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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 18, 2004 as doi:10.1096/fj.03-1050fje. |
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Division of General Internal Medicine, Department of Internal Medicine, Innsbruck University Hospital, Innsbruck, Austria
1Correspondence: Department of Internal Medicine, University of Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria. E-mail: Christian.Wiedermann{at}uibk.ac.at
SPECIFIC AIMS
In Alzheimer’s disease (AD), progressive dementia and neurodegeneration are associated with a complex pathological lesion made up of neurofibrillary tangles and aggregated extracellular protein deposits, known as senile plaques, together surrounded and infiltrated by activated microglial cells. Amyloid-ß (Aß), a heterogeneous 39-43 amino acid, self-aggregating peptide produced by sequential cleavage of amyloid precursor protein (Aß-PP) by the enzymes ß-secretes and 
-secretase, is central in the pathogenesis of the disease. It may induce neurotoxicity indirectly through its proinflammatory property, which includes the possibility of induction of chemotaxis of mononuclear phagocytes as well as the production of cytokines and reactive oxygen species by microglial cells, monocytes, and neutrophils.
It has been proposed that chemotaxis of mononuclear cells toward Aß occurs via activation of the formyl peptide receptor like-1 (FPRL1). FPRL1 is considered to be the low affinity formyl-met-leu-phe (fMLP) receptor in human leukocytes but also to interact with prion protein and Aß; FPRL1 signals via Gi-proteins and hence is a prototypical G-protein-coupled, seven transmembrane receptor (GPCR). Chemotactic responses to ligation with prion protein have been shown to depend on activation of sphingosine-1-phosphate (S1P) signaling.
PRINCIPAL FINDINGS
We show that migration of monocytes toward Aß is sphingosine kinase dependent and that the immunomodulatory drug FTY720, an agonist on S1P receptors after its phosphorylation by sphingosine kinase, is able to inhibit monocyte chemotaxis toward Aß. We demonstrate that Aß regulates S1P receptor expression in human monocytes and that the neuropeptides SN, SP, CGRP, and VIP arrest monocytes that migrate toward high Aß concentrations.
1. Aß-, Aß precursor protein-, FTY-, and FTY-P-induced chemotaxis of human monocytes
Aß and Aß-PP induce chemotaxis in human monocytes, with a maximum response at 1 pmol/L.
To investigate whether FTY and FTY-P could induce monocyte chemotaxis, experiments with untreated human monocytes were performed resulting in concentration-dependent migration toward FTY and FTY-P (Fig. 1
).
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2. Inhibition of Aß and Aß precursor protein-induced monocyte migration by neuropeptides
To delineate whether monocyte-attracting neuropeptides influence Aß- and Aß-PP-induced monocyte locomotion, cells were exposed to bombesin, CGRP, SP, SN, or VIP and chemotaxis toward Aß and Aß-PP was tested. All neuropeptides inhibited chemotaxis concentration dependently.
3. Effects of signaling enzyme inhibitors on Aß-induced migration
Protein kinase C inhibitor GFX, the tyrosine kinase inhibitor tyrphostin-23, and PI3K inhibitor WTN were used to block signaling enzymes; involvement of G-proteins was tested with PTX, known to induce Gi proteins, and CTX, which induces Gs proteins. Migration of monocytes toward Aß and Aß-PP was inhibited by GFX, WTN, tyrphostin, and IBMX. In an analysis of G-protein involvement, activation of migration in Aß and Aß-PP was significantly affected by PTX but not by treatment with CTX.
4. Inhibition of Aß-, Aß-PP-, FTY720-, and FTY720-P-induced monocyte migration by N,N-dimethylsphingosine and DL-threo-dihydrosphingosine
To determine whether S1P-dependent signaling mechanisms are involved in migration of human monocytes toward the peptides, cells were pretreated with the sphingosine kinase inhibitors, DMS also known to interfere with protein kinase C, and the more selective inhibitor DL-threo-dihydrosphingosine at different concentrations. fMLP was used as a control attractant. Treatment with the inhibitors diminished Aß- and Aß-PP-induced chemotaxis whereas fMLP-induced chemotaxis was not affected.
To test the requirement for sphingosine kinase signaling, inhibitors were tested in FTY720- and FTY720-P-induced chemotaxis. Pretreatment of monocytes with DMS and DL-threo-dihydrosphingosine at diminished the chemotactic response of FTY720 and FTY720-P, supporting the role of sphingosine kinase signaling in FTY720- and FTY720-P-induced chemotaxis.
5. Inhibition of Aß-induced but not chemokine-induced migration of human monocytes by FTY720
To test whether FTY720 is able to inhibit Aß-induced chemotaxis, monocytes were preincubated with FTY720. After washing, chemotaxis toward Aß, MIP-1ß, MCP-1, and MCP-3 was investigated. FTY720 inhibited Aß-induced chemotaxis but had no influence on classical chemokine-induced migration of monocytes (Fig. 2
).
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6. Inhibition of FTY720 phosphorylation restores Aß-induced chemotaxis
Since it is well known that FTY720 has to be phosphorylated by sphingosine kinase to become biologically active on S1PRs, we tested whether FTY720 was able to inhibit Aß-induced chemotaxis after blocking sphingosine kinase by the sphingosine kinase inhibitor DMS. After pretreatment with FTY720, and with or without cotreatment with DMS, cells were allowed to migrate toward Aß and Aß-PP. Pretreatment with either DMS or FTY alone inhibited migration of the cells whereas cotreatment with both restored the chemotactic effect of Aß and Aß-PP, suggesting that FTY720 has to be phosphorylated by sphingosine kinase to inhibit Aß-induced chemotaxis.
7. S1P receptor mRNA expression is regulated by Aß
To investigate the role of Aß in S1PR 1, S1PR 3, S1PR 2, S1PR 4, and S1PR 5 mRNA expression, monocytes were incubated with various concentrations of the peptide. After incubation, RT-PCR was performed and equal amounts of cDNA were subjected to agarose gel electrophoresis. Induction of S1PR 2 and S1PR 5 mRNA in Aß-treated cells was observed.
CONCLUSIONS
In vitro data from the present study demonstrate that human monocyte migration toward Aß and Aß-PP involves activation of sphingosine kinase, which causes cell movement via S1P-dependent mechanisms. Most effects of S1P are mediated by a family of specific GPCRs, named S1PRs. S1P-induced chemotaxis is dependent on the activation of small Rho-GTPases, phosphoinositol-3-kinase, and tyrosine kinase whereas sphingosine kinase activity is induced by protein kinase C. After activation by protein kinase C, sphingosine kinase translocates to the plasma membrane, accompanied by the extracellular appearance of S1P. This may subsequently modulate S1PR function. Our results indicate that Aß- and Aß-PP-induced signaling in monocyte migration activates tyrosine kinase, PKC, and PI3K. Thus, our observations are compatible with the fact that Aß and Aß-PP signal through FPRL1 and activate sphingosine kinase with a GPCR-specific signaling pattern different from other prototypical FPRL1-ligands. FPRL1-mediated, sphingosine kinase-dependent mechanisms may affect progression of inflammation surrounding the Aß plaques.
Our biochemical and functional data show that sphingosine kinase activity and S1PR expression are regulated by Aß and Aß-PP, which, in combination with FTY720, may lead to cellular arrest. FTY720 is an immunomodulatory drug highly efficacious in models of transplantation and autoimmune disease. Conversion of FTY720 to its monophosphate appears to be essential for the drugs’s effects on lymphocyte homing to lymph nodes, since FTY720 is a potent agonist on four S1PRs. It is reported that in vivo phosphorylation of FTY720 occurs by sphingosine kinase. FTY720 has been reported to stimulate T cell chemotaxis. We observed FTY720- and FTY720P-induced chemotaxis of monocytes. This chemotactic response seems to be mediated mainly via sphingosine kinase since inhibition of this enzyme with DMS and the more specific inhibitor DL-threo-dihydrosphingosine inhibits FTY720 as well as FTY720-P-induced chemotaxis.
To exclude the possibility that only phosphorylation of FTY720 is sphingosine kinase dependent, FTY720-P was used as a chemoattractant in the presence of sphingosine kinase inhibitors. FTY720- and FTY720-P-induced chemotaxis was decreased after inhibition of sphingosine kinase, suggesting an important role for this enzyme not only in phosphorylation, but also in direct migration. That FTY720-induced inhibition of Aß-chemotaxis is diminished by concomitant inhibition of sphingosine kinase indicates that this effect of FTY720 is exerted by the phosphorylated metabolite of FTY720. Concomitant treatment of monocytes with DMS and FTY720, leading to inhibition of FTY720 phosphorylation by sphingosine kinase, restores Aß-induced migration. These observations confirm the necessity of FTY720 phosphorylation to become active. As FTY720 is inactive at the S1PR 2, a receptor that inhibits chemotaxis, a direct migration-inhibiting effect of FTY720 via S1PR 2 is unlikely. Since FTY720 itself is a chemoattractant for monocytes, it is more likely that FTY720 inhibits Aß-induced migration by heterologous receptor deactivation. Similar to some agonists of GPCRs, S1P can activate growth factor tyrosine kinase receptors in the absence of growth factors, suggesting that receptor tyrosine kinases may be activated through receptor cross-talk and that monocyte chemotaxis toward Aß might be inhibited. It was reported that after pretreatment with FTY720, the number of T lymphocytes was dramatically reduced in the spinal cord of animals with experimentally induced autoimmune encephalitis.
S1PRs may be important coreceptors for FPRL1 in Aß-induced chemotaxis. Thus, whether S1P stimulates or inhibits chemotaxis may depend not only on the concentration of S1P, but on the respective receptor expression.
In conclusion, our data suggest that treatment of monocytes with FTY720 results in an inhibition of their migration toward Aß in a sphingosine kinase-dependent mechanism. Changes in S1PR mRNA levels by Aß underline the important role of S1P in Aß-induced monocyte migration and interference with FTY720 as an agonist on S1PRs. These observations together strengthen the role of S1P in inflammatory cell migration toward Aß. Whether treatment of patients with FTY720 might slow down the progression of plaque formation in AD requires further study.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1050fje; doi: 10.1096/fj.03-1050fje
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