Kynurenic acid triggers firm arrest of leukocytes to vascular endothelium under flow conditions

Abstract

Recent studies have demonstrated that kynurenic acid (KYNA), a compound produced endogenously by the interferon-gamma-induced degradation of tryptophan by indoleamine 2,3-dioxygenase, activates the previously orphaned G protein-coupled receptor, GPR35. This receptor is expressed in immune tissues, although its potential function in immunomodulation remains to be explored. We determined that GPR35 was most highly expressed on human peripheral monocytes. In an in vitro vascular flow model, KYNA triggered the firm arrest of monocytes to both fibronectin and ICAM-1, via beta(1) integrin- and beta(2) integrin-mediated mechanisms, respectively. Incubation of monocytes with pertussis toxin prior to use in flow experiments significantly reduced the KYNA-induced monocyte adhesion, suggesting that adhesion is triggered by a G(i)-mediated process. Furthermore, KYNA-triggered adhesion of monocytic cells was reduced by short hairpin RNA-mediated silencing of GPR35. Although GPR35 is expressed at slightly lower levels on neutrophils, KYNA induced firm adhesion of these cells to an ICAM-1-expressing monolayer as well. KYNA also elicited neutrophil shedding of surface L-selectin, another indicator of leukocyte activation. Taken together, these data suggest that KYNA could be an important early mediator of leukocyte recruitment.

FIGURE 1.

Tryptophan catabolism pathway. Schematic diagram of tryptophan catabolism. Tryptophan is converted to N-formylkynurenine by IDO, and is then deformylated to form kynurenine. Kynurenine can be converted to kynurenic acid via kynurenine aminotransferases I and II (KAT), or converted via alternative pathways to anthranilic acid or quinolinic acid, a NAD precursor.

FIGURE 2.

GPR35 expression in human cells measured by QPCR. RNA was harvested from freshly isolated leukocyte subtypes and HUVECs, and receptor expression was determined with real-time QPCR using SYBR Green fluorescence relative to that of ROX reference dye to determine the transcript copy number; this figure was then normalized to the copy number of the housekeeping gene, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as previously performed (40, 41). Data shown are mean ± S.E. from at least 3 donors. A, GPR35 levels in leukocyte subtypes and HUVECs. B, GPR35 expression in monocytes is comparable with that of the chemokine receptor CXCR2, and substantially greater than CCR3.

FIGURE 3.

Kynurenic acid induces firm arrest of monocytes on fibronectin through a mechanism that is β₁ integrin-mediated and G_i-coupled. Interaction of monocytes with a fibronectin-coated surface was studied at 1.0 dyne/cm². Adhesion was quantified 8 min after the introduction of the indicated compound. A, KYNA induces firm arrest of monocytes on fibronectin (**, p < 0.005), whereas tryptophan (Trp) and AA treatment does not differ from background. B, pretreatment with the α₄ integrin-blocking antibody HP2/1 interferes with KYNA-induced adhesion (◇, p < 0.05 when compared with KYNA adhesion), whereas pretreatment with isotype-matched control antibody has no effect (*, p < 0.05 relative to untreated). Pretreatment of monocytes with PTX also significantly reduced KYNA-induced arrest. n ≥ 2 for all conditions shown.

FIGURE 4.

Kynurenic acid induces firm arrest of monocytes on ICAM-1-expressing HUVECs in a dose-dependent fashion via a β₂ integrin-mediated mechanism. HUVECs were transduced with adenoviral ICAM and cultured for 48 h. Interaction of monocytes with the ICAM-1-expressing monolayer was studied at 1.75 dyne/cm². Adhesion was quantified 5 min after the introduction of the indicated compound. Arrested cell counts were normalized to the level of adhesion of untreated monocytes in each experiment. A, KYNA induces firm arrest of monocytes to ICAM-1-expressing HUVECs in a dose-dependent fashion (*, p < 0.05; ***, p < 0.0005), whereas AA treatment does not differ from background. B, pre-treatment of monocytes with the β₂ integrin-blocking antibody TS1/18, but not the α_vβ₃-blocking antibody LM609, abolished KYNA-induced binding of monocytes, as did pretreatment with PTX (◇, p < 0.05; ◇ ◇ ◇, p < 0.005). n ≥ 3 for all conditions shown.

FIGURE 5.

Silencing of GPR35 reduces KYNA-induced adhesion to ICAM-1-expressing HUVECs. THP-1 cells were infected with lentivirus containing shRNA sequences to either green fluorescent protein or GPR35. Adhesion of puromycin-selected cells to ICAM-1-expressing HUVEC monolayers was studied at 1.75 dyne/cm². A, mRNA expression of GPR35, measured by real-time QPCR as described above, is significantly reduced after silencing with GPR35-specific shRNA. B, THP-1 cells with reduced GPR35 expression show no significant firm arrest relative to baseline after KYNA treatment (*, p < 0.05 relative to baseline). The KYNA-treated adhesion was normalized to the baseline adhesion for each cell strain. n ≥ 3 for both untreated and KYNA-treated conditions in both cell strains.

FIGURE 6.

Kynurenic acid induces firm arrest of neutrophils on ICAM-1-expressing HUVECs in a β₂ integrin-mediated process without induction of an endothelial cell response. HUVECs were transduced with adenoviral ICAM and cultured for 48 h. Interaction of neutrophils with the ICAM-1-expressing monolayer was studied at 1.75 dyne/cm². Adhesion was quantified 5 min after the introduction of the indicated compound. Arrested cell counts were normalized to the level of adhesion of untreated neutrophils in each experiment. A, KYNA at 300 nm and 1 μm induces firm arrest of neutrophils on ICAM-1 (*, p < 0.05; **, p < 0.005), whereas 1 μm AA does not. B, pretreatment with the β₂ integrin-blocking antibody TS1/18 abolishes KYNA-induced adhesion to the monolayer (◇ ◇, p < 0.005 compared with 1 μm KYNA adhesion), whereas pretreatment with an isotype-matched control antibody has no effect. C, treatment of the HUVEC monolayer with 1 μm KYNA followed by rigorous washing resulted in no increased adhesion of neutrophils relative to control. n ≥ 3 for all conditions shown.

FIGURE 7.

Kynurenic acid induces activation of neutrophils. Freshly isolated neutrophils were incubated with the indicated compounds for 30 min at room temperature, and the resulting cleaved L-selectin in the medium was measured with an enzyme-linked immunosorbent assay. In each experiment, data were normalized to the level of soluble L-selectin measured in the untreated sample. KYNA significantly increased soluble L-selectin at all concentrations tested (*, p < 0.05; **, p < 0.005), whereas tryptophan and anthranilic acid elicited no change. Data represent means from at least four separate experiments.