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Rheumatology 2001; 40: 1089-1096
© 2001 British Society for Rheumatology

Original Papers

Low levels of nitric oxide (NO) in systemic sclerosis: inducible NO synthase production is decreased in cultured peripheral blood monocyte/macrophage cells

Y. Allanore, D. Borderie1,*, P. Hilliquin*, A. Hernvann1, M. Levacher2, H. Lemaréchal1, O. G. Ekindjian1 and A. Kahan

Departments of Rheumatology A,
1 Biochemistry A and
2 Pharmacology, Cochin Hospital, René Descartes University, Paris 75014, France


   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. To investigate nitric oxide (NO) production and inducible NO synthase expression by cultured peripheral blood mononuclear cells (PBMC) in patients with systemic sclerosis (SSc).

Methods. Eighteen patients with SSc were compared with two control groups: 16 patients with rheumatoid arthritis (RA) and 23 patients with mechanical sciatica. Nitrate was determined by fluorimetry in plasma and by spectrophotometry in supernatants. Inducible NO synthase (iNOS) was detected in cultured PBMC by immunofluorescence, immunoblotting and flow cytometry with or without treatment of the cells with interleukin (IL) 1ß+ tumour necrosis factor {alpha} (TNF-{alpha}), IL-4 or interferon {gamma} (IFN-{gamma}) from day 1 to day 5.

Results. NO metabolite concentrations were lower in SSc patients (mean±s.e.m. 34.3±2.63 µmol/l) than in RA (48.3±2.82 µmol/l; P<0.02) and sciatica (43.3±5.24 µmol/l; P<0.03) patients. iNOS was detected in cultured monocytes in all three groups but induction occurred on day 1 in RA, day 2 in sciatica and only on day 3 in SSc, whatever the stimulus.

Conclusions. The concentrations of NO metabolites are decreased in SSc patients and the metabolism of these compounds in PBMC is altered. Low levels of NO, a vasodilator, may be involved in vasospasm, which is critical in SSc. This may have therapeutic implications.

KEY WORDS: Nitric oxide, Mononuclear cells, Systemic sclerosis.


   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 References
 
Systemic sclerosis (SSc) is a multisystem connective tissue disorder characterized by vascular lesions and collagen accumulation. Its pathogenesis is unclear, but its vasospastic propensity is of importance. It is involved in Raynaud's phenomenon and also in cardiac and renal lesions [1]. Study of the vascular endothelium has shown that there is an increase in the synthesis and release of the vasoconstrictor peptide endothelin in SSc [2]. Recent studies have suggested that, in SSc, the endothelium may fail to release nitric oxide (NO) upon cold stimulation [3]. NO normally acts as a vasodilator. In vascular diseases, deficient endothelial NO production responses are involved in hypertension, atherosclerosis and vasospasm [4, 5].

Monocyte–endothelial cell interactions are essential factors in the development of vascular disease [6]. In SSc, mononuclear cells may affect vascular lesions and fibrotic processes by direct cell–cell interactions and by the production of various molecules [7], such as cytokines, collagen and glycosaminoglycans [8]. Histological studies of cutaneous scleroderma lesions have revealed early endothelial injury and mononuclear cell infiltration [9]. Mononuclear cells have been found in cutaneous tissue and in the perivascular spaces around small vessels [10]. The migration of mononuclear leukocytes across endothelial cell monolayers in tissue culture is promoted by fibroblasts via a mechanism that depends on monocyte chemoattractant protein 1 [11].

NO is synthesized by several types of NO synthase (NOS) [12]. The inducible type 2 NOS (iNOS) is a cytotoxic effector molecule and may act as an immunoregulator [13]. Several reports have provided evidence for the existence of a NO pathway in human mononuclear cells. The ligation of the CD23 receptor may also play an important regulatory role in the process [14]. iNOS is induced in these cells by various stimuli, including interferon {gamma} (IFN-{gamma}), interleukin 4 (IL-4) and tumour necrosis factor {alpha} (TNF-{alpha}) [14]. Several cytokines are involved in SSc: TNF-{alpha}, interleukin (IL) 1ß, IL-2, IL-4, IL-6 and IL-8 concentrations are high in serum, and mononuclear cells produce large amounts of TNF-{alpha}, IL-1ß and IL-4 [15, 16]. In contrast, the IFN-{gamma} concentration is low in SSc patients [16, 17].

We investigated whether the level of NO production by peripheral blood mononuclear cells (PBMC) was low in SSc, as this might contribute to the vasodilatory abnormalities observed in this disease. We determined NO metabolites in plasma and PBMC supernatants, and iNOS synthesis in PBMC that were untreated or treated with various cytokines.


   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 References
 
Patients
Eighteen patients with SSc were recruited (Table 1Go). All fulfilled the criteria of the American College of Rheumatology [18]. Thirteen patients had diffuse cutaneous SSc and five had limited cutaneous SSc according to the criteria of LeRoy et al. [19]. Clinical, laboratory and treatment data were collected at the time when blood samples were drawn (Table 1Go).


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  		TABLE 1. Characteristics of SSc patients

 
We investigated two control groups. Sixteen patients with rheumatoid arthritis (RA) (Table 2Go) constituted a positive control group, as there is excess NO production and iNOS synthesis in this disease [20]. All patients fulfilled the American Rheumatism Association 1987 revised criteria for RA [21]. Twenty-three patients with mechanical sciatica constituted a negative control group (Table 2Go). Non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids were authorized but stopped 24 h before blood collection. The use of disease-modifying anti-rheumatic drugs was stable for at least 3 months in the SSc and RA patients. Calcium channel inhibitors were authorized. No specific diet was fed to the patients, all of whom were hospitalized in the same hospital department during a 6-month period (from November 1998 to April 1999).


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  		TABLE 2. Characteristics of the three groups of patients and healthy controls

 
Five healthy volunteers agreed to give blood samples for the determination of NO metabolites.

Blood samples
Thirty millilitres of blood was collected into EDTA (ethylenediamine tetraacetate) from each patient. Informed consent was obtained from each patient before participation. Assistance Publique Hôpitaux de Paris promoted the study. The protocol was approved by the Cochin Hospital Ethics Committee.

PBMC cultures
Mononuclear cells were separated by centrifugation (25 min, 150 g) through Ficoll/Hypaque (Eurobio, France) (specific gravity=1.077). Cells were washed twice with RPMI 1640 solution (Eurobio, Les Ulis, France). The cell population consisted, on average, of 20% monocytes and 80% lymphocytes. Cells were cultured at density 5x105 per 6-mm diameter microtitre plate well in 0.2 ml of medium for supernatant measurements, and 5x105 per 5 ml of medium for iNOS analysis. The medium used was RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum (Boehringer Ingelheim, Gagny, France) with or without IFN-{gamma} (100 IU/ml), TNF-{alpha} (5 ng/ml), IL-1ß (10 ng/ml), IL-4 (5 ng/ml), IL-1ß+TNF-{alpha}, IL-1ß (10 ng/ml)+ TNF-{alpha}+IFN-{gamma}, lipopolysaccharide (10 ng/ml) (cytokines were human recombinant forms; all were obtained from Sigma Aldrich, St Louis, MO, USA). The cells were cultured for 5 days and then the supernatant was removed for nitrite and nitrate determination.

Determination of nitrite and nitrate
In aqueous solution, NO is rapidly oxidized to nitrite by oxygen. In blood, NO may be further oxidized to nitrate by haemoglobin [12].

The nitrite concentration of supernatants was determined by a fluorimetric assay. In acid solution, nitrites are transformed into NO, which combines with diaminonaphthalene to give naphthotriazole, a fluorescent molecule. Determinations were performed at 360 nm (excitation wavelength) and 450 nm (emission wavelength) (FluoroCount 96; Pharmacia, Amersham Biotech, Amersham, UK). The nitrite concentration was calculated from a sodium nitrite standard curve.

The nitrate concentration was determined in plasma and supernatants with a spectrophotometric assay using oxidation catalysed by cadmium metal (Oxis; Bioxytech, Portland, OR, USA), which converts nitrate into nitrite. The total nitrite determined therefore corresponds to NO production. Protein was first removed from the medium by incubation with zinc sulphate. Cadmium metal was then added and the medium was incubated overnight, with shaking in the dark. The next day, the solution was mixed and sulphanilamide (Sigma Aldrich) and N-1 naphthyl ethylenediamine (Pierce, Oud Beijerland, The Netherlands) were added. These compounds form a coloured complex with nitrate, which can be determined by spectrophotometry (Dynatech Instruments, Guernsey) as described by Green et al. [22].

The detection limit for the nitrite and nitrate assays was 0.04 µmol/l.

iNOS expression in cultured PBMC
Immunofluorescence
Cells were cultured on glass slides. They were fixed and permeabilized by incubation with an acetone/methanol mixture (v/v) at -20°C for 10 min and then rehydrated in 1% bovine serum albumin in phosphate-buffered saline (PBS). The slides were incubated for 1 h at 37°C with mouse anti-iNOS monoclonal antibody (Interchim, USA) diluted 1:500 in PBS or normal mouse immunoglobulin as a negative control. Slides were then incubated with a fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin G antibody (Interchim, Montluçon, France) diluted 1:100 in PBS.

Immunoblot assay
A monoclonal anti-NOS2 antibody (Transduction Laboratories, USA) at a dilution of 1/1000 and substrate Opti 4-cn (Bio-Rad, Hercules, CA, USA) were used. Immunoblot results were classed as positive if a band was detected at about 130 kDa.

Flow cytometry
To quantify NOS expression in cultured mononuclear cells, we used fluorescence-activated cell sorting (FACS) analysis to evaluate the amounts of intracellular iNOS protein and CD23 membranous receptor as previously described (23). Monoclonal anti-CD23 antibody conjugated with R-phycoerythrin (15 µl per ml of solution for 20 min) was used. Cells were permeabilized (FACS permeabilizing solution; Becton-Dickinson, Lincoln Park, NJ, USA) and washed with PBS, then incubated with monoclonal anti-iNOS antibody conjugated with fluorescein isothiocyanate (Transduction Laboratories, San Jose, CA, USA) (1/100 dilution in PBS; 1 h at room temperature). A minimum of two thousand labelled monocytes were analysed per sample, with a FACScan cytofluorometer using Lysis II software (Becton-Dickinson). The monocyte/macrophage population was defined on the basis of size and granularity.

Statistical analysis
Results were analysed with non-parametric tests (Mann–Whitney and Kruskal–Wallis) (Statview software). P values less than 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 References
 
Total plasma nitrite concentration after nitrate reduction
The concentrations of NO metabolites were lower in SSc patients (mean±S.E.M. 34.3±2.63 µmol/l) than in patients with RA (48.3 µmol/l±2.82, P<0.02) or sciatica (43.3±5.24 µmol/l, P<0.03) (Fig. 1Go). There was no significant difference between limited cutaneous SSc (34.1 µmol/l; n=5) and diffuse cutaneous SSc (34.4 µmol/l; n=13). Nitrate concentration (mean±S.E.M.) was 40.8±1.2 µmol/l in the group of healthy volunteers.



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  		FIG. 1. Plasma nitrate concentration (µmol/l) in the three groups of patients and healthy controls.

 

NO metabolites in PBMC supernatants
The supernatant was analysed on day 5 of culture. Regardless of the stimulant used, nitrate and nitrite were not detected in any of the PBMC supernatants from patients, controls and healthy controls.

iNOS synthesis in cultured PBMC
iNOS was detected in cultured PBMC by immunofluorescence with or without IL-1ß+TNF-{alpha}, IL-4 or IFN-{gamma} from day 1 to day 5. We found that monocyte/macrophage cells produced iNOS but induction occurred on day 1 in RA, day 2 in sciatica and on day 3 in SSc, with or without stimulation (Fig. 2Go). The greatest production of iNOS was obtained with IFN-{gamma} for all groups studied. Expression persisted in all groups on day 5. Lymphocytes did not produce significant amounts of iNOS.



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  		FIG. 2. Immunofluorescence of iNOS in SSc.

 
Immunoblots were done from day 1 to day 5 of culture, with or without IL-1ß+TNF-{alpha}, IL-4 and IFN-{gamma}. The results obtained were consistent with the immunofluorescence results for iNOS produced from day 1 in RA, day 2 in sciatica and day 3 in SSc, regardless of the culture conditions. Figure 3Go shows iNOS synthesis after stimulation with IFN-{gamma} in SSc and RA patients.



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  		FIG. 3. iNOS immunoblot for PBMC treated with IFN-{gamma} from day 1 to day 5. 1, 3, 5 and 7: RA; 2, 4, 6 and 8: SSc; 9: molecular weight marker; 10: iNOS positive control (130 kDa).

 
Five consecutive samples from each population were tested by flow cytometry for iNOS and expression of the CD23 receptor from day 0 to day 5, with or without IL-1ß+TNF-{alpha}, IL-4 and IFN-{gamma}. The five consecutive SSc patients recruited were patients 11–15 (Table 1Go). These patients were representative of the SSc group as a whole in terms of total nitrite concentrations (32.7 µmol/l, no significant difference). Cytometry results are expressed as the percentage of monocyte/macrophage cells containing iNOS in Fig. 4Go. Lymphocytes did not produce iNOS. In SSc patients, iNOS induction was delayed until day 3 in all culture conditions, and was maximal if IFN-{gamma} was added (37% of monocytes labelled), with a decrease after day 3 with or without IL-1ß+TNF-{alpha}. In the RA group, iNOS was induced in all cases on day 1 of culture, and its synthesis increased until day 5. For the sciatica group, induction occurred on day 2, intermediate between SSc and RA, with or without cytokine. Significant differences were observed (Fig. 4Go): on day 1, without additive or with IL-4 or IFN-{gamma}; on day 2, without cytokine and with IL-1ß+TNF-{alpha}; and on day 5, without cytokine and with IL-1ß+TNF-{alpha}. Levels of iNOS were higher in RA patients than in patients with sciatica on day 1 for cultures treated with IFN-{gamma} or IL-1ß+TNF-{alpha}, and on day 2 if IFN-{gamma} was added to cultures (Fig. 4Go).



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  		FIG. 4. Kinetics (day 1 to day 5) of iNOS production measured by flow cytometry in cultured PBMC without additive (a) and with IL-4 (b), IFN-{gamma} (c) or IL-1+TNF-{alpha} (d). Results are percentages of labelled PBMC. Five patients of each group (SSc, RA, sciatica) were tested for all the additives.

 
The expression of the CD23 receptor by monocytes/macrophages was analysed by flow cytometry. On day 0, CD23 receptor levels were highest in RA patients. Percentages of positive mononuclear cells (mean±S.E.M.) were 5.9±1.4 in SSc patients, 18.3±5.5 in RA patients and 5.8±1.3 in sciatica patients (P<0.03). On subsequent days, regardless of the cytokine added, there was no significant difference between the various groups.

Figure 5Go shows a typical dot-plot analysis in which SSc and RA patients were compared. Induction of iNOS was delayed in SSc patients (FL-1, right skew).



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  		FIG. 5. Kinetics (days 1–13) of iNOS production and CD23 expression in cultured PBMC treated with IFN-{gamma}; flow cytometry analysis (dot–plot). F1-1, iNOS, FL-2, CD23 receptor. A, systemic sclerosis, B, rheumatoid arthritis.

 


   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
References
 
The data presented in this study demonstrate concomitant decreases in plasma NO concentration and iNOS synthesis in cultured peripheral blood monocyte/macrophage cells in SSc patients.

Total plasma nitrite concentration after nitrate reduction was lower in SSc than in RA and sciatica patients. Subgroup analysis showed no difference between limited and diffuse SSc forms. Total plasma nitrite concentrations in five healthy volunteers were similar to those in patients with sciatica, which is consistent with this group being an appropriate control group. Nitrite levels in RA patients were consistent with those usually observed: Grabowski et al. [24] reported levels of 38±14 µmol/l, and Hilliquin et al. [25] reported 61.5±32.7 mol/l. Plasma nitrite concentration was higher in RA patients than in patients with sciatica, but this difference was not statistically significant. Various treatments, such as NSAIDs [26], corticosteroids [25] and methotrexate [27], decrease NO production. In our study, the decrease in NO production in SSc patients was not related to these treatments because SSc patients received a lower dose of corticosteroids than the RA patients (3.7 mg/day in SSc patients, 7.4 mg/day in RA patients) and very few patients received NSAIDs (two patients in the SSc group, five in the RA group and seven in the sciatica group) or methotrexate (one in the SSc group, nine in the RA group and none in the sciatica group). The effect of calcium channel inhibitors is unclear [28, 29]. We compared the subgroups of patients with SSc or sciatica who were taking calcium channel inhibitors. Total nitrite concentration was 33.9 µmol/l in the subgroup of SSc patients treated with calcium channel inhibitors (n=12) and 50.4 µmol/l in the sciatica subgroup (n=7). Therefore, the decrease in nitrite concentration in SSc patients was not due to this treatment. Our results are consistent with those of Kahaleh's group [30], who measured plasma NO levels by chemiluminescence and showed lower levels in SSc patients (25.8±1.3 µmol/l) than in healthy controls (35.5±2.1 µmol/l). Yamamoto et al. [31] reported higher nitrite levels in SSc patients (47.8±17 vs 25.6±10.3 µmol/l in healthy controls); this was unexpected because nitrite concentrations are usually between 1 and 7 µmol/l [32]. Indeed, in blood, nitrite is rapidly oxidized to nitrate in the presence of haemoglobin [33].

In SSc, endothelial cells fail to produce NO, possibly because of lower levels of expression of the NO synthase gene [3, 34]. We focused on PBMC because they infiltrate early SSc lesions and produce numerous mediators [7, 10], and because the release of NO from these cells may influence local vascular phenomena.

We first measured NO production by means of nitrite and nitrate assays in PBMC supernatants after 5 days of culture with various regulators. We were unable to detect NO production in SSc patients, control patients and healthy volunteers. NO production has been demonstrated clearly in rodent macrophages [35] in culture, but many investigators have failed to demonstrate NO production by human monocytes in culture or have detected very low levels [36, 37]. However, NO synthase production and activity have been established [17, 38]. Additional missing cofactors or post-translational regulation may be necessary for efficient iNOS activity. However, Yamamoto et al. [39] reported an increase in NO production by PBMC stimulated by IL-1ß in SSc patients.

We then analysed iNOS induction in PBMC by flow cytometry, which made possible a coupled study of the CD23 receptor. The synthesis of iNOS in cultured mononocyte/macrophage cells was demonstrated in all groups by various methods: immunofluorescence, immunoblot assay (iNOS had its usual molecular weight of around 130 kDa) and flow cytometry. In SSc patients, iNOS induction was delayed until day 3 of culture regardless of the regulator added, and in most cases decreased after 5 days of culture to become almost undetectable in untreated cells or in cells treated with IL-1ß+TNF-{alpha}. The persistently high level of iNOS expression on day 5 in RA patients is consistent with a previously report [20], suggesting that iNOS expression increases in RA. Expression of iNOS was not studied in healthy controls, thus not allowing any conclusion in this population. In all three groups of patients, IFN-{gamma} was the most powerful inducer of iNOS. The concentration of this cytokine is low in SSc patients [13, 14], which is consistent with the notion that the level of iNOS synthesis is low in this disease. Activation of the NO pathway has been demonstrated in human monocytes by ligand binding to the CD23 receptor [17]. Flow cytometry analysis showed that CD23 expression in RA patients was high on day 0, which could account for the early induction of iNOS. We demonstrated that delayed iNOS induction was not attributable to a decrease in CD23 receptor expression in SSc. The efficiency and ligation of the CD23 receptor in SSc should therefore be checked.

The involvement of NO in SSc is a matter of debate. Some reports suggest that NOS activity could be increased in SSc, in PBMC [40] and in the skin [41], but the site studied and disease activity may affect the results. In contrast, the notion that NO production is deficient in SSc is supported by the preliminary results of Kahaleh et al. [30, 34] and by a recent study showing that intra-arterial infusions of L-arginine and sodium nitroprusside significantly decrease the incidence of laboratory-induced Raynaud's phenomenon in scleroderma patients [42]. In addition, calcium channel inhibitors, which are important in SSc [1, 43, 44], may release NO [29, 45].

Conclusion
Vascular impairment, and in particular, generalized vasospastic propensity, are major features of the pathophysiology of SSc. Our results, together with those of Kahaleh and colleagues [3, 30, 34], suggest that NO production is low in SSc. Thus, low levels of vasodilator may be involved in the tendency towards vasospasm. If confirmed, these results suggest that it may be effective to use NO donors to treat SSc. Longitudinal studies are required in order to determine the relationship of NO metabolism with disease activity and progression.


   Acknowledgments
 
Y.A. received awards from Société Française de Rhumatologie and Fonds d'Etude et de Recherche du Corps Médical des Hôpitaux de Paris.


   Notes
 
Correspondence to: A. Kahan, Rhumatologie A, Hôpital Cochin, 27 rue du Faubourg Saint-Jacques, 75014 Paris, France. Back

* These authors contributed equally to the work. Back


   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 24 May 2000; Accepted 13 March 2001


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