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Published ahead of print on March 15, 2007, doi:10.1165/rcmb.2006-0262OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 37, pp. 105-112, 2007
© 2007 American Thoracic Society
DOI: 10.1165/rcmb.2006-0262OC

Alveolar Macrophages from Normal Subjects Lack the NOS-Related System y+ for Arginine Transport

Bianca Maria Rotoli, Valeria Dall'Asta, Amelia Barilli, Raffaele D'Ippolito, Annalisa Tipa, Dario Olivieri, Gian C. Gazzola and Ovidio Bussolati

Department of Experimental Medicine, Section of General and Clinical Pathology, and Department of Clinical Sciences, Section of Respiratory Diseases, University of Parma, Parma, Italy

Correspondence and requests for reprints should be addressed to Bianca Maria Rotoli, Ph.D., Sezione di Patologia Generale e Clinica, Dipartimento di Medicina Sperimentale, Università di Parma, Via Volturno, 39, 43100 Parma, Italy. E-mail: biancamaria.rotoli{at}unipr.it


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Systems y+ and y+L represent the main routes for arginine transport in mammalian cells. While system y+ activity is needed for the stimulated NO production in rodent alveolar macrophages (AM), no information is yet available about arginine transport in human AM. We study here arginine influx and genes for arginine transporters in AM from bronchoalveolar lavage of normal subjects. These cells express the y+-related genes SLC7A1/CAT1 and SLC7A2/CAT2B, as well as the y+L genes SLC7A7/y+LAT1 and SLC7A6/y+LAT2. However, compared with human endothelial cells, AM express much less SLC7A2 mRNA and higher levels of SLC7A7 mRNA. Granulocyte macrophage colony-stimulating factor or IFN-{gamma} do not change the expression of any transporter gene, while lipopolysaccharide induces SLC7A2/CAT2B. Under all the conditions tested, leucine inhibits most of the arginine transport in the presence of Na+ and N-ethylmaleimide, an inhibitor of system y+, is completely ineffective, indicating that system y+L operates most of the arginine influx. Comparable results are obtained in AM from patients with interstitial lung disease, such as Nonspecific Interstitial Pneumonia (NSIP), although these cells have a higher SLC7A1 and a lower SLC7A7 expression than AM from normal subjects. It is concluded that AM from normal subjects or patients with NSIP lack a functional transport system y+, a situation that may limit arginine availability for NO synthesis. Moreover, since mutations of SLC7A7/y+LAT1 cause Lysinuric Protein Intolerance, a disease often associated with AM impairment and alveolar proteinosis, the high SLC7A7 expression observed in human AM suggests that y+LAT1 activity is important for the function of these cells.

Key Words: alveolar proteinosis • Lysinuric Protein Intolerance • NO • system y+ • system y+L



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Alveolar macrophages do not transport Arg through the nitric oxide synthase–related CAT transporters but, rather, through y+LAT1 and 2. Since y+LAT1 mutations underlie some cases of alveolar proteinosis, these data suggest that y+LAT1 activity is required for alveolar macrophage function.

 
Alveolar macrophages (AM) are resident cells that play several important roles in airway homeostasis (1). They are responsible for the first line defense of lower respiratory tract against airborne pathogens, provide surfactant catabolism, and are involved in complex regulatory interplays with airway epithelial cells, as well as with endothelial cells, lymphocytes, and fibroblasts of the alveolar septa. Defective granulocyte macrophage colony-stimulating factor (GM-CSF) signaling to AM has been implicated in the pathogenesis of alveolar proteinosis (2, 3) and alterations in the same pathway have been described in other pathological conditions (4, 5).

It is well documented that AM produce a wide array of mediators, such as various cytokines, while it is still controversial if and under what conditions they produce nitric oxide (NO), although NO output from resting AM is believed to be very low (6). The cationic amino acid arginine is the obliged substrate of all the isotypes of nitric oxide synthase (NOS), and in several models of rodent macrophagic cells, NOS2-dependent NO production, induced by lipolysaccharide (LPS) and IFN-{gamma}, requires the uptake of extracellular arginine through transport system y+, and, in particular, through the inducible isoform CAT2B (7, 8). Consistently, several reports have documented CAT2B expression in alveolar macrophages from animal models (9–13) and, most recently, CAT2B transporter has been found to play a critical role in regulating AM activity in the mouse (14).

No investigation has thus far concerned arginine transport in human AM and it is not known whether these cells take up the cationic amino acid through system y+ or some other transport systems described to operate arginine transport in mammalian cells (see Ref. 15 for review). We have recently shown that in human monocytes from peripheral blood system y+ activity is marginal and most of arginine transport is due to system y+L, with a clear-cut expression of both y+LAT1 and y+LAT2 transporters (16). Interestingly, the gene for y+LAT1, SLC7A7, is mutated in patients affected by Lysinuric Protein Intolerance (17, 18), a condition often associated with pulmonary alveolar proteinosis and with alterations of AM (19).

Those data prompted us to extend our investigation to human AM. Here we show that AM from normal subjects are not endowed with system y+ activity, although they express mRNA for CAT transporters and induce CAT2B mRNA, but not NO production, after stimulation with LPS. On the contrary, human AM exhibit a clear-cut expression of SLC7A7/y+LAT1 and SLC7A6/y+LAT2 along with an evident arginine transport through system y+L. Comparable findings have been also obtained in AM from patients with Nonspecific Interstitial Pneumonia (NSIP). These results suggest that lack of a significant activity of system y+-related transporters in human AM may be a limiting factor for NO production by these cells.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Human AM Isolation from Bronchoalveolar Lavage and Culture Conditions
Sixteen normal subjects (9 male and 7 female; age range, 30–78 yr; nonsmokers [n = 12] and smokers [n = 4]), and 7 patients with NSIP (5 male and 2 female; age range, 33–93 yr) were enrolled by the Section of Respiratory Diseases, Department of Clinical Sciences, of the University of Parma. NSIP was diagnosed on the basis of histologic (temporal homogeneity of fibrosis, variable inflammation, rare fibroblastic foci) and high-resolution computed tomography (CT) (bilateral lower lobe reticular abnormalities, variable traction bronchiectasis, rare honeycombing fibrosis) criteria (20). The lavage procedure was performed to exclude bronchial carcinoma in patients with unexplained dyspnea or cough or hemophtysis who were free of acute respiratory tract infections. After written informed consent and local anesthesia of the bronchial tree, fiberoptic bronchoscopy and bronchoalveolar lavage (BAL) were performed as previously described (21). Briefly, the bronchoscope was wedged into a segment of the right middle lobe and three 50-ml aliquots of sterile saline solution, warmed at 37°C, were instilled into the subsegmental bronchus. In patients with NSIP, the bronchoscope was wedged in the segment including the larger fibrotic zone. Fluid was gently aspirated immediately after each aliquot had been introduced and collected into a sterile container.

The fluid obtained was filtered through two layers of sterile surgical gauze and centrifuged (200 x g, 10 min). The whole BAL pellet was washed once in RPMI 1640, centrifuged at 200 x g for 10 min, and resuspended in RPMI 1640 supplemented with 10% endotoxin-free FBS, 2 mM glutamine, 10U/mL penicillin, and 10µg/ml streptomycin. Cells were plated in 12-well or 96-well dishes and allowed to adhere (37°C, 5% CO2) within 3 h of the lavage procedure. After 2 h, the medium was renewed to remove nonadherent cells and AM were used immediately or cultured in RPMI supplemented as above described for the times indicated in each figure. Cell viability exceeded 95%, as assessed by trypan blue exclusion. Of the 23 samples obtained, 4 BALs from normal subjects and 2 BALs from patients with NSIP were excluded for the presence of blood contamination or poor cellularity. The remaining 12 BALs (7 from males and 5 from females; 9 nonsmokers and 3 smokers) from normal subjects and 5 BALs from patients with NSIP (4 from males and 1 from a female) were used for the experiments.

L-Arginine Influx
For transport studies cells were seeded on 96-well dishes. AM, washed once with a modified bicarbonate-free EBSS buffered at pH 7.4 with 20 mM Tris/HCl, were incubated for 1 min in 50 µl of the same solution containing [3H]-arginine (4 µCi/ml, 100 µM). In this interval of time arginine uptake approached linearity (results not shown).

The components of L-arginine influx referable to the operations of systems y+L and y+ were estimated as follows (see Ref. 16 for discussion): system y+L was estimated as the leucine-inhibitable component, calculated from the difference between total influx and the influx measured in the presence of 2 mM leucine in the presence of sodium; system y+ was calculated from the difference between the influx measured in the presence of 2 mM leucine, and the influx measured in the presence of both 2 mM leucine and 2 mM lysine in the presence of sodium.

The experiment was terminated by two rapid washes (< 10 s) in cold PBS, and cell monolayers were extracted in 50 µl ethanol. The radioactivity in cell extracts was determined with Microbeta Trilux (Perkin Elmer, Monza, Italy). Extracted cells were then dissolved with 0.5% sodium deoxycholate in 1 M NaOH, and protein content was determined directly in the well using a modified Lowry procedure as previously described (22). In the experiments in which Na+-independent transport was to be measured, a modified Na+-free EBSS (NMG-EBSS) was used. In this solution NaCl and NaH2PO4 were replaced, respectively, by N-methyl-D-glucamine and choline salts. Amino acid influx was expressed as nmoles per mg of protein per min.

Reverse Transcription and Polymerase Chain Reaction
For the expression studies, AM were seeded onto 12-well trays. Total RNA was isolated with RNeasy Mini Kit (QIAGEN S.p.a., Milano, Italy). Reverse transcription was performed as described previously (16).

For PCR (32 cycles), 100 ng of single-strand cDNA from each sample were amplified with 10x HotMaster Taq Buffer (Eppendorf s.r.l., Milano, Italy) and 0.2 mM each dNTPs along with proband primers and 1.25 U of HotMaster Taq Polymerase (Eppendorf s.r.l.). Primer sequences for SLC7A1, SLC7A2, SLC7A6, SLC7A7, and GAPDH were reported previously (16). The primers for NOS2 were 5' TCT GTT CAA GAC CAA ATT CCA CC 3' (forward) and 5' CGG GGA CTC ATT CTG CTG C 3' (reverse). Images of the electrophoresed cDNAs were recorded with a DS Cooled Camera Head DS-5Mc (Nikon Instruments, Cusago, Italy). GAPDH primers were also added in the amplification mixture.

For qPCR, 30ng of cDNA from each sample were amplified in a total volume of 25 µl with 2x Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen s.r.l., San Giuliano Milanese, Milano, Italy), along with the following forward and reverse primers (5 pmol each) for the genes: 5' CTT CAT CAC CGG CTG GAA CT 3' and 5' GGG TCT GCC TAT CAG CTC GT 3' for SLC7A1; 5' TTC TCT CTGCGC CTT GTC AA 3' and 5' CCA TCC TCC GCC ATA GCA TA 3' for SLC7A2, CAT2B; 5' ACA GAC CTT CCA GGA GAA TG 3' and 5' GCA GTT CAG TGA TCG TAC AG 3' for IL1B; 5' TCT GTT CAA GAC CAA ATT CCA CC 3' and 5' CGG GGA CTC ATT CTG CTG C 3' for NOS2; 5' TAG ACC CTT TCT CCT CCA GGA GAC G 3' and 5' GCT GGG GGT CTC CCG GCA AAA GGT 3' for TGFB1; 5' AGC CTC AAG ATC ATC AGC AAT G 3' and 5' CAC GAT ACC AAA GTT GTC ATG GA 3' for GAPDH. These primers were designed according to the known sequences reported in GenBank with the help of Primer 3 program (23). The primers for SLC7A7 and SLC7A6 were purchased from Applied Biosystems (Monza, Italy) as TaqMan Gene Expression Assay (cat. N° Hs00374417_m1 and Hs00187757_m1). Quantitative PCR was performed in a 36-well Rotor Gene 3000 (Rotor-Gene 3000, version 5.0.60; Corbett Research, Mortlake, Australia). For all probands each cycle consisted of a denaturation step at 95°C for 15 s, followed by separate annealing (30 s, 57 °C) and extension (15 s, 72°C) steps. Fluorescence was monitored at the end of each extension step. A no-template control was included in each experiment. At the end of the amplification cycles a melting curve analysis was added. Data of gene expression were indexed to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the formula (24):

Formula 1(Eq. 1)
where {Delta}CT = CTGAPDH – CTproband gene.

Nitrite Production
Before uptake experiments and RNA extraction, culture medium was collected for the determination of nitrite production. Nitrite formation in the culture media of AMs was determined through a fluorimetric approach, based upon the production of the fluorescent molecule 1-(H)-naphtotriazole from 2,3-diaminonaphthalene (DAN) in acid environment, as described previously for endothelial cells (22). Fluorescence was determined with Victor2 1420 Multilabel Counter (Perkin Elmer).

Statistical Analysis
Transport data are expressed as mean ± SD. Significance was assessed with a two-tailed t test for unpaired data. In all cases, probability values were considered significant if they were < 0.05.

Materials
All reagents, including LPS (serotype 055:B55), were from Sigma (Milano, Italy) unless otherwise indicated. RPMI 1640 and endotoxin-free FBS (Euroclone) were purchased from Celbio (Pero, Italy). [L-2,3,4-3H]Arginine (45–70 Ci/mmol) was obtained from Perkin-Elmer. GM-CSF (RELIAtech GmbH) and IFN-{gamma} were purchased from Vinci-Biochem (Firenze, Italy) and 2,3-diaminonaphthalene (DAN, Molecular Probes) from Invitrogen (San Giuliano Milanese, Milano, Italy).


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Characterization of L-Arginine Transport in Human AM
The characterization of arginine transport was performed in adherent AM after 2 h of incubation in RPMI. In these freshly isolated, resting AM, arginine transport was 1.15 ± 0.099 nmol/mg of protein/min (mean ± SEM, n = 12; range, 0.64–2.00). No obvious correlation between arginine influx and sex or age was demonstrated. The transport values observed in AM from the three smoking subjects (1.06 ± 0.16, 1.20 ± 0.11, and 0.89 ± 0.06 nmol/mg of protein/min, means ± SD, n = 5) were close to the overall mean.

Since at least four distinct membrane transport systems can perform arginine transport in mammalian cells (15), a discrimination of the various components was performed according to the strategy already used in other cell models (16, 22, 25). The results of this analysis are shown in Figure 1A.


Figure 1
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  		Figure 1. Characterization of L-arginine influx in human AM. Freshly isolated AM, seeded for 2 h in complete RPMI, were employed. (A) Cells were washed in EBSS or Na+-free NMG-EBSS, as indicated. Arginine uptake was then assayed with 1-min incubations in the same solution employed for washing, supplemented with 100 µM L-[3H]arginine in the absence (open bars, total uptake) or in the presence of 2mM L-leucine, 2 mM L-leucine + 2 mM L-lysine, or 1 mM NEM, as indicated. Points are means of five independent determinations, with SD indicated in a representative experiment. The experiment was repeated in AM isolated from 12 different subjects with comparable results. (B) Dispersion plot of arginine transport in human AM. The plot shows, for each AM population isolated from different subjects, the values of saturable arginine uptake (calculated as the difference between the total uptake and the uptake obtained in the presence of 2 mM leucine and 2 mM lysine) and of the leucine-inhibitable component (calculated as the difference between the total uptake and the uptake obtained in the presence of 2 mM leucine). (C) RT-PCR analysis was performed as described in MATERIALS AND METHODS, and the amplification products, obtained with the primers of the indicated genes (see MATERIALS AND METHODS), are shown. Thirty-two cycles of amplification were employed for all transcripts. The experiment was repeated in cells isolated from 5 different subjects with comparable results.

 
In human AM, arginine transport was comparable in the absence or in the presence of sodium, thus excluding a significant contribution of sodium-dependent transport systems, such as B0,+. The addition of leucine, a substrate of system y+L, inhibited arginine influx by more than 70% only in the presence of sodium, indicating that most of the arginine uptake occurred through this route. On the contrary, lack of inhibition by leucine in the absence of sodium excluded a significant contribution of the broad specificity, sodium-independent system b0,+. In the presence of sodium, the addition of 2 mM lysine to leucine did not produce a further decrease of arginine transport, thus indicating that the contribution of system y+ was negligible under these conditions. Consistently, either in the presence or in the absence of sodium, NEM, an inhibitor of system y+ but not of system y+L (26), was ineffective in inhibiting arginine transport. The residual influx of arginine detected in the presence of 2 mM leucine and 2 mM lysine was referable to low-affinity transport pathways not distinguishable from diffusive routes. Thus, system y+L, identifiable as the leucine inhibitable portion of transport, accounted for most of the saturable transport of arginine in human AM, a finding consistently observed in all the isolates (Figure 1B).

Figure 1C shows the RT-PCR analysis of arginine transporters expressed in human AM 2 h after isolation. Among system y+-related genes, only the expression of SLC7A1, coding for the CAT1 transporter, was evident, whereas SLC7A2B transcript (CAT2B transporter) was barely detectable and no expression of SLC7A2A (CAT2A transporter) was detected. On the contrary, all the genes for y+L-related transporters were clearly detectable: SLC3A2, which codes for system y+L heavy chain CD98/4F2hc; SLC7A7, coding for the light chain y+LAT1; and SLC7A6, for the alternative light chain y+LAT2.

Effect of LPS and IFN-{gamma} on L-Arginine Transport
Figure 2A, shows the discriminated activity of the L-arginine transport routes, determined as above described, and the expression of genes encoding for system y+ and system y+L (Figure 2B) in human AM incubated for 24 h in the absence or in the presence of LPS, IFN-{gamma}, or both. As in freshly isolated cells, system y+L accounted for most of the arginine influx, even in the presence of either LPS or IFN-{gamma}. In parallel, the effect of the same compounds on the expression of the genes for transporters was assessed with quantitative real-time PCR. No significant change was detected under these conditions for any gene, although LPS-treated cells consistently exhibited an increase in abundance of SLC7A2/CAT2B transcript that, however, did not reach statistical significance.


Figure 2
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  		Figure 2. Effect of LPS and IFN-{gamma} on the activity and expression of arginine transporters in human AM. AM were incubated for 24 h in complete RPMI in the absence (control) or in the presence of LPS (1 µg/ml), IFN-{gamma} (20 ng/ml), or both, as indicated. (A) L-Arginine uptake was performed as described in the legend for Figure 1. System y+L (open bars): Leucine-inhibitable component of arginine influx, calculated as the difference between total influx and the influx measured in the presence of 2 mM leucine. System y+ (solid bars): the component of arginine influx calculated as the difference between the influx measured in the presence of 2 mM leucine and the influx measured in the presence of both 2 mM leucine and 2 mM lysine. Bars are means of five independent determinations with SD indicated. The experiment was repeated in cells isolated from four different subjects, with comparable results. (B) The qRT-PCR analysis of SLC7A1/CAT1, SLC7A2/CAT2B, SLC7A6/y+LAT2, and SLC7A7/y+LAT1 mRNA is shown. The values are normalized to that of GAPDH. Data are means ± SD of two separate experiments, each performed in duplicate.

 
IL-1beta is a cytokine, produced by activated AM, with important functional consequences on alveolar epithelial cells (27–29). In order to investigate the activation status of AM during the isolation and culture procedures adopted here, IL-1beta mRNA was quantified after 24 h of culture. Figure 3 shows that IL-1beta expression was markedly induced by LPS, thus demonstrating that at this time point AM maintained a good functional competence. IFN-{gamma} treatment was unable to induce IL-1beta expression.


Figure 3
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  		Figure 3. Effect of LPS and IFN-{gamma} on the expression of IL-1beta in human alveolar macrophages. AM were incubated in complete RPMI for 24 h in the absence or in the presence of LPS (1 µg/ml) or IFN-{gamma} (20 ng/ml) before RNA extraction. IL-1beta RNA expression was normalized to GAPDH. Data are means ± SD of four independent experiments, each performed in duplicate.

 
Effect of GM-CSF on Arginine Transport and Nitrite Production
GM-CSF promotes the survival and the differentiation of AM and modulates the biological functions of these cells (30–33).

To evaluate the effect of GM-CSF on the expression and function of arginine transporters in AM, cells were maintained in a complete growth medium in the absence or in the presence of 10 ng/ml GM-CSF for 6 d. LPS was added in a subset of cells during the last 24 h of incubation either in the presence or in the absence of the colony-stimulating factor.

Compared with arginine influx observed after 24 h of culture (see Figure 2), the activity of system y+L, obtained in AM cultured for 6d, was significantly higher (1.54 ± 0.154 versus 0.72 ± 0.085 nmol/mg of protein/min, n = 3, P < 0.05) and constituted, also at this time point, the predominant transport route for arginine (Figure 4A), while the contribution of system y+ was marginal. Neither the 6d-incubation in the presence of GM-CSF nor the 24-h treatment with LPS had any significant effect on either system y+L or y+ transport components.


Figure 4
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  		Figure 4. Effect of LPS on the activity and expression of arginine transporters in AM cultured in the presence and absence of GM-CSF. Cells were cultured for 6d in the absence or in the presence of GM-CSF (10 ng/ml) or, for the last 24 h, of LPS (1 µg/ml), as indicated. (A) The components of arginine uptake have been defined in the legend for Figure 2. Bars are means of five independent determinations with SD indicated. The experiment was repeated in cells isolated from four different subjects, with comparable results. (B) The results of qRT-PCR analysis of the indicated genes have been normalized to GAPDH. Data are means ± SD of three independent experiments, each performed in duplicate. *P < 0.05 versus control, untreated cells.

 
The evaluation of transporter gene expression (Figure 4B) indicated that, while SLC7A1 expression was not modified, a significant induction of SLC7A2/CAT2B expression was observed in cells incubated with LPS. In GM-CSF–primed cells LPS produced a more evident induction of the transporter gene, while the colony stimulating factor alone did not produce a significant increase of SLC7A2B transcript. No significant change in the expression of either SLC7A6 or SLC7A7 genes, encoding for the system y+L transporters y+LAT2 and y+LAT1, was observed in cells treated with GM-CSF, LPS, or both.

Under the same conditions, treatment with LPS, but not with GM-CSF, produced a huge induction of IL-1beta mRNA, which was nearly 50-fold more abundant in AM incubated with the endotoxin compared with untreated cells (Figure 5A). On the contrary, nitrite accumulation in the culture medium, an indicator of NO output of AM, was comparably low under all the conditions tested (Figure 5B). Consistently, AM expressed barely detectable levels of NOS2 mRNA (Figures 5C and and 5D).


Figure 5
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  		Figure 5. Nitrite production and expression of NOS2 and IL-1beta in AM cultured in the presence and absence of GM-CSF: effect of LPS. Cells were cultured for 6 d in the absence or in the presence of GM-CSF (10 ng/ml) and, for the last 24 h, of LPS (1 µg/ml), as indicated. (A) IL-1beta mRNA expression was analyzed by qRT-PCR (see MATERIALS AND METHODS) normalized to GAPDH expression. Data are means ± SD of three independent experiments, each performed in duplicate. (B) After the indicated treatments, nitrite concentration in the incubation medium was determined as described in MATERIALS AND METHODS. The values are mean of five determinations with SD indicated. The experiment was repeated in AM isolated from three different patients, with comparable results. (C) NOS2 mRNA expression was analyzed by qRT-PCR (see MATERIALS AND METHODS) and normalized to GAPDH expression. Data are means ± SD of two independent experiments, each performed in duplicate. A549 cells, pretreated for 24 h with IFN-{gamma} (20 ng/ml), TNF-{alpha} (10 ng/ml), and IL-1beta (10 ng/ml), were used as a positive control for NOS2 expression. (D) RT-PCR analysis was performed as described in MATERIALS AND METHODS and the amplification products, obtained with the primers of the indicated genes (see MATERIALS AND METHODS), are shown. Thirty cycles of amplification were employed for all transcripts. The experiment was repeated in cells isolated from three different subjects with comparable results. A549 cells were used as a positive control.

 
Arginine Transport and Expression of Genes for Arginine Transporters in AM from Patients with NSIP
To verify if our observations on arginine transport in AM from normal subjects were confirmed in cells derived from patients with interstitial lung disease, we measured the influx of the cationic amino acid in AM freshly isolated from patients with NSIP. In these cells arginine transport was not significantly different from the values obtained in normal AM (mean 1.19 ± 0.093 nmol/mg of protein/min, n = 5; range, 0.90–1.41). Also, in AM from patients with NSIP arginine transport was markedly inhibited by 2 mM leucine, and lysine did not produce further inhibition, indicating that system y+L was predominant (Figure 6A). In these cells the expression of NOS2 was very low and insensitive to a 24-h treatment with LPS (Figure 6B). Consistently, LPS treatment did not cause an increase in nitrite accumulation in the culture medium (not shown).


Figure 6
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  		Figure 6. Arginine transport, NOS2 expression in AM obtained by patients with NSIP. (A) Arginine uptake was assessed in freshly-isolated AM from patients with NSIP, seeded for 2 h in complete RPMI, as described for AM from normal subjects in the legend for Figure 1. Points are means of values obtained from five independent determinations for each of five distinct AM isolations, with SEM indicated. (B) Cells were cultured for 24 h in complete medium in the absence or in the presence of LPS (1 µg/ml). NOS2 mRNA expression was analyzed by qRT-PCR (see MATERIALS AND METHODS) and normalized to GAPDH expression. Data are means ± SD of two independent experiments, each performed in duplicate. A549 cells, treated as described in the legend for Figure 5, were used as a positive control.

 
The relative expression of genes for arginine transporters was compared in five AM preparations from patients with NSIP and in three AM preparations from normal subjects. The results (Table 1) indicate that AM from patients with NSIP expressed significantly less SLC7A7 mRNA, while they had higher levels of SLC7A1 and SLC7A6 mRNA. The levels of SLC7A2/CAT2B mRNA were comparable in AM from normal subjects and patients with fibrosis. When compared with human umbilical vein endothelial cells, a cell type in which arginine transport has been thoroughly characterized and both systems y+ and y+L are functional (22), AM from normal subjects and from patients with NSIP had a much lower expression of SLC7A2/CAT2B and a higher expression of SLC7A7/y+LAT1. Interestingly, NSIP AM exhibited higher TGF-beta1 expression than AM from normal subjects, with an expression, normalized to GAPDH mRNA according to Eq.1, of 516 ± 51 (n = 5) versus 334 ± 31 (n = 3, P < 0.05).


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  		TABLE 1. RELATIVE EXPRESSION OF GENES FOR ARGININE TRANSPORTERS IN AM AND HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
This report represents the first demonstration that human AM from normal subjects do not take up arginine through system y+, the transport activity due to the operation of CAT transporters and related to NOS activity. In these cells, instead, most of arginine transport (> 90% of the saturable uptake) occurs through system y+L, a NOS-unrelated transport mechanism. Lack of system y+ operation is observed in freshly isolated AM, as well as in cells cultured for up to 6d, and is maintained even if AM have been incubated with GM-CSF, LPS, or IFN-{gamma}, stimuli that are able to induce this transport activity in macrophages from animal models (7, 34, 35). These results suggest that monocytic/macrophagic cells from humans and animals present obvious differences as far as the regulation of arginine transport is concerned.

However, human AM express mRNA for CAT1 transporter under all the experimental conditions adopted and, moreover, cells cultured for 6d and treated with LPS markedly overexpress the mRNA for the inducible transporter SLC7A2/CAT2B. The discrepancy between the expression of CAT transporters at mRNA level and the lack of a detectable transport activity of system y+ may be explained either by low levels of absolute expression or by the absence of carrier protein production or membrane targeting. The first hypothesis is consistent with the exceedingly low levels of relative expression of SLC7A2/CAT2B detected in AM compared with human endothelial cells, as shown in Table 1. In contrast, it seems unlikely for SLC7A1/CAT1, since the expression of this transporter, relative to that of GAPDH, is comparable in AM and in endothelial cells, where the operation of CAT transporters is readily detected (22). As for the second possibility, literature evidence, obtained in nonmacrophagic models, indicates that the trafficking of CAT transporters to the plasma membrane is regulated by PKC activity and suppressed when PKC is activated (36, 37). Therefore, it is possible that in human AM from normal subjects CAT1 protein is expressed but remains in the intracellular compartment. However, a formal validation of this hypothesis requires large numbers of AM, exceeding those obtainable from BAL or, at least, a reliable anti-human CAT1 antibody that is, at present, unavailable. In any case, lack of transport activity of CAT transporters should not be referred to cell dedifferentiation during the culture procedure, since AM maintain the competence for IL-1beta production in response to LPS treatment up to 6d of culture.

Nitrite determination, performed under conditions of maximal stimulation of system y+–related genes, indicated that human AM have a very low, LPS- and GM-CSF–insensitive NO production and, consistently, do not express NOS2 at appreciable levels. These data are in agreement with the observations of other groups (38, 39), which indicate that arginine metabolism and NO production differ markedly between animal and human macrophagic cells. For instance, at variance with what observed in murine monocytic cells, IFN-{gamma} failed to enhance growth inhibition of mycobacteria by human AM, the NOS inhibitor NMMA had no effect on macrophagic functions, and neither NOS2 mRNA nor NO could be detected in human AM stimulated with LPS and IFN-{gamma} or even mycobacteria (40). The divergent behavior of rodent and human AM is not caused by limited availability of extracellular L-arginine, since the supplementation of the culture medium with the cationic amino acid does not trigger NO production in human AM (41). However, from studies in rodent cells, it is known that, at variance with other cells (42), macrophages markedly depend upon the transport of extracellular arginine to the intracellular compartment through the inducible transporter SLC7A2/CAT2B for NOS2-dependent NO synthesis (8). Moreover, the expression of NOS2 and CAT2B appear strictly linked. In rat alveolar macrophages, for example, NOS2 and CAT2B are simultaneously induced in an NF-{kappa}B–dependent manner by LPS and IFN-{gamma} (11) and are inhibited by polyamines (13). Those data suggest that arginine flux through CAT2B is functionally linked to NOS2 activity. If this is the case, the absence of a significant activity of CAT transporters would lead to an insufficient fuelling of NOS2 even in the presence of fairly high concentrations of extracellular arginine.

In pulmonary fibrosis, as well as in other interstitial lung diseases, AM are markedly activated (43–48), and some evidence exists that their NO production is increased (49). We present here results obtained with AM derived from patients with NSIP, a pulmonary fibrosis. The expression of arginine transporters in these cells is significantly modified with respect to AM from normal subjects, with SLC7A1/CAT1 expression doubled and SLC7A7/y+LAT1 lowered in AM from patients with NSIP (Table 1). It is possible that these changes in gene expression are the results of the activated phenotype of these cells, demonstrated by the higher expression of TGF-beta1, a cytokine produced by AM activated via the alternative pathway (50) as observed in pulmonary fibrosis (51). Nevertheless, arginine transport is still accounted for by the activity of system y+L in AM from patients with NSIP (Figure 6A). These cells have low, LPS-insensitive NO output and NOS2 expression, indicating that the fibrotic environment is not sufficient to trigger NOS2-dependent NO synthesis in AM. NOS2 expression and evidence for NO production was reported in AM from patients with idiopathic pulmonary fibrosis (49) but these cells were infected with BCG in vitro, suggesting that additional stimuli are needed for the expression of NO-synthetic machinery in AM from patients with fibrosis.

It should be stressed that the results presented here may not be necessarily extendible to cells obtained from subjects affected by other pathological conditions. Indeed, an effective NOS2 expression by AM has been reported in systemic sclerosis (52) or acute respiratory distress syndrome (53). In this last situation, although NO production by AM has not been directly demonstrated, high levels of nitrites/nitrates are detected in bronchoalveolar lavage and immunocytochemical positivity for protein nitrosylation has been observed in AM (53). It will be, therefore, interesting to assess if, in these situations, system y+ transport activity is expressed by human AM and, in this case, to define what kind of signals trigger its functional expression.

In the absence of a functional system y+, system y+L represents the major route for arginine transport in human AM. The findings obtained by other groups (54, 55) and by our laboratory (16) in blood monocytes are thus extended to a type of human macrophages differentiated in vivo. Human AM exhibit a clear-cut expression of the y+L-related genes SLC7A7 and SLC7A6 that code for the two alternative light chains of the transporter, y+LAT1 and y+LAT2. In particular, SLC7A7 relative expression is higher in AM than in endothelial cells, suggesting that the expression of this transporter is a marker of cells of monocytic lineage. However, at variance with blood monocytes (16), human AM exhibit no stimulation of SLC7A7 expression of system y+L activity upon treatment with IFN-{gamma}. Given that system y+L, at least in epithelial models, mediates arginine efflux in exchange with extracellular neutral amino acids and sodium (25, 56), the prevalent arginine flux across AM membrane appears to be outwardly directed. Mutations of SLC7A7/y+LAT1 are responsible for Lysinuric Protein Intolerance (LPI) (17, 18) and are associated with a severe, GM-CSF–insensitive form of pulmonary alveolar proteinosis (PAP) with apparent alterations in AM (19). It is possible, therefore, that the absence of y+LAT1 expression and/or activity interferes with AM-dependent surfactant catabolism, thus mimicking the defects in GM-CSF signaling pathway that underlie most cases of PAP (57). The definition of the functional role of system y+L in human AM may, therefore, contribute to the definition of LPI and PAP pathogenesis.


   Footnotes
 
This work was supported by MIUR, PRIN "Lysinuric protein intolerance: generation and analysis of a conditional mouse model and of human cellular models."

Originally Published in Press as DOI: 10.1165/rcmb.2006-0262OC on March 15, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form July 18, 2006

Accepted in final form January 17, 2007


   References
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 Abstract
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 MATERIALS AND METHODS
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 References
 

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