Elevated plasma concentrations of nitric oxide, soluble thrombomodulin and soluble vascular cell adhesion molecule-1 in patients with systemic lupus erythematosus

C. Y. Ho, C. K. Wong, E. K. Li1, L. S. Tam1 and C. W. K. Lam

Department of Chemical Pathology and
1 Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, NT, Hong Kong


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. To investigate the correlations among plasma concentrations of nitric oxide (NO), soluble thrombomodulin (sTM) and vascular cell adhesion molecule (sVCAM-1), and whether these three molecules are associated with renal involvement in patients with systemic lupus erythematosus (SLE).

Methods. Plasma NO concentrations of 73 SLE patients (35 with renal disease, RSLE patients; 38 without renal disease, SLE patients) and 28 age- and sex-matched healthy control subjects were measured by the non-enzymatic Griess assay, and sTM and sVCAM-1 by enzyme-linked immunosorbent assay.

Results. In RSLE patients, plasma nitrite concentrations were significantly higher than in control subjects (P=0.009) and correlated positively with plasma sTM, plasma creatinine and urea (all P<0.05). Plasma sTM and sVCAM-1 concentrations were significantly elevated in RSLE and SLE patients (both P<0.0001) compared with controls. Plasma sTM was significantly correlated with plasma sVCAM-1, and both were correlated with plasma creatinine and urea and the SLE Disease Activity Index (all P<0.05).

Conclusion. Elevated plasma NO, sTM, and sVCAM-1 concentrations have significant intercorrelations and are strongly associated with renal involvement in SLE.

KEY WORDS: Systemic lupus erythematosus, Nitric oxide, Thrombomodulin, Vascular cell adhesion molecule, Glomerulonephritis.


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown aetiology affecting multiple organ systems. The role of vascular injury in the pathogenesis of SLE has been well described [13]. Circulating immune complexes of autoantibodies and self-antigens are deposited in the vascular walls of SLE patients and activate the complement pathways. The resulting complement products stimulate leucocytes to injure the vascular endothelium, leading to blood vessel destruction and organ injury (e.g. glomerulonephritis and vasculitis). It has been suggested that expression of adhesion molecules and overproduction of nitric oxide (NO) primes the vascular endothelium for subsequent injury, as reflected by increased release of thrombomodulin (TM) into the circulation [46].

NO is a biological messenger that mediates many physiological functions and pathological processes. It plays a vital role in host defence and immunity by modulating inflammatory responses. NO is synthesized from L-arginine, by constitutive NO synthase (cNOS) and inducible NO synthase (iNOS) [7, 8]. It is readily transformed into nitrite and nitrate, which are excreted into the urine. Expression of iNOS in response to phlogistic stimuli can lead to a sustained production of high concentrations of NO with cytotoxic effects [9]. Recent studies have shown that excessive NO production may play a pivotal role in the pathogenesis of SLE. In murine models, NO is found to be involved in the pathogenesis of arthritis and glomerulonephritis [10, 11]. Increased production of NO has been documented in rheumatoid arthritis [12, 13] and SLE [5, 14]. Up-regulated expression of iNOS was found in the kidneys of patients with active glomerulonephritis, including those with lupus [5].

TM is a cell-surface glycoprotein located at the luminal surface of the vascular endothelium and acts as a membrane-bound, high-affinity thrombin receptor [15]. It down-regulates coagulation by acting as a cofactor of thrombin-catalysed activation of protein C. Its soluble form (sTM) is detected in plasma and urine after endothelial injury, and has been reported as a predictor of thrombotic crisis [16]. TM has been used widely as a marker of microvascular endothelial injury and thrombotic events in various diseases, such as disseminated intravascular coagulation [17], multiple sclerosis [18] and rheumatic diseases [19]. Previous studies have reported that elevated sTM is associated with the disease activity of SLE, nephritis and vasculitis [6, 2022]. TM is thus considered to be a specific marker of endothelial cell injury.

Adhesion molecules, including vascular cell adhesion molecule-1 (VCAM-1), E-selectin and intercellular adhesion molecule-1 (ICAM-1), are essential for cellular interactions, and play an important role in the activation and adhesion of cells [23]. Up-regulated expression of adhesion molecules on leucocytes and vascular endothelium leads to the adherence of inflammatory cells to the blood vessel wall and their subsequent extravasation [3, 23]. Soluble adhesion molecules have been detected in plasma and thus serve as useful markers of both leucocyte and endothelial cell activation in different diseases, such as autoimmune disorders, including rheumatoid arthritis, vasculitis and SLE [2427]. A long-term study has revealed that elevated levels of soluble VCAM-1 (sVCAM-1), but not of soluble ICAM-1 (sICAM-1) and soluble E-selectin (sE-selectin), in SLE sera correlate positively with disease activity [28]. Plasma sVCAM-1 concentration is significantly elevated in patients with active lupus nephritis of WHO classes III and IV, and is decreased during remission [28, 29]. These results suggest that sVCAM-1 may be a promising marker for monitoring patients with lupus nephritis.

In this study, we measured the plasma concentrations of NO (as nitrite), sTM and sVCAM-1 and investigated their intercorrelations and their correlation with disease activity and renal function (in terms of plasma creatinine and urea concentrations) in SLE patients with and without renal disease.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
SLE patients, control subjects and blood samples
Seventy-three Chinese SLE patients were recruited at the Rheumatology Out-patient Clinic of the Prince of Wales Hospital, Hong Kong. The diagnosis of SLE was established according to the 1982 revised American Rheumatism Association criteria [30] and disease activity was evaluated with the SLE Disease Activity Index (SLEDAI) [31]. The patients were divided into two groups: 35 SLE patients with renal disease (RSLE group) and 38 SLE patients without renal disease (SLE group). Twenty-eight sex- and age-matched healthy Chinese volunteers were recruited as controls. Standard routine laboratory tests were used to measure the plasma creatinine and urea concentrations. Twenty millilitres of EDTA (ethylenediamine tetraacetate)-anticoagulated venous peripheral blood was collected from each patient and control subject. Plasma samples were preserved at -70°C for subsequent assays. The protocol was approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong and informed consent was obtained from all participants.

Assay for NO
Plasma NO in patient with SLE and control subjects was measured as nitrite concentration because NO is converted rapidly to nitrites and nitrates. The Bioxytech NO non-enzymatic assay kit (Oxis International, Portland, OR, USA) was used for measuring the plasma total nitrite concentration. Briefly, plasma samples were deproteinized by precipitation in zinc sulphate. After centrifugation, the supernatants and the nitrite standards were mixed overnight with 0.5 g granular cadmium beads for reduction of nitrate to nitrite. Following removal of cadmium beads, the supernatants were mixed with the Griess reagent in a 96-well flat-bottomed microtitre plate and the absorbance of developed colour was measured at 540 nm with a microtitre plate reader.

Assays for sTM and sVCAM-1
Plasma sTM and sVCAM-1 concentrations of SLE patients and control subjects were measured by enzyme-linked immunosorbent assay (ELISA) using reagent kits from Diaclone Research (Besançon, France) and R & D Systems (Minneapolis, MN, USA) respectively.

Statistical analyses
Because plasma concentrations of nitrite, sTM and sVCAM-1 were not in a Gaussian distribution, the Mann–Whitney rank sum test was used to assess the differences between patients and control subjects. Results were expressed as median (interquartile range). Spearman's rank correlation test was used to assess the correlations between the variables. All analyses were performed using the Statistical Package for the Social Sciences (SPSS) statistical software for Windows, version 9.0 (SPSS, Chicago, IL, USA). A probability (P) value less than 0.05 was considered as indicating a significant difference.


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
SLE patients and control subjects
The age, sex, SLEDAI score, duration of diagnosis, plasma creatinine and urea concentrations and drug treatment of the study populations are summarized in Table 1Go. Thirty-five SLE patients with renal disease (RSLE patients; 34 females and 1 male, mean±S.D. age 39.1±10.1 yr, range 20–59) and 38 SLE patients without renal disease (SLE patients; 38 females, 39.3±10.8 yr, range 20–67) were recruited. The period since the diagnosis of SLE at the time when patients were evaluated for this study was 12.4±6.3 yr (range 1.7–26.6) for RSLE patients and 9.0±6.8 yr (range 0.3–25.6) for SLE patients. The SLEDAI scores of RSLE and SLE patients were 7.9±5.9 (range 0–20) and 2.8±5.6 (range 0–32) respectively. The plasma creatinine concentrations of RSLE and SLE patients were 105.2±73.0 and 68.2±11.7 µmol/l respectively (normal range 44–107). The plasma urea concentrations of RSLE and SLE patients were 8.5±5.7 and 4.7±1.3 mmol/l respectively (P<0.0001, RSLE vs SLE); the normal range is 3.4–8.9 mmol/l. Patients were being treated with the following drugs, alone or in combination: prednisolone (RSLE, 6.7±5.1 mg daily, 100.0% of patients; SLE, 3.2±6.6 mg daily, 50.0% of patients), hydroxychloroquine (RSLE, 87.5±99.0 mg daily, 40.0%; SLE, 112.0±108.0 mg daily, 63.2%), azathioprine (RSLE, 20.0±30.0 mg daily, 37.1%; SLE, 9.0±30.0 mg daily, 13.2%) or cyclosporin A (RSLE, 20.9±43.5 mg daily, 25.7%; SLE, 0 mg daily, 0%). Twenty-eight sex- and age-matched normal control (NC) subjects (27 females and one male, aged 38.5±7.9 yr, range 22–51) were recruited. There was no significant difference among the ages of the RSLE, SLE and NC subjects (all P>0.05), and all the three groups were sex-matched.


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TABLE 1. Characteristics of RSLE and SLE patients and normal control (NC) subjects

 

Plasma concentrations of nitrite, sTM and sVCAM-1
As shown in Fig. 1Go, plasma concentrations of nitrite, sTM and sVCAM-1 were significantly higher in RSLE patients than in NC subjects [median (interquartile range) nitrite concentration 291.2 (183.4–465.4) vs 172.7 (105.1–249.7) µM, P=0.009; sTM, 2.0 (1.3–5.3) vs 0.4 (0.0–0.8) ng/ml, P<0.0001; sVCAM-1, 611.5 (433.8–1051.0) vs 316.0 (277.1–336.6) ng/ml, P<0.0001]. Plasma concentrations of sTM and sVCAM-1 were also significantly higher in SLE patients than NC subjects [sTM, 1.7 (1.1–2.4) vs 0.4 (0.0–0.8) ng/ml, P<0.0001; sVCAM-1, 457.4 (323.6–743.4) vs 316.0 (277.1–336.6) ng/ml, P<0.0001]. However, although the plasma nitrite concentration in SLE patients was slightly higher than in NC subjects, the difference was not significant (P=0.075). Plasma concentrations of sVCAM-1 in RSLE patients were significantly higher than in SLE patients (P=0.036) (Fig. 1CGo).



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FIG. 1. Plasma concentrations of nitrite (A), sTM (B) and sVCAM-1 (C) in RSLE, SLE and control subjects (NC). The median is indicated by the horizontal bar and each symbol represents an individual. Differences between RSLE or SLE patients and the control subjects were tested with the Mann–Whitney rank sum test.

 

Correlations among plasma nitrite, sTM, sVCAM-1, creatinine, urea and SLEDAI
In RSLE patients, plasma nitrite showed a positive correlation with sTM (r=0.543, P=0.004), creatinine (r=0.410, P=0.034) and urea (r=0.685, P<0.0001) (Table 2Go). Plasma sTM showed a strong correlation with sVCAM-1 (r=0.728, P<0.0001), creatinine (r=0.646, P<0.0001), urea (r=0.534, P=0.001) and SLEDAI (r=0.410, P=0.016). Plasma sVCAM-1 also correlated significantly with creatinine (r=0.394, P=0.021), urea (r=0.360, P=0.034) and SLEDAI (r=0.694, P<0.0001) (Table 2Go).


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TABLE 2. Correlations among disease activity parameters in RSLE patients

 
In SLE patients, plasma sVCAM-1 correlated with sTM (r=0.334, P=0.043) and SLEDAI only (r=0.541, P=0.0004) (Table 3Go).


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TABLE 3. Correlations among disease activity parameters in SLE patients

 


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Overproduction of NO could contribute to tissue injury, given its capacity to increase vascular permeability, generate toxic free radicals such as peroxynitrite, and induce cytotoxicity [9]. Belmont et al. [5] found that endothelial cells overexpress iNOS during periods of active SLE, and are therefore a potential cellular source of excess NO production. Measurement of total nitrite confirmed elevation of the plasma NO concentration in RSLE patients compared with controls (Fig. 1AGo). This finding is similar to those of previous studies, which reported increased serum nitrite levels in SLE patients with active nephritis [5, 14, 32].

Due to the extremely short half-life of NO (milliseconds in most instances), its production cannot be measured easily in vivo. Metabolites of NO, including nitrite and nitrate, are widely used as indicators, and measurements of them are influenced by rates of both production and clearance. We found statistically significant correlations of plasma nitrite with creatinine and urea (Table 2Go). Elevated plasma creatinine and urea concentrations are commonly used for the assessment of impaired glomerular function, which may be caused by circulating nephritogenic autoantibodies and immune complexes [33]. These results suggest that plasma nitrite might be related to the renal disease found in RSLE patients (e.g. lupus nephritis). These patients might have increased expression of iNOS, in particular in the vascular endothelium of the kidneys. Alternatively, blood urea nitrogen and creatinine might also be sources of plasma nitrite and nitrate.

TM is an endothelial cell-membrane glycoprotein released upon damage of the endothelial cells [16]. Elevated sTM levels have been shown in patients with SLE, in particular those with lupus nephritis [6, 21, 22]. Our results demonstrate that plasma concentrations of sTM in both RSLE and SLE patients were significantly elevated when compared with control subjects (Fig. 1BGo). Recent studies have shown that serum or plasma sTM can be a reliable marker, with advantages over established serological parameters, to indicate SLE disease activity [6, 21, 34, 35]. We found a significant correlation between plasma sTM and SLEDAI in RSLE patients (Table 2Go), confirming that sTM is a promising marker reflecting SLE disease activity.

As sTM is excreted by the kidneys, increased concentrations are observed in renal failure and parallel elevated serum creatinine concentrations [20, 21, 36]. Statistically significant correlations of plasma sTM with creatinine and urea were observed in the RSLE patients, suggesting that reduced creatinine and urea clearance due to renal failure might account in part for the elevation of plasma sTM. It has been suggested that elevated sTM levels reflect endothelial cell damage rather than activation, because endothelial cell–leucocyte adhesion and interaction after the activation of inflammatory cytokines might result in the release of TM from the endothelial cell surface [37]. We found that plasma nitrite significantly correlated with plasma sTM concentrations in RSLE patients but not in SLE patients, suggesting that increased NO production during endothelial cell activation might be closely associated with the endothelial damage, as reflected by elevated sTM levels, in particular in the kidneys of RSLE patients.

Activated endothelial cells are a major source of VCAM-1, being shed from the cell surface into the circulation during endothelial activation or damage. Previous studies have reported that increased serum levels of sVCAM-1 correlate with disease activity [24, 29, 38]. Our present results show that plasma sVCAM-1 concentrations in RSLE and SLE patients were significantly higher than in control subjects (Fig. 1CGo) and correlated strongly with SLEDAI (Tables 2Go and 3Go). These results suggest that the sVCAM-1 level might be useful in monitoring SLE patients, especially those with renal diseases. In RSLE patients, significant correlation between plasma sVCAM-1 and creatinine or urea revealed that renal failure due to tissue inflammation might result in enhanced VCAM-1 expression and hence the increased sVCAM-1 level (Table 2Go). Although elevated serum or plasma levels of sICAM-1 and sE-selectin have been demonstrated in other studies, their correlations with disease activity vary [2528, 39]. This indicates that, among the soluble adhesion molecules, sVCAM-1 seems to be the best parameter of SLE activity.

Our findings showed that plasma sVCAM-1 correlated strongly with sTM concentration in both RSLE and SLE patients, which highlights its role as a vasculopathy marker during thrombotic events (Table 2Go). Plasma levels of the endothelial molecules studied (NO, sTM and sVCAM-1) correlated with creatinaemia and uraemia in RSLE patients, reflecting the in vivo endothelial activation and damage in renal failure of SLE. As observed by us, the plasma concentration of NO, sTM or sVCAM-1 in our cohort of patients did not correlate with the dosages of the drugs listed in Table 1Go.

In conclusion, elevated concentrations of circulating NO, sTM and sVCAM-1 can serve as indicators of endothelial activation and/or damage, which may occur in the pathogenesis of SLE with thrombotic and/or renal involvement. Further studies are being conducted to assess the clinical utility of these parameters.


    Acknowledgments
 
This study was supported by a Direct Grant for Research from The Chinese University of Hong Kong and a donation from Zindart (De Zhen) Foundation Ltd, Hong Kong.


    Notes
 
Correspondence to: C. W. K. Lam, Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, NT, Hong Kong. E-mail: waikeilam{at}cuhk.edu.hk Back


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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 11 April 2002; Accepted 10 June 2002