Expression of functional GPR35 in human iNKT cells
Research highlights
► Human invariant NKT (iNKT) cells express GPR35 at both gene and protein levels. ► GPR35 internalize in human iNKT cells following receptor activation. ► Specific GPR35 agonists significantly reduce the release of IL-4, but not that of IFN-γ. ► GPR35 couples Gi/o proteins in human iNKT cells.
Introduction
G protein-coupled receptor (GPCR)35 is one of the “orphan” GPCR [1] that shares homology with subtypes of the purinergic (GPR23/P2Y9) [2], nicotinic acid (HM74) [3], and lysophosphatidylinositol (GPR55) [4] receptors. The chromosomal mapping of GPR35 and its association with a number of human diseases have been largely investigated [5], while its expression and pharmacological characterization remains to be fully defined.
The identification of l-kynurenic acid (KYNA) [6], an endogenous product of the l-tryptophan catabolism (kynurenine pathway), and 2-acyl lysophosphatidic acid (LPA) [7], an endogenous molecule generated via a phospholipase A1-dependent pathway from PA, as selective ligands for GPR35 led to its deorphanization and characterization. GPR35 preferentially couples with Gi/o proteins in GPR35-transfected Chinese hamster ovary cells [6], as well as in cells constitutively expressing this receptor (e.g., rat sympathetic [8] and dorsal root ganglion neurons [9], human monocytes [10]).
The expression analysis of human and mouse GPR35 revealed that it is predominantly expressed in immune and gastrointestinal systems. Among immune cells, GPR35 is highly expressed in human CD14+ monocytes, T cells, neutrophils, and dendritic cells (DC), with lower expression levels in B cells, eosinophils, basophils, and platelets [6], [10].
To the best of our knowledge the expression of GPR35 in a separate lineage of T lymphocytes, named invariant natural killer T (iNKT) cells, has not yet been investigated.
iNKT cells are a unique autoreactive cluster of differentiation 1 (CD1)d-restricted T cell subpopulation, characterized by the expression of an invariant T cell receptor (TCR) alpha chain, (Vα14–Jα18 in mice, and Vα24–Jα18 in humans), in combination with typical surface receptors of natural killer (NK) cells [11].
In mice, iNKT cells are most frequent (20–30%) among liver T lymphocytes, while they only constitute 0.4–1% of the total T cells in thymus, bone marrow, spleen, lymph node, and intraepithelial lymphocytes. In humans, iNKT cells constitute approximately only a 0.1–0.2% of the peripheral blood T lymphocytes, and iNKT cells are present in the spleen, liver, thymus, bone marrow, and peripheral lymph nodes [12].iNKT cells recognize, through their semi-invariant TCR, glycolipid antigens, such as α-galactosylceramide (α-GalCer) and its analogs, presented by antigen presenting cells (APC) in the context of CD1d presenting molecules [13]. After TCR ligation, iNKT cells promptly produce a large amount of the T helper (Th)1 and Th2 cytokines [e.g., interferon (IFN)-γ and interleukin (IL)-4] [12]. The cytokine burst from activated iNKT cells triggers DC maturation, NK cell proliferation, and expression of B cell activation markers, these events together contributing to adaptive immune responses. On the other hand, iNKT cells display important cytotoxic activities, which are essential for immune responses [14].
Recently, evidences on the role of environmental factors, such as histamine [15], IL-33 [16], and prostaglandin D2 [17], as well as of costimulatory molecules (e.g., CD80/86–CD28, and CD154–CD40) [13], in specifically activating iNKT cells have been reported. Our group [18] showed that the culture medium from human monocytic cell lines (THP-1), expressing the kynurenine pathway and releasing micromolar concentrations of l-kynurenine and KYNA following α-GalCer stimulation, is able to stimulate mouse iNKT hybridoma cells to release IL-2. These data suggested the possibility that iNKT cells express specific surface receptors for the kynurenines.
The aim of this study was to examine whether human iNKT cells express GPR35 and to study the effects induced by selective activation of this receptor (receptor localization and cytokine release).
Section snippets
Reagents
RPMI-1640, l-glutamine, and fetal bovine serum (FBS) were from EuroClone (Celbio, Europe). Ficoll-Hystopaque PLUS, kanamycin, sodium pyruvate, MEM amino acid solution, KYNA, IL-2, pertussis toxin (PTX), 1,4-dihydro-5-(2-propoxyphenyl)-7H-1,2,3-triazolo[4,5-d]pyrimidine-7-one (zaprinast), paraformaldehyde, and GenElute™ mammalian total RNA miniprep kit were from Sigma Aldrich (Milan, Italy), enzyme-linked chemiluminescence (ECL) Plus Western blotting detection system from Amersham (GE
GPR35 gene and protein expression in human iNKT cells
To determine whether GPR35 is expressed in human iNKT cells we carried out RT-PCR analysis. A RT-PCR product of the appropriate size (80 bp) was detected in both human iNKT cells and resting PBMC, used as positive controls of the receptor expression [6], [10] (Fig. 1A). Omission of reverse transcriptase during the RT-PCR (negative controls; NC) abolished the amplification of these PCR products (Fig. 1A).
The expression of the GPR35 protein was investigated by Western blotting on human iNKT cells
Discussion
In this study, we demonstrate that: (1) human iNKT cells express GPR35 at both gene and protein levels; (2) GPR35 mostly localizes in the plasma membrane of human iNKT cells, while it internalizes in punctate intracellular structures following specific receptor activation; (3) the specific activation of GPR35 by selective receptor agonists (KYNA or zaprinast) significantly reduces the release of IL-4, but not that of IFN-γ; (4) GPR35 couples Gi/o proteins in human iNKT cells.
A large bulk of
Acknowledgments
We thank Prof. G. De Libero, University Hospital Basel, Basel (CH) for providing human iNKT cells and human CD1d-transfected THP-1 line, and Prof. L. Panza, University of “Piemonte Orientale”, Novara (Italy) for providing α-GalCer. We are grateful to Prof. P. Dellabona, DIBIT “San Raffaele” Scientific Institute, Milano (Italy) for giving us the opportunity to learn the methods for human iNKT cell isolation and culture.
This study was supported by grants from University of “Piemonte Orientale
References (38)
- et al.
Discovery of three novel G-protein-coupled receptor genes
Genomics
(1998) - et al.
Identification of p2y9/GPR23 as a novel G protein-coupled receptor for lysophosphatidic acid, structurally distant from the Edg family
J. Biol. Chem.
(2003) - et al.
Identification of GPR55 as a lysophosphatidylinositol receptor
Biochem. Biophys. Res. Commun.
(2007) - et al.
Kynurenic acid as a ligand for orphan G protein-coupled receptor GPR35
J. Biol. Chem.
(2006) - et al.
GPR35 is a novel lysophosphatidic acid receptor
Biochem. Biophys. Res. Commun.
(2010) - et al.
GPR35 is a functional receptor in rat dorsal root ganglion neurons
Biochem. Biophys. Res. Commun.
(2008) - et al.
Kynurenic acid triggers firm arrest of leukocytes to vascular endothelium under flow conditions
J. Biol. Chem.
(2009) - et al.
Modulation of invariant natural killer T cell cytokine responses by indoleamine 2,3-dioxygenase
Immunol. Lett.
(2008) - et al.
The immunoregulatory role of CD1d-restricted natural killer T cells in disease
Clin. Immunol.
(2004) - et al.
ICOS costimulates invariant NKT cell activation
Biochem. Biophys. Res. Commun.
(2005)