Immunodeficiency - The Thymus
“Patients with Down’s syndrome suffer from frequent infections and have an increased mortality in infectious diseases compared to a normal population. Several laboratory studies have demonstrated abnormalities of cell-mediated and humoral immune capacity and of phagocyte function.” (Björkstén et al, 1980). As zinc is vital for the functioning of the immune system (Meek, 1996) it makes sense to consider the role of zinc when questioning why the DS immune system is weak, and how it can be supported. It has been found that young animals and humans, when receiving insufficient zinc, exhibit:
rapid thymic atrophy
decreased production of thymic hormones
impaired lymphocyte proliferation after phytomitogen stimulation
a) decreased number of, and dysfunction of, T-lymphocytes b) abnormal T-helper and/or suppressor cell function
deficiency of natural killer cells
significantly reduced antibody and cell-mediated responses
delayed hypersensitivity reaction
decreased spleen and lymph nodes
generalised defective development of lymph tissue
(Franceschi et al, 1988; Stabile et al, 1991; Brigino et al, 1996)
Therefore the first step is to investigate whether DS people also exhibit any of these dysfunctions. In fact DS individuals have many complex immune disturbances (Opitz and Gilbert-Barness, 1990) into which research is only beginning to make inroads.
It is in the thymus that immature T-cells differentiate and mature and thymic hormones are produced which perform such functions as inducing the expression of T-cell markers and modulating T-cell function. In healthy individuals, much of the thymic tissue is replaced by fat and connective tissue after puberty, and by maturity the thymus has atrophied — though it is still continuing to function. However in DS subjects there is rapid and premature thymic atrophy (Fabris et al, 1993), suggesting a reason for at least some of the characteristic immune weakness.
Measurement of thymulin — a thymic hormone which induces expression of several T-cell markers and modulates T-cell function — has shown significantly lowered levels in DS (Franceschi et al, 1988; Licastro et al, 1994; Brigino et al 1996). Franceschi et al differentiated between DS normozincaemic children and hypozincaemic children, and found that both groups had low thymulin levels.
DS is undoubtedly concurrent with a significantly reduced ability of lymphocytes when stimulated with mitogens (Björkstén et al,1980; Franceschi et al, 1988; Lockitch, 1989; Stabile et al, 1991; Fabris et al, 1993; Licastro et al, 1994)
A number of papers report a reduction in T-lymphocyte numbers and in distribution of subsets, though they are sometimes contradictory in how the distribution varies from normal (Noble and Warren, 1982; Franceschi et al, 1988; Fabris et al, 1993; Licastro et al, 1994; Brigino et al, 1996). In 1990, Serra and Neri reported “reduced expansion of T-cell precursors in the thymus, which results in incomplete T-cell subsets, a lowered response to antigens, and consequently a profound impairment of T-cell-mediated immunity”. Lockitch et al (1989) and Stabile et al (1991) found normal numbers of total lymphocytes and of total T-cells. Lockitch et al report finding lower T-lymphocyte, and lower T helper cell and T suppressor cell counts in earlier research. Interestingly Stabile used the most closely age-matched controls and subjects; unfortunately Lockitch does not give mean age or standard deviation for their subjects or controls. Lockitch did not investigate subset distribution, but Stabile found only elevated CD8+ (precursor of cytotoxic T-cells) levels and a raised ratio of CD8+ to CD4+ (precursor of T-helper cells).
Fabris et al (1993) reports raised levels of natural killer (NK) cells and lowered NK cell activity. Stabile also found elevated levels of NK cells. Most other papers investigating DS and the role of zinc in immune potency do not mention NK cells, but Noble and Warren observed normal NK cell activity and Lockitch et al found normal NK cell numbers.
No research was found directly investigating the activity of DS antibodies in relation to zinc status. Franceschi et al report low levels of B-cells, and both Stabile and Lockitch found normal levels.
Only Licastro et al and Björkstén et al measured delayed hypersensitivity reactions, a class of allergic reaction where either CD4+ or CD8+ T cells secrete cytokines that activate allergen-eating macrophages. Both used harmless skin tests, and both found that their DS subjects had a diminished response.
And 9. There is no work, at the time of writing, which investigates the development and functioning of lymph and spleen tissue in DS subjects in relation to zinc.
The significance of the thymus
“The majority of immune alterations observed in DS subjects seem to depend on defective thymic function.” (Fabris et al, 1993). This would include low levels of thymulin; a reduction in and/or disruption to the subset division of, T-lymphocytes; a reduction in B-lymphocytes (the proliferation of which is controlled by the T helper cells) and diminished delayed hypersensitivity — all part of what is known as the adaptive immune system. Napolitano et al (1990) claim that low zinc levels are responsible for the early atrophy of the thymus. One obvious link between the thymus and low zinc status is that zinc is required to transport vitamin A from the liver, and vitamin A is necessary for the growth hormone which maintains the thymus (Meek, 1996).
The importance of functional assays
Before the effects of zinc supplementation on these parameters is considered, a ground-breaking piece of research by Fabris et al in 1984 must be considered. Having realised that plasma concentrations of thymic factors are not necessarily a reliable index of the functional activity of the gland, the investigators measured the levels of active thymulin and the levels of thymulin inhibitory activity in young DS subjects and in healthy controls. They then added zinc sulphate in vitro and assayed again. The finding was that the DS individuals and the normal subjects over 50 years old had an inverse correlation between plasma active thymulin and thymulin-inhibitory activity. In normal people up to 20 years old thymulin levels were highest of all those measured and thymulin-inhibitory activity was not detected in healthy subjects until they reached 30. Conversely thymulin-inhibitory activity was high even in the youngest DS children. In both the elderly people and DS subjects plasma zinc was below the normal range for healthy adults. Once zinc sulphate was added to the plasma samples from both these groups the active thymulin levels become the same as those found in healthy young adults and the thymulin-inhibitory activity completely disappeared. These inhibitory factors have not yet been identified, though some experiments suggest an anti-thymulin anti-body is involved. However, as Fabris et al point out, this is highly unlikely in their work, as there is a strict inverse correlation between the thymulin and the thymulin inhibitory activity, which is reversed by the addition of zinc sulphate. Their interpretation is that the inhibitory substance is in fact biologically inactive thymulin, which is still able to bind to thymulin receptor sites, and that the thymulin is activated by zinc. This is a very neat assumption, which accords with other research. Interestingly, though the DS active/inactive thymulin ratio was completely corrected by the addition of zinc sulphate, in the normal subjects this was only partial, suggesting that in physiological ageing other factors interfere with thymulin turnover. The overall picture is that DS people’s thymuses do produce sufficient thymulin, that insufficient zinc is available to activate it, and, importantly, simply measuring levels of thymulin in the plasma was not telling the whole story. This paper plainly shows that measuring levels and counting numbers is not the same as assaying what is biologically available. In deed Lockitch et al acknowledge in their paper that “lymphocyte number and subset distribution are relatively static indexes of immune system capability and that functional assays such as in vitro antibody or interleukin production may be more sensitive indicators for future studies.”
Does zinc improve the functioning of the immune system in Down’s syndrome?
Franceschi et al found that zinc significantly raised active thymulin and lowered inactive thymulin, echoing Fabris’s findings, but Brigino found no increase in thymulin levels despite normalised cellular zinc levels. These findings can not really be compared as Brigino used only 5 subjects, all of whom presented with recurrent infections such as pneumonias and chronic otitis media. It is quite possible that such conditions, which did improve with supplementation, were utilising the extra available zinc. Franceschi et al used 18 subjects and Fabris et al used 72, all off whom were basically healthy.
Zinc supplementation appears to increase lymphocyte proliferation (Stabile et al, Licastro et al, Brigino et al), which may be because zinc is essential for cell division. This is because DNA polymerase is the enzyme central to DNA replication, and cannot function without zinc.
The aforementioned three papers also all found a reduced incidence of recurrent infections. Lockitch et al however, found no improvement in the frequency of infections. It is this author’s opinion that the methods of assessing frequency of infectious episodes prior to and after zinc supplementation were far too inexact to be useful. For example Licastro et al asked parents to remember the number and type of infections their child had had the previous year and Stabile et al give no indication how they collected the information. The parents in Lockitch’s investigation were instructed in detail how to fill in an infection log, which lends more credence to this research, but still relied on parental judgement on what would be considered normal. Possibly more objective means of assessing day-to-day infectious status, such as a daily temperature chart, should be investigated.
Other papers found improvements such as increased T-lymphocytes (Franceschi et al), improved skin responsiveness (Björkstén), or improved utilisation of interleukin 2 (IL-2) (Licastro et al), but until these tests are repeated it is not possible for this author to confidently comment on their validity. Lockitch et al found no improvement in any of the parameters they measured, and indeed found that lymphocyte proliferation decreased even further. The researchers themselves suggest that “although low doses raise serum zinc values, a much higher intake is needed to correct cellular deficiencies,” but Licastro et al propose the possibility that “the zinc supplementation was given for a longer period (6 months in the [Lockitch] study). Prolonged zinc administration (6 months versus [Licastro’s] 4 months) might suppress immune functions. An excessive zinc intake has indeed been shown to impair… lymphocyte [proliferation] in humans, polymorphonuclear migration response to the chemotactic factors and granulocyte phagocytosis of opsonized bacteria.”
Thus it is still unclear what would be the basic zinc dosage to work from (moving up or down until an optimum dose is found). Most researchers based dosage on body weight, whether by os or a proportion of their own devising, sometimes referring to elemental weight of the supplement and sometimes referring to the compound weight. Others stuck with a set dose for all their subjects. Björkstén used 600mg (135mg elemental zinc) for all, meaning that the youngest, who at 8 years old would have been receiving about 24mg if dosed by os, were receiving a huge amount compared to those in Franceschi et al’s and Licastro et al’s papers. Lockitch’s dosages did not rise correspondingly as weight rose, which may partly explain her failure to repeat other papers’ successes. Perhaps only one or two of her subjects were receiving sufficient zinc to make a difference? The other paper to have little success with zinc supplementation was that of Stabile et al, and it should be noted that these investigators only gave zinc to the DS children with low serum zinc status. This author considers normal serum zinc to be a poor indicator to whether the DS immune system needs more zinc; perhaps if all the children in Stabile’s research had been supplemented there would have been a higher proportion of children benefiting?
The risk of serious infectious disease is very real for the DS child, and, in this author’s opinion, the research to date shows that zinc has a role to play in supporting the adaptive immune system and reducing infectious episodes. Zinc activates thymulin, and may increase lymphocyte proliferation, restore delayed hypersensitivity, and normalise other immune parameters. As too little zinc will not have the ideal effect and too much can suppress immunity, finding an optimum dosing regime is of immediate importance. This author also wonders whether long-term zinc supplementation, started early, would slow thymus atrophy and if this would increase the T-lymphocyte pool. Perhaps other lymph tissues would also benefit from long term supplementation? Before such research can ethically take place it is vital that the optimum dosage for DS infants is set. The other aspect of this research that needs to be improved is the method of assessing infectious episodes, finding more objective methods that can be used daily by parents.