Circulating soluble adhesion molecules in ANCA-associated vasculitis

Jordi Ara,1, Eduard Mirapeix1, Pilar Arrizabalaga1, Rosa Rodriguez1, Carlos Ascaso2, Rosa Abellana2, Josep Font3 and Alexandre Darnell1

1 Nephrology Service 2 Biostatistical and Epidemiological Unit and 3 Autoimmune diseases Unit, Hospital Clínic, Universitat de Barcelona, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain



   Abstract
 Top
 Abstract
 Introduction
 Subjects
 Methods
 Other laboratory studies
 Results
 Discussion
 Conclusions
 References
 
Background. To evaluate whether changes in concentrations of soluble (s) E-selectin, sP-selectin, sL-selectin, intercellular adhesion molecule 1 (sICAM-1), and vascular cell adhesion molecule 1 (sVCAM-1) reflect disease activity in patients with ANCA-associated vasculitis and whether serum levels of these adhesion molecules are related to the degree of renal failure in patients with chronic renal failure (CRF).

Subjects and methods. A sandwich ELISA was used to measure these soluble adhesion molecules in (i) sera from 20 patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (10 patients with Wegener's granulomatosis (WG) and 10 patients with microscopic polyangiitis (MPA)), obtained at the time of diagnosis and during the remission period; (ii) sera from 40 patients with CRF not undergoing haemodialysis.

Results. At the time of diagnosis, serum levels of sE-selectin, sICAM-1 and sVCAM-1 (88±42 ng/ml, 437±184 ng/ml, 1720±1174 ng/ml respectively) were significantly higher in patients with ANCA-associated vasculitis than in healthy controls (P<0.0001, P=0.002 and P=0.001 respectively). Serum sP-selectin values did not differ from those obtained in normal donors. In contrast, sL-selectin levels (940±349 ng/ml) were significantly lower in patients than those recorded in healthy controls (P<0.0001). A significant decrease in concentrations of sE-selectin, sP-selectin, sICAM-1, and sVCAM-1 was observed between active and remission phases (P<0.0001, P=0.002, P=0.001 and P=0.001 respectively). No significant differences were observed in sL-selectin levels between active and remission phases. sL-selectin concentrations (802±306 ng/ml) during the remission phase remained lower than those observed in healthy controls (P<0.0001). No correlation was observed between serum creatinine and sE-selectin, sP-selectin, sICAM-1 and sVCAM-1 in patients of the CRF group. A slight negative correlation was established between creatinine and sL-selectin concentration.

Conclusions. Increased serum levels of sE-selectin, sICAM-1, and sVCAM-1 and decreased levels of sL-selectin in active ANCA-associated vasculitis, and the normalization of sE-selectin, sICAM-1, and sVCAM-1 during the remission phase suggest that the concentration of soluble levels of these adhesion molecules reflects disease activity. The decrease in sP-selectin levels between active and inactive phases also suggest that this receptor may reflect clinical activity.

The lack of correlation between serum levels of sE-selectin, sP-selectin, sICAM-1, and sVCAM-1 and the degree of renal failure in patients with CRF suggests that the mechanism of clearance of these molecules is not renal.

Keywords: ANCA-associated vasculitis; disease activity; microscopic polyangiitis; soluble adhesion molecules; Wegener's; granulomatosis



   Introduction
 Top
 Abstract
 Introduction
 Subjects
 Methods
 Other laboratory studies
 Results
 Discussion
 Conclusions
 References
 
Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA) are two different forms of small-vessel vasculitis with renal involvement, associated with antineutrophil cytoplasmic antibodies (ANCA). The glomerular lesions of these diseases are pathologically similar, being characterized by focally and segmentally variable glomerular inflammation, necrosis, and crescent formation [1].

The development of vascular and glomerular infiltrates requires dynamic interactions between leukocyte surface receptors and their ligands on the endothelial cell surface. This process can be divided into three steps: (i) initial interaction of leukocytes on activated endothelium mediated by selectin (E-selectin, P-selectin and L-selectin) and carbohydrate ligands; (ii) leukocyte activation with firm adhesion to endothelial cells mediated by interaction between integrins and immunoglobulin superfamily molecules (ICAM-1, VCAM-1); and finally, (iii) leukocyte extravasation into the surrounding tissue [2,3].

Circulating forms of selectins and immunoglobulin superfamily adhesion molecules can be detected in serum, plasma, and other body fluids. These molecules may derive from proteolytic cleavage of membrane-anchored forms or from alternatively spliced transcripts that lack a transmembrane domain [4]. In vitro studies have shown that these soluble forms appear in the supernatant of activated leukocytes or cytokine-stimulated endothelial cells [5,6]. The amount of soluble adhesion molecules released in vitro in activated endothelial cell supernatant correlates with their levels of cell surface expression [5].

Elevated levels of circulating adhesion molecules have been detected in disorders where leukocyte/ endothelial cell interactions play a significant role, such as infections, atherosclerosis, neoplasms, chronic inflammatory diseases and vasculitis, and have been considered a consequence of endothelial and/or immune activation [713]. Some authors have suggested a role of soluble adhesion molecules as markers for disease activity in autoimmune disease and vasculitis [1119].

In this study we measured circulating levels of soluble E-selectin, P-selectin, L-selectin, ICAM-1 and VCAM-1 in patients with ANCA-associated vasculitis such as Wegener's granulomatosis and microscopic polyangiitis, as well as in patients with chronic renal failure (CRF), in order to determine: (i) whether changes in soluble adhesion molecules reflect disease activity; (ii) whether serum levels of the adhesion molecules studied are related to the degree of renal failure in a group of patients with stable-phase CRF.



   Subjects
 Top
 Abstract
 Introduction
 Subjects
 Methods
 Other laboratory studies
 Results
 Discussion
 Conclusions
 References
 
Patients
Vasculitis group
Twenty patients with ANCA-associated vasculitis undergoing regular follow-up at our hospital between 1993 and 1997 were studied. Ten patients had WG and 10 had MPA. Diagnosis of MPA and Wegener's granulomatosis was based on the criteria accepted at the Chappel-Hill conference [20]. Diagnosis of WG necessitates a suitable clinical manifestation supported by either confirmatory histological or ANCA serological findings or both. All patients had renal-biopsy-proven vasculitis. Clinical, analytical and serological features at diagnosis are summarized in Table 1Go.


View this table:
[in this window]
[in a new window]
 
Table 1. Clinical and analytical features of patients included

 
All patients were treated with immunosuppressive drugs that included steroids (1 mg/kg) with swift subsequent tapering, and cyclophosphamide (2 mg/kg) for the induction of remission, and cyclophosphamide plus low doses of steroids in the remission period. Pulses of methylprednisolone (three pulses of 1 g) were added to the induction treatment in patients with severe renal involvement.

Serum from the 20 patients was obtained at the time of diagnosis (pre-treatment) and during the remission period (between 3 and 6 months after diagnosis). Two patients required haemodialysis during the active phase. The serum samples were obtained prior to start this treatment. Whenever serum samples were obtained, the absence of any other disease, such as infection or neoplasia, was confirmed.

CRF group
In order to ascertain whether there was any link between soluble forms of adhesion molecules and degree of renal failure as measured by creatinine levels, we examined forty patients with CRF, not undergoing haemodialysis (22 male and 18 female, mean age 64.85±9.55 years (range 44-79 years). The group consisted of 10 patients with creatinine levels of between 1.3 and 3 mg/dl, 10 with levels of between 3.1 and 5 mg/dl, 10 with levels of between 5.1 and 7 mg/dl, and finally 10 with levels of between 7.1 and 9 mg/dl. The aetiology of CRF in these patients was nephroangiosclerosis (27, 67.5%), diabetic nephropathy (three, 7.5%), tubulointerstitial nephropathy (six, 15%), and non-ANCA-related glomerular nephropathy (four, 10%). None of these patients had neoplasia, liver disease, or concomitant infections.

Control group
Control serum samples were obtained from 20 healthy volunteers, eight men and 12 women, with a mean age of 43.25±12.51 years (range 25–65 years). All subjects had normal hepatic and renal function.

In all cases, serum was aliquoted, frozen at -70°, and stored until measurement of adhesion molecules. All samples were obtained with the prior consent of patients.



   Methods
 Top
 Abstract
 Introduction
 Subjects
 Methods
 Other laboratory studies
 Results
 Discussion
 Conclusions
 References
 
Assessment of vasculitis activity
Patients were considered active at the time of diagnosis prior to the beginning of corticosteroid and immunosuppressive therapy. Vasculitis activity was assessed using clinical and biological parameters. Clinical activity was assessed using the Birmingham Vasculitis Activity Score (BVAS) [21]. This is a validated method that measures the degree of disease involvement in nine organs or systems. According to the BVAS, patients with a score above 5 were considered active. Biological activity was assessed measuring serum C reactive protein (CRP) concentrations (normal values below 0.8 mg/dl).

Remission was defined as the absence of clinical activity using the BVAS list (score below 5), supported by normal CRP levels. Renal remission was defined as the absence of microhaematuria or haematic cell cast, together with improved or stable renal function [21].

Measurement of soluble adhesion molecules
Levels of sE-selectin, sL-selectin, sP-selectin, sICAM-1, and sVCAM-1 were analysed using sandwich ELISA following manufacturer's instructions, using commercially available kits from R&D Systems, Avingdon, UK. All measurements were performed in duplicate. Briefly, a horseradish peroxidase-conjugated monoclonal antibody against adhesion molecules (sE-selectin, sP-selectin, sL-selectin, sICAM1, and sVCAM1) was added to microtitre plates coated with murine monoclonal IgG antibody recognizing a different epitope of the corresponding molecule. After incubation with patient and control samples and standards at appropriate dilutions, the colour reaction was developed with tetramethylbenzidine, and plates were read on an automated multiscanner at 450 nm (reference wavelength 600 nm).

The calculated intra-assay coefficient of variation was 2.73% for s-E-selectin, 1.6% for sP-selectin, 0.92% for sL-selectin, 2% for sICAM-1 and 3.27% for sVCAM-1.



   Other laboratory studies
 Top
 Abstract
 Introduction
 Subjects
 Methods
 Other laboratory studies
 Results
 Discussion
 Conclusions
 References
 
ANCA assays
ANCA were assayed by IFI and ELISA, both at diagnosis and on remission. IFI was performed according to the method described by Wilk et al. [22]. Briefly, it consists in determining indirect immunofluorescence on total leukocytes fixed on glass slides with ethanol. IFI was considered positive at a serum dilution of 1/20. MPO-ELISA was performed as follows: microtitre plates were sensitized overnight with 1.5 µg of purchased human MPO (Calbiochem, La Jolla, USA) at 4 °C in a carbonate buffer, pH 9.6. Plates were washed twice with 0.05% phosphate-buffered saline (PBS)–Tween 20 and blocked with 1% PBS–BSA. Patient and control sera were tested at a dilution of 1/100 in 0.05% PBS–Tween 20 with added 1% gelatin. After incubation for 1 h at room temperature, a 1/2000 dilution of goat anti-human IgG conjugated with alkaline phosphatase was applied (Dako, Barcelona, Spain). Colour absorbance was read at 405 nm with an MR 5000/7000 microplate reader (Dinatech).

Results were expressed as arbitrary units by reading off a standard curve, and the normal range was determined by the mean plus 2 SD obtained from 50 normal blood donors. The quantitative anti-PR3 ELISA was performed using a commercial kit (Diagnostika GMBH, Hamburg, Germany). Sera below 10 IU were considered negative.

Measurement of CRP
Serum CRP concentrations were measured by standard nephelometry. Values below 0.8 mg/dl were considered normal.

Statistical analysis
All data are expressed as mean±SD. The normality hypothesis of the variables was tested using the Kolmogorov–Smirnov test. Comparisons of serum adhesion molecule levels between WG and MPA groups in active and inactive phases were analysed by covariance analysis (ANCOVA). A paired Student test was used to compare differences between serum adhesion levels during active and inactive phases. Differences between two independent groups were analysed by Student t-test for independent samples. Correlation between variables were evaluated by Pearson's correlation coefficient. The cut-off point between active and inactive phases was calculated by ROC curve. The sensitivity and specificity of the cut-off point were estimated using Stata program version 5.0 (Stata, Santa Mónica, California, USA).

Statistical analyses were performed using SPSS program version 6.0 (SPSS Inc. Chicago, USA). Differences were taken to be significant at P<0.05.



   Results
 Top
 Abstract
 Introduction
 Subjects
 Methods
 Other laboratory studies
 Results
 Discussion
 Conclusions
 References
 
Circulating soluble levels of adhesion molecule levels in WG and MPA
Serum levels of the adhesion molecules studied (sE-sel, sL-sel, sP-sel, ICAM-1, VCAM-1) at the time of diagnosis (active phase) and during remission (inactive phase) for patients in the WG and MPA groups are summarized in Figure 1Go.



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 1. Serum levels of sE-selectin, sP-selectin, sL-selectin, sICAM-1, and sVCAM-1 in patients with WG and MPA, and in healthy control group. Box plots indicate the overall range (error bars), 25-75% range (boxes) and median value (horizontal lines) of serum levels of adhesion molecules.

 
No significant differences were found between WG and MPA with regard to levels of the adhesion molecules in the active phase. A positive correlation was recorded between all adhesion molecules studied and phases of the disease (active and inactive) (sE-selectin r=0.26 P=0.0009; sL-selectin r=0.26, P=0.0021; sP-selectin r=0.48 P=0.004; sICAM-1 r=0.26 P=0.024; sVCAM-1 r=0.62 P=0.001), although no differences were encountered between WG and MPA. Thus it was assumed that serum levels of the five adhesion molecules studied were not significantly different for the two groups, either in the active or in the inactive phases; henceforth, results are expressed for a single unified vasculitis group.

Circulating soluble adhesion molecules in unified vasculitis group
sE-selectin
Mean sE-selectin levels during the active phase were significantly higher than those recorded in healthy controls (t=4.10, P<0.0001). These levels fell significantly on clinical remission (t=4.94, P<0.0001). Mean decrease between phases was 37.157 ng/ml (95% CI:21.42; 52.89). No significant differences were observed in sE-selectin levels between patients in remission and healthy controls (P=0.951) (Figure 2Go).



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 2. Serum levels of sE-selectin, sP-selectin, sL-selectin, sICAM-1, and sVCAM-1 in patients with vasculitis, and in healthy control group. Box plots indicate the overall range (error bars), 25-75% range (boxes) and median value (horizontal lines) of serum levels of adhesion molecules.

 

sL-selectin
Mean sL-selectin levels in vasculitis patients during the active phase were significantly lower than those recorded in healthy controls (t=6.99, P<0.0001). No significant difference was observed in sL-selectin levels between active and inactive phases (t=1.88, P=0.075). Concentrations during the remission phase remained significantly lower than those observed in controls (t=8.71, P<0.0001; Figure 2Go).

sP-selectin
sP-selectin levels were significantly higher in the active than in the inactive phase (t=3.55, P=0.002). Mean decrease between phases was 56.97 ng/ml (95% CI, 23.36; 90.57). No significant difference in concentrations was observed, however, between active phase and controls (t=-1.01, P=0.322) or between inactive phase and controls (t=1.42, P=0.164; Figure 2Go).

sICAM-1
Mean sICAM-1 levels during the active phase were significantly higher than those recorded in healthy controls (t=-3.42, P=0.002). A significant decrease in concentration was recorded between active and inactive phases (t=3.83, P=0.001). Mean decrease between phases was 127.45 ng/ml (95% CI, 57.72; 197.18). No significant differences were observed for this adhesion molecule between patients in remission and healthy controls (t=-0.76, P=0.454; Figure 2Go).

sVCAM-1
Mean sVCAM-1 levels during the active phase were significantly higher than those recorded in healthy controls (t=3.70, P=0.001). A significant decrease in concentration was recorded between active and inactive phases (t=4.02, P=0.001). Mean decrease between phases was 496.48 ng/ml (95% CI, 238.17; 754.80). No statistically significant differences were observed for this adhesion molecule between inactive-phase patients and healthy controls (t=-2.18, P=0.054; Figure 2Go).

ROC curves
Area of the ROC curve for sE-selectin was 0.786 (standard error 0.073). The proposed cut-off point was 78.6 ng/ml, yielding a sensitivity of 55% (IC 95% (31.6-76.9%)) and a specificity of 90% (IC 95% (68.3-98.5%)). For sICAM-1, the area of the ROC curve was 0.740 (standard error 0.079). The proposed cut-off point of 269.2 ng/ml gave a sensitivity of 95% (IC 95% (75.1-99.2%)) and a specificity of 50% (IC 95% (27.2-72.8%)).

Correlation between serum adhesion molecule levels and renal function, ANCA, CRP, and BVAS
Renal function
A positive correlation was recorded between VCAM-1 and creatinine levels during the active phase (r=0.5171, P=0.02), though not between other adhesion molecules and this variable.

To determine the possible link between the fall in adhesion molecule levels (sE-selectin, sP-selectin, sICAM-1, and sVCAM-1) from active to inactive phase and the decrease in creatinine concentrations, Pearson's correlation coefficient was calculated for decreases in adhesion molecules (active-phase adhesion molecule concentrations minus inactive-phase adhesion molecule concentrations) and decreases in creatinine in the same phases (active-phase creatinine minus inactive-phase creatinine). Results failed to suggest any link between the two decreases.

Correlation between differing creatinine values and:

ANCA
A positive correlation was recorded between and sE-selectin, s-Pselectin, ICAM-1, and sVCAM-1 and ANCA levels (r=0.47, r=0.34, r=0.55, r=0.36 respectively)

CRP/BVAS
No correlation was observed between CRP and serum adhesion molecule levels during the active phase, or between molecule levels and the BVAS score.

Correlation between different adhesion molecules
No correlation was observed between serum levels of different adhesion molecules during active-phase vasculitis.

CRF group
Mean serum levels of sE-selectin, sICAM-1 and sVCAM-1 were significantly higher than those recorded in healthy controls (t=3.11, P=0.003; t=4.54, P=0.0001; t=7.06, P=0.0001 respectively). No significant differences were observed in sP-selectin levels between the CRF group and the healthy control group (t=0.87;P=0.388). Finally, mean sL-selectin levels in this group were significantly lower than those observed in the healthy control group (t=2.35, P=0.022).

No correlation was observed between serum creatinine and the variables sE-selectin (r=-0.06, P=0.71), sP-selectin (r=-0.32, P=0.06), sICAM-1 (r=0.08, P=0.61) and sVCAM-1 (r=0.33, P=0.061). A negative correlation was established between creatinine and sL-selectin (r=-0.32, P=0.04).



   Discussion
 Top
 Abstract
 Introduction
 Subjects
 Methods
 Other laboratory studies
 Results
 Discussion
 Conclusions
 References
 
Serum concentrations of soluble ICAM-1, VCAM-1, and E-selectin in patients with active-phase, ANCA-positive, small-vessel vasculitis (WG and MPA) were higher than those recorded in controls. Concentrations of these molecules fell sharply on clinical remission. Serum L-selectin levels in both active and inactive phases were lower than controls; no significant inter-phase differences were observed. Finally, active-phase P-selectin levels were significantly higher than inactive-phase values, although no significant difference in serum levels was recorded between active-phase vasculitis patients and healthy controls.

ICAM-1 is basally expressed in significant amounts in a limited number of cell types, including monocytes and endothelial cells and it is widely inducible, or upregulated, on many cells including lymphocytes, monocytes, dendritic cells, epithelial cells, and endothelial cells under appropriate stimuli [23]. VCAM-1 has a more restricted distribution, mainly in the endothelium, and E-selectin is exclusively expressed by endothelial cells [24].

Increased circulating levels of adhesion molecules ICAM-1, VCAM-1 and E-selectin, have been considered a consequence of endothelial cell activation, particularly in the case of E-selectin [5,25]. Both ICAM-1, and VCAM-1 are also expressed and released by activated lymphocytes and macrophages; therefore, their increase may also reflect immune activation.

Elevated soluble adhesion molecule levels have been demonstrated by ELISA in the sera of patients with vasculitis [26]. Some initial studies included patients with a variety of diseases and are therefore difficult to interpret. [8,11,27]. In giant-cell arteritis, high levels of ICAM-1 and normal values of E-selectin, and VCAM-1 are reported during the active phase. Circulating ICAM-1 concentrations clearly correlate with clinical activity [13]. Increased levels of ICAM-1, VCAM-1, and E-selectin have been shown in patients with active PAN [12]. Increased ICAM-1 and E-selectin levels also have been demonstrated in Kawasaki disease [28,29], with higher levels of ICAM-1 in patients with coronary-artery lesions. Furukawa et al. [28] suggest that serum ICAM-1 level is an important predictor for the severity of vascular damage in Kawasaki disease. In Wegener's granulomatosis, ICAM-1 and VCAM-1 levels, but not E-selectin levels, are significantly elevated and correlate with disease activity. [10]. Tesar et al. [30] show that patients with MPA in active phase (n=5) had elevated levels of ICAM-1 and VCAM-1.

Histological examination of the kidney confirms that adhesion molecules ICAM-1 and VCAM-1 are over-expressed in patients with ANCA-positive, small-vessel vasculitis. [31] and there is some evidence that suggests a high degree of correlation between histological expression of ICAM-1 and its serum concentrations [32].

In our study, serum concentrations of sE-selectin, sICAM-1, and sVCAM-1 in patients with active phase of ANCA-positive, small-vessel vasculitis were higher than those recorded in controls. Concentrations of these molecules fell sharply on clinical remission. This behaviour is consistent when the two groups (WG and MPA) are viewed separately. Given the sample size used here, it cannot be stated unequivocally that the two groups are the same in term of soluble adhesion molecules, although descriptive statistics and inter-group correlations strongly support a high degree of similarity.

The restriction of E-selectin to activated endothelium makes this adhesion receptor an attractive marker of endothelial cell activation. Initial studies in vasculitis patients have yielded conflicting results. While some authors report high levels of E-selectin in various types of vasculitis [13,33], others find normal levels [10]. For example, Stegeman et al. [10] report normal mean soluble E-selectin levels in a group of 22 active-phase WG patients. This study shows that patients with generalized WG had significantly higher E-selectin levels than patients with localized WG. No patients with localized WG were included in the present study, which might account for the high E-selectin levels recorded in the active phase. Other authors report a progressive increase in serum E-selectin concentrations prior to the onset of clinical relapse [10]. Our results suggest a clear relationship between serum E-selectin levels and clinical activity in both WG and MPA.

Previous studies of WG, and the present study of MPA and WG show that ICAM-1 levels mirror the clinical course of the disease. The successful prevention of crescentic glomerulonephritis with anti-ICAM-1 antibodies has been demonstrated experimentally [34]. These findings suggest that this adhesion molecule may play a major role in the development of inflammatory lesions, and thus may in future furnish helpful markers of disease activity in patients with ANCA vasculitis. The results obtained with ROC curves here indicate that sE-selectin levels and sICAM-1 levels may provide a valuable tool for differentiating between active- and inactive-phase patients.

sVCAM-1 levels also showed good correlation with clinical activity. A positive correlation was found here between serum creatinine levels in active-phase patients and serum VCAM-1 levels. At the same time, no correlation was observed between VCAM-1 and creatinine in the CRF group. It is felt that this molecule may be a useful marker of histological activity in tissue [31]. If the present results are confirmed, serum VCAM levels may be valuable as an indicator of the severity of renal vasculitis.

After starting immunosuppressive treatment, when patients are in the inactive phase, E-selectin, ICAM-1, and VCAM-1 levels were significantly lower than those recorded during the active phase, and no differences were observed between inactive-phase patients and healthy controls. This decrease in adhesion molecule levels may be linked to the treatment; it has been shown that corticoids decrease the production of cytokines, thus possibly reducing adhesion molecule expression or function [35]. It has been demonstrated in vitro that corticosteroids directly decrease endothelial ICAM-1 and E-selectin expression [36].

In some patients, inactive-phase adhesion molecule levels, though significantly lower than those recorded during the active phase, were still considerably higher than control values (particularly VCAM-1). Preliminary results for various groups suggest that although adhesion molecules decrease on clinical remission, they may not actually return to normal [12]. These findings might reflect a persistent exposure of endothelial cells to a mild remaining inflammatory microenvironment.

As indicated earlier, there was a good correlation between clinical activity and levels of circulating soluble adhesion molecules. However, there was no evidence of correlation between CRP values and BVAS score with levels of soluble adhesion molecules. In contrast, a strong correlation was noted between ELISA-measured ANCA levels and concentrations of the five soluble adhesion molecules.

P-selectin is a membrane glycoprotein located in alpha granules of platelets and Weibel–Palade bodies of endothelial cells. When platelets or endothelial cells are activated by agonists such as thrombin or histamine, P-selectin is rapidly translocated to the cell surface and can be a receptor for leukocytes at sites of inflammation [37].

P-selectin has not been fully studied in human glomerulonephritis. Segawa et al. [38] showed that P-selectin was associated with both glomerular and interstitial leukocyte accumulation in human proliferative glomerulonephritis, and might be expressed by two distinct mechanisms that are the activated platelets in glomeruli and de novo expression in the interstitial lesions. Soluble sP-selectin may be useful marker for predicting in situ P-selectin expression and could reflect vascular damage and local platelet activation [38]. This is the first report about serum sP-selectin levels in patients with ANCA-associated vasculitis. We showed that sP-selectin levels were higher in the active phase of vasculitis than in the control group but the difference was not significant. P-selectin probably plays a role in the very early stages of leukocyte/endothelial cell interaction, and at the time of diagnosis all patients had well-established lesions of several weeks’ standing [37,39]. A significant decrease in concentrations of this adhesion molecule was recorded between the active and the inactive phase of the disease. This fact suggests that sP-selectin may correlate with clinical activity.

L-selectin is a cell surface receptor on granulocytes, lymphocytes, and monocytes, which is responsible for the initial contact of leukocytes with the endothelium [40]. The extracellular domain of L-selectin is proteolytically shed from leukocytes following cellular activation in vitro [41]. The shed form is functionally active, and at high concentrations can inhibit attachment to the endothelium [42]. Patients with sepsis and HIV infection showed markedly elevated L-selectin levels in serum [6]. In contrast to these diseases, patients who progressed to adult respiratory distress syndrome (ARDS) had significantly lower plasma L-selectin levels, and a good correlation was found between low values of L-selectin and indices of subsequent lung injury and mortality [43]. This is the first report about serum levels of L-selectin in patients with ANCA-associated vasculitis. We showed that patients with MPA and WG (active and inactive phases) had low levels of sL-selectin. This apparent paradox has also been observed in Kawasaki disease [6], systemic sclerosis, and other vasculitides [44].

Low levels of sL-selectin in patients with vasculitis may be due to reduced expression in leukocytes, reduced shedding or increased internalization of cell bound L-selectin. sL-selectin may also bind with stronger than usual affinity to possible up-regulated levels of its ligand(s) on the endothelial cell (i.e. gly-CAM-1 and/or MAdCAM-1). The implications of the present findings (low levels of sL-selectin in vasculitis) are unclear. sL-selectin retains functional activity [42], so that if the soluble molecule does have a physiological role in inhibiting adhesion, it may be speculated that low levels indirectly promote the adhesion process, and this may be related to the presence of leukocytes in the vessel wall and/or injury to the endothelium.

In the present study-group, inflammatory disease was concurrent with renal failure. The question of whether elevated levels of adhesion molecules in the blood are due to an increase in production and/or to a reduction in elimination remains at present unanswered, and the mechanisms of clearance of these molecules are largely unknown.

Like other authors [11,32], we observed no significant correlation between creatinine levels and serum levels of sE-selectin, sP-selectin, sICAM-1, and sVCAM-1 in patients with CRF. The lack of correlation between the degree of renal failure and sE-selectin, sICAM-1 and sVCAM-1 levels may be due to the high molecular weight (approximately 100 kDa) of these proteins.

Patients with active vasculitis had worse renal function than inactive patients, but no relationship was observed between serum concentrations of adhesion molecules and the degree of renal failure in these patients, except for active-phase VCAM-1. No statistically significant correlation was observed between the degree of improvement of renal function in these patients and the decrease in levels of adhesion molecules (sE-selectin, sP-selectin, sICAM-1, and sVCAM-1) observed between active phase and remission.

Low levels of sL-selectin were observed in active and inactive phases of vasculitis, and normal values of E-selectin, P-selectin, ICAM-1, and VCAM-1 were observed in remission patients although some of these patients had impaired renal function.

Only three patients with active vasculitis and normal renal function were included in this study and all of them had elevated levels of sE-selectin, sICAM-1 and sVCAM-1 in the active phase and normal values of these soluble adhesion molecules in the remission period.



   Conclusions
 Top
 Abstract
 Introduction
 Subjects
 Methods
 Other laboratory studies
 Results
 Discussion
 Conclusions
 References
 
Patients with active-phase WG and MPA share the same serological pattern of expression for the five adhesion molecules studied.

The increase in E-selectin, ICAM-1 and VCAM levels in active-phase vasculitis and the return to normal values during the inactive phase suggest that the serum concentration of soluble forms of these adhesion molecules reflect disease activity. The difference in serum sP-selectin levels between active and inactive phases also suggest that this protein may reflect clinical activity.

In patients with stable-phase CRF, no relationship was observed between serum E-selectin, P-selectin, ICAM-1, and VCAM-1 levels and the degree of renal failure as measured by serum creatinine.

Further research is required to determine whether there is any correlation between expression of adhesion molecules in tissue and serum levels of those molecules, and to ascertain the value of serum concentrations in monitoring activity and identifying patients with low activity and no clinical symptoms.



   Acknowledgments
 
Jordi Ara is a research award recipient from SEN and Hospital Clínic i Provincial de Barcelona. This work was supported by grants from FIS (96/2104).



   Notes
 
Correspondence and offprint requests to: Dr Jordi Ara, Nephrology Service, Hospital Clínic, Barcelona, C/Villarroel 170, E-08036 Barcelona, Spain. Back



   References
 Top
 Abstract
 Introduction
 Subjects
 Methods
 Other laboratory studies
 Results
 Discussion
 Conclusions
 References
 

  1. Falk RJ, Jeannette JC. Renal vasculitis. In: Neilson EG, Couser WC, eds. Immunologic Renal Disease. Lippincott–Raven, Philadelphia, 1997; 1099–1119
  2. Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: The multistep paradigm. Cell1994; 76: 301–314[ISI][Medline]
  3. Carlos TM, Harlan JM. Leukocyte–endothelial adhesion molecules. Blood1994; 84: 2068–2101[Abstract/Free Full Text]
  4. Gearing AJH, Newman W. Circulating adhesion molecules in disease. Immunol Today1993; 14: 506–512[ISI][Medline]
  5. Leeuwenberg J, Smeets E, Neefjes JJ et al. E-selectin and ICAM-1 are released by activated human endothelial cells in vitro. Immunology1992; 77: 543–549[ISI][Medline]
  6. Spertini O, Schleiffenbaum B, Whitewen C, Ruiz P, Tedder T. ELISA for quantitation of L-selectin shed from leukocytes in vivo. J Immunol Methods1992; 156: 115–123[ISI][Medline]
  7. Ornaldi Q, Loscalzo J. Adhesion molecules in atherosclerosis. In: Paul LC, Issekutz TB, eds. Adhesion Molecules in Health and Disease. Marcel Decker, New York, 1997; 401–427
  8. Janssen RA, Luqmani RA, Gordon C et al. Correlation of blood levels of soluble vascular cell adhesion molecule-1 with disease activity in systemic lupus erythematosus and vasculitis. Br J Rheumatol1994; 33: 1112–1116[ISI][Medline]
  9. Mrowka C, Sieberth HG. Circulating adhesion molecules ICAM-1, VCAM-1 and E-selectin in systemic vasculitis: marked differences between Wegener's granulomatosis and systemic lupus erythematosus. Clin Invest1994; 72: 762–768[ISI][Medline]
  10. Stegeman CA, Tervaert JWC, Huitema MG, Jong PE, Kallerberg CGM. Serum levels of soluble adhesion molecules ICAM-1, VCAM-1 and E-selectin in patients with Wegener granulomatosis. Arthritis Rheum1994; 37: 1228–1235[ISI][Medline]
  11. Pall AA, Adu D, Drayson M, Taylor CM, Richards NT, Michael J. Circulating soluble adhesion molecules in systemic vasculitis. Nephrol Dial Transplant1994; 9: 770–774[Abstract]
  12. Collvinent B, Grau JM, López-Soto A et al. Circulating soluble adhesion molecules in patients with classical polyarteritis nodosa. Br J Rheumatol1997; 36: 1178–1183[ISI][Medline]
  13. Collvinent B, Vilardell C, Font C et al. Circulating soluble adhesion molecules in patients with giant cell arteritis. Correlation between sICAM-1 concentrations and disease activity. Ann Rheum Dis1999; 58: 189–192[Abstract/Free Full Text]
  14. Wellicome SM, Kapahi P, Mason JC, Lebranchu Y, Yarwood H, Haskard DO. Detection of a circulating form of vascular cell adhesion molecule-1: raised levels in rheumatoid arthritis and systemic lupus erythematosus. Clin Exp Immunol1993; 92: 412–418[ISI][Medline]
  15. Mason JC, Kapahi P, Haskard DO. Detection of increased levels of circulating adhesion molecule-1 in some patients with rheumatoid arthritis but not in patients with systemic lupus erythematosus. Arthritis Rheum1993; 26: 519–527
  16. Mackhold KP, Kiener HP, Graninger W, Graninger WB. Soluble intercellular adhesion molecule-1 in patients with rheumatoid arthritis and systemic lupus erythematosus. Clin Immunol Immunopathol1993; 68: 74–78[ISI][Medline]
  17. Heufelder AE, Bahn RS. Soluble intercellular adhesion molecule-1 in sera of patients with Grave's ophthalmopathy and thyroid diseases. Clin Exp Immunol1993; 92: 296–302[ISI][Medline]
  18. Cush JJ, Rothlein R, Lindsley HB, Mainolfi EA, Lipsky PE. Increased levels of ICAM-1 in the sera of patients with rheumatoid arthritis. Arthritis Rheum1993; 36: 1098–1102[ISI][Medline]
  19. Seth R, Raymond FD, Makgoba NW. Circulating ICAM-1 isoforms: diagnostic prospects for inflammatory and immune disorders. Lancet1991; 338: 83–84[ISI][Medline]
  20. Jennette JC, Falk RJ, Andrassy K et al. Nomenclature of systemic vasculitis. Proposal of an International Consensus Conference. Arthritis Rheum1994; 37: 187–192[ISI][Medline]
  21. Bacon A, Moots RJ, Exley A, Luqmani R, Rasmussen N. Vital assessment of vasculitis. Clin Exp Rheumatol1995; 13: 275–278[ISI][Medline]
  22. Wiik A, Rasmussen N, Weislander J. Methods to detect autoantibodies to neutrophilic granulocytes. In: Van Venrooig WJ, ed. Manual of Biological Markers of Disease. Kluwer Academic Publishers, Dordrecht, 1993; 1–14
  23. Lebranchu Y, Valentin JF, Büchler M. Measurement of soluble adhesion molecules in biological fluids. In: Paul LC, Issekutz TB, eds. Adhesion Molecules in Health and Disease. Marcel Decker, New York, 1997; 87–121
  24. Abelda SM, Wayne Smith C, Ward PA. Adhesion molecules in inflammatory injury. FASEB J1994; 8: 504–512[Abstract/Free Full Text]
  25. Pigott R, Dillon LP, Hemingway IH, Gearing AJH. Soluble forms of E-selectin, ICAM-1 and VCAM-1 are present in the supernatants of cytokine activated cultured endothelial cells. Biochem Biophys Res Commun1992; 187: 584–589[ISI][Medline]
  26. Cohen Tervaert JWC, Kallerberg CGM. Cell adhesion molecules in vasculitis. Curr Opin Rheumatol1997; 9: 16–25[Medline]
  27. John S, Neumayer H, Weber M. Serum circulating ICAM-1 levels are not useful to indicate active vasculitis or early renal allograft rejection. Clin Nephrol1994; 42: 369–375[ISI][Medline]
  28. Furukawa S, Imai K, Matsubara T et al. Increased levels of ICAM-1 in Kawasaki disease. Arthritis Rheum1993; 35: 672–677[ISI]
  29. Nash MC, Shah V, Dillon MJ. Soluble cell adhesion molecules and von Willebrand factor in children with Kawasaki disease. Clin Exp Immunol1995; 10: 13–17
  30. Tesar V, Masek JE, Jirsa M et al. Influence of plasma exchange on serum levels of cytokines and adhesion molecules in ANCA positive renal vasculitis. Blood Purif1998; 16: 72–80[ISI][Medline]
  31. Rastaldi MP, Ferrario F, Tunesi S, Yang L, Dá mico G. Intraglomerular and interstitial leukocyte infiltration, adhesion molecules, and interleukin-1 expression in 15 cases of ANCA associated renal vasculitis. Am J Kidney Dis1996; 27: 48–57[ISI][Medline]
  32. Lhotta K, Schögl A, Kronenberg F, Joannidis M, König P. Soluble intercellular adhesion molecule-1 (ICAM-1) in serum and urine: correlation with renal expression of ICAM-1 in patients with kidney disease. Clin Nephrol1997; 48: 85–91[ISI][Medline]
  33. Spronk PE, Bootsma H, Huitema PC, Limburg PC, Kallerberg CGM. Levels of soluble VCAM-1, soluble ICAM-1 and soluble E-selectin during disease exacerbations in patients with systemic lupus erythematosus, a long prospective study. Clin Exp Immunol1994; 97: 439–444[ISI][Medline]
  34. Nishikawa K, Guo YJ, Miyasaka M et al. Antibodies to ICAM-1/ lymphocyte function-associated antigen-1 prevent crescent formation in rat autoimmune glomerulonephritis. J Exp Med1993; 177: 667–677[Abstract]
  35. Boupas DT, Chrousos GP, Wilder RL, Cupps TR, Ballow JE. Glucocorticoid therapy for immune-mediated disease: Basic and clinical correlates. Ann Intern Med1993; 119: 1198–1208[Abstract/Free Full Text]
  36. Cronstein DT, Kimmel SC, Levin RI, Martiniuk F, Weissman GA. Mechanism for the antiinflammatory effects of corticosteroids the glucocorticoid receptor regulates leukocyte adhesion to endothelial cells and expression of endothelial–leukocyte adhesion molecule 1 and intercellular adhesion molecule 1. Proc Natl Acad Sci USA1992; 89: 9991–9995[Abstract]
  37. McEver RP. Selectins. Curr Opin Immunol1994; 6: 75–84[ISI][Medline]
  38. Segawa C, Akashi W, Takaeda M et al. In situ expression and soluble form of P-selectin in human glomerulonephritis. Kidney Int1997; 52: 1054–1063[ISI][Medline]
  39. Beliacqua MP, Nelson RM. Selectins. J Clin Invest1993; 91: 379–387[ISI][Medline]
  40. Spertini O, Luscinskas FW, Kansas GS. Leukocyte adhesion molecule-1 interacts with an inducible endothelial cell ligand to support leukocyte adhesion and transmigration. J Immunol1991; 147: 2565–2573[Abstract/Free Full Text]
  41. Kishimoto TK, Jutila MA, Berg EL, Butcher EC. Neutrophil MAC-1 and MEL-14 adhesion proteins inversely regulated by chemotactic factors. Science1989; 245: 1238–1241[ISI][Medline]
  42. Schleiffenbaum B, Spertini O, Tedder TF. Soluble L-selectin is found in human plasma at high levels and retains functional activity. J Cell Biol1992; 119: 229–238[Abstract]
  43. Donnelly S, Haslett C, Dransfield I. Role of selectin in development of adult respiratory distress syndrome. Lancet1994; 344: 215–218[ISI][Medline]
  44. Blann AD, Sanders PA, Herrick A, Jayson MIV. Soluble L-selectin in the connective tissue diseases. Br J Haematol1996; 95: 192–194[ISI][Medline]
Received for publication: 7. 6.99
Revision received 11. 7.00.