Kynurenic Acid Triggers Firm Arrest of Leukocytes to Vascular Endothelium under Flow Conditions^*

EXPERIMENTAL PROCEDURES

RESULTS

To assess the potential role of KYNA in leukocyte adhesion processes, we first measured the level of GPR35 mRNA in several leukocyte cell types, as well as in endothelial cells. GPR35 was most highly expressed in monocytes, with significant expression in neutrophils as well. By contrast, QPCR experiments suggested virtually no expression in endothelial cells (Fig. 2A). The level of GPR35 expression on monocytes was less than that of the prototypical monocyte chemokine receptor, CCR2, although comparable with that of CXCR2 (Fig. 2B). Activation of both of these receptors results in robust monocyte adhesion (3) and also contributes to monocyte trafficking in atherosclerosis in vivo (21, 22). GPR35 expression greatly exceeded that of CCR3, a chemokine receptor that is highly expressed on basophils and eosinophils but present only at very low levels on monocytes (23).

To test whether KYNA was sufficient to induce β₁ integrin-mediated firm arrest under physiological flow conditions, we employed a flow chamber to pass monocytes over a fibronectin-coated surface. At a shear stress of 1.0 dyne/cm², KYNA treatment resulted in a 13-fold increase in the number of adherent cells as compared with baseline, slightly higher than adhesion induced by the CCR2 agonist MCP-1. Tryptophan and AA, two metabolites closely related to KYNA that do not bind GPR35 (9), had no effect on the adhesion of monocytes (Fig. 3A). Pretreatment of the monocytes with HP2/1, an α₄ integrin-blocking antibody that interferes with α₄β₁-mediated arrest, significantly reduced the number of adherent cells induced by KYNA exposure. By contrast, pretreatment with an isotype-matched control antibody did not result in any reduction in KYNA-induced firm arrest (Fig. 3B).

Many chemokine-triggered effector functions in leukocytes are dependent on the G protein α_i subunit of the G protein heterotrimer (24). To assess whether the effects of KYNA on monocyte adhesion involved this pathway, we pretreated cells with PTX prior to introducing them into the flow chamber. PTX exerts its effects by ADP-ribosylating the α subunit of the G protein heterotrimer, thus preventing the complex from coupling with receptors on the surface of the cell (25). Incubation of monocytes with PTX prior to use in flow experiments significantly reduced the KYNA-induced adhesion of monocytes to fibronectin (Fig. 3B), confirming that this adhesion is triggered by a G_i-mediated process.

Interactions between activated β₂ integrins and intercellular adhesion molecule (ICAM)-1 are also sufficient to confer monocyte arrest under flow conditions (3). We therefore examined the adhesion of monocytes to endothelial monolayers expressing ICAM-1 using an adenoviral expression system. We have previously demonstrated that this system is extremely sensitive to adhesive triggers (3). KYNA treatment of monocytes in this system increased adhesion in a dose-dependent fashion, with significant arrest observed as low as 300 nm, while treatment with 1 μm anthranilic acid elicited no effect (Fig. 4A). This is significantly lower than the concentration necessary to induce adhesion to fibronectin surfaces, consistent with previous reports that firm arrest of leukocytes on adhesion molecule-expressing endothelial monolayers (as opposed to purified protein immobilized on coverslip) is highly sensitive to chemoattractant agonists (3). To verify the specificity of the observed interactions, we pretreated the leukocytes with the β₂ integrin-blocking antibody, TS1/18. Pretreatment with this antibody, but not an α_vβ₃-blocking antibody, LM609, abolished the KYNA-induced response (Fig. 4B). As with adhesion to fibronectin, pretreatment of monocytes with PTX significantly reduced adhesion to ICAM-1-expressing monolayers, confirming G_i involvement (Fig. 4B). Although KYNA-triggered response of monocytes on fibronectin was greater than the MCP-1-triggered response, the reverse was observed with the ICAM-1 monolayers.

As KYNA and MCP-1 would both be expected to be present under certain inflammatory circumstances (and potentially other chemokines as well), we tested how the addition of both agonists modulated adhesion. Simultaneous treatment with both MCP-1 and KYNA resulted in an additive, but not synergistic effect on firm arrest (data not shown).

To confirm that the observed adhesion was indeed due to activation of GPR35 by KYNA, we used shRNA to generate THP-1 cells with reduced expression of GPR35. GPR35-silenced cells showed a marked reduction in GPR35 mRNA expression relative to control cells infected with an anti-green fluorescent protein lentivirus (Fig. 5A). Green fluorescent protein-silenced control THP-1 cells retained a level of KYNA-triggered firm arrest relative to baseline that is similar to that observed in freshly isolated human leukocytes. By contrast, cells with reduced GPR35 expression did not show a significant increase in firm arrest after KYNA treatment (Fig. 5B).

Because GPR35 was also readily detected in human neutrophils, we tested whether KYNA was sufficient to trigger adhesion of this leukocyte subset as well. Because neutrophil adhesion under flow is predominantly dependent on β₂ integrin-ICAM-1 interactions, we also flowed neutrophils over ICAM-1-expressing HUVECs. At a shear stress of 1.75 dyne/cm², 300 nm KYNA was sufficient to trigger statistically significant increases in neutrophil adhesion (Fig. 6A), whereas treatment with 1 μm AA did not increase adhesion above background levels. In this system as well, pretreatment of the neutrophils with the β₂ integrin-blocking antibody TS1/18 abrogated the effects of KYNA on leukocyte arrest.

To verify that KYNA-induced arrest is a result of leukocyte, and not endothelial activation, we treated the HUVEC monolayers for 15 min with KYNA in perfusion buffer and then washed the monolayers prior to placement in the flow chamber. As treatment of the monolayers resulted in no increase of neutrophil adhesion over background (Fig. 6C), KYNA appeared to be exerting its effects on the neutrophils.

We also examined the cleavage of L-selectin from the surface of neutrophils as an indicator of the degree of cellular activation (26). After a 30-min incubation with 100 nm of the known activating agent IL-8, a significant increase in cleaved L-selectin over baseline was observed (Fig. 7), consistent with previous results (27). Treatment with KYNA resulted in shedding of L-selectin from neutrophils in a dose-dependent fashion, showing a greater than 2-fold increase in shed L-selectin over baseline at a concentration of 10 mm, and a significant increase in the cleaved molecule even at the lowest KYNA dose tested (100 μm) (Fig. 7). Additionally, we tested two other metabolites from the tryptophan metabolic pathway that are known not to activate GPR35 (9): tryptophan and anthranilic acid. At a concentration of 1 mm, neither compound elicited an increase in L-selectin shedding above the background level (Fig. 7).

DISCUSSION

We found that KYNA is capable of activating multiple subsets of leukocytes, eliciting firm arrest in these cells through both β₁ and β₂ integrin-mediated adhesion. This adhesion was PTX-sensitive, which suggests that it occurs through a G_i-coupled pathway. By contrast, two related compounds that are known not to bind GPR35, tryptophan and anthranilic acid (9), did not evoke activation or adhesion of leukocytes, consistent with a GPR35-mediated mechanism. Silencing of GPR35 expression using shRNA significantly reduced KYNA-induced firm arrest of a monocytic cell line to ICAM-1-expressing surfaces, confirming GPR35 involvement in this process.

The ability of KYNA to trigger adhesion is consistent with other studies demonstrating unanticipated roles for small molecules in immune modulation. Medium chain fatty acids, particularly capric acid, undecanoic acid, and lauric acid, are able to activate GPR84, a receptor that is significantly up-regulated in lipopolysaccharide-stimulated monocytes. Treatment with these compounds amplifies the effect of lipopolysaccharide treatment on secretion of the cytokine IL-12p40 by RAW264.7 cells (28).

The baseline concentration of KYNA in human plasma is ∼25 nm (29–31), which is insufficient to evoke leukocyte adhesion in our studies. During inflammation, however, interferon-γ induction of IDO leads to the degradation of tryptophan and subsequent generation of catabolites including KYNA. As such, the concentration of KYNA could be expected to reach the micromolar range (32–34), a concentration sufficient to evoke significant adhesion of both monocytes and neutrophils to ICAM-1-expressing endothelial cells in our studies, particularly near the site of generation. Vascular endothelial cells have been demonstrated to convert kynurenine to KYNA, so localized regions with sufficient KYNA concentration to elicit a leukocyte response may be present in the vasculature.

Our studies indicate that KYNA can evoke firm adhesion at concentrations as low as 300 nm. This is significantly less than the EC₅₀ of 39.2 μm previously reported for KYNA activation of human GPR35 overexpressed on Chinese hamster ovary cells (9). However, we would expect that the receptor expressed in its endogenous context would behave differently than when overexpressed in a transformed cell line. The coupling of GPRs to chimeric G proteins for Ca²⁺ mobilization assays may significantly increase the measured EC₅₀ for some ligands, relative to assays utilizing native couplings (35, 36); differences up to 50-fold have been reported (37). Additionally, the arrest of leukocytes on ICAM-1-expressing endothelial cells is a highly sensitive assay (3), and significant adhesion may be seen with ligands at less than half-maximal activation concentration.

Reports have indicated that KYNA can reduce the lipopolysaccharide-stimulated secretion of the pro-inflammatory cytokines tumor necrosis factor-α and interferon-γ over long (18–72 h) time periods (9, 38). This would suggest a down-modulation of the immune response; however, other types of acute assays were not performed. Although immunosuppression is consistent with evidence that tryptophan metabolites generated after the induction of IDO act in an anti-inflammatory capacity (15), the data presented here suggest that the situation may in fact be more complex. Given the ability of KYNA to elicit early stage arrest of leukocytes, tryptophan catabolism by IDO may have a rapid pro-inflammatory phase, followed by a later anti-inflammatory period. Further investigation into the kinetic response of leukocytes to KYNA will be necessary to better understand both the pro- and anti-inflammatory actions of KYNA on specific leukocyte subsets.

shRNA-mediated decrease in GPR35 expression was accompanied by a concordant reduction in KYNA-triggered adhesion of monocytic cells. We were also interested to find that GPR35-deficient cells grew in clusters, although the cells could be easily dissociated by gentle pipetting for functional assays. The spectrum of chronic effects of GPR35 deletion in vitro and in vivo thus remains the subject of future investigation.

Also worth considering is the potential effect of KYNA on individuals with renal insufficiency. Because the primary method of excretion of tryptophan catabolites is through the kidneys, renal insufficient patients have chronically elevated levels of KYNA. A recent study has shown that uremic patients exhibit pre-dialysis serum levels of KYNA greater than 300 nm; this elevation is only partially reduced by hemodialysis, with KYNA remaining at ∼10 times the baseline level (39). As patients with renal insufficiency suffer from an increased rate of cardiovascular disease, further investigation into the consequences of chronically elevated KYNA in these patients may be merited.

In summary, our studies have shown that KYNA is capable of eliciting firm arrest of both neutrophils and monocytes under physiological flow conditions in an in vitro model. Clearly, multiple factors contribute to the adhesion process in vivo that are not present in this system, and additional studies in animal models are warranted.

Supplementary Material