Activated NK cells produce various cytokines and thus play an important immunomodulatory role in the production of antibodies. The marker for NK cells, NK1.1 in mice and NKR-P1 on human and rat cells (Lanier et al., 1994), is also expressed on a subset of T-lymphocytes, the NKT cells (Bendelac et al., 1997; Kronenberg, 2005). NK cells are known as an early source of IFNg, whereas NKT cells have been shown to rapidly secrete IL-4 and IL-10 as well as IFNg. Understanding the development, functionality, and activation potential of NK cells and NKT cells is currently of great interest both in relationship to antibody production and tumor immunity.

NK and NKT cells in spleen and peripheral blood were investigated in a study of aging rats fed diets that differed only in VA contents (marginal, adequate, and supplemented) from the time of weaning, throughout their life time (Dawson and Ross, 1999; Dawson et al., 1999). The study was designed to cover a wide range of VA consumption but to exclude states of clinically evident VA deficiency or toxicity. Rats fed these diets showed distinct differences in VA status, ranging from a state of marginal VA deficiency (depleted liver VA stores, reduced serum retinol, but normal growth, which became progressively lower as the rats aged), to normal VA status (normal plasma retinol and tissue VA reserves that gradually accumulated with age), to a state of excessive VA accumulation in the VA-supplemented group (elevated plasma retinol and tissues stores, which increased with age, but no overt toxicity) (Dawson and Ross, 1999; Dawson et al., 1999). To quantify NK and NKT cells, peripheral blood mononuclear cell (PBMC) and splenocytes were costained with antibodies against NKR-P1 and CD3 and analyzed by flow cytometry. Marginal VA status was associated with a reduction in the number of NK cells in peripheral blood and a lower percentage of NK cells compared total PBMCs (Dawson et al., 1999). The reduction in NK cells in VA-marginal rats is consistent with previous reports of low NK cells and reduced cytotoxicity in VA-deficient rats (Zhao et al., 1994). Conversely, VA supplementation and aging increased the percentage and number of NK cells above the values in VA-adequate rats (Dawson et al., 1999). Overall, the percentage of NK cells was significantly reduced in VA-marginal rats and increased in VA-supplemented rats, and the effect of diet was greater in old-aged rats (Fig. 2A). In contrast to NK cells, the percentage and number of NKT cells were both increased in peripheral a reciprocal manner compared to NK cells; age was also a factor for NKT cells. Data from Dawson and Ross (1999). The percentage of NKT cells was inversely correlated with the ratio of CD4:CD8 T-cells in peripheral blood (not shown, Dawson and Ross, 1999). (C) Neonatal mice were treated in a short-term (3 days after priming with tetanus toxoid) study with RA (days —1,0, 1, and 2 before cells were analyzed on day 3), PIC (day 0 only), or both RA + PIC. Total NK1.1+ cells, CD3+ NK1.1+ (NKT), and CD3— NK1.1+ (NK cells) were analyzed by flow cytometry after double staining with fluorescently labeled anti-NK1.1 and anti-CD3 antibodies. Data from Ma and Ross (2005).

blood of VA-marginal rats, but NKT cells did not differ between VA-adequate and VA-supplemented rats (Dawson and Ross, 1999) (Fig. 2B). The proportion of CD3+ cells (total T-cells) expressing NKR-P1 increased significantly with age, consistent with the higher proportion of these cells among total T-cells in adult humans compared to infants [15-40% in adults versus <5% in infants (Lanier et al., 1994)]. Overall in this long-term study in rats, marginal VA deficiency significantly increased the absolute number and the proportion of NKT cells relative to NK cells (Dawson and Ross, 1999; Dawson et al., 1999), especially as rats aged, and the marginal VA status became more tenuous (with serum declining from 0.97, to 0.63, to 0.38 in VA-marginal young, middle-aged, and old rats, respectively) (Dawson et al., 1999). Given the cytokine-producing function of NKT cells, it is tempting to speculate that an increase in NKT cells could be a compensatory mechanism that helps to counteract a reduced overall capacity for cytokine production in VA deficiency. However, it was reported earlier that IFNg production is elevated in splenocytes of VA-deficient mice (Carman and Hayes, 1991). An increase in the production of IFNg by activated NKT cells might explain, in part, the dysregulated antibody responses of VA-deficient mice and rats. However, IFNg production was not observed to differ in this study of VA-marginal, adequate, and VA-supplemented aging rats, which may suggest that retinol must be nearly completely depleted before IFNg production becomes dysregulated.

NK cell cytolytic activity in spleen and blood, measured as lysis of Yac-1 target cells, was proportional to the number of NK cells, and was therefore lower in VA-marginal rats, and in older rats. NK cell lytic efficiency (activity per NK cell) also fell with age but it was not affected by VA status. All groups showed a similar increase in NK cell lytic function when peripheral blood cells were incubated with IFNa (Dawson et al., 1999), a cytokine known to be released early in the response to viruses and to be a potent activator of NK cells (Asselin-Paturel and Trinchieri, 2005). This result suggests that the cell surface receptor activated by IFNa and the signaling pathways involved in NK cell proliferation and increased cytotoxicity are intact and functionally equivalent in rats of different ages and VA status. Although IL-2 production by peripheral blood mononuclear cells did not differ significantly with VA status, IL-2 production by splenocytes was lower in VA-marginal rats in each age group (Dawson et al., 1999). Overall, this study identified VA status as a factor in maintaining the NK to NKT cell ratio and suggested that even marginal VA deficiency, especially with advancing age, is a risk factor for reduced NK cell function. Unfortunately, it was unknown at the time this study was conducted that a substantial proportion of the lymphocytes residing in the liver are NKT cells (Wick et al., 2002), and thus this population was not analyzed. It would be of interest to characterize liver NKT cells in relationship to VA status because NKT cells are implicated in the rapid production of cytokines, especially

IFNg and IL-4, which are both important in regulating adaptive immune responses, and as NKT cells in the liver may be important in the surveillance of virally infected or abnormal cells passing through the liver sinusoids (Kawamura et al., 1999; Wick et al., 2002).

Short-term studies were conducted of VA-adequate rats treated with RA, coincident with immunization with tetanus toxoid (DeCicco et al., 2001). RA alone, in an 11-day study, did not significantly increase the NK cell population in spleen or blood, but did increase the number of NK-cells induced by PIC, known to stimulate NK cell proliferation. In a more comprehensive study of the lymphocyte populations in VA-adequate adult mice (Ma et al., 2005), RA was not a significant factor for the number of NK1.1+ NK cells, but, as in adult VA-adequate rats, RA further increased the positive effect of PIC on the NK cell population. RA also was a positive regulator of the CD3+ NK1.1+ (NKT) population, and it increased the NKT to NK cell ratio. Furthermore, the NKT to NK cell ratio in adult mouse spleen was positively correlated with the ratio of IL-4 to IFNg mRNAs in the same tissue, which was higher in RA-treated mice, suggesting that an elevation of NKT cells could be a factor in the increase in Th2/type 2 antibody production (ratio of IgG1 to IgG2a) in the same animals (Ma et al., 2005), which is likely to be driven in part by IL-4. As discussed above, the ability of RA to promote type 2 immune responses has been a consistent finding in a number of studies. In neonatal mice treated at the time of antigen priming with RA and/or PIC (Ma and Ross, 2005), the population of NK1.1+ cells in the spleen was less than half that in adult spleen (NK cells, 2.08% versus 4.33%; NKT cells, 0.7% versus 1.36%, respectively). Yet even with these small numbers, it was observed that RA combined with PIC rapidly increased the percentage and number of splenic NK and NKT cells (Fig. 2C). The changes in cell populations observed in normal rats (DeCicco et al., 2001) and mice (Ma and Ross, 2005; Ma et al., 2005) after short-term treatment with RA are likely to be due to a rapid release of cells already near maturation, or to changes in cell-surface molecules that could result in changes in the number of cells in certain compartments. It is interesting that RA has been shown to influence the homing of mouse T-cells to the gut (Iwata et al., 2004), through expression of integrins and other factors.

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