Neuroprotectin D1/protectin D1 stereoselective and specific binding with human retinal pigment epithelial cells and neutrophils
Introduction
Photoreceptor cells (cones and rods) are differentiated neurons that contain the phototransduction apparatus in the tightly organized outer segment membrane discs. These are the richest membranes containing the omega-3 fatty acid family member, docosahexaenoic acid (DHA) [1], as an acyl chain of phospholipids. Photoreceptor outer segment membranes are continuously assembled at the base of the outer segments throughout life, while at their tips, the oldest discs are shed and phagocytized by retinal pigment epithelial (RPE) cells. In turn, RPE cells recycle DHA and all-trans retinol (vitamin A, the precursor of the visual pigment chromophore) back to the photoreceptors. Recently, DHA was shown to be the precursor of neuroprotectin D1 (NPD1). NPD1 formation is upregulated in RPE cells during oxidative stress [2] or upon exposure to neurotrophins [3] and, in turn, promotes homeostasis and cell survival [1].
The retinal pigment epithelium (RPE) and photoreceptors comprise a region of the retina immersed in an environment prone to oxidative stress due to high oxygen consumption, a high metabolic rate, significant exposure to light, and an active flux of polyunsaturated fatty acids. Enhanced oxidative stress is involved in age-related macular degeneration and other retinal degenerations [1]. The ability of the retinal pigment epithelium to increase NPD1 production when confronted with excessive oxidative stress is a newly defined protective mechanism [4]. Moreover, the photoreceptor–phagocytosis enhancement of RPE cell survival (when confronted with oxidative stress) underlies the generation of NPD1 [3]. NPD1 biosynthesis is observed in response to injury in several instances, such as in RPE cells challenged by oxidative stress [1], [2], [3], in A2E oxirane accumulation [3], during ischemia–reperfusion in the brain [5] and kidneys [6], and in human brain cells in culture undergoing apoptosis upon exposure to beta amyloid peptide [7].
Resolving inflammatory exudates biosynthesize the D-series resolvins and protectins (i.e., 10,17-diHDHA) from DHA [8], [9]. NPD1/PD1 is a stereochemically defined 10,17-diHDHA molecule [10] that matches the endogenous product and possesses both specific and potent anti-inflammatory as well as modulatory actions in the immune [9], [10], [11], [12] and inflammatory [13], [14] systems. For example, NPD1/PD1 is anti-inflammatory, stopping neutrophil infiltration and T cell migration in vivo [10], [11]. Thus, NPD1/PD1 is active in both the neural and immune systems. We coined the term protectin D1 for when NPD1/PD1 is biosynthesized by immune cells [10]; when NPD1/PD1 is specifically biosynthesized by neural and neural ectoderm cells, the prefix neuro is added (i.e., NPD1).
Here, we identify a 16,17-epoxytriene as a key intermediate in NPD1 biosynthesis and demonstrate the potent and stereospecific bioactivity of NPD1 with ARPE-19 cells. Using tritium-labeled NPD1 ([3H]-NPD1) and stereoisomers of NPD1, methyl esters, and structurally related compounds, we demonstrate high-affinity stereoselective binding of NPD1 with both human ARPE-19 cells and isolated human neutrophils.
Section snippets
Materials and methods
The studies reported here have been reviewed and approved by the Institutional Animal Care and Use Committee at the Louisiana State University Health Sciences Center, New Orleans and the Harvard Medical Area Standing Committee on Animals, Boston (Protocol #02570). Synthetic NPD1, NPD1-Me, NPD1 stereoisomers, and RvD1-Me were prepared in stereochemically pure form via total organic synthesis [10].
NPD1 biosynthesis in ARPE-19 cells involves a 16,17-epoxide intermediate
ARPE-19 cells were incubated with 17S-hydro(peroxy)docosa-4Z,7Z,10Z,13Z,15E,19Z-hexaenoic acid, and the incubations were stopped with the addition of 10-vol/vol excess acidic methanol. Following extraction, LC–MS/MS analyses were used to identify whether methoxy-trapping products were generated from the proposed epoxide-containing intermediate in these cells [2], [7] as observed in exudates, glial cells and T cells [8], [9], [11]. The identified methoxy-trapping products from ARPE-19 cells are
Discussion
Here we demonstrate high-affinity stereoselective binding of NPD1 in ARPE-19 cells and human neutrophils. For this purpose, we prepared [3H]-NPD1, NPD1 isomers, and structurally-related compounds to test their actions with ARPE-19 cells as in acute inflammation and resolution [10].
NPD1 is a potent bioactive product biosynthesized from the omega-3 fatty acid DHA that is endowed with anti-inflammatory and pro-survival bioactivity generated in response to H2O2/TNFα-triggered oxidative stress [2],
Acknowledgements
We thank M.H. Small for expert assistance in manuscript preparation and Dr. N. Chiang (BWH-HMS) for calculating the Kd values for human PMN.
This work was supported by National Institutes of Health Grants R01 EY005121 and P20 RR016816 (N.G.B.), and Grants GM38765 and P50 DE016191 (C.N.S.).
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- 1
Current affiliation: Graduate School of Pharmaceutical Sciences, University of Tokyo, Japan.
- 2
Both laboratories contributed equally and C.N.S. and N.G.B. share senior authorship.