Arginase 2 is expressed by human lung cancer, but it neither induces immune suppression, nor affects disease progression

Abstract

In human prostate cancer, Arginase 2 (ARG2) and nitric oxide synthase (NOS) are concomitantly expressed by tumor cells, and induce tumor immune escape via peroxynitrite-dependent Tyrosine nitrosylation. Since there were no data regarding this immune suppressive mechanism in other tumor types, and an evaluation of its clinical relevance in human tumors had still to be provided, we have investigated presence and clinical relevance of ARG2 and NOS expression in lung cancer. No evidence of NOS expression was found, no significant NOS enzymatic activity was detected. Instead, ARG2 protein was expressed by tumor cells. In a cohort of 120 patients, the amount of ARG2-positive tumor cells was significantly higher in small cell lung cancers (SCLC) than in non-small cell lung cancers (NSCLC). Large cell undifferentiated carcinomas had twice ARG2 than the other NSCLC subtypes. ARG2 expression was increased in Grade 3 tumors, as compared to Grades 1 and 2. However, no relationship was found with tumor size and stage, and with patient survival. Indeed, the enzyme was active, since the Arginine catabolite Ornithine was produced, but Arginine depletion was not attained. In addition, nitrotyrosine was not found in tumor tissue. Accordingly, when tumor cells isolated from lung cancer were incubated with activated autologous T cells, no inhibition of proliferation was detected. Our results indicate that ARG2 is expressed in lung cancer, but it does not induce tumor immune escape and does not affect disease progression, most probably due to the lack of concomitant NOS expression. © 2008 Wiley-Liss, Inc.

The regulation of amino acid metabolism plays a fundamental role in modulating immune response. In particular, adequate levels of Arginine in the extracellular milieu are crucial to T cell activation and proliferation.1

Arginine levels are regulated by 2 enzymes, degrading the amino acid through different pathways: arginase (ARG) hydrolyzes arginine to ornithine and urea, whilst nitric oxide synthase (NOS) oxidizes arginine to citrulline and nitric oxide (NO). Analysis of ARG and NOS expression is quite complex, since both enzymes have isoforms, that are differently expressed in human normal tissues. NOS can be expressed in 3 isoforms, eNOS, nNOS and iNOS. eNOS and nNOS are constitutively expressed, mainly by endothelial cells and neurons, respectively, whilst iNOS expression can be induced in macrophages, epithelial and endothelial cells. ARG exists in 2 isoforms, ARG1 and ARG2. In humans, ARG1 is expressed mainly in liver, but is also found in granules of neutrophil granulocytes.2, 3 Contrary to mice, that can express ARG1 in macrophages and in myeloid-derived suppressor cells, human monocytes and macrophages neither express ARG1 nor ARG2.3 In humans, ARG2 is expressed mainly in kidney cells, within mitochondria, where it is involved in the biosynthesis of polyamines.

It has been proposed that ARG activity, by itself, is able to induce immune suppression. The enzyme, by depleting the extracellular milieu of Arginine, modulates the expression of CD3ζ chain in T lymphocytes, therefore inducing inhibition of T cell proliferation.4 Instead, the role of NO in T cells remains controversial, and whether or not it regulates lymphocyte activation is unclear. Actually, NO produced in T lymphocytes by endogenous eNOS potentiates T cell receptor signaling.5 However, it has to be noted that when ARG and NOS are concomitantly expressed, apoptosis of activated T lymphocytes could ensue. In fact, in an arginine-depleted microenvironment, such as in Arginine-free medium, NOS generates superoxide ion, that reacts with NO to form peroxynitrite.6 Peroxynitrite, through nitration of tyrosine residues, is able to inhibit activation-induced protein tyrosine phosphorylation in T lymphocytes and prime them to undergo apoptosis.7 It has been proposed that, under conditions of arginine depletion resulting from ARG activity, peroxynitrite produced by NOS could cause T cell apoptosis.8 The strong immune suppressive activity exerted by the concomitant activity of ARG and NOS suggests to evaluate the role of these enzymes in tumor-associated immune suppression.

So far, the presence of arginine-catabolizing enzymes in human solid tumors and their role in inducing tumor immune escape remain to be fully elucidated. Initial investigations have provided evidences for the presence of either ARG or NOS in tumor tissues; however they have also supplied conflicting results on whether tumors express more or less enzyme than the corresponding normal tissue.9–12 A more recent paper indicates that in human prostate cancer ARG2 and iNOS are coexpressed in tumor cells, and both enzymes participate in local peroxynitrite-dependent immune suppression.13 However, no data have been provided so far on the existence of such immune suppressive mechanism in human tumors other than prostate carcinoma. Furthermore, the clinical significance of ARG2 and iNOS in human tumors has not been evaluated yet. The finding that this mechanism facilitates tumor progression may have relevant implications for tumor immunotherapy.

Aim of this study was to investigate incidence and clinical relevance of expression of ARG2 and NOS in lung cancers. In these tumors an effective immune escape takes place, and tumor-infiltrating lymphocytes (TIL) are quiescent, nonproliferating, anergic cells,14–17 but the underlying immune suppressive mechanism has not been identified.

ARG, arginase; NO, nitric oxide; NOS, nitric oxide synthase; NSCLC, non-small cell lung cancer; SCLC, small cell lung cancer; TIL, tumor-infiltrating lymphocytes.

Material and methods

Patients

Specimens for ARG2 immunohistochemistry were obtained from the patients with lung carcinoma undergoing surgery between September 1984 and June 2004 at Policlinico S. Matteo, University of Pavia, Italy (79 cases), Policlinico S. Martino, University of Genoa, Italy (19 cases), and Ospedale S.Croce e Carle, Cuneo, Italy (22 cases). None of them had received therapy before surgery. A summary of clinical data from these patients is provided in Table I. Follow-up was interrupted on March 2006.

Table I. Clinical Data of Patients

Parameter	n (%)
Median age, years: 61 (35–82)
Sex
M	102 (85)
F	18 (15)
Histologic type
Neuroendocrine tumor	9 (7.5)
Small cell carcinoma	9 (7.5)
Non small cell carcinoma	111 (92.5)
Adenocarcinoma	68 (56.7)
Squamous cell carcinoma	32 (26.7)
Bronco-alveolar carcinoma	5 (4.2)
Large cell undifferentiated carcinoma	6 (5)
Stage
IA	32 (26.7)
IB	25 (20.8)
IIA	9 (7.5)
IIB	23 (19.2)
IIIA	27 (22.5)
IIIB	–
IV	4 (3.3)
Grading
1	19 (15.8)
2	60 (50)
3	41 (34.2)
T
1	43 (35.8)
2	61 (50.8)
3	16 (13.3)
N
0	58 (48.3)
1	33 (27.5)
2	29 (24.2)
M
0	79 (65.8)
1	4 (3.3)
X	37 (30.8)
Median survival, months: 48 (3–215)
Overall survival at March 2006
Alive (months 18–215)	19 (15.8)
Dead (months 3–194)	111 (92.5)
Survival at 5 years	35 (29.2)

Cell separations were performed on peripheral blood and on surgical sample obtained, upon informed consent, from 11 randomly chosen patients with non-small cell lung cancer (NSCLC), undergoing surgery at Ospedale Santa Croce e Carle, Cuneo, Italy.

The study was performed in the frame of the Protocol “Monocentric Phase II clinical trial of multimodal treatment by adoptive immunotherapy with LAK and TIL cells, and IL-2, in patients with locally advanced non small cell lung cancer, Stage IIIa/IIIb,” approved by the Regione Piemonte Ethical Committee.

Cell separations

Peripheral blood mononuclear cells were isolated by centrifugation on Fycoll-Hypaque (Biochrom KG, Berlin, Germany). PBL were collected after removal of adherent cells, and cultured in culture medium (RPMI 1640 supplemented with 10% FCS and 2 mM L-Glutamine, all from Biochrom KG).

At the time of surgery 2 specimens were taken, one of neoplastic tissue, and one of normal lung tissue, as described elsewhere.18 On both samples a confirmatory histological analysis was retrospectively performed. Part of each sample was frozen for Western blot and for RT-PCR analyses, and part was processed for cell separation, as previously described.19 Briefly, tissues were dissociated by sterile mechanical dissection and enzymatic digestion. Afterward, cell suspension was filtered and layered on Ficoll-Hypaque. CD14+ cells were then enriched by MACS technology (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), following manufacturer's instructions. Briefly, cells were incubated with 50 μg human immunoglobulins to block Fc Receptors, then MACS CD14 Micro Beads were added. Afterward, cells were washed, and applied to an LS Separation Column, attached to a Midi MACS Separation Unit. Purity was checked by flow cytometry analysis using an anti CD11c MoAb.

Western blot analysis

Frozen tumor tissue, normal lung tissue, and frozen cell pellets of purified CD14+ or CD14− cell fractions were homogenized with a potter in lysis buffer (10 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.1% SDS, 1 mM PMSF, 10 μg/ml Leupeptin, and 18 μg/ml Aprotinin, all from Sigma-Aldrich, St. Louis, MO) on ice. Cell lysates were then quantified using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). A standard Western blot analysis was then performed. Briefly, 50–100 μg of each sample was run on 8% polyacrilamide gel. In the case of eNOS and ARG2 analysis, gel was blotted onto a Hybond-C membrane (Amersham Biosciences, Pittsburg, PA); in the case of iNOS, gel was blotted onto an Immobilon P membrane (Millipore, Billerica, MA). Membranes were saturated and then incubated for 2 hr at room temperature with the following primary antibodies: rabbit anti-Arginase II antibody diluted 1:200 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-eNOS antibody diluted 1:2,500 (BD Biosciences), mouse anti-iNOS antibody diluted 1:5,000 (R&D Systems, Minneapolis, MN), mouse anti-β actin antibody diluted 1:5,000 (Sigma-Aldrich). After washing, the blot was stained with a 1:1,500 dilution of peroxidase-conjugated anti-rabbit or anti-mouse Ig antiserum (DAKO, Glostrup, Denmark). Bands were visualized by ECL system (Amersham Biosciences).

Histology and immunohistochemistry

Histology was performed on Hematoxilin/Eosin stained slides, and on Alcian Blue/Shiff's periodic acid pH 2.5 stained slides, when better characterization of histological subtype was needed. Tumors were classified according to WHO in NSCLC and neuroendocrine lung cancers.20 In our cohort the latter tumors were all small cell lung cancers (SCLC). For lung cancers the following histological parameters were evaluated: differentiation grade (well, moderate, poor differentiation); TNM and stage21; mitotic rate, expressed as number of mitosis detected in 10 high power field (40×); peri- and intra-tumor inflammation, scored as 0 = scattered lymphocytes and plasma cells, 1 = mild, 2 = moderate, 3 = marked inflammation, with nodular aggregates of inflammatory cells; intra-tumoral lymphocyte, granulocyte and macrophage infiltration, scored as 0 = absent, 1 = mild, 2 = moderate, 3 = marked; tumoral necrosis, scored as 0 = absent, 1 = focal necrosis limited to few cells, 2 = necrosis of large groups of cells, 3 = necrosis involving 30% or more of tumor mass; vascular invasion, scored as 0 = absent, 1 = presence of neoplastic emboli in lymphatic or venous vessels. Besides 120 primary lung tumors, 40 metastatic, tumor-draining, lymph nodes from 15 cases of NSCLC were also analyzed for ARG2 expression.

Routine immunohistochemical staining for ARG2 was performed using an automated stainer (BenchMark XT, Ventana Medical Systems, Tucson, AR). Briefly, after thermal pretreatment for antigen retrieval, the anti-ARG2 antiserum at 1:100 dilution was added, and staining was performed using avidin-biotin-peroxidase complex technique and 3-3′ diaminobenzidine (DAB) chromogen.

For negative control, the same tissue was stained by replacing the primary antibody with preimmune rabbit serum. Sections of prostate adenocarcinoma, positive for ARG2, were used as positive control. Percentage of ARG2-positive tumor cells, intensity (weak vs. strong) of staining, and distribution (focal vs. diffuse) of staining were recorded.

The rest of immunohistochemistry was performed manually.

To perform double staining for ARG2 and CD68, slides were hydrated, and antigen retrieval was performed by heating slides in citrate buffer, pH 6.0, in a microwave. After incubation for 15 min with the peroxidase block from EnVision Kit (Dako, Glostrup, DK), samples were washed and nonspecific binding was prevented by incubating samples for 30 min in PBS containing 0.05% Tween 20, 5% bovine serum albumin (BSA, Sigma-Aldrich) and 0.5 mg/ml human IgG (Ig Vena N, Sclavo, Siena, Italy). Afterward, the anti-ARG2 rabbit antiserum at 1:100 dilution in PBS plus 0.05% Tween 20 and 1% BSA was added. After incubation for 1 hr at 37°C, slides were washed and incubated for 30 min at room temperature with the anti-rabbit, HRP-conjugated, secondary antibody from EnVision Kit (Dako). Slides were washed, and then stained with the AEC chromogen and the AEC substrate of the Ultravision Detection System (Lab Vision, Fremont, CA). After washing, slides were incubated for 40 min at 37°C with an anti human CD68 mouse monoclonal (Dako) 1:100. Slides were washed and incubated for 30 min at room temperature with a biotin-conjugated goat anti mouse IgG (BioSpa, Milan, Italy) 1:600. After washing slides were washed and incubated for 10 min at room temperature with Alkaline Phosphatase-conjugated Streptavidin (BioSpa) 1:300. After washing, staining was performed with Fast Blue plus 2.5 mg/ml Levamisole (Sigma). A mild counterstaining with hematoxylin was performed.

To detect the different isoforms of NOS, samples from 20 randomly chosen patients were hydrated; afterward, antigen retrieval, peroxidase blocking and nonspecific binding blocking were performed as indicated above. The following primary antibodies were used: rabbit anti iNOS (Chemicon-Millipore, Billerica, MA) 1:5,000, mouse anti eNOS (BD Biosciences, San Jose, CA) 1:500, mouse anti nNOS (BD Biosciences) 1:100. Negative controls were run in parallel with pre-immune rabbit serum and purified mouse Ig, respectively. After incubation for 1 hr at 37°C, slides were washed and incubated for 30 min with either 1:300 biotin-conjugated goat anti rabbit (BioSpa), or with 1:500 biotin-conjugated goat anti mouse (BioSpa). After washing, 1:300 HRP-conjugated streptavidin (BioSpa) was added, and slides were stained with AEC System.

To assess the specificity of anti-iNOS staining, immunohistochemistry was also performed in the presence of the blocking peptide. Briefly, the C-terminal 19 amino acid peptide used as immunogen was synthesized (Tib Molbiol, Berlin, Germany). The peptide, at a concentration of 0.2 μg/ml was incubated for 30 min at room temperature with the rabbit anti iNOS antiserum in PBS containing 0.05% Tween 20 and 1% BSA. Afterward, the mix was added to slides of either lung cancer or HCV-infected liver, used as positive control, and staining was performed as described above.

For nitrotyrosine staining, an anti-nitrotyrosine rabbit policlonal antibody (Upstate, Lake Placid, NY) was used, at 1:100 dilution. Negative controls were run in parallel with pre-immune rabbit serum. After 1 hr at 37°C, slides were washed and incubated for 30 min with 1:300 biotin-conjugated goat anti rabbit (BioSpa). After washing, 1:300 HRP-conjugated streptavidin (BioSpa) was added and slides were stained with AEC System. Slides from prostate carcinoma were used as positive control.

Counterstaining was performed with hematoxylin.

Evaluation of histology and immunohistochemistry was performed by the same pathologist (L.M.).

Confocal microscopy

NIH-H82, a SCLC cell line, and cell suspensions from 2 NSCLC were used to investigate intracellular location of the enzyme. ARG2 expression was assessed by Western blot. Cells were incubated for 45 min in culture medium plus 100 nM MitoTracker Deep Red 633 FM (Invitrogen, Carlsbad, CA), washed, fixed in precooled methanol at room temperature for 10 min, washed again, and cytocentrifuged on glass slides. After blocking for 30 min with 10% normal mouse serum, samples were incubated for 1 hr with 1:20 goat anti human ARG2 (Santa Cruz), washed, and incubated for 30 min with 1:50 FITC-conjugated swine anti goat IgG (Invitrogen). After mounting, slides were examined using a laser-scanning FV500 microscope equipped with 488, 543 and 633 nm lasers, and coupled to an inverted IX81 platform (all from Olympus Optical, Tokio, Japan). Digital images were acquired with Fluoview 4.3b software program.

T cell proliferation

T cell proliferation studies were performed on phytohemagglutinin (PHA)-activated PBL by standard ³H-thymidine incorporation technique. Briefly, 1 × 10⁶/ml PBL were incubated for 1 hr with PHA M form (Life Technologies, Grand Island, NY) at a final concentration of 10 μl/ml. Afterward, cells were washed twice, resuspended in culture medium, and seeded in round bottom 96-well plates at 5 × 10⁴ cells/well. For experiments of coincubation with intact cells, autologous CD14− cells from tumor tissue were added, at CD14− to PBL ratios up to 12 to 1. The day after, 10 U/ml IL-2 (Chiron BV, Amsterdam, The Netherlands) were added. Forty-eight hours after PHA stimulation an aliquot of supernatant was collected for mass spectrometry evaluation of arginine and ornithine, and 1 μCi ³H-thymidine (Amersham Biosciences), together with IL-2, were added. For experiments of coincubation with cell lysate, CD14− cells from tumor underwent 7 rounds of freezing and thawing. Cell lysate was then incubated with activated PBL, either in the presence or in the absence of 50 μM Nor-NOHA (Calbiochem, Merck KGaA, Darmstadt, Germany) and tests were performed as indicated above. Thymidine incorporation was measured in a β-Counter after further 24 hr. Tests were performed in triplicate.

Evaluation of ARG2 activity

Arginase activity was evaluated by tandem mass spectrometry, measuring the decrease in arginine, and the increase in ornithine. Supernatants of T cell proliferation tests were collected after 48 hr of incubation. To precipitate proteins, 150 μl of ice-cold 5% sulfosalicylic acid in water were added, incubated for 30 min at 4°C, and spun at 18,000g for 10 min. Supernatants were collected, and analysis was performed by the LC-ESI-MS/MS method. Thirty microlitre of each sample were spotted on Grade 903 Schleicher & Schuell filter paper (Whatman, Brentford, UK). From the spotted samples, 3-mm disks were excised, and extracted with a solution of CH₃OH/H₂O containing stable-isotope-labeled internal standards: ¹³C-arginine and d₂-ornitine (Neogram Amino Acid and Acylcarnitine Tandem Mass Spectrometry Kit, PerkinElmer). After butylation, samples were reconstituted with mobile phase and injected by flow injection analysis mode in an API 2000 ESI-Tandem Mass Spectrometer coupled with a 200 Autosampler and a Pump 200 (PerkinElmer, Wellesley, MA). Analytes were monitored in positive ion mode using the turbon ion sprayer interface. MS/MS analysis was calibrated by infusion of a tuning solution containing deuterated internal standards. Arginine and ornitine were analyzed scanning for multiple reaction monitoring, with their specific transition Q1→Q3: Arg 231→70; Orn189→70 amu. Analytes and their internal standards were identified by sorting their respective mass-to-charge ratio.

Isolation of RNA and RT-PCR

Expression of iNOS, eNOS, and nNOS was evaluated by RT-PCR on CD14+ cells and on CD14− cells isolated from tumor tissue and from normal lung tissue. Total RNA was isolated using the RNAeasy mini Kit (Quiagen, Valencia, CA) and digested with DNAse following manufacturer instructions. After spectrophotometric quantification, 1.2 μg of total RNA were reverse transcribed in a 20-μl reaction mix with oligo-dT primers and Moloney murine leukemia virus RNAse H- reverse transcriptase (Promega, Madison, WI). PCR was performed on 5 μl of cDNA in 20-μl total reaction volume with primers and experimental conditions indicated in Table II. Amplification of βActin was run as control for cDNA quality and quantity. PCR products were electrophoresed on 1.5% agarose gels and stained with ethidium bromide. To ensure the identity of the PCR-amplified fragments, the size of each amplified mRNA fragment was compared with DNA standards (Invitrogen).

Table II. Primers and Experimental Conditions for RT-PCR

Gene	Primers (5′→3′)	Annealing	Cycles	Base pairs	Reference
S, sense; AS, antisense.
iNOS	primers 1: S: TCCgAggCAAACAgCACATTC	65°C	40	462	22
	AS: gggTTgggggTgTggTgATgT
	primers 2: S: gCAAtgAATggggAAAAAgA	61°C	40	265	–
	AS: TggCCTTATggTgAAgTgTg
eNOS	S: CCAgCTAgCCAAAgTCACCAT	55°C	40	350	23
	AS: gTCTCggAgCCATACAggATT
nNOS	S: TTgggggCCTgggATTTCTgg	55°C	40	456	24
	AS: CgTTggCATgggggAgTgAgC
βactin	S: ggCATCgTgATggACTCCg	60°C	40	600	25
	AS: gCTggAAggTggACAgCgA

Evaluation of nitrite production

Supernatants of CD14+ cells isolated from tumor tissue were collected after 48 hr, and evaluation of nitrite production was performed using the Nitrate/Nitrite Fluorometric Assay Kit (Cayman Chemical, Ann Arbor, MI), following manufacturer's instructions. Standard curve was prepared by diluting standards in culture medium, to reach the same dilution of our samples. Afterward, manufacturer's instructions were followed exactly. No Nitrate Reductase was added, and measurement of nitrite content was performed by evaluating conversion of 2,3-diaminonaphtalene to 1(H)-naphtotriazole with a fluorimeter.

Statistical analysis

Between groups, comparisons were carried out using the non parametric Mann-Whitney or the Kruskal-Wallis test for 2 or more than 2 groups, respectively. Survival analysis was performed using the proportional hazard Cox model.

Results

Analysis of ARG2 expression in primary lung cancer

ARG2 expression was initially investigated by Western blot analysis. Different amounts of ARG2 were found among tumor tissues, whilst no enzyme could be detected in the corresponding normal lung tissues (Fig. 1a). ARG2 expression was then evaluated, by immunohistochemistry, in 120 cases of lung cancer. The ARG2-specific antibody stained tumor cells, and the enzyme expression greatly varied among the samples, ranging from utterly negative to intense and diffuse (Fig. 1b). Cells in the tumor-associated stroma were not stained; in particular, double immunohistochemistry for CD68 and ARG2 showed CD68-positive cells within stroma, but no colocalization with ARG2 was found in any of the 7 cases examined (Fig. 1c). The percentage of ARG2 positive tumor cells per each of the 120 patients is indicated in Figure 1d. In detail, 21 samples did not express ARG2, in the remaining cases the percentage of ARG2-positive tumor cells ranged from 1 to 90%.

Figure 1. Analysis of ARG2 expression in lung cancers. (a) Western blot analysis of ARG2 expression from tumor tissue of 5 NSCLC patients and from normal lung tissue from the first 2 patients. Normal kidney is shown, as positive control. (b) Immunohistochemistry for ARG2 on formalin-fixed, paraffin-embedded samples of NSCLC. A positive sample, with diffuse staining of tumor cells, is shown on the upper panel and a negative one is shown on the lower panel. (c) Double immunohistochemistry for ARG2 and CD68. The Arg2-expressing cells, stained in red, were all in tumor parenchyma, whilst CD68-positive cells, stained in blue, were scattered within stromal septa. (d) Hundred and twenty patients were analyzed for ARG2 expression by immunohistochemistry. The percentage of ARG2-positive tumor cells is shown per each of them. (e) Confocal microscopy for ARG2 and mitochondria was performed on NIH-H82 cells and on cells isolated from 2 NSCLC. Mitochondria were labelled by MitoTracker Far Red; ARG2 was stained by indirect immunofluorescence, using a FITC-conjugated secondary antibody. Left, right and central panels show staining for ARG2, mitochondria and both, respectively. Sample from a NSCLC is shown.

To examine intracellular localization of the enzyme, confocal microscopy was performed with an anti-ARG2 antibody, together with a fluorescent probe targeting mitochondria, the organelles that in normal tissues contain ARG2. A SCLC cell line and 2 cell suspensions from NSCLC tissue were used. No colocalization was found, indicating that within tumor cells the enzyme is located outside mitochondria (Fig. 1e).

Correlation of ARG2 expression with clinical and histological parameters

The percentage of ARG2-positive cells and the intensity and distribution of staining were then compared with histological type and subtype, stage, grading, TNM, tumor size, extent of, respectively, lymphocyte, macrophage and granulocyte infiltrate, mitotic rate, extent of necrosis, of inflammation, of vascular invasion, and overall survival. A statistically significant difference in ARG2 expression was found between NSCLC and SCLC, with the latter tumors having, as an average, twice the number of ARG2-expressing tumor cells of the formers (Table III). Among NSCLC, large cell undifferentiated carcinomas expressed more ARG2 than the other subtypes but, due to the paucity of the formers, statistical significance was not achieved. An increase in average ARG2 expression was also found when Grade 3 tumors were compared with Grades 1 and 2 (Table III). Instead, no significant correlations were found when the remaining parameters were investigated. In particular, the amount of ARG2-expressing cells did not affect patient's overall survival (Cox analysis: HR = 1.002, 95%CI = 0.989–1.014, p = 0.769). Likewise, comparisons between ARG2 expression and the remaining parameters always gave p values above 0.5. In particular, no relationship was found between ARG2 expression and stage at diagnosis, the parameter that, on the basis of Cox analysis, most affected patient survival. No significant correlations were also found when all the above mentioned clinical and histological parameters were compared with intensity and distribution of ARG2 staining.

Table III. Comparison Between Some Clinical Parameters and ARG2 Expression

Another point we addressed was the contribution of ARG2 expression to metastasis formation. In this regard we have compared, in 15 cases of NSCLC, the percentage of ARG2-positive tumor cells in metastatic, tumor-draining lymph nodes with the percentage of ARG2-positive tumor cells in the primary tumor. Data (10.0 ± 17.1 and 8.5 ± 12.3, respectively, p = 0.773), albeit resulting from a small number of cases, argue against a role of ARG2 expression in metastasis formation.

Analysis of enzymatic activity and immune suppressive effect of ARG2

The evidence of ARG2 expression in tumor tissue without impact on tumor progression prompted us to investigate the reason for the lack of effect.

ARG2 exerts the immune suppressive effect via its enzymatic activity: the complete depletion of Arginine from extracellular milieu induces inhibition of lymphocyte proliferation.1 Therefore, we investigated if ARG2 expressed by tumor cells was enzymatically active, and if its activity inhibited T cell proliferation. We thus compared the level of ARG2 expression in CD14− cells with the Arginase activity displayed, and with the effect that these cells had on T cell proliferation (Fig. 2a). Tests were performed with the CD14− cell fraction from tumors because: (i) this fraction was enriched in ARG2+ tumor cells, (ii) we wanted to get rid of the effect exerted by macrophages on lymphocyte proliferation, and (iii) we wanted to prevent errors due to ARG1 release by tumor-infiltrating granulocytes: in these cells, activation resultingfrom tissue migration induces up-regulation of membrane CD14.26–28 ARG2 expression was investigated by Western blot in CD14− cells isolated from 3 NSCLC patients (Fig. 2a, left panel), and confirmed by immunohistochemistry, that showed that all of them had at least 20 % of ARG2-positive tumor cells (data not shown). In parallel, PHA-activated PBL from the patients were cultured in the presence or in the absence of autologous CD14− cells from tumor, and ARG activity and T cell proliferation were evaluated. ARG activity was checked after 48 hr by measuring arginine and ornithine levels in supernatants (Fig. 2a, central panel). Production of ornithine and catabolism of arginine could be detected in samples containing CD14− cells from tumor, but not in samples with lymphocytes only. In each of the former samples, the amount of ornithine produced and of Arginine catabolized paralleled the level of ARG2 expressed, as evaluated by Western blot in the same CD14− cells from tumor: it was minimal in the 2 samples with low ARG2 and it was abundant in the sample with high ARG2 expression. This finding rules out the possibility of ARG1-expressing cells contaminating our CD14− cell samples.

Figure 2. Evaluation of the effect of either intact or lysed CD14− cells from tumor tissue on lymphocyte proliferation. (a) Three cases were analyzed. On the left panel the ARG2 expression, as assessed by Western blot on CD14− cells isolated from tumor tissue, is shown. In the central panel the concentrations of arginine and ornithine in supernatant of PHA activated PBL in the presence or in the absence of intact autologous CD14− cells isolated from tumor, and in cell culture medium are indicated. Measurements were performed by Tandem Mass Spectrometry after a 48-hr culture. In the right panel the effect of intact CD14− cells from tumor on proliferation of PHA-activated autologous PBL at 12:1 ratio is shown. ³H Tymidine was added to the cultures used for Arginine and Ornithine assay, and Tymidine incorporation was assayed after 24 hr. PBL proliferation in the presence of autologous CD14− cells from tumor is indicated as percentage of the proliferation of the same PBL alone. (b) Three cases were analyzed. Per each case the percentage of ARG2-positive tumor cells, as detected by immunohistochemistry, is indicated. CD14− cells isolated from tumor were lysed, and cell lysate was added to PHA-activated PBL either in the absence or in the presence of the ARG2 inhibitor Nor-NOHA. Cell proliferation was assessed as indicated above.

Addition of CD14− cells from tumor altered proliferation of PHA-treated autologous lymphocytes, but in a way that could not be related to the amount of ARG2 expressed, or with the arginase activity displayed (Fig. 2a, right panel). Actually, in 2 out of 3 cases, coculture with CD14− cells increased lymphocyte proliferation, while only in 1 case proliferation was partially inhibited. In particular the sample with the highest ARG2 expression in CD14− cells was the one that most increased T cell proliferation. As a whole, mitogen-induced T cell proliferation showed no relationship with ornithine and arginine levels in supernatants.

The system of incubation of activated PBL together with CD14− cells from tumor possibly does not fully reflect the situation within tumor stroma in vivo, where tumor cell apoptosis or necrosis is frequently present, with potential release of ARG2 into microenvironment. To mimic this phenomenon, activated PBL were incubated with cell lysate of CD14− cells from tumor, and to evaluate the role of ARG2, tests were performed in the presence or in the absence of the ARG2 inhibitor Nor-NOHA4 (Fig. 2b). T cell proliferation was not inhibited by cell lysates, nor increased by further addition of Nor-NOHA. These results suggest that ARG2 released by disrupted tumor cells is not effective in inhibiting T cell proliferation.

Analysis of the expression of the 3 NOS isoforms

The lack of any suppressive effect that we observed, may depend on the requirement, for an effective immune suppression, of the concomitant expression of ARG2 and any of NOS isoforms. We therefore studied NOS isoforms expression in 20 randomly chosen cases of NSCLC, using different techniques.

Western blot analysis, performed on CD14+ and CD14− cells from both tumor tissue and normal lung tissue, showed no bands corresponding to iNOS protein in any of the samples tested (Fig. 3a). Instead, iNOS was clearly detectable in A549 cells treated with IFNγ, run in a parallel lane as positive control.29 RT-PCR was also performed on the same samples, using 2 different sets of primers for iNOS. No specific mRNA was detected in tumor samples, while IFNγ-activated A549 cells showed strong iNOS mRNA expression (Fig. 3b). Immunohistochemistry showed scattered cells in the stroma, that reacted with the anti-iNOS antibody; however reactivity did not disappear in the presence of the synthetic iNOS peptide used as immunogen, indicating a non-specific binding (data not shown).

Figure 3. Analysis of iNOS expression in lung cancers. (a) Western blot analysis was performed on CD14+ and CD14− cells from both tumor tissue and normal lung tissue. The results with CD14+ and CD14− cells from tumor of 3 patients is shown. The remaining samples gave identical results. iNOS could be detected in positive control, i.e. A549 cells treated for 24 h with 50 U/ml IFNγ. (b) RT-PCR analysis was performed on CD14+ and CD14− cells from both tumor tissue and normal lung tissue. Two different sets of iNOS-specific primers were used. No iNOS transcripts could be detected. The results with CD14+ cells from tumor and from normal lung tissue of 2 patients is shown. The remaining samples gave identical results. iNOS mRNA could be detected in IFNγ-treated A549 cells.

In addition, eNOS-positive cells could not be detected in tumor tissue by immunohistochemistry, by Western blot analysis, and by RT-PCR (data not shown).

Also nNOS expression could not be detected in our samples, both by immunohistochemistry and by RT-PCR (data not shown).

Analysis of peroxynitrite production and nitrotyrosine generation

The results obtained with different techniques on NOS expression do not support the possibility of NOS isoform(s) being expressed and peroxynitrite being produced in lung tumors. This issue is crucial, since peroxynitrite-dependent nitrosylation of Tyrosine residues, resulting from NOS enzymatic activity, should lead to T cell apoptosis.7, 30 To fully elucidate this point, productions of peroxynitrite and of nitrotyrosine were assayed.

Peroxynitrite was evaluated in supernatant of CD14+ cells and on CD14− cells from tumor tissue from 7 randomly chosen cases of NSCLC. Nitrite concentrations in supernatants from all the CD14− samples were all below the sensitivity threshold of the technique (data not shown), whilst 4 out of 7 CD14+ samples had measurable, albeit negligible, nitrite levels (Fig. 4a).

Figure 4. Evaluation of peroxynitrite production and nitrotyrosine generation. (a) Peroxinitrite production was assessed in supernatants of CD14+ cells from tumor by a fluorometric technique. Asterisks indicate samples with nitrite concentration below the sensitivity threshold of the method (30 nM nitrite). (b) Nitrosylation of Tyrosine residues was evaluated by immunohistochemistry. Nitrotyrosine was not detected in any of 7 samples of NSCLC and of 9 samples of SCLC. One case of SCLC is shown. In the inset the positive control, i.e. prostate adenocarcinoma.

The assay for peroxynitrite could not be performed on SCLC, since these tumors do not usually undergo surgery, and no fresh SCLC tissue was available. However, the presence of nitrotyrosine could be investigated by immunohistochemistry on paraffin-embedded samples of the 9 SCLC of our cohort, besides the 7 NSCLC mentioned above. No nitrotyrosine was detected in any sample, indicating that no concomitant activity of ARG2 and NOS was present in vivo at the tumor site (Fig. 4b). This finding differs from prostate carcinoma, which was regarded as a positive control for immunohistochemical detection of nitrotyrosines (Fig. 4b).13

Discussion

We have evaluated the possible existence of immune suppression resulting from Arginine catabolism, and the potential clinical relevance of this tumor immune escape mechanism in lung cancer.

Our data show that ARG2 is expressed by lung cancer tumor cells and that ARG2 expression is higher in more aggressive histological types. Indeed, SCLC significantly expressed more ARG2 than NSCLC, and among the latter tumors the enzyme was more expressed in large cell undifferentiated carcinomas. Furthermore, the average ARG2 expression increased in Grade 3 tumors, as respect with Grades 1 and 2. In this regard, ARG2 expression could be linked to de-differentiation phenomena occurring during tumor transformation.

However, we failed to detect any effect of ARG2 expression on tumor progression and metastasis formation. In particular, the level of enzyme did not correlate with patient survival, and no relationship was found with stage at diagnosis, the parameter most affecting survival.

The fact that ARG2 expression in lung cancer has no impact on tumor progression could be explained by the lack of an effective immune suppressive activity in vivo. As a matter of fact, the tumor-cell enriched CD14− fraction was unable to inhibit proliferation of autologous T lymphocytes in vitro in 2 cases out of 3, and in the third case inhibition did not depend on ARG2 activity (Fig. 2a). When CD14− cell were lysed, to mimic tumor cell necrosis in vivo, T cell proliferation was neither inhibited by addition of cell lysate, nor increased by further addition of the ARG2 inhibitor Nor-NOHA (Fig. 2b).

The lack of inhibition was not due to an insufficient number of CD14− cells added to activated lymphocytes, since the PBL:CD14− cells ratio used in the experiments (1:12) was within the range of frequency of TIL in lung tumors.31, 32 It has also to be noted that the Arginase-dependent down-regulation of CD3ζ chain and the consequent inhibition of T cell proliferation occur only in an arginine-free microenvironment,1, 33 a condition never attained in our experiments. In no case arginine concentration in supernatants dropped close to zero. Instead, arginine levels were only slightly reduced by CD14− cells from tumor, but still remained in the upper range of arginemia in healthy individuals.4 Indeed, a complete depletion of free arginine could hardly be achieved in tissues: cells can get the amino acid from the circulating pool of free arginine. Arginine may also become available from protein catabolism, or it can be synthesized in large amounts by certain cells, such as kidney epithelial cells. Even in end-stage renal failure patients, arginine serum level, albeit reduced to the half, remains in the range of 50 μmol/l.34 It could be hypothesized that the ectopic intracellular localization of the enzyme in lung cancer cells could affect its activity. Arginine uptake by mitochondria results in arginine concentration within the organelles that, depending on species, ranges from several to many times the concentration outside.35, 36 Thus, the extra-mitochondrial localization of ARG2 in tumor cells could result in decreased activity due to reduced substrate availability.

As a whole, our results indicate that an immune escape mechanism based on arginine deprivation, resulting from the sole ARG2 activity in lung cancer is unlikely.

However, we cannot rule out the possibility that tumor-infiltrating, ARG1-expressing cells could cooperate with ARG2-expressing tumor cells in inducing arginine depletion in lung cancer tissue. In humans, ARG1 is expressed, among leukocytes and leukocyte-derived cells, by neutrophil granulocytes only. In these cells the enzyme is stored in an inactive form within intracellular granules, and gets activated after extracellular release.2 Indeed, a role of granulocytes in inducing ARG1-dependent immune escape has been suggested for human renal cancer in a recent review.37 Further studies are required in order to asses if ARG1 and ARG2 are concomitantly active in lung cancer, and if a reduction in arginine level sufficient to induce inhibition of T cell proliferation is achieved.

A different scenery could be envisaged if any of the different isoforms of NOS should also be expressed. In this case, the 2 enzymes could cooperate in scavenging Arginine from extracellular milieu. We have looked for the expression of the 3 NOS isoforms in lung cancer, but neither nNOS, nor eNOS, nor iNOS were detected. Furthermore, enzymatic activity was negligible. As a matter of fact, production of peroxynitrite by CD14+ cells from tumor never exceeded 170 μM; in comparison, mouse macrophages generate under the same conditions 100 times more peroxynitrite.38 Consequently, no nitrosylation of Tyrosine residues could be detected in tumor tissue samples; in particular, no nitrotyrosine could be detected in SCLC, the tumor type having the highest expression of ARG2. Therefore, the possibility of a concominant activity of ARG2 and NOS, which is required for maximal immune suppressive effects related to T cell apoptotis, can be ruled out.7 Altogether, our data indicate that an immune suppressive mechanism based on concomitant expression of ARG2 and NOS does not occur in lung cancer, which may possess some other mechanisms of tumor immune escape.

This finding also implies that therapies aimed at boosting anti-tumor immune response in lung cancer do not have to face this potentially powerful immune escape mechanism. Nonetheless, the presence of ARG2 in lung tumors suggests that caution should be used when drugs increasing arginine catabolism are taken into account for lung cancer therapy. Pegylated recombinant human arginase and arginine deiminase, an arginine-catabolizing enzyme from Mycoplasma, are effective in inhibiting proliferation of tumor cell lines, and have been proposed for anti-cancer therapy.39, 40 Indeed, phase I-II trials in patients with melanoma or hepatocellular carcinoma have shown that arginine deiminase has anti-tumor activity and tolerable side effects.41, 42 Should the drugs be used for lung cancer, the concomitant effect of endogenous ARG2 and exogenous pegylated arginase or arginine deiminase could induce severe Arginine depletion and potentially result in local immune suppression.

Certainly, our data do not rule out the possibility that arginine catabolism-based immune escape might play a relevant role in other tumors. In prostate cancer ARG2 and iNOS are co-expressed by tumor cells, and both enzymes participate in local peroxynitrite-dependent immune suppression.13 In this case the altered arginine catabolism could have an impact on tumor progression. The different findings obtained in lung tumors indicate that the problem of the immune suppression based on arginine catabolism should be addressed independently for each tumor type.

Acknowledgements

T.P. is recipient of a FIRC fellowship.