2.1 Cell cultures

Human monocytes were isolated from buffy coats of normal, healthy donors, provided by the Unit of Immunohematology and Transfusion of the Azienda Ospedaliero-Universitaria of Parma (local ethics committee approval # 43899, 03/12/2015), as previously described.11 Monocyte-derived macrophages (MDM) were obtained by incubating monocytes for 6 d in RPMI1640 supplemented with 10% endotoxin-free foetal bovine serum (FBS) and 50 ng/mL of recombinant human GM-CSF. Normal human fibroblasts, obtained from a 15-year-old healthy donor,12 were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) with 4 mmol/L glutamine. Human renal proximal tubular epithelial cells (HRPTEpC), purchased from Sigma-Aldrich (Italy), were grown in REGM medium, as indicated by the manufacturer; only cells between first and third passages were used. Caco-2 intestinal epithelial cells were obtained from American Type Culture Collection (ATCC) and routinely grown in DMEM. Polarized Caco-2 monolayers were established by seeding cells at a density of 3 × 10⁵/mL on polyester (PET) membrane Transwell^® inserts (12 mm diameter, 0.4 µm pore size, Falcon) in 24-well plates. About 200 μL and 700 µL culture media were added to the apical and basolateral compartments of the insert, respectively. Cell cultures were employed 7 days after seeding when monolayers exhibited high transepithelial electrical resistance (TEER values > 600 Ohm·cm² using an epithelial voltmeter (EVOM, World Precision Instruments)). The integrity of cell monolayers was preserved after the experiments. EpiIntestinal™, a model of human small intestinal tissue (SMI-100), was supplied by MatTek Corporation (Ashland) and cultured with medium provided by the manufacturer. Alveolar epithelial A549 and Calu-3 cells, deriving from a human lung adenocarcinoma, were obtained from ATCC and cultured in high glucose DMEM and in Eagle's Minimum Essential Medium (EMEM), respectively. K562, a chronic myeloid leukaemia cell line, was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH and maintained as suspension cultures in RPMI1640 medium. Lymphoblasts, kindly provided by Dr G. Sebastio (Department of Paediatrics of the University Federico II of Naples; Italy), were grown in suspension in RPMI1640 medium. Human CD3⁺ lymphocytes were a kind gift of Dr Laura Passerini (San Raffaele Telethon Institute for Gene Therapy, Milan, Italy). EpiAirway™ system, a 3-D human-derived tracheal/bronchial epithelial cells (AIR-200-PE6.5), was supplied by MatTek Corporation and cultured as indicated by the manufacturer. Unless otherwise specified, the growth medium for all these cell types was supplemented with 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin; all cells were routinely cultured under physiological conditions (37.5°C, 5% CO₂, 95% humidity).

2.2 Gene silencing

Gene silencing was performed employing HiPerFect Transfection Reagent, AllStar Negative Control (scrambled siRNA, Cat.# SI03650318) and FlexiTube short interfering RNAs (SLC7A6/y+LAT2 siRNA, Cat.# SI00058681 and SLC7A7/y+LAT1 siRNA, Cat.# SI00058660) by Qiagen®. Fibroblasts were transfected according to the protocol provided by the manufacturer. For MDM, isolated monocytes (3 × 10⁶/mL) were maintained in antibiotic-free RPMI1640 medium containing 50 ng/mL GM-CSF for 72 hours, then washed with PBS and incubated in serum-free medium containing 60 µL/mL HiPerFect Transfection Reagent and 2 nmol/L (final concentration) scrambled or specific siRNA; after 6 hours, 0.3 volumes of complete growth medium were added. Cells were employed after 96 hours.

2.3 RT-qPCR analysis

Total RNA was isolated with GeneJET RNA Purification Kit and reverse transcribed with RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific); the amount of the genes of interest (SLC7A6/y+LAT2 and SLC7A7/y+LAT1) was, then, determined using TaqMan® Gene Expression Assays by Thermo Fisher Scientific (Cat# Hs00187757_m1, Hs00909952_m1, and Hs03855120_g1, respectively), and expressed as numbers of mRNA molecules upon normalization to that of the housekeeping gene (RPL15, Ribosomal Protein Like 15).13

2.4 L-Arginine uptake

In mammalian cells, four distinct transport mechanisms, namely systems y⁺, y⁺L, b^0,+ and B^0,+, mediate cationic amino acid uptake.5 System y⁺ operates a sodium-independent and membrane potential–dependent unidirectional transport specific for cationic amino acids, while the other three transporters accept both cationic and neutral amino acids.14 ATB^0,+ is a highly concentrative system, being energized by the transmembrane gradients of Na⁺ and Cl⁻, as well as by membrane potential. System b^0,+ is Na⁺-independent and mediates the obligatory exchange of cationic amino acids and cystine with neutral amino acids with 1:1 stoichiometry. ATB^0,+ and b^0,+are expressed in the apical membrane of epithelial cells. Lastly, the ubiquitary expressed system y⁺L transports cationic amino acids in the absence of sodium and neutral amino acids in the presence of the cation. Under physiological conditions, it operates as an efflux route by exporting cationic amino acids from inside the cells in exchange with neutral amino acids plus sodium; this system is localized at the basolateral side of epithelial cells. The features of these transport systems are resumed in Figure 1.

For uptake studies, cells, cultured onto 96-well trays, were washed twice at 37°C with Earle's Balanced Salt Solution (EBSS, composition in mmol/L: 117 NaCl, 1.8 CaCl₂, 5.3 KCl, 0.9 NaH₂PO₄, 0.8 MgSO₄, 5.5 glucose, 26 Tris/HCl, pH 7.4), and incubated for 1 minute in the same solution containing [³H]arginine (50 µmol/L, 5 μCi/mL) in the absence or presence of leucine (2 mmol/L) or leucine plus lysine (2 mmol/L). For the measurements of arginine uptake in the absence of sodium, a modified EBSS (NMG-EBSS) was employed in which NaCl and NaH₂PO₄ were replaced by N-methyl-D-glucamine and choline salts, respectively. Uptake was terminated by the removal of uptake solution followed by three rapid washes with cold 300 mmol/L urea. Cell monolayers were extracted in ethanol and radioactivity counted in a Wallac Microbeta Trilux2 liquid scintillation spectrometer (Perkin Elmer). L-arginine uptake was normalized for protein content, determined directly in each well by using a modified Lowry method.15 Uptake is expressed as nmol/mg of protein/min. The activity of system b^0,+ was calculated as the difference between arginine transport in the absence of inhibitors (total) and the transport measured in the presence of leucine in the absence of sodium. System y⁺L was calculated as the difference between arginine transport in the absence of inhibitors (total) and the transport measured in the presence of leucine in the presence of sodium, after the subtraction of b^0,+ quote, when present. System y⁺ activity was determined as the quote of transport further inhibited by lysine in the presence of sodium.

The apparent kinetic parameters K_m (Michaelis constant) and V_max (maximum transport rate) of arginine uptake were calculated by non-linear regression fitting according to the Michaelis-Menten equations:

(1)

for a single saturable component, where v is the initial influx, V_max is the maximal influx and K_m is the Michaelis constant.

2.5 Determination of arginine transcellular fluxes in polarized Caco-2 cells

Caco-2 cell monolayers, grown in Transwell^® (24-well plates) to confluence, were washed with Earle's Balanced Salt Solution (EBSS) at 37°C and incubated, at the apical side, with 100 µL EBSS containing [³H]arginine (50 µmol/L, 10 µCi/mL); the basolateral compartment was incubated in 550 µL EBSS, in the absence or in the presence of 10 mmol/L leucine. At the times indicated, 100 µL of the solution in the basolateral compartment was collected and replaced with an equal volume of fresh EBSS solution. Radioactivity in each sample was counted in a Wallac Microbeta Trilux2 liquid scintillation spectrometer (Perkin Elmer).

2.6 Statistics

Prism^® 5.0 GraphPad software has been employed for the statistical analysis; statistical significance was calculated with Student's t test for unpaired data, unless stated otherwise. Differences were considered significant when P < .05.

2.7 Materials

Endotoxin-free foetal bovine serum (FBS) was purchased from EuroClone (Italy); L-[2,3,4-3H]Arginine monohydrochloride (43 Ci/mmol) was purchased from Perkin-Elmer (Italy) and GM-CSF from Vinci-Biochem (Italy). Unless otherwise stated, Sigma-Aldrich (Italy) was the source of all the other chemicals.

3.1 Biochemical features of y+LAT1 and y+LAT2 transporters

In a previous contribution, we showed that the activity of system y⁺L was impaired in LPI monocytes and alveolar macrophages, but readily detectable in fibroblast-like mesenchymal cells obtained from an LPI patient, as well as in fibroblasts from healthy donors.7 Based on the analysis of gene expression, we there suggested that a normal expression of SLC7A6/y+LAT2 could compensate the mutation of SLC7A7/y+LAT1 in LPI fibroblasts conferring them normal arginine uptake, whereas the predominant expression of y+LAT1 in monocytes and macrophages could explain the defective transport observed in LPI cells.

In order to verify this hypothesis, we here first tried to discriminate the relative contribution of the two transporters in fibroblasts and monocytes-derived macrophages (MDM), by selectively silencing one or the other gene. Data obtained (Figure 2) indicate that the expression of both transporters was efficiently reduced by the specific siRNA, both in MDM (panel A) and in fibroblasts (panel D). In MDM, SLC7A6 gene silencing resulted in a slight reduction of total arginine transport (panel B), whereas a more marked inhibition (more than 65%) was observed in SLC7A7-silenced cells. Since the presence of leucine almost completely suppressed arginine uptake under any condition, we can conclude that in MDM, the activity of system y⁺L, calculated as the quote of arginine uptake inhibited by leucine (panel C), is only marginally hindered by SLC7A6 siRNA, while almost completely abolished upon SLC7A7 silencing. Conversely, in fibroblasts only the down-regulation of SLC7A6 proved effective in reducing arginine transport, while SLC7A7 silencing did not (panel E); moreover, since leucine exerted its inhibitory effect in SLC7A7-, but not in SLC7A6-silenced cells (panel F), we can conclude that system y⁺L-mediated arginine transport in these cells is likely accounted for by y+LAT2 protein.

Starting from these findings, we next addressed the biochemical features of the transporters by performing the kinetic analysis of arginine transport in silenced cells; more precisely, we measured the kinetic constants of system y⁺L (calculated as the difference between total transport and the transport measured in the present of leucine) in MDM silenced for SLC7A6 (hence measuring the activity of the sole y+LAT1) and in fibroblasts silenced for SLC7A7 (hence measuring the activity of the sole y+LAT2). Results, presented in Figure 3, indicate that the Michaelis constants (K_m) of the two alternative proteins are very similar (0.182 ± 0.035 mmol/L and 0.145 ± 0.028 mmol/L for MDM and fibroblasts, respectively) and, thus, that they have a comparable affinity for arginine. V_max values are, instead, different (3.822 ± 0.24 and 1.479 ± 0.09 nmol/mg of protein/min for MDM and fibroblasts, respectively), suggesting differences in the number of functional transporters on the membrane or in their intrinsic activity due to in-trans substrate availability.

3.2 Tissue-specific pattern of expression of SLC7A6/y+LAT2 and SLC7A7/y+LAT1

Next, we addressed the differential expression of SLC7A6/y+LAT2 and SLC7A7/y+LAT1 in different tissues through a quantitative RT-qPCR analysis of the two genes in various cell models. As shown in Figure 4, SLC7A7 was impressively expressed in MDM and intestinal Caco-2 cells, and relatively high in human renal proximal tubular (HRPTEpC) and in small intestine epithelial cells (EpiIntestinal™); with the exception of THP-1 monocytic cell line, the gene was undetectable in all other models. On the contrary, the expression of SLC7A6 was almost comparable in all cells tested, with CD3⁺ T lymphocytes showing the highest mRNA levels.

3.3 Arginine transport in renal and intestinal epithelial cells

Data of gene expression are consistent with the notion that beside immune cells, intestine and kidney are the main targets of LPI defect; however, little is thus far known about system y⁺L activity in human renal and intestinal cells. Figure 5 shows the characterization of arginine transport in primary renal proximal tubular epithelial cells (HRPTEpC) and in Caco-2 cells, the representative in vitro model of the intestinal barrier. The relative contribution of each transporter has been assessed through a methodology previously used in other cell models.16 Arginine uptake was comparable in the absence and in the presence of sodium in both renal (panel A) and intestinal (panel C) cells, thus excluding a significant contribution of sodium-dependent transport systems, such as B^0,+. In the absence of sodium, leucine significantly inhibited arginine uptake, demonstrating that system b^0,+ is operative in these cells. Moreover, since the inhibitory effect of leucine was much more evident in the presence of the cation, we can conclude that also system y⁺L, typically inhibited by neutral amino acids only in the presence of sodium, is functional. Under this experimental condition, the addition of 2 mmol/L lysine to leucine further lowered arginine transport in HRPTEpC, indicating the operation of system y⁺ in these cells; on the contrary, the lack of inhibition by lysine in Caco-2 cells indicates that the contribution of system y⁺ in this model is at most marginal. In summary, arginine uptake in renal cells is mediated by the activity of systems y⁺L, b^0,+ and y⁺ (panel B), while only systems y⁺L and b^0,+are operative in the intestinal model (panel D).

In these latter, the transcellular flux of arginine has been also monitored in polarized monolayers; to this end, cells grown on inserts were incubated, at the apical side, in the presence of labelled arginine and, at the basolateral side, in EBSS, either in the absence or presence of leucine. As demonstrated in panel E, the efflux of arginine through the basal side of the monolayer was trans-stimulated by the presence of leucine, indicating that, in the intestinal barrier, system y⁺L likely cooperates to the absorption of the amino acid by exchanging intracellular arginine with extracellular leucine plus sodium.