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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 19, 2004 as doi:10.1096/fj.04-1652fje. |
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Department of Immunology, Weizmann Institute of Science, Rehovot, Israel; and
* Beer Yaakov Mental Health Center, Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel
1 Correspondence: Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: sara.fuchs{at}weizmann.ac.il
SPECIFIC AIMS
Our aim was to examine a hypothesis on regulation of peripheral T cells by dopamine (DA). Recent studies showed that activated T cell blasts can cross the blood-brain barrier (BBB) due to high expression of adhesion molecules. We examined whether DA receptors could have a special function on blasts as they enter the brain, focusing on the D3 dopamine receptor (D3R) as a representative dopaminergic receptor.
PRINCIPAL FINDINGS
1. D3 dopamine receptor is functionally coupled to G-protein in T cells
To demonstrate the functionality of dopamine receptor on T cells, we measured cAMP accumulation in resting T cells and blasts after dopaminergic activation with quinpirole, a D2/D3 agonist. D3 dopamine receptor (D3R) has been shown to couple to inhibitory and stimulatory G-proteins (Gi and Gs) in COS cells. We measured the coupling of D3R to each pathway independently, using the selective inhibitors pertussis toxin (for Gi) and cholera toxin (for Gs). We demonstrated that D3R in T cells couples to Gi and Gs pathways, resulting in changes in cAMP accumulation. In blasts, D3R exhibited a pattern of specific coupling to Gi and Gs similar to that in resting T cells, but changes in cAMP accumulation were greater in blasts than in resting T cells. D3R mRNA levels were also much higher in blasts than in resting T cells, signifying elevated basal expression of this receptor.
2. Dopaminergic stimulation of T cells affects cytokine expression
To determine whether D3R activation in resting T cells and blasts affects immunological properties of these cells, we tested its effect on cytokine expression. RT-PCR analysis of IFN-
, IL-4, and IL-10 was performed. As seen in Fig. 1
A, dopaminergic activation had no effect on cytokine levels of resting T cells. mRNA levels of all three cytokines were undetectable in resting T cells before quinpirole treatment and after activation. Blasts, on the other hand, responded to dopaminergic treatment by changes in cytokine expression. Before exposure to quinpirole, blasts expressed high levels of IL-4 and IL-10 and undetectable levels of IFN-. After quinpirole, IL-4 and IL-10 were reduced to undetectable levels and IFN- levels increased. The change in cytokine profile of blasts after quinpirole was abrogated upon addition of the D3R inhibitor U-maleate (Fig. 1B
), indicating that the effect is mediated through D3R.
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3. Dopaminergic stimulation affects both Th1 and Th2 cells
A switch in cytokine expression from IL-4 and IL-10 to IFN- is usually associated with a Th2 to Th1 shift of CD4 T cells. However, as IFN- is also secreted by CD8 T cells, the switch could be attributed to CD8 T cell activation. To determine which subset of cells participates in this change, CD4 and CD8 T cells were isolated. Blasts were then induced from the two subsets of cells and the effect of D3R activation with quinpirole on their cytokine expression was measured (Fig. 1C
). For CD4 blasts, the switch in cytokine profile was the same as observed for the total blast population: a switch from pronounced IL-4 and IL-10 levels and negligible IFN- levels to negligible IL-4 and IL-10 and pronounced IFN- levels. CD8 blasts, on the other hand, had undetectable mRNA levels of all three cytokines prior to treatment and demonstrated increased levels of only IFN- in response to quinpirole. These findings suggest that, when exposed to dopamine, CD4 blasts undergo a Th2 to Th1 shift, and CD8 blasts are triggered to produce IFN-.
4. Dopaminergic stimulation of T cells affects membrane molecules expression
Expression of integrins and other receptors mediating cell activation and trafficking is another fundamental aspect of the immune cell response. We examined the effect of D3R activation of resting T cells and blasts on three surface markers: VLA-4, involved in blast recruitment and crossing of the BBB; CD25, a classical activation marker; and CXCR3, a principal inflammatory chemokine receptor involved in trafficking of T cells to the central nervous system (CNS) (Fig. 1D
). Resting T cells exhibited low expression levels of all three molecules, whereas blasts, due to their increased activation state, expressed them more intensely. Dopaminergic activation did not significantly affect the expression of the three molecules in resting T cells. However, exposure of blasts to dopaminergic ligand led to an increase in CD25 expression and a decrease in CXCR3 whereas VLA-4 levels remained unchanged. The increase in CD25 and the decrease in CXCR3 expression were abrogated by the dopaminergic antagonist U-maleate (Fig. 1D
).
5. Dopaminergic effects on T cells in in vivo models
We next asked whether a change in the brain could affect immune cells in vivo and whether these effects could be transferred to the peripheral immune system. Two model systems were tested. In the first, rats were injected with L-Dopa/carbidopa, known to elevate dopamine levels exclusively in the CNS, and their peripheral T cells were analyzed. Higher brain dopamine levels were verified using HPLC. We found that peripheral T cells from rats treated for 1 (Fig. 2
) or 3 wk (data not shown) with L-Dopa/carbidopa had features similar to dopaminergically activated human blasts: namely, higher mRNA expression of INF- and D3R, and higher levels of VLA-4 and CD25 compared with control groups.
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Schizophrenia can serve as a natural human model for a CNS-restricted increase in DA. We compared cAMP accumulation after quinpirole treatment in T cells from schizophrenia patients and healthy controls. We observed a higher increase in quinpirole-induced cAMP accumulation in T cells from schizophrenia patients than in healthy individuals (79% and 9%, respectively). This is similar to the increase in cAMP accumulation seen in blasts vs. resting T cells from healthy humans.
6. Changes in cytokine expression can be induced by soluble factors
To elucidate the mechanism by which increased DA levels in the CNS can affect peripheral T cells, we have examined whether blasts can affect peripheral T cells through soluble factors they secrete. We tested the effect of supernatants isolated from blasts treated or not with quinpirole on resting T cells. Supernatants were separated from blasts derived from human lymphocytes that had been incubated with or without quinpirole, followed by incubation in quinpirole-free medium to remove quinpirole. These blast supernatants were incubated with freshly isolated resting T cells and their effect on mRNA levels of IFN-, IL-4, IL-10 in the reacted resting T cells was measured. Supernatant-induced alterations in the cytokine profile of the resting T cells reflected the cytokine profile of the blasts from which the supernatant was derived, indicating that soluble factors secreted by blasts can affect T cells.
CONCLUSIONS AND SIGNIFICANCE
The existence of receptors for neurotransmitters on immune cells is still enigmatic. It was commonly assumed that immune cells do not cross the BBB, and so can encounter neurotransmitters only in the periphery. Thus, in earlier studies only peripheral mechanisms for neurotransmitter effects on T cells were considered. It was recently shown that highly activated T cells, termed blasts, can cross the BBB due to high expression of adhesion molecules and regardless of antigen specificity. Based on these findings, we propose a hypothesis that D3 dopamine receptors on T cells participate in brain regulation of peripheral immune cells. We show that, after dopaminergic activation, blasts undergo fundamental immunological changes, which may be transmitted to the periphery either by these cells leaving the CNS or via messenger-soluble factors (see Fig. 3
). Throughout this study, only blasts (and not resting T cells) were shown to be affected by dopaminergic ligands. Thus, it seems that dopaminergic regulation is mediated through activated T cell blasts able to cross the BBB.
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Our findings are supported by earlier studies of experimental allergic encephalomyelitis. These reported findings present the same pattern of changes in T cells after invasion to the CNS as we demonstrated in dopaminergically activated blasts.
Our in vitro findings are further supported by in vivo experiments. Peripheral T cells isolated from rats injected with L-Dopa/carbidopa demonstrated immunological properties identical to dopamine-induced human blasts. The same phenomena were observed with peripheral T cells isolated from schizophrenia patients. Schizophrenia was chosen since the prevailing theory is it results from excessive dopamine activity in specific brain regions, thus serving as a human model for increased brain dopamine levels. A decrease in D3R mRNA levels and IFN- secretion has been reported in peripheral lymphocytes from Parkinson’s disease patients. These features are exactly the opposite of what was found in schizophrenia and with dopaminergic-activated blasts, and they suggest that the proposed mechanism functions when CNS DA levels are decreased, as in Parkinson’s disease.
We propose the existence of a "brain to T cells" pathway by which the brain could manipulate immune cell via affecting activated cells that have invaded the brain (Fig. 3)
. The fact that the organ that not long ago was considered immune privileged now appears to interact with and affect immune cells suggests a conceptually new pathway of neuroimmune interactions. Although the above model does not provide a detailed mechanism for transferring blasts’ cytokine characteristics to T cells, it suggests that DA-activated blasts could trigger T cells to mimic their properties by soluble factors.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1652fje;
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