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. 2003 Dec;134(3):538–544. doi: 10.1111/j.1365-2249.2003.02320.x

Serum nitric oxide (NO) levels in systemic sclerosis patients: correlation between NO levels and clinical features

K TAKAGI 1, Y KAWAGUCHI 1, M HARA 1, T SUGIURA 1, M HARIGAI 1, N KAMATANI 1
PMCID: PMC1808884  PMID: 14632763

Abstract

Vascular damage in systemic sclerosis (SSc) may be a factor in the abnormal regulation of several vasoactive agents. It has been well confirmed that plasma endothelin-1, as a vasoconstrictive factor, is elevated in patients with SSc. However, it is still controversial whether the levels of serum nitric oxide (NO), a strong vasodilator, are increased or decreased in SSc patients compared to healthy donors. In this study, we measured the levels of serum NO metabolites in SSc patients and determined the contribution of the excessive production of NO synthase (NOS)-2 by skin fibroblasts to NO synthesis. Serum NO levels of 45 patients with SSc were significantly higher than those of 20 healthy volunteers. In addition, some clinical features of SSc (the extent of skin fibrosis, short disease duration, and the complication of active fibrosing alveolitis) were all correlated positively with the levels of NO metabolites in SSc patients. To evaluate the levels of NOS-2 produced by skin fibroblasts, skin fibroblast cultures were established from SSc patients and healthy volunteers. Reverse transcription-polymerase chain reaction indicated that NOS-2 mRNA was spontaneously expressed in cultured fibroblasts derived from SSc patients, but not in those derived from healthy normal controls. Immunohistochemical staining also showed that NOS-2 proteins were detected in SSc fibroblasts but not in normal fibroblasts. The production of NO by cultured fibroblasts was visualized directly by a reagent (DAF-2 DA) used for the fluorescent detection of NO. Cultured SSc fibroblasts were capable of NO synthesis in culture media containing l-arginine, whereas normal fibroblasts (with no expression of NOS-2) did not synthesize detectable NO. These observations indicate that NO production is increased markedly in early-stage diffuse cutaneous SSc patients with active fibrosing alveolitis, and that constitutive NOS-2 expression in SSc fibroblasts may contribute to increased NO production.

Keywords: fibroblasts, fibrosis, nitric oxide, systemic sclerosis

INTRODUCTION

Systemic sclerosis (SSc) is a connective tissue disease characterized by tissue fibrosis and vascular endothelial damage [1]. We and other investigators have described the aberrant production of cytokines and growth factors that may lead to tissue fibrosis in SSc [2–5]. Histopathological characteristics of the affected skin of SSc patients include increased collagen deposition in the dermis, infiltration of mononuclear cells and abnormal thickening of vessel walls [1]. The most pronounced vascular finding is severe intimal fibrosis with various degrees of intimal thickening, which is sometimes associated with the narrowing and obliteration of vessels [1].

Endothelin (ET) and nitric oxide (NO) have been implicated in this vascular damage, which is related to tissue fibrosis in SSc patients [6,7]. In a previous study, we demonstrated that plasma levels of ET-1 in SSc patients were significantly higher than those in normal healthy individuals [7]. Because ET-1 is both a strong vasoconstrictor and a growth factor for fibroblasts, we proposed that increased levels of ET-1 might play a key role in the induction of vascular endothelial damage and tissue fibrosis. In contrast, NO is an endothelial-derived relaxing factor (EDRF), synthesized from l-arginine by NO synthase (NOS) [8,9], which can counteract the function of ET-1. Three isoforms of NOS have been identified [10]: NOS-1, NOS-2, and NOS-3. Both NOS-1 and NOS-3 are expressed constitutively in several kinds of cells, while NOS-2 is not expressed without stimuli; however, its expression is induced drastically by multiple stimuli, such as interferon (IFN)-γ, tumour necrosis factor (TNF)-α, interleukin-1 (IL-1) and bacterial lipopolysaccharide (LPS). An immunohistochemical study revealed that NOS-2 is present in macrophages, lymphocytes and vascular endothelial cells. In addition to its role as a vasodilator, NO can mediate free radical-related tissue injuries. Recently, it was reported that NO exhibited cytotoxicity [11] and antineoplastic activity [12]. Furthermore, NO has been reported to act as a potent cytotoxic effector of tissue damage in autoimmune disorders such as rheumatoid arthritis [13] and multiple sclerosis [14].

The dual functions of NO seem to be both beneficial (as a vasodilator) and harmful (as a cytotoxic effector) in regard to the clinical manifestations of SSc. Indeed, a controversy exists as to whether NO levels in SSc patients are elevated or depressed [6,15–19]. In this study, we sought to determine whether increased NO levels are associated with various clinical subsets of SSc patients, and to assess the contribution of fibroblasts in skin lesions to NO synthesis.

MATERIALS AND METHODS

Patients

Fifteen patients with limited cutaneous SSc (lSSc) and 30 patients with diffuse cutaneous SSc (dSSc) were recruited, all of whom were admitted to the Aoyama Hospital of Tokyo Women's Medical University (Table 1). Informed consent was obtained from all patients enrolled in this study. All SSc patients met the criteria of the American College of Rheumatology [20] and the patients with renal dysfunction were excluded. Patients were classified as having either diffuse or limited cutaneous SSc according to the classification of LeRoy et al. [21]. We also divided all patients into two clinical subsets according to the length of disease duration. Those with early-stage SSc had been diagnosed within the past 2 years, and those with late-stage SSc had been diagnosed more than 2 years ago. The presence of fibrosing alveolitis (FA), a complication of SSc, was determined by high-resolution computed tomography (CT) of the chest. The pulmonary function of each patient was evaluated by measurement of the flow volume curve (FVC). In this study, active FA (aFA) was assessed based on the findings of a ground-glass appearance of the chest CT and recent deterioration in the FVC. No specific diet was fed to the patients while hospitalized. Twenty healthy volunteers, who had no history of a diet and had never smoked, agreed (with informed consent) to give blood samples for use as normal controls.

Table 1.

Characteristics of patients with systemic sclerosis (SSc) and healthy normal control (NC)

SSc NC
No. studied individuals 45 20
Age, years
 Mean ± s.d. 45·7 ± 12·7 40·8 ± 9·7
 Range 18–72 26–65
No. females/males 39/6 17/3
No. with diffuse/limited SSc 30/15
 Age, years
  Mean ± s.d. 43·7 ± 13·8/49·6 ± 9·5
No. with early/late SSc 26/19
 Age, years
  Mean ± s.d. 41·9 ± 12·3/52·6 ± 9·2
No. with active fibrosing alveolitis 12 (all are diffuse SSc)
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Determination of serum NO

Because serum NO is degraded quickly into nitrite and nitrate, we used a colorimetric assay kit (Oxis, Portland, OR, USA) to measure the total levels of these NO metabolites, as an indicator of NO levels. This kit employs a nitrate reductase for enzymatic reduction of nitrate to nitrite, prior to measurement of the nitrite levels using the Griess reagent. Using this system, the measured nitrite level represents the total of the levels of both NO metabolites (nitrate and nitrite).

Fibroblast cultures

Fibroblasts were prepared by an explant method from the affected skin of nine dSSc patients and the normal skin of seven healthy donors, obtained by skin biopsies, as described previously [7]. The fibroblasts were cultured using DMEM (gibco, Grand Island, NY, USA) with 10% fetal bovine serum (Sigma, St Louis, MO, USA) and penicillin/streptomycin (gibco). Cells from the third to fifth passages were used in the present study.

Reverse transcription–polymerase chain reaction (RT-PCR)

After the fibroblasts achieved confluency in 100-mm dishes, the medium was changed to a serum-free medium (QBSF-51, Sigma) for an additional 72 h, after which total RNA was extracted using RNA Zol (Biotex, Houston, TX, USA). RT-PCR was performed using an RNA PCR kit (Perkin-Elmer Cetus, Norwalk, CT, USA). Briefly, 1 µg of total RNA from each sample was reverse transcribed to cDNA; the RT reaction was performed at 42°C for 30 min in a total volume of 20 µl; 2 µl of each RT solution was then used for PCR. Thirty-five cycles of PCR were performed with a set of primers for NOS-2 and β-actin. The sequences of the primers were as follows: NOS-2 (985–1177 bp from the transcription start site): sense: 5′-AGTGATGGCAAGCACGACTTC-3′, antisense: 5′- ACGGCCATTGGCCTGCAGGACC-3′; β-actin: sense: 5′-AAGAGAGGCATCCTCACCCT-3′, antisense: 5′-TACATGGCTGGGGTGTTGAA-3′. The PCR products were electrophoresed in a 2% agarose gel, stained with 1% ethidium bromide, and visualized under ultraviolet light. β-actin was used as an internal control.

Immunohistochemical study

Cells were analysed at 60% to 80% confluency in chamber slide glasses. Cultured fibroblasts were fixed with 2% paraformaldehyde at 4°C for 30 min and rinsed briefly in water and then in phosphate-buffered saline (PBS). The cells were then blocked with 2·5% horse serum (Vector, Burlingame, CA, USA) in PBS for 10 min at room temperature. The slides were incubated with the primary antibody against NOS-2 (N-20, Santa Cruz, Santa Cruz, CA, USA) or non-immune rabbit IgG (Vector) at a concentration of 10 µg/ml in PBS for 60 min at room temperature. After extensive washing with PBS, biotin-labelled horse polyclonal antirabbit IgG (Vector) was incubated for 10 min at room temperature. After washing with PBS, the slides were incubated in a streptavidin/peroxidase complex working solution for 5 min (Vector). Streptavidin/peroxidase complexes were developed by 0·5 mg/ml diaminobenzidine (DAB, Sigma) with 0·1% hydrogen peroxide.

NO production of cultured fibroblasts

The production of NO by cultured fibroblasts was measured using cell-permeable diaminofluorescein-2 diacetate (DAF-2 DA, Daichi Pure Chemicals, Tokyo, Japan). Fibroblasts were cultured in serum-free medium in six-well culture plates. Before reaching 80% confluency, the cells were cultured in serum-free medium with or without 10 ng/ml of recombinant IL-1α (R&D Systems, Minneapolis, MN, USA) for 12 h. After washing with PBS, 10 µm DAF-2 DA in PBS was added and incubated for 1 h at 37°C. The DAF-2 DA is hydrolysed to DAF-2 by esterase in the cytosol. After washing, cells were incubated with 10 mm l-arginine (Sigma) with or without 100 µm N-nitro-l-arginine methyl ester (l-NAME, provided by Sigma) in PBS for 10 min and observed by fluorescence microscopy. NO provides the third nitrogen to form a triazo ring from the two amino groups of the nonfluorescent DAF-2, and converts it to diaminotriazolofluorescein (DAF-2T), which is monitored at 488 nm excitation and 540 nm emission.

Statistical analysis

Results are expressed as mean ± standard deviations (s.d.). Statistical comparisons were performed using the Mann–Whitney U-test. Results were considered significant at P <0·05.

RESULTS

Serum NO levels in patients with SSc

Serum NO metabolites were significantly (P < 0·005) higher in SSc patients (131·2 ±76·6 µm, n = 45) than those in healthy controls (74·0 ± 32·4 µm, n = 20). The concentrations of NO metabolites in SSc patients showed a very wide range (25–330 µm) and many serum samples in SSc patients indicated low levels of NO metabolites, similar to the levels in normal controls. These results suggest that NO metabolite levels might be dependent upon the individual clinical characteristics of each patient. As shown in Fig. 1, we investigated NO levels in several clinical subsets of SSc patients (extent of skin thickening, fibrosing alveolitis and disease duration). Serum NO metabolite levels were significantly higher in dSSc patients than in lSSc (dSSc patients: 151·1 ± 81·8 µm; lSSc: 91·3 ± 45·1 µm, P < 0·05). There was no significant difference in NO metabolite levels between lSSc patients and normal controls. We also examined serum NO levels in SSc patients with or without active fibrosing alveolitis (aFA). Serum NO levels were significantly higher in SSc with aFA than those without aFA (188·3 ± 88·6 versus 110·5 ± 60·9 µm, P < 0·005). Serum levels of NO metabolites were significantly higher in early-stage SSc patients than in late-stage SSc patients (early stage: 172·1 ± 73·6 µm, late stage: 78·1 ± 31·7 µm, P < 0·001). NO metabolite levels were also determined in each subset of dSSc, classified by disease duration or the presence of aFA (Fig. 2). A subset of dSSc with high NO metabolite levels was associated with early phase and the presence of aFA. Moreover, the levels of NO metabolites in early-stage dSSc patients with aFA were the highest among all groups studied (223·9 ± 70·5 µm), and were significantly higher than those in all dSSc patients (P < 0·05).

Fig. 1.

Fig. 1

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Serum NO metabolite levels in clinical subsets of patients with systemic sclerosis (SSc) and normal healthy donors (NC). Patients were classified as having either diffuse or limited SSc according to the classification of LeRoy et al. The presence of active fibrosing alveolitis (aFA) was determined by high-resolution computed tomography of the chest and the measurement of the flow volume curve. The patients with early-stage SSc had been diagnosed within the past 2 years.

Fig. 2.

Fig. 2

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Serum NO metabolite levels in various subsets of diffuse SSc (dSSc) patients. We divided all dSSc patients (All) into two clinical subsets according to the activity of fibrosing alveolitis and disease duration. Early-stage dSSc with active fibrosing alveolitis (aFA) showed significantly higher levels of serum NO metabolites than all dSSc patients. Early-stage SSc (EO) was defined as the patients who had been diagnosed within the past 2 years.

NOS-2 expression in cultured fibroblasts

NOS-2 is a NO synthase that is induced in many types of cells by proinflammatory cytokines and a variety of other inflammatory mediators. To explore the mechanism of elevated serum NO levels in SSc patients, we examined the expression of NOS-2 mRNA by RT-PCR in cultured fibroblasts derived from five early-stage dSSc, four late-stage dSSc and seven normal controls. NOS-2 mRNA was spontaneously expressed in fibroblasts from early-stage and late-stage SSc patients, but not in those from normal controls (Fig. 3).

Fig. 3.

Fig. 3

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Levels of expression of NOS-2 mRNA in cultured fibroblasts. Spontaneous expression of NOS-2 mRNA was detected in early-stage SSc fibroblasts (S1–S5) and late-stage SSc fibroblasts (S6–S9), and was not detected in normal controls (N1–N7). Early-stage SSc was defined as the patients who had been diagnosed within the past 2 years. β-actin was used as an internal control. These are representative findings for three independent experiments.

We next examined the expression of NOS-2 protein in cultured fibroblasts from five SSc patients and five normal controls. Staining for NOS-2 was detected in all SSc fibroblasts, but was undetected or very faint in normal fibroblasts. Representative results are shown in Fig. 4.

Fig. 4.

Fig. 4

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Expression of NOS-2 in cultured fibroblasts. Fibroblasts were cultured in chamber-slide glasses. After paraformaldehyde fixation, cells were incubated with antibodies against NOS-2, and binding visualized by indirect peroxidase assay. These are representative findings for independent experiments using fibroblasts from five SSc patients and five normal controls. (a) SSc fibroblast, (b) normal fibroblast.

Detection of real-time NO production by cultured fibroblasts

Cultured fibroblasts derived from three SSc patients and three normal controls were investigated for NO production using the DAF-2 DA system. DAF-2 fluorescence was detected in SSc fibroblasts (Fig. 5a). Fluorescence was not detectable in the absence of l-arginine (Fig. 5b) or in the added presence of L-NMMA (Fig. 5c). Conversely, DAF-2 fluorescence was not detected in normal fibroblasts in the presence of l-arginine (Fig. 5d), but fluorescence was detectable in cells pretreated with IL-1α (Fig. 5e).

Fig. 5.

Fig. 5

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Representative results showing DAF-2-associated fluorescence in SSc and normal fibroblasts. The production of NO by cultured fibroblasts was measured using diaminofluorescein-2 diacetate (DAF-2 DA). Fibroblasts from SSc patient (a–c) and healthy donor (d) and (c) were cultured in serum-free medium in six-well culture plates. Ten µm of DAF-2 DA were added and incubated for 1 h at 37°C. After washing, cells were incubated with 10 mm l-arginine with or without 100 µm N-nitro-l-arginine methyl ester (l-NAME) in PBS for 10 min and observed by fluorescence microscopy. (a) and (d): with l-arginine alone, (b): without l-arginine nor l-NAME, (c): with both l-arginine and l-NAME, (e): with l-arginine alone after the pretreatment with IL-1α. These experiments were performed using the different fibroblast lines from three SSc patients and three healthy donors. Similar results were obtained in all experiments.

DISCUSSION

In the present study, we determined that serum NO levels were significantly elevated in patients with SSc as compared to healthy normal controls. In previous reports, Kahaleh et al. [15] and Allanore et al. [16] described low NO levels in SSc patients. However, Yamamoto et al. [17] and Andersen et al. [18] found increased NO levels, which is consistent with our observations. The discrepancy in these results could be explained by disease stage, severity of tissue fibrosis and various circumstances of endothelial damage. Therefore, we further investigated serum NO levels in various clinical subsets of SSc. Patients were first classified into dSSc or lSSc, based on the extent of skin fibrosis. The NO serum levels in lSSc patients showed no significant difference from those of normal controls. This suggests that the increased NO serum levels found in the SSc patients as a whole reflects the increase in NO synthesis in dSSc patients alone. However, a subset of dSSc patients had low serum levels of NO. We next compared NO serum levels between patients in the early and late stages of the disease. Serum NO levels in early-stage SSc patients were strikingly higher than those of late-stage SSc patients. Interestingly, there was no significant difference in serum NO levels between normal controls and late-stage SSc patients. Finally, we examined NO serum levels in patients with SSc complicated by aFA, a condition in which many inflammatory mediators are reported to be involved [22]. In SSc patients with interstitial lung disease (ILD), the key factor in SSc mortality [23], several research groups have reported elevated levels of exhaled NO [24–26]. These findings strongly suggest that NO synthesis may be elevated in the lung tissues of SSc patients with FA. We found that serum NO levels were elevated in dSSc patients with aFA. Notably, NO serum levels in early-stage dSSc patients with aFA were especially high. In contrast, NO serum levels in late-stage lSSc without aFA were less than those in normal controls (data not shown). Moodley et al. indicated that exhaled NO is elevated in SSc patients with early subclinical ILD, as demonstrated by abnormal data from bronchoalveolar lavage fluids [27]. Based on those findings, our observations strongly suggest that serum NO levels are elevated in early-stage SSc patients with severe tissue fibrosis (skin and lung), but are not elevated in late-stage SSc patients with limited skin fibrosis or without aFA. Further analysis revealed that NO metabolites were not elevated in the serum of SSc patients with inactive FA compared to healthy controls. We therefore speculate that serum NO levels may be a sensitive marker of the early stages of the development of severe tissue fibrosis in SSc patients, although a longitudinal and prospective study is needed to confirm this.

Because NO production is catalysed by a series of NOS enzymes, it was thought that increased NO levels might result from the excessive production of NOS enzymes, especially NOS-2. The expression of NOS-2 was reported to be induced by inflammatory mediators such as cytokines and bacterial lipopolysaccharide [10]. In this study, NOS-2 was produced spontaneously by cultured SSc fibroblasts, suggesting that increased serum NO levels might reflect in part the elevated expression of NOS-2 by fibroblasts derived from SSc patients. Although spontaneous NOS-2 production is lower than NOS-1 or NOS-3 production in various tissues, stimulation by inflammatory mediators can up-regulate NOS-2 production to a level 100-fold that of NOS-1 or NOS-3 [28]. The fibroblasts of SSc patients were reported to produce increased amounts of IL-1α[29] and IL-6 [30], which are known to up-regulate NOS-2 in endothelial cells and fibroblasts. Our observation revealed that NO synthesis was detected in the culture of normal fibroblasts pretreated with IL-1α. It reflects that IL-1α can induce NOS-2 in normal fibroblasts. Those results suggest that the increased levels of NOS-2 in SSc fibroblasts might result from the aberrant production of IL-1α.

Nitric oxide is a strong vasodilator and thus can inhibit the biochemical effect of ET-1 on vascular tonus [8,9]. However, ET-1 induces NOS-2 production through the ET receptor on endothelial cells [31], which results in the eventual synthesis of NO. Presumably, the ET-1/NO system is a very important biological control for vascular tone homeostasis. In a previous study, plasma levels of ET-1 were found to be significantly higher in SSc patients than in normal healthy individuals [7]. It is possible that these increased levels of ET-1 contribute to the disorders associated with endothelial damage in SSc (i.e. Raynaud's phenomenon and pulmonary hypertension). In contrast, the increase in NO in fibrosing tissues might counteract the effect of vasoconstrictive agents, including ET-1. We suspected that the overproduction of NO may be a form of biological feedback neutralizing the effect of ET-1. However, serum NO levels were higher in dSSc patients with severe fibrosis of several internal organs than in lSSc patients. The discrepancy between these results could be explained by a concentration of bioactive NO that is insufficient to inhibit vasoconstrictive agents in SSc patients. It is inferred from our observations that use of a high concentration of a stable analogue of NO could be a novel strategy for the treatment of the early phase of dSSc.

Although NO appears to improve the clinical manifestations of SSc, it has also been reported to be associated with tissue injuries in states of inflammation [32] and autoimmunity [33]. Gilkeson et al. found that NO could play an important role in the development of glomerulonephritis and synovitis in a study of MRL-lpr mice using the NOS-2 gene knock-out technique [34]. Another study showed that NOS inhibitors protected against the destruction of islet cells in mice with experimental diabetes induced by streptozocin [35]. Taken together, these findings show that tissue injuries after inflammation and autoimmunity are induced by the excessive production of NO.

The mechanism by which excessive NO production affects tissue injury is, however, still unclear. It is possible that the peroxynitrate anion (ONOO–), which is a powerful oxidizing agent, is synthesized by NO and O2− [36,37], suggesting that the formation of ONOO– may mediate tissue injury in states of inflammation and autoimmunity. Another important biological function of NO may be the regulation of apoptosis. It has been reported that excessive synthesis of NO results in the apoptosis of rat enterochromaffin-like cells [38], and also that NO functions as a signal for triggering apoptosis in thymocytes [39]. Because Sgonc et al. observed that endothelial cells undergo apoptosis in the fibrotic tissues of SSc patients [40], they speculated that the induction of apoptosis played a critical role in endothelial damage in SSc. Since excessive NO synthesis can induce apoptosis of cells, it appears that endothelial damage in SSc may result from the apoptosis induced by NO. Thus, the overproduction of NO in the fibrotic lesions of SSc can contribute to endothelial damage, leading to tissue fibrosis.

In summary, we found increased serum NO levels in early-stage dSSc with aFA, but not in lSSc or late-stage SSc patients. We conclude that serum NO is an important marker of the early phases of dSSc, which can be attributed to increased NOS-2 production by SSc fibroblasts; however, further investigation is needed to discover whether NO is beneficial or harmful to SSc patients.

Acknowledgments

We gratefully acknowledge the financial support received from a research grant by the Uehara Memorial Foundation in Japan, which partially funded this study.

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