hPepT1 mediates bacterial tripeptide fMLP uptake in human monocytes

Abstract

Here, we examined hPepT1 expression in the monocytic cell line, KG-1. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that hPepT1 is expressed in KG-1 cells, while cDNA cloning and direct sequencing confirmed the sequence of KG-1 hPepT1 (accession number, AY634368). Immunoblotting of cell lysates from KG-1 cells or macrophages isolated from human peripheral blood revealed a B100 kDa immunoreactive band mainly present in the membrane fraction. Uptake experiments showed that the transport of 20 lM radiolabeled Gly-Sarcosine ([ 14 C]Gly-Sar) in KG-1 cells was Na þ , Cl À dependent and disodium 4,4 0 -diisothiocyanatostilbene-2,2 0 -disulfonate (DIDS)-sensitive. In addition, hPepT1 activity was likely to be coupled to a Na þ /H þ exchanger, as evidenced by the fact that [ 14 C]Gly-Sar uptake was not affected by the absence of Na þ when cells were incubated at low pH (5.2). Interestingly, hPepT1-mediated transport was reduced in KG-1 cells incubated at low pH as it was also observed in nonpolarized Caco2-BBE cells. This pattern of pH-dependence is due to a disruption of the driving force of hPepT1-mediated transport events. This was supported by our finding that nonpolarized cells, Caco2-BBE cells and KG-1 cells, have an increased permeability to H þ when compared to polarized Caco2-BBE cells. Finally, we showed that hPepT1 is responsible for transporting fMLP into undifferentiated and differentiated (macrophage-like) KG-1 cells. Together, these results show that hPepT1 is expressed in nonpolarized immune cells, such as macrophages, where the transporter functions best at the physiological pH 7.2. Furthermore, we provide evidence for hPepT1-mediated fMLP transport, which might constitute a novel immune cell activation pathway during intestinal inflammation.

PepT1-Mediated Tripeptide KPV Uptake Reduces Intestinal Inflammation

“…We previously showed that human monocytes express a functional hPepT1 protein (18). Our work demonstrates for the first time that the human Jurkat T cell line also expresses a functional hPepT1 protein able to transport KPV into the cytosol where it can accumulate and inhibit inflammatory signaling pathways and subsequent cytokine secretion.…”

“…Although human PepT1 is not expressed in normal colonic epithelial cells (10,16,17), we detected its expression at the apical membrane of epithelial cells in chronically inflamed colon (17). Interestingly, we have also shown that immune cells, such as macrophages, which are in close contact with the lamina propria of the intestine, also express PepT1 at their membranes (17,18).…”

“…Similar results were found in Jurkat cells. Indeed the Km is ~700 μM and only one study reported kinetic experiments in immune cells showing that the Km of hPepT1 for its substrates Gly-Sar and fMLP were ~2 mM (18).…”

Pannexin-1-Mediated Intracellular Delivery of Muramyl Dipeptide Induces Caspase-1 Activation via Cryopyrin/NLRP3 Independently of Nod2

“…It has been reported that the plasma membrane transporter, PepT1, can translocate MDP into the cytosol of host cells (51,52). PepT1 is expressed primarily at the apical membrane of intestinal epithelial cells (53), although some expression has been reported in human monocytes (54). However, there is no evidence that PepT1 functions as a transporter of MDP in monocytic cells.…”

NLR proteins: integral members of innate immunity and mediators of inflammatory diseases

“…However, chronically inflamed colonic epithelial cells do express PepT1 [40]. In addition, hPepT1 is also expressed in normal human macrophages and human monocytic KG-1 cells, where it has been shown to mediate transport of di-and tripeptides [41]. Therefore, PepT1 remains as a possibility in terms of MDP transport.…”