Interferon-γ Increases hPepT1-Mediated Uptake of Di-Tripeptides Including the Bacterial Tripeptide fMLP in Polarized Intestinal Epithelia

Abstract

Interferon-gamma causes a global phenotypic switch in intestinal epithelial function, in which enterocytes become immune accessory cells. The phenotypic switch is characterized by a down-regulation of membrane transporters and up-regulation of immune accessory molecules in intestinal epithelial cells. However, the effect of interferon-gamma on the intestinal epithelia di-tripeptide hPepT1 transporter has not been investigated. In this study we demonstrate that 1) interferon-gamma increases di-tripeptide uptake in dose- and time-dependent manner in model intestinal epithelia (Caco-2 BBE cell monolayers), 2) the increase in di-tripeptides induced by interferon-gamma is hPepT1 mediated, 3) interferon-gamma does not affect the hPept1 expression at the mRNA and protein levels 4) interferon-gamma increases the intracellular pH and consequently enhances the H+-electrochemical gradient across apical plasma membrane in model intestinal epithelia (Caco2-BBE monolayers). We suggest that interferon-gamma could increase the hPepT1 mediated di-tripeptides uptake in inflamed epithelial cells. Under these conditions, interferon-gamma will increase the intracellular amount of such diverse prokaryotic and eucaryotic small di-tripeptides in inflamed epithelial cells. The intracellular accumulation of such di-tripeptides may be important in enterocytes becoming immune accessory cells.

hPepT1 mediates bacterial tripeptide fMLP uptake in human monocytes

“…This hypothesis is supported by (1) our findings that nonpolarized Caco2-BBE cells and KG-1 cells underwent drastic decreases of intracellular pH following acidification of the incubation buffer, while intracellular pH of polarized Caco2-BBE cells remained constant and (2) a previous report from our group showing that an increase of intracellular pH induced by interferon-gamma (IFNg) in polarized Caco2-BBE cells enhanced hPepT1-mediated uptake. 16 The fact that intracellular pH of polarized Caco2-BBE cells did not undergo any major changes after acidification of the apical pH is consistent with the ability of the intestinal epithelium to maintain an apical-to-intracellular pH gradient. Impermeability to acid of apical cell membrane has already been described for alveolar epithelial cells.…”

“…The human enterocyte-like Caco2-BBE cell line [16][17][18][19] were grown in high glucose Dulbecco's Vogt-modified Eagle's media (DMEM, Invitrogen) supplemented with 14 mmol/l NaHCO 3 , 10% (v/v) heat-inactivated FBS and 1.5 mg/ml plasmocin. Cells were kept at 371C in 5% CO 2 and 90% humidity, and the medium was changed every other day.…”

“…15 Consistent with this, the ED 50 of the fMLP receptor is in the nM range, 12 allowing passive movement of fMLP across the tight junctions of the epithelium under normal conditions. However, during inflammation, colonic tissues express hPepT1, 9,16 leading to two main consequences. First, the intracellular accumulation of fMLP induces an epithelial inflammatory response, including activation of the NF-kB pathway and immune accessory molecules such as MHC class I.…”

Activation of epithelial CD98 glycoprotein perpetuates colonic inflammation

“…Caco2-BBE [16][17][18][19] cells (between passages 30 and 50) were grown in high glucose Dulbecco's Vogt Modified Eagle's Media (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 14 mmol/l NaHCO 3 , and 10% newborn calf serum. Cells were incubated at 371C in 5% CO 2 and 90% humidity, and the medium was changed daily.…”

“…Previous work showed that the cell polarity is not affected under these treatment conditions. 19 At the end of the incubation period, we investigated CD98 protein expression using confocal microscopy and Western blotting. Confocal microscopy of confluent Caco2-BBE cell monolayers revealed that the staining for immunoreactive CD98 in control cells was localized to the basolateral domain, mainly in the tight junction area (Figure 1a).…”