In Lysinuric Protein Intolerance system y+L activity is defective in monocytes and in GM-CSF-differentiated macrophages

Abstract

Background: In the recessive aminoaciduria Lysinuric Protein Intolerance (LPI), mutations of SLC7A7/y+LAT1 impair system y+L transport activity for cationic amino acids. A severe complication of LPI is a form of Pulmonary Alveolar Proteinosis (PAP), in which alveolar spaces are filled with lipoproteinaceous material because of the impaired surfactant clearance by resident macrophages. The pathogenesis of LPI-associated PAP remains still obscure. The present study investigates for the first time the expression and function of y+LAT1 in monocytes and macrophages isolated from a patient affected by LPI-associated PAP. A comparison with mesenchymal cells from the same subject has been also performed.

Methods: Monocytes from peripheral blood were isolated from a 21-year-old patient with LPI. Alveolar macrophages and fibroblastic-like mesenchymal cells were obtained from a whole lung lavage (WLL) performed on the same patient. System y+L activity was determined measuring the 1-min uptake of [3H]-arginine under discriminating conditions. Gene expression was evaluated through qRT-PCR.

Results: We have found that: 1) system y+L activity is markedly lowered in monocytes and alveolar macrophages from the LPI patient, because of the prevailing expression of SLC7A7/y+LAT1 in these cells; 2) on the contrary, fibroblasts isolated from the same patient do not display the transport defect due to compensation by the SLC7A6/y+LAT2 isoform; 3) in both normal and LPI monocytes, GM-CSF induces the expression of SLC7A7, suggesting that the gene is a target of the cytokine; 4) GM-CSF-induced differentiation of LPI monocytes is comparable to that of normal cells, demonstrating that GM-CSF signalling is unaltered; 5) general and respiratory conditions of the patient, along with PAP-associated parameters, markedly improved after GM-CSF therapy through aerosolization.

Conclusions: Monocytes and macrophages, but not fibroblasts, derived from a LPI patient clearly display the defect in system y+L-mediated arginine transport. The different transport phenotypes are referable to the relative levels of expression of SLC7A7 and SLC7A6. Moreover, the expression of SLC7A7 is regulated by GM-CSF in monocytes, pointing to a role of y+LAT1 in the pathogenesis of LPI associated PAP.

Figure 1

Phase contrast image of fibroblast-like mesenchymal cells obtained from the LPI subject. Cells were isolated from WLL fluid, as described in Methods. × 100

Figure 2

Chest HRCT. Panel A. Lung CT scan obtained before WLL (December 2008). Bilateral, diffuse ground glass of lower pulmonary lobes, with thickening of intra- and inter-lobular septa (crazy paving pattern). Panel B. Lung CT scan obtained after inhaled rGM-CSF trial (April 2010), showing a marked bilateral improvement in lower lobe involvement; nevertheless, lung density appears still increased.

Figure 3

Characterization of L-arginine influx in LPI monocytes. Normal and LPI monocytes, isolated as described in Methods, were washed in EBSS. Panel A. Arginine uptake was assayed by 1 min incubation in EBSS supplemented with L-[³H]-arginine (50 μM; 4 μCi/ml) in the absence (total uptake) or in the presence of leucine (2 mM) or leucine + lysine (both 2 mM) as indicated. For normal cells, data are means ± SEM of 9 independent experiments (n = 9 normal donors), each performed in quadruplicate. For the LPI patient, data are means ± SD of 4 determinations obtained in a representative experiment, repeated twice with comparable results. *** p < 0.001 vs total; NS, Not Significant vs +Leucine; ns, not significant vs total; ^# p < 0.05 vs +Leucine. Panel B. Arginine transport values of each subject (n = 9 normal donors; n = 2 determinations in the LPI patient) were employed to calculate system y⁺L and system y⁺ transport activity. System y⁺L: difference between total uptake and the uptake obtained in the presence of 2 mM leucine; system y⁺: difference between the influx measured in the presence of 2 mM leucine and that measured in the presence of 2 mM leucine + 2 mM lysine.

Figure 4

System y⁺L and y⁺ activities in alveolar macrophages (AM) and in fibroblasts obtained from the LPI subject. Arginine uptake was assayed in AM or fibroblasts from different healthy donors (n = 3) or from the LPI patient (2 experiments on AM and 3 on fibroblasts) and system y⁺L and system y⁺ transport activities were determined as described in Figure 3..

Figure 5

Expression of SLC7A6 and SLC7A7 in monocytes, alveolar macrophages (AM) and fibroblasts obtained from the LPI subject. After RNA extraction and reverse transcription, cDNA was employed as template for qPCR with SLC7A6/y+LAT2 and SLC7A7/y+LAT1 primers. mRNA level, normalized for RPL15 gene, is expressed as number of molecules (see Methods). For normal cells, data are means ± SEM of 9 (monocytes) or 3 (AM and fibroblasts) independent experiments. For the LPI patient, bars are means ± SD of 6 (monocytes and fibroblasts) or 4 (AM) determinations.

Figure 6

Effect of GM-CSF on the differentiation of LPI monocytes to macrophages. Normal and LPI monocytes were differentiated to Monocyte-Derived Marcophages (MDM) by 5 d incubation in RPMI supplemented with rGM-CSF (10 ng/ml). Panels A and B. Phase contrast images of normal (A) and LPI (B) MDM. × 100. Panel C. Induction of macrophage differentiation markers in normal and LPI MDM. In freshly isolated (control) or rGM-CSF-treated (MDM) monocytes RNA was extracted and cDNA employed as template for qPCR with primers for the indicated genes. The values are normalized to that of RPL (see Eq 1 in Methods). For normal cells, data are means ± SEM of 5 independent experiments (n = 5 normal donors). For the LPI patient, data are means ± SD of 4 determinations.

Figure 7

Effect of GM-CSF on System y⁺L activity and transporters expression. Normal and LPI monocytes were differentiated to Monocyte-Derived Marcophages (MDM) as described in Figure 6. Panel A. The activities of system y⁺L and system y⁺ were determined as described in Figure 3, both in freshly isolated (control) and in rGM-CSF-treated (MDM) monocytes. For normal cells, data are means ± SEM of 6 independent experiments (n = 6 normal donors). For the LPI patient, data are means ± SD of 4 determinations in a representative experiment that was repeated twice with comparable results. ***p < 0.001 MDM vs control. Panel B. In the same conditions SLC7A6 and SLC7A7 expression were determined as numbers of molecules of mRNA indexed to RPL mRNA (see Methods). For normal cells, data are means ± SEM of 6 independent experiments (n = 6 normal donors). For the LPI patient, data are means ± SD of 4 determinations. * p < 0.05 MDM vs control.