis antigenemia in children with protein-calorie malnutrition, to quote but a few examples. Pathological Autopsy findings from children with kwashiorkor have revealed grossly atrophied thymus glands with the lymphoid elements and Hassal’s corpuscles replaced by fibrous tissue.6 The peripheral lymph nodes,6 tonsils, and spleen* are seen to be small in malnourished children.
In Vivo Skin Tests The cell-mediated immunity (CMI) is evaluated in vivo by intradermal skin testing. In order to respond positively to a skin test antigen such as purified protein derivative (PPD) or monilia, one must have lymphocytes that can be sensitized, that proliferate upon re-exposure to the same antigen, and a normal inflammatory response. The percentage of positive tuberculin skin tests is significantly lower in children with protein-calorie malnutrition (PCM) than in a control population. The same response has also been noted to monilia, streptococcal antigen, tricophyton, phytohemagglunin (PHA), mumps, virus, and keyhole limpet hemocyanin. A suppression of delayed cutaneous hypersensitivity has been seen in malnourished adults. The malnourished child is neither able to be sensitized when exposed to a new antigen, nor develop a normal inflammatory response to a skin test irritant.’ TABLE 2 shows the response in studies done by us to various in vivo antigens in healthy and malnourished ~ h i l d r e n . ~ In Vitro Studies
In vivo findings were confirmed by in vitro studies with antigen and mitogenic stimulation of lymphocytes. Peripheral blood lymphocytes from malnourished children reacted poorly to stimulation with mitogens such as P H A and pokeweed. Lymphocyte transformation, which was determined by blast cell transformation, and the uptake of tritiated thymidine improved with nutritional recovery. There may also be certain inhibitory factors in the plasma of malnourished children such as a , globulins and
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Delayed Cutaneous Hypersensitivity
( n = 50) Protein-energy malnutrition ( n = 50)
36" (7.8 k 1.9)*
(15.3 k 3.6)
11 (3.9 t 1.1)
14 (4.1 t 2.8)
"Number showing positive response (induration 2 5 mm). 'Figures in parentheses refer to the mean k SD diameter of induration for the entire group.
C-reactive proteins which are elevated. Since the malnourished child has deficiencies of protein, calories, vitamins, and minerals in addition to superimposed infection, it is difficult to determine which factors actually cause the depression of cell-mediated immune response. However, as recovery of cell-mediated immune response is noted long before complete nutritional recovery, the depressed skin test reactivity in malnourished children may be explained by the depressed number and responsiveness of the thymus-dependent (T) lymphocytes, specially CD4 and helper cells (FIG.2 ) . Phagocytes Polymorphonuclear leucocytes and macrophages are phagocytic cells. Their functions are arbitrarily divided into (i) chemotaxis and (ii) engulfment. Post phagocytic
FIGURE 2. Number of T4 (CD4+) helper cells in undernourished subjects, before and after recovery, and in controls.
CHANDRA & SUDHAKARAN: IMMUNE RESPONSES
events may be classified as (1) phagocytic vacuole formation, (2) microbial killing, and (3) metabolic changes. All these events may be affected in various ways in the malnourished individual. Chemotaxis is the active migration of phagocytes towards a chemotactic stimulus using in vivo techniques (Rebuck skin window technique). This was seen to be increased in polymorphonuclear leucocytes but decreased in monocytes from malnourished patients." Using in vitro techniques, there was an initial delay in the migration of cells which, however, reached control values within a few hours. Phagocytosis does not seem to be markedly affected in malnutrition. In the post-phagocytic phase, neither vacuole formation and degranulation nor microbial killing appears to be diminished in the malnourished child. Among the enzymatic changes seen, decreased iodination, low lactate and glycolysis activity, and decreased levels of oxidases and myeloperoxidases have been reported in some cases. However, these findings are not universal and cannot be said to be a major factor in the malnourished child's decreased host resistance.
Humoral Response The B lymphocyte is responsible for immunoglobulin production. The competency of this system is judged by the following tests. (i) B cell enumeration. In the malnourished child B cell numbers are normal or elevated, which may account for the normal or elevated levels of immunoglobulin titers. Differing opinions are present as to the cause of this. One view ascribes it to the chronic underlying infection present in most malnourished children. Others credit it to suppression of the T suppressor population, resulting in uncontrolled nonspecific antibody production.' (ii) Serum immunoglobulins. The majority of malnourished children have either normal or elevated levels of circulating IgA, IgM, and IgG. Elevated IgE levels may be due to parasite overload. (iii) Antibody responses. Serum antibody responses to antigens such as yellow fever vaccine, influenza vaccine, and typhoid vaccine have been shown to be impaired in malnutrition. However, the responses to others like limpet hemocyanin, lipopolysaccharide, measles, poliovirus, tetanus, and diphtheria toxoid were adequate. The precise cause for decreased responsiveness may be difficult to determine as many factors play a role, namely, dose of antigen, route of administration, severity of malnutrition, presence of infection, and so forth. (iv) Secretory immunoglobulins: Depressed secretory IgA responses are seen in malnourished children. Depressed levels have been demonstrated in the nasopharyngeal and salivary secretions of these children. This reduced mucosal immunity may lead to endemic spread and frequent occurrence of gram-negative septicemia. Complement System In studies carried out in malnourished children, most of the complement components in both classical and alternate pathways were depressed (except C4) with the mean levels being lower in patients of kwashiorkor as compared to marasmic persons.' There was also depression of the hemolytic complement system (TABLE3).
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In conclusion, it has been demonstrated by several investigators that malnutrition affects several immune parameters. in vivo and in vitro cell-mediated immunity is depressed. Though circulating immunoglobulins are elevated, the ability to respond to an antigenic stimulus is compromised by the nutritional status of the person. Secretory immunoglobulins are depressed but recover with improved nutrition. Individual complement proteins and hernolytic complement activity are decreased and the chemotactic and phagocytic activities of P M N leukocytes remain normal.
PYRIDOXINE AND IMMUNE RESPONSES Since pyridoxine is required for normal nucleic acid and protein synthesis as well as for cellular proliferation, pyridoxine deficiencies would cause profound effects on the immune system. Among the effects of severe deficiency of this vitamin are (1) changes in the structure and cellular content of lymphoid organs, (2) inhibition of humoral responsiveness to a variety of test antigens, and (3) an inhibition of various cellmediated immune responses. It has also been seen that pyridoxine deficiency in the diet of pregnant rats may lead to reduced immunological responsiveness and alterations in the lymphoid organs of the offspring. As the effects of deficiency due to any one factor are difficult to elucidate in human beings, a lot of the work in the deficiency states of a single nutrient like pyridoxine is done in experimental animals. There have been a few studies in human beings, but by and large, most of our conclusions are drawn from work done on laboratory animals.
Human Studies There are very few studies on the effects of pyridoxine deficiency in human beings. Wayne et al. (1958) reported that experimentally induced vitamin B, deficiency in human subjects did not interfere with antibody production in response to typhoid vaccine or the A and B blood group substances.” Hodges et al. (1962) maintained humans on a deficient diet for five weeks; a slight impairment of antibody response to tetanus toxoid and typhoid vaccine was noted.” Fisher (1959) was unable to detect any change in skin allograft survival times in pyridoxine-deficient humans.” Dobbelstein el al. (1 974) reported that lymphoid cells from vitamin B,-deficient uremic patients exhibited diminished cell-mediated immune responses.I4 In more recent studies on patients undergoing renal dialysis, it was seen that pyridoxine could correct reduced immunocompetence in certain case^,"^^^ Pre-existent lymphopenia was not corrected, but the number of lymphocytes possessing neither E rosette nor aggregated IgG markers increased markedly from < 1% to >20% after supplementation. Similarly, T lymphocyte rosette formation and in vivo response to mitogenic stimulation by PHA and CoA were normalized by pyridoxine administration. In these studies, no effect was seen on phagocytic or bactericidal activity or polyrnorphonuclear leucocytes. The reduction of nitro blue tetrazolium which was decreased in three of six patients returned to normal after pyridoxine administration. A significant increase in chemotactic response to immune complexes or bacteria was noted in four of six patients given pyridoxine supplementation.
"Mean t SEM. 'Number of individuals showing positive test.
Total Hemolytic Complement Activity CH,, (units/ml)
61 + 9
C3 (mg/dl) 143
Circulating C3 Split Products
Complement Hemolytic Activity and Concentration of Various Components
Healthy (n = 40) Protein-energy malnutrition (n = 40)
+ 17 6 9 + 11
Pathway Hemolytic Activity (W control)
Factor B (mg/dl)
Circulating Immune Complexes
356 i 2 3z
E > R > 56
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A recent study in elderly persons examined the effect of pyridoxine supplementation on lymphocytes.” After one and two months of supplementation with pyridoxine (PN), plasma pyridoxal phosphate (PLP) levels increased by 195 88 n M and 201 f 84 nM, respectively. Lymphocyte proliferation increased significantly in response to PHA and Staphylococcus aureus (Cowain I). For PN-treated subjects with low presupplemental plasma PLP, lymphocyte blastogenesis also increased significantly in response to concanavalin A. Percentages of T 3 + and T4 + but not T8 + cells increased significantly in PN-treated subjects. These results suggest that improving vitamin B, status is important in stimulating immunocompetence in the elderly.
The Efect of Pyridoxine Dejciency on the Structural and Cellular Content of the Thymus and Other Lymphoid Organs One of the earliest studies was done by Stoerk et at. in 1947 who showed that pyridoxine deficiency caused a severe withering of the thymus in rats.I8 These organs were virtually depleted of lymphocytes and consisted almost entirely of epithelial cells
TABLE 4. Organ Weights
Group Vitamin B, deficient Pair-fed control P “Mean + standard deviation. ’Not significant.
Number of Animals
151 t 29“ 233 * 25
314 t 39 369 + 46
and strorna. There was also atrophy of the lymph nodes and a loss of corticomedullary differentiation. Mushett et al.,I9 Ott,20 and Emerson” showed that vitamin B, deficiency and the administration of its antagonist deoxypyridoxine caused lymphoid atrophy and thymic necrosis in dogs, a decreased ratio of spleen to body weight in chicks, and loss of lymphoid elements in the spleen, thymus, and lymph nodes of monkeys. When neonatal chicks were fed a vitamin B, deficient diet for two weeks, significant atrophy of the spleen, thymus, and bursa of Fabricus was seen.22Liver weights were unaffected. In a series of experiments carried out by usz3on SpragueDawley rats in control and experimental settings, similar findings were obtained (TABLE 4). The ultrastructure is significantly altered (FIG. 3). Under the electron microscope, Perkins et al. (1978) studied the thymus of rats fed vitamin B,-deficient diets.z4 After two weeks, there was a decreased density of small lymphocytes in the cortex, increased macrophage activity, and a significant number of plasma cells (unlike normal circumstances when few plasma cells are present). After a period of six weeks, the cortex was virtually depleted of small lymphocytes and
CHANDRA & SUDHAKARAN. IMMUNE RESPONSES
FIGURE 3. Ultrastructure of spleen (a) control (b) vitamin B, deficient animal. Original magnification x6384; reduced by 40%.
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contained predominantly epithelial cells. The thymus capsule had also increased in size and contained aggregates of small lymphocytes. The medullar regions appeared to be unaffected, though. The diet also did not affect the cytoarchitecture of the spleen and lymph nodes. A study by Willis Carr (1978) on rats fed vitamin B,-deficient diets also showed depletion of lymphocytes, mostly in the cortical areas. At the electron microscopic level, however, no gross deviation was seen.25 Cassel et al. (1978) demonstrated that the bone marrow from pyridoxine-deficient rats had reduced numbers of neutrophils, erythroid cells, and small lymphocytes.26On returning the rats to a normal diet, complete recovery of cellularity occurred. In a study by Blalock et al. (1984) in which only marginal deficiency of vitamin B, was induced in chicks, there was no overt morphological damage to primary and secondary immunologic organs.27 The lymphocytes that are depleted from various lymphoid organs belong to two subpopulations of small lymphocytes, namely short-lived small lymphocytes made up of predominantly B lymphocytes, and long-lived small lymphocytes which are mainly T cells. It was seen that the cells that were depleted from the thymus and bone marrow of pyridoxine-deficient animals were mainly short-lived small lymphocytes (SLSL) and those found to be depleted from the thoracic duct lymph and blood were mainly long-lived small lymphocytes (LLSL).28The loss of SLSLs was postulated to be due to an inhibition of their proliferation and accelerated rate of d e s t r ~ c t i o nHowever .~~ the loss of LLSLs was probably the result of a loss of thymic function as the formation and maintenance of LLSL may be dependent on a humoral factor produced by thymic epithelial cells which is most probably impaired in vitamin B, d e f i ~ i e n c y . ~ ~
The InfIuence of Pyridoxine on Humoral Immune Response A large number of studies have been carried out to show that deficiency of vitamin B, may lead to impaired antibody production in experimental animals. Stoerk et al. (1946, 1947) observed a suppression of the formation of circulating antibodies to sheep red cells in rats fed a vitamin B,-deficient diet for six week^.^'.^' Axelrod et al. (1947) reported similar findings in rats immunized with human red blood cells.32Other studies have subsequently demonstrated that pyridoxine deficiency is consistently accompanied by an inhibited antibody formation to a large number of antigens. In all of these studies, animals fed a deficient diet for 2-10 weeks with or without the addition of deoxypyridoxine showed a reduction in the levels of circulating antibodies following immunization with diphtheria t o ~ o i d influenza ,~~ or B typho~urn,’~ C. K r i t ~ h e r i , ~murine , typhus rickettsia^,^' H. pertussis, Salmonella p u l l ~ r u m , poly ’ ~ G I u Lys ~ ~ 33 Tyr’5.39The reduction in circulating antibody cells was also accompanied by a reduction in antibody-forming cells in the spleens of such pyridoxine-deficient animals.40Axelrod also demonstrated that the secondary response to diphtheria toxoid was diminished to a greater extent than was the primary response to this antigen in pyridoxine-deficient animals. However the anaphylactic reaction in guinea pigs was not altered when serum albumin was used as the immunizing or challenging dose.4’ Marginal deficiency of pyridoxine in chicks26resulted in significant reduction in antibody levels to sheep red blood cells (SRBC) and relative levels of IgM and IgG during the peak and degradation phases of the primary response.
CHANDRA & SUDHAKARAN: IMMUNE RESPONSES
Using the Jerne plaque assay, we found decreased number of IgM-producing spleen cells in vitamin B,-deficient rats sensitized with sheep red blood cells (TABLE c i 23 2).
The results of various experiments indicate that vitamin B, deficiency, both in uivo and in vitro, results in a decreased biosynthesis of D N A and R N A especially mRNA.42i43The inhibition of nucleic acid synthesis was specifically linked to a decreased production of “C” fragments from serine. It was concluded that inhibition of nucleic acid synthesis is the most likely mechanism whereby pyridoxine deficiency may interfere with cellular multiplication and protein biosynthesis, and hence with normal antibody production. Gershoff et al. even suggested that the effects of pyridoxine deficiency in hindering antibody synthesis may be partially reversed by serine, and to a lesser extent, by glycine s ~ p p l e m e n t a t i o n . ~ ~ Influence of Pyridoxine on Cell-Mediated Immune Responses Lack of vitamin B, from diets of experimental animals results in a suppression of various cell-mediated immune responses. The earliest studies of Axelrod et al. (1 958) and Fisher et al. (1958) reported that rats fed deficient diets retained allogenic skin
Plaque-Forming Cell Response
Number of Rats
Vitamin B, deficient Pair-fed
Background 0.49 0.21
Direct PFC per Spleen x 10’ 5th Day PostImmunization 21.7 49.7