The mechanisms whereby vitamin A stimulates the immune system are poorly understood. In the current study, we attempted to elucidate the potential mechanisms of action of all-trans retinoic acid (atRA) on proliferation of human T lymphocytes. We found that physiological levels of atRA potently augmented T cell proliferation when added in combination with common T cell-stimulating agents. This was reflected in a time- and concentration-dependent stimulation of the cell cycle machinery. The presence of atRA led to elevated levels of cyclin D3, -E, and -A, decreased levels of p27Kip1, increased activity of cyclin-dependent kinase 2, and enhanced phosphorylation of the retinoblastoma protein (pRB). The atRA-mediated changes in the cell cycle machinery were late events, appearing after 20 h of stimulation, indicating that the effects of atRA were indirect. atRA did not alter the expression of the high-affinity IL-2R. However, the level of IL-2 secreted by T cells was strongly enhanced by atRA. rIL-2 was able to substitute for the effects of atRA on the cell cycle machinery and on DNA synthesis, and blocking the IL-2R markedly inhibited atRA-induced cell proliferation and pRB phosphorylation. A retinoic acid receptor (RAR)-selective agonist and 9-cis-RA had the same potency as atRA on T cell proliferation and IL-2 secretion, whereas a retinoid X receptor-selective agonist had only marginal effects. Furthermore, a RAR-selective antagonist completely suppressed T cell proliferation and pRB phosphorylation induced by atRA. Taken together, these results suggest that atRA stimulates the cell cycle machinery and proliferation of normal human T cells by increasing IL-2 secretion through mechanisms involving RARs.

Foreign CpG-DNA from viruses and bacteria can activate memory B cells through binding to toll-like receptor 9, and this pathway has been hypothesized to be involved in the continuous activation of memory B cells ensuring life-long humoral immunity. In this study, we demonstrate that retinoic acid (RA) is a potent coactivator of this pathway in human B cells. RA enhanced the CpG-mediated proliferation of CD27 ؉ memory B cells, and the proliferative response was accompanied by increased immunoglobulin (Ig) secretion indicative of plasma-cell formation. The RA-induced proliferation was preceded by enhanced expression of cyclin D3, and both the expression of cyclin D3 and the induced Ig secretion were found to be dependent on IL-10. Of importance, RA increased the CpG-induced phosphorylation of ERK1/2, p38MAPK, and IB as early as 30 minutes after stimulation. By using specific inhibitors, all the RA-mediated events, including proliferation, cyclin D3 expression, IL-10 se- IntroductionVitamin A is important for an optimal functioning of the immune system, but the mechanisms involved are not fully understood. A part of the increased resistance to infection has been attributed to improved epithelial integrity, but a direct effect on cells of the immune system is also established. [1][2][3] We and others have shown that all-trans retinoic acid has a stimulatory role in T lymphocytes. [4][5][6][7][8][9][10][11] However, there has been some controversy regarding the importance of vitamin A for B-cell activity. Some reports document that retinoids are required for B-cell activity 12 and have the potential to enhance differentiation and antibody responses of B cells, [13][14][15][16] and that B-cell responses are impaired in vitamin A-deficient rats. 17 We have previously shown that RA inhibits the proliferation of human and murine B-cell precursors 18 as well as peripheral-blood B cells stimulated via B-cell receptor (BcR). 19 We also demonstrated that the inhibition of B-cell proliferation was accompanied by inhibition of the cell-cycle machinery driving the cells from G 1 to S phase. 20 In the primary immune response, naive B cells proliferate and differentiate into plasma cells that secrete antibodies mainly of the immunoglobulin-M (IgM) class. During this process, somatic hypermutation and isotype switching take place, producing highaffinity antibodies of the IgG class. 21 Some B cells embark on a different program to become long-lived memory B cells, which are characterized by lower threshold for activation and differentiation. 22 In the past 2 decades, it has become clear that B cells can be activated by DNA from bacteria and viruses. 23 The discrimination between human and bacterial/viral DNA is based on the fact that CpG dinucleotides are underrepresented and generally methylated in vertebrate DNA, while they are present at expected frequency and are unmethylated in bacteria and viruses. 23,24 Unmethylated CpG DNA is recognized by toll-like receptor 9 (TLR9), which is primarily expressed in memory B cells and plas...

Our study aimed to investigate, in vivo, the relationship between vitamin A status and NF-kappaB activity, a transcription factor central in regulating inflammatory and immune responses. We used a novel transgenic murine NF-kappaB-luciferase reporter model that enabled molecular imaging of NF-kappaB activity in live mice via an intensified image-capture apparatus. Whole-body luminescence, which reflects overall NF-kappaB activity, was elevated 2.2-fold in vitamin A-deficient (VAD) mice compared with control mice. Specifically, NF-kappaB activity in VAD mice was increased 1.8-fold in the lymph nodes and 1.4-fold in the thymus and, NF-kappaB induction in UVB radiation-exposed skin was also enhanced in VAD mice compared with control mice. The administration of all-trans retinoic acid to VAD mice resulted in a transient reduction in NF-kappaB activity and, conversely, a single dose of the RAR-pan-antagonist, AGN 194310, administered to control mice, led to a marked, transient induction of whole-body luminescence. Our results suggest that vitamin A status, and vitamin A itself, affects NF-kappaB activity in vivo and that the elevated NF-kappaB activity in VAD may be a mechanism underlying some of the features of VAD syndrome.

At the end of an immune response, most activated T cells spontaneously undergo programmed cell death (apoptosis). In the present study we show that all-trans retinoic acid (atRA), a major vitamin A metabolite, can inhibit the spontaneous apoptosis of activated human T lymphocytes in vitro. Isolated peripheral blood T lymphocytes were activated by 12-O-tetradecanoyl phorbol 13-acetate and cultured for up to 11 days without any further stimuli. With time, a gradual increase in cell death was observed. This spontaneous death of activated T cells was apoptotic, as demonstrated by cell shrinkage, DNA fragmentation and depolarization of the mitochondrial membrane. In the presence of physiological concentrations of atRA, the percentage of T cells exhibiting these apoptotic features was significantly reduced. After 5 days of stimulation, the percentage of TUNEL+ T cells decreased from 28 to 12% in the presence of atRA. The anti-apoptotic effect of atRA was mimicked by the retinoic acid receptor (RAR)-selective agonists 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid and AM-580, and totally abrogated by the RAR-selective antagonist Ro 41-5253. Cytokines of the IL-2 family have been shown to improve the survival of activated T cells. Strikingly, we found that the ability of atRA to inhibit apoptosis was significantly correlated with its ability to increase the production of IL-2. Furthermore, a blocking anti-IL-2 receptor antibody completely abrogated the anti-apoptotic effect of atRA. Together, these results suggest that retinoic acid inhibits spontaneous apoptosis of activated T lymphocytes through a RAR-dependent increase in IL-2 production.

Summary Interleukin‐2 (IL‐2) is an essential cytokine for T‐lymphocyte homeostasis. We have previously reported that all‐trans retinoic acid (atRA) enhances the secretion of IL‐2 from human peripheral blood T cells in vitro, followed by increased proliferation and inhibition of spontaneous cell death. In this study we used a transgenic IL‐2 gene luciferase reporter model to examine the effects of atRA in vivo. In contrast to the observations in human T cells, we found an overall reduction in luciferase‐reported IL‐2 gene expression in mice treated with atRA. Whole‐body luminescence of anti‐CD3‐treated and non‐treated mice was reduced in mice receiving atRA. Accordingly, after 7 hr, IL‐2 gene expression was on average 55% lower in the atRA‐treated mice compared with the control mice. Furthermore, mice fed a vitamin A‐deficient diet had a significantly higher basal level of luciferase activity compared with control mice, demonstrating that vitamin A modulates IL‐2 gene expression in vivo. Importantly, the atRA‐mediated inhibition of IL‐2 gene expression was accompanied by decreased DNA synthesis in murine T cells, suggesting a physiological relevance of the reduced IL‐2 gene expression observed in transgenic reporter mice.