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Rheumatology 1999; 38: 1301-1304
© 1999 British Society for Rheumatology

Letters to the Editor

Nitric oxide synthesis in peripheral blood mononuclear and polymorphonuclear cells from patients with systemic sclerosis

G. Cavallo 1, L. Sabadini, L. Rollo, M. Catenaccio, S. Lorenzini, N. Pipitone and R. Marcolongo

Istituto di Reumatologia, Università di Siena, Siena and
1 Wellcome Research Laboratory, Pomezia, Italy

Correspondence to: R. Marcolongo, Istituto di Reumatologia, Policlinico `Le Scotte', 53100 Siena, Italy.

Sir, Nitric oxide (NO) is a free radical synthesized from L-arginine by NO synthase (NOS) [1]. The endothelial isoform of NOS (eNOS) synthesizes small amounts of NO that induce peripheral vasodilatation, whilst the nearly ubiquitous inducible isoform (iNOS) produces large amounts of NO with pro-inflammatory action [2–4]. NO is thought to play a role in a number of inflammatory processes, including rheumatoid arthritis [5] and systemic lupus erythematosus [6]. The effect of NO in these conditions is not fully elucidated, but it is generally felt that it can contribute to maintaining inflammation [5, 6]. Controversial data have been reported on NO synthesis in systemic sclerosis (SSc). Both reduced NO synthesis by eNOS [7] and increased NO synthesis by iNOS [8] have been reported as occurring in the skin of SSc patients. Reduced NO synthesis by endothelial cells (EC) could be a cause of the manifestations of vascular insufficiency observed in SSc patients, including Raynaud's phenomenon. On the other hand, excess NO produced by iNOS could promote inflammation [4, 9] and possibly fibrosis [10]. Normal 24 h urinary excretion [11] and slightly elevated serum levels of nitrite [12], a relatively stable breakdown product of NO, have been reported in patients with SSc. However, whilst serum concentrations of nitrite may to some extent reflect NO production in the EC and at sites of tissue inflammation, such as the affected skin, little is known about NO production by blood cells. We read with interest the paper by Yamamoto et al. [8], which demonstrates that peripheral blood mononuclear cells (PBMNC) from patients with SSc release increased amounts of NO as compared with controls after interleukin-1 stimulation, but not in baseline condition. Here, we would like to report the results of a study similar in design. The aim of our study was to quantify the activity of NOS in PBMNC and polymorphonuclear cells (PMN) from 11 patients (all females, mean age 54.8 yr) who fulfilled the preliminary criteria of the American College of Rheumatology for SSc [13] and eight sex- and age-matched controls. Disease duration was 8.7 ± 6.0 yr (mean ± S.D.). All patients had skin involvement (limited in six patients and diffuse in the remaining), whilst internal organs were affected in all patients. Exclusion criteria were current infections or neoplasms, renal failure, or treatment with prednisone equivalent in excess of 7.5 mg/day. Venous blood was drawn from all patients into a heparinized syringe and separated by density-gradient centrifugation (Polymorphoprep, Nicomed, Oslo, Norway). Separated cells were washed twice with Hepes buffer (0.1 M, pH 7.2), resuspended in the same buffer containing 0.1 mM dithiothreitol, and homogenized by sonication (twice) for 20 s. After centrifugation at 105000 g for 30 min at 4°C, the supernatant was incubated with AG50X8 (100 mg of exchange resin for 1 ml of supernatant) for 5 min at 4°C to deplete endogenous L-arginine [14]. As NO reacts with oxygen in neutral aqueous solution, generating nitrite in a time-, L-arginine- and NADPH-dependent way [15], nitrite was determined as an index of NOS activity. The enzymatic activity was measured as described elsewhere [16]. Briefly, 500 µl of cytosolic preparations were mixed with 20 µl of valine (to inhibit arginase), 12 µl of CaCl2 and 12 µl of MgCl2 , to obtain, respectively, 20 mM, 0.2 mM and 1 mM final concentrations. The reaction mixture was equilibrated for 15 min at 37°C; subsequently, a further 25 µl of L-arginine (0.1 mM final concentration) and 25 µl of NADPH (0.1 mM final concentration) were added. The reaction was stopped by freezing the reagents after 25 min at 37°C and nitrite was quantified by HPLC using a 2000i Dionex pump with an ASA Dionex Ionpac 37014 column and a UV detector at 208 nm. To verify the specificity of NOS activity by nitrite measurement, L-NG monomethyl arginine acetate (L-NMMA), an inhibitor of all isoforms of NOS, was added to cytosolic preparations. L-NMMA at 0.1 mM inhibited nitrite production in all preparations. Cytosolic protein content was quantified by a micromethod based on the Folin–Ciocalteu reaction [17]. Our method of nitrite quantification in cell supernatants is similar to that described elsewhere [18, 19], the main difference being that HPLC, instead of the Griess reaction, was used. Amino acids were obtained from Fluka (Switzerland). Sodium nitrite and nitrate and other salts (sodium borate, sodium phosphate, calcium chloride, magnesium chloride) were purchased from Merck (Germany). L-NMMA acetate (Wellcome Foundation, UK) was a gift from Dr Salvador Moncada. The results were expressed as the median plus the range. The Mann–Whitney two-tailed test was used for between-group comparisons. Spearman rank order correlation test was used for between-group correlations. P <0.05 and R >0.6 were considered significant.

NOS activity was expressed as nitrite production (ng/mg of protein) following addition of a standard amount (0.1 mM) of L-arginine, the substrate of NOS, to the cytosols of PBMNC and PMN, respectively. PBMNC NOS activity was 107.5 (259.5–0.025) in SSc patients and 20.0 (49–3.25) in normal controls (P=0.052), whilst PMN NOS activity was 98.3 (407–0.025) in SSc patients and 32.2 (65.2–0.025) in normal controls (P=0.137). There was a statistically significant correlation between PBMNC and PMN NOS activity (R=0.84, P<0.01).

Mononuclear cells (MNC), mostly T cells of the helper/inducer phenotype, predominate in the dermis infiltrate in the inflammatory stages of SSc [20], and show excessive functional activity in the peripheral blood of SSc patients [21]. Our finding of a trend for increased NO synthesis in scleroderma PBMNC is in agreement with the data reported by Yamamoto et al. [8], and could lend further support to the hypothesis that MNC play a role in the pathogenesis of SSc. However, it is also possible that PBMNC may differ from the MNC found at sites of active tissue lesions. If this is the case, increased NO synthesis by PBMNC could simply reflect the degree of cell activation associated with increased production of pro-inflammatory cytokines.

We found no significant difference in PMN NOS activity between the SSc patients and the controls. PMN activation has been described in patients with SSc [22], particularly in those with active pulmonary fibrosis [23], whilst increased iNOS expression has been observed in the neutrophils from the lungs of patients with idiopathic pulmonary fibrosis [24], whose interstitial lung disease is very similar to that seen in SSc [25]. In this study, PMN NOS activity was comparable in SSc patients and in the controls, but values were much more scattered in the SSc group as compared with the latter, with seven patients out of 11 having PMN NOS values above the cut-off value of the upper quartile of the control group (in five cases at least twice as high). The activation of PMN NOS found in some of our SSc patients may be in keeping with the reported involvement of PMN in scleroderma, but it is also possible that it may be simply related to circulating factors stimulating the PMN.

Thus, our results seem to confirm that NO synthesis is dysregulated in SSc, as suggested by Yamamoto and others [8, 26]. As a potent vasodilator, and a pro-inflammatory agent, NO appears to be a double-edged sword whose potential effects in SSc could be both beneficial and detrimental, depending on the concentration and the local environment. Recent studies carried out at sites of active lesions are shedding new light on the pathogenic relevance of NO in SSc in both regards [8, 27, 28], possibly opening new avenues in the treatment of SSc [25].

The authors wish to thank Ms M. A. Chessa and Mr Walter Pensa for their skilful technical assistance.

Accepted 21 June 1999

References

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  6.  Belmont HM, Levartovsky D, Goel A et al. Increased nitric oxide production accompanied by the up-regulation of inducible nitric oxide synthase in vascular endothelium from patients with systemic lupus erythematosus. Arthritis Rheum 1997;40:1810–6.[Web of Science][Medline]
  7.  Kahaleh MB, Fan PS. The persistence of vasospastic and platelet adhesive properties for scleroderma microvascular endothelial cells in vitro. Arthritis Rheum 1996;39:S235 (Abstract).
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  19.  Carreras MC, Catz SD, Pargament GA, Del Bosco CG, Poderoso JJ. Decreased production of nitric oxide by human neutrophils during septic multiple organ dysfunction syndrome. Inflammation 1994;18:151–60.[Medline]
  20.  Roumm AD, Whiteside TL, Medsger TA, Rodnan GP. Lymphocytes in the skin of patients with progressive systemic sclerosis. Arthritis Rheum 1984;27:645–53.[Medline]
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  28.  Fajac I, Kahan A, Menkès CJ, Dessanges JF, Dall'Ava-Santucci J, Dinh-Xuan AT. Increased nitric oxide in the exhaled air in patients with systemic sclerosis. Clin Exp Rheumatol 1998;16:547–52.[Medline]
 

Reply

T. Yamamoto

Department of Dermatology, Tokyo Medical and Dental University, School of Medicine, Tokyo, Japan

We appreciate the interest of Cavallo et al. regarding our recent article on increased production of nitrite in PBMC of SSc patients [1]. Their work indicates that nitric oxide synthase (NOS) in PBMC is activated in SSc patients. We agree that NO synthesis is dysregulated in SSc patients. As they stated, increasing studies are being compiled suggesting that circulating mononuclear cells are activated which release increased amounts of NO in SSc patients. On the contrary, in vitro studies demonstrate that transforming growth factor beta or platelet-derived growth factor, which are representative fibrogenic cytokines, suppress NO production [2]. In fact, it is also reported that steady-state NO is decreased in SSc patients [3].

As there is a heterogeneity in the population of SSc patients, it will be important to assess the NO level in association with disease activity (duration from onset, limited or diffuse, oedematous or sclerotic phase, etc.) to determine whether NO is involved in the early inflammatory phase or sclerotic phase in future studies. Further studies are needed to clarify whether NO acts as a merit or demerit for SSc, which might lead to the treatment of SSc, as they described.

We have recently observed that bleomycin-stimulated PBMC show growth-stimulatory effects on fibroblasts [4], with enhanced NO release [5]. These effects were considered to be mediated by macrophages. Bleomycin is a representative drug which induces fibrosis. We hope that it might be a clue about the role of NO in the induction of fibrosis.

References

  1.  Yamamoto T, Sawada Y, Katayama I, Nishioka K. Increased production of nitric oxide stimulated by interleukin-1 ß in peripheral blood mononuclear cells in patients with systemic sclerosis. Br J Rheumatol 1998;37:1123–5.
  2.  Schini VB, Durante W, Elizondo E et al. The induction of nitric oxide synthase activity is inhibited by TGF-beta 1, PDGFAB and PDGFBB in vascular smooth muscle cells. Eur J Pharmacol 1992;216:379–83.[Web of Science][Medline]
  3.  Kahaleh MB, Fan PS. The persistence of vasospastic and platelet adhesive properties for scleroderma microvascular endothelial cells in vitro. Arthritis Rheum 1996;39:S235.
  4.  Yamamoto T, Katayama I, Nishioka K. Fibroblast proliferation by bleomycin stimulated peripheral blood mononuclear cell factors. J Rheumatol 1999;26:609–15.[Web of Science][Medline]
  5.  Yamamoto T, Katayama I, Nishioka K. Nitrite production in mouse 3T3 fibroblasts by bleomycin-stimulated peripheral blood mononuclear cell factors. Clin Exp Rheumatol 1999;17:343–6.[Medline]

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