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Clin Exp Immunol. Jul 2005; 141(1): 183–188.
PMCID: PMC1809423

Sustained activation of neutrophils in the course of Kawasaki disease: an association with matrix metalloproteinases

Abstract

Kawasaki disease (KD) is an acute febrile syndrome of childhood, characterized by vasculitis of the medium-sized arteries. White blood cell counts and the inflammatory parameter C-reactive protein (CRP) are known to be elevated in the acute phase of the disease. In this study we investigated the course of inflammatory cell type-specific parameters in KD over a longer period of time. Plasma levels of human neutrophil elastase (HNE), matrix metalloproteinases-2 and -9 (MMP2, MMP9), and neutrophil gelatinase-associated lipocalin (NGAL), macrophage neopterin and CRP were measured. Plasma samples were collected in the acute, subacute and early convalescent stage, and three months after the onset of disease. Median CRP and neopterin normalized within two weeks. In contrast, six weeks and three months after onset of disease, levels of HNE were still elevated, with median values of 163 ng/ml and 156 ng/ml, respectively (control children median < 50 ng/ml; for all time-points P < 0·0001). Values of NGAL correlated with the levels of HNE (r = 0·39, P = 0·013). These results demonstrate a longer state of neutrophil activation in KD than was previously assumed. The potential relationship between this prolonged neutrophil activation, coronary artery lesion formation and their persistence, as well as the risk of premature atherosclerosis warrants further evaluation.

Keywords: Kawasaki disease, neutrophil, elastase, metalloproteinase, neopterin

Introduction

Kawasaki disease (KD) is an acute febrile syndrome of unknown aetiology. KD represents a vasculitis of childhood, affecting medium-sized arteries, in particular coronary arteries. In approximately 25% of affected children, the vasculitis will lead to coronary artery lesions (CAL), as can be detected by echocardiography. KD is the leading cause of acquired heart disease in children in the developed countries. Diagnosis is made on clinical criteria [1,2]. Therapy consists of a single, high dose of intravenous immunoglobulins (IVIG) and aspirin. When administered within the first 10 days of illness, this therapy reduces the incidence of CAL to less than 5%[3,4].

Laboratory features show an increased level of C-reactive protein (CRP) and an elevated white blood-cell count (WBC) in the acute phase of the disease, consisting mainly of polymorphonuclear neutrophils (PMNs) with a typical left-shift. Pathological postmortem studies show oedema of the endothelial cells and inflammation of the adventitial layer of the coronary arteries at the early stage of the disease. The infiltrating cells initially consist mainly of neutrophils, followed in time by macrophages [5]. In this study we investigated the activity of the inflammatory cells in the peripheral blood over time. For this purpose, we assessed the levels of human neutrophil elastase (HNE) and macrophage neopterin as markers for neutrophil and monocyte/macrophage activity, respectively.

Matrix metalloproteinases (MMPs) are enzymes that are able to degrade components of the extracellular matrix, such as collagen, fibronectin and laminin. In pathophysiological conditions, MMPs function in vascular remodelling during wound healing and inflammation [6]. The proteolytic activity is counteracted by a set of natural inhibitors, the tissue inhibitors of MMPs (TIMPs). Discordant levels of MMPs and TIMPs have been implicated in various vascular diseases such as giant-cell arteritis, aortic aneurysm formation [7]. MMP9 can be released by many cell types, among which neutrophils. Neutrophils also release a typical molecular form of MMP9 associated with lipocalin (NGAL) with a characteristic molecular mass of 135 kD in zymograms. Recent studies have shown elevated plasma concentrations of certain MMPs in KD patients in the acute and subacute phase of the disease [8,9]. To define the time-relationship to disease activity and the risk of developing vascular lesions we measured these inflammatory parameters at several time points over at least three months.

Subjects and methods

Study design and sample collection

The diagnosis of KD was based on criteria and echocardiographic scoring, according to the guidelines of the Kawasaki Disease Research Committee in Japan from 1984. Coronary artery dilatations with a diameter > 3 mm for children < 5 years of age (> 4 mm for children > 5 years) or > 1·5 times the adjacent vessel diameter were scored as coronary artery lesions (CAL). Standard therapy consisted of a single IVIG infusion (2 g/kg in 8–12 h) in combination with oral aspirin (80–100 mg/kg divided over 4 equal doses). Initially, 28 patients participated in this study (17 boys and 11 girls, age: 2 months−11 years). Blood was collected at sequential time points. A group of 18 afebrile children (Ten boys and eight girls, age: 6 months−12 years.) served as controls. Controls were children with congenital heart diseases, such as ventricular and atrial septum defects, who were hospitalized for elective cardiac surgery. When blood was collected for preoperative screening, an extra sample was withdrawn for this study. Informed consent was obtained from all parents/caregivers of the participants in the study. The study was approved by the medical-ethical committee of the Academic Medical Centre (AMC), Amsterdam, the Netherlands.

Plasma proteins

The assay for CRP was performed as described before [10], with polyclonal antibody to CRP (KH 61: 2 µg/ml) as the capture mAb, and biotinylated anti-CRP mAb (5G4) as detecting antibody. Results were related to a commercially available standard (Behringwerke, Marburg, Germany) and expressed in mg/l. The detection limit for CRP was 0·1 mg/l.

Plasma levels of HNE/α1-antitrypsin-complex were assessed with a sandwich-type ELISA [11]. Polyclonal anti-HNE antibodies were used as catching antibodies and biotinylated monoclonal antibody AT15 against complexed human α1-antitrypsin as detecting antibody. Results were related to an in-house standard and expressed in ng/ml. The detection limit for HNE/α1-antitrypsin was 5·0 ng/ml. Inter- and intra-assay variability was assessed and never exceeded 5%.

Plasma levels of neopterin were assessed with a commercially available ELISA (Brahms, Berlin, Germany). The detection limit for this assay was 2·5 nmol/ml.

Zymography

Gelatinase activity of the circulating MMPs was assessed by zymography. Plasma samples were diluted 1 in 5 with sample buffer yielding a final 5·25% (w/v) of SDS and run on SDS-polyacrylamide (10%; w/v) gels containing 0·2% (w/v) gelatin (Sigma, St Louis, MO) for separation by molecular mass. This relatively high amount of SDS was required to obtain distinct bands. After electrophoresis, the gels were washed extensively in 50 m m Tris-HCl, pH 7·5, containing 5 m m CaCl2, 0·02% (w/v) NaN3 and 2·5% (v/v) Triton X-100, to remove SDS. Next the gels were incubated overnight at 37°C in the same buffer, but now with 1% (v/v) Triton X-100, to facilitate degradation of gelatine by gelatinases. Subsequently, the gels were stained with PhastGel™ Blue R (Amersham Pharmacia Biotech AB; Upsala, Sweden) and destained in a solution of 7% (v/v) acetic acid and 20% (v/v) methanol.

Gelatinase activity in plasma is mainly due to the presence of MMP9, neutrophil-associated MMP9 (NGAL) and MMP2, and appears, after staining of the gels, as clear bands due to proteolysis against a blue background where gelatin has remained intact. With this method molecular forms of MMP9 and MMP2 that are not physiologically activated, i.e. latent MMP9 and MMP2, also show gelatinase activity. Purified metalloproteinases, among which were MMP2 and MMP9 (Roche; Mannheim, Germany) combined with prestained molecular weight marker (Bio-Rad, Richmond, CA, USA), had been used previously to identify the different protein bands with gelatinase activity. The gelatinase activities were expressed in arbitrary units (AU), upon quantification by densitometry. The interassay variations between separate gels were corrected by a standard amount of collagenase (Clostridium histolyticum type 1 A; Sigma) displaying gelatinase activity, run as control in parallel on each gel.

Statistical analysis

Differences between groups and healthy controls were analysed where appropriate with the Mann—Whitney or the t-test. A two-tailed P-value less than 0·05 was considered to be significant. Statistical analysis was performed with the SPSS package for Windows, version 10·0 (SPSS inc.).

Results

Levels of CRP

Levels of CRP showed a pattern consistent with findings in other reports [8,9], i.e. very high levels in the first week: median 62 mg/l, soon returning to normal at three weeks (Fig. 1). After six weeks and three months, CRP levels had completely normalized in all KD patients, with a median of = 1·0 mg/l, respectively. For the afebrile controls the median plasma level of CRP was = 1·0 mg/l, which is in accordance with the normal value for children. These findings suggest a rapid decline of the acute phase response in KD.

Fig. 1
Course of C-reactive protein (CRP) in Kawasaki disease. Plasma levels were measured in patients with KD on several time points after onset of disease, and in controls. Statistically significant differences versus controls, calculated with the Mann—Whitney ...

Levels of HNE-α1 antitrypsin

In contrast to CRP, the levels of HNE-α1 antitrypsin in plasma remained high for a prolonged period (Fig. 2). In the acute phase of the disease, levels were at a median of 216 ng/ml in the first week. These values only slowly normalized. After six weeks and three months median HNE-α1 antitrypsin levels were still 163 ng/ml and 156 ng/ml, respectively. For comparison, the median level in a group of age-matched afebrile controls was 50 ng/ml. The levels of HNE-α1 antitrypsin in KD patients were significantly higher than those in the afebrile controls at all time-points (P < 0·0001).

Fig. 2
Course of HNE-α1-antitrypsin in Kawasaki disease. Plasma levels were measured in patients with KD on several time points after onset of disease, as well as in age-matched controls. Even after 3 months, statistically significant differences versus ...

Levels of neopterin

To assess activation of macrophages, we investigated plasma levels of neopterin. The course of neopterin resembles that of CRP, i.e. elevated during the acute phase, to return rapidly to normal values thereafter (Fig. 3).

Fig. 3
Course of neopterin in Kawasaki disease. Plasma levels were measured in patients with KD on several time points after onset of disease. Only at week 1 levels were elevated compared to normal values (< 10·0 nmol/ml, P < 0·001). ...

This suggests activation of monocytes/macrophages in the acute phase, but not in the subacute stage and convalescence.

Activity of MMPs and NGAL

We additionally assessed gelatinase activities by zymography, of which a typical example is shown (Fig. 4). In plasma samples MMP2, MMP9 and neutrophil gelatinase-associated lipocalin (NGAL) were readily detected. Levels of MMP9 did not show a significant increase during the course of KD in our cohort (Fig. 5). In contrast, levels of NGAL were elevated at week 1, week 3 and 6 (Fig. 6). The sustained activation of NGAL resembled the increased levels of HNE-α1 antitrypsin demonstrating prolonged neutrophil activation. NGAL, but not MMP9 and MMP2, correlated significantly with levels of HNE-α1 antitrypsin (r = 0·39, P = 0·013, Fig. 7). No significant or trendwise correlation was seen between levels of NGAL and those of CRP or neopterin, indicating no correlation with the acute phase of KD. Also levels of MMP9 did neither correlate with those of CRP or neopterin nor with those for NGAL. Taken together these data suggest that MMP9 is derived from other cells than neutrophils and macrophages and does not relate to the acute phase of the disease.

Fig. 4
Zymogram of plasma samples from KD patients. Lanes 1–5, and lane 8 are analyses of plasma samples from patients. In lanes 6 and 7, the gelatinolytic activity of control collagenase (Clostridium histolyticum type 1 A) and of a culture supernatant ...
Fig. 5
Course of MMP9 in Kawasaki disease. Levels were measured in patients with KD on several time points after onset of disease and in controls. There was no significant elevation at any time point. Horizontal bars represent medians.
Fig. 6
Course of neutrophil-derived gelatinase (NGAL) in the course of Kawasaki disease. Levels were measured in patients with KD on several time points after onset of disease and in controls. Levels were elevated at week 1 (P < 0·05), week 3 ...
Fig. 7
Correlation between the levels of neutrophil-derived gelatinase (NGAL) and HNE-α1-antitrypsin levels in Kawasaki disease. Levels of HNE-α1-antitrypsin were correlated, by means of the Spearman-correlation test, with the expression of NGAL. ...

MMP2 was present in plasma as its 68 kD proform. Its actual presence in AU was not significantly different between the various time points (data not shown). MMP2 is most abundant and constitutively secreted in a latent form by many cell types of mesenchymal origin [12]. It is unique among MMPs in that it is not activated by enzymatic cleavage with proteinases such as elastase, cathepsin G, plasmins or urokinase, but instead is activated only on the cell-surface by recently identified membrane type (MT)-MMPs [13,14].

Discussion

Both the clinical course of KD as well as the evolution of routine inflammatory parameters such as CRP suggest a resolution of the disease activity within 4–6 weeks after administration of IVIG [15]. Our CRP and neopterin data corroborate these findings. However, plasma levels of HNE-α1 antitrypsin were elevated for a prolonged period. HNE is secreted by neutrophils upon activation and inactivated by its main inhibitor in plasma and extravascular fluid, α1-antitrypsin. Free HNE has a half-life of seconds, whereas in a complexed and inactivated form, its half-life in plasma is about 60 min [16]. Complexes of HNE and α1-antitrypsin are therefore better markers for activation of neutrophils in vivo than elastase. Macrophages produce elastase to a minor extent when compared to the massive release by activated neutrophils.

The concomitant presence of NGAL further supports a persistent activation of neutrophils and suggests an inflammatory process that proceeds longer than previously assumed in KD. The course of the macrophage-specific marker neopterin was not elevated after the acute phase. Neopterin is produced by monocytes and macrophages, primarily upon induction by cytokines, for instance interferon-gamma [17,18]. It is considered a marker for inflammatory activity or cellular immunity, with a half-life in the circulation of approximately 90 min, and is used to monitor the course of infections, malignant disease, and autoimmune or inflammatory processes. Iizuka et al. [19] have previously described concentrations of neopterin to be increased in the urine of patients with KD, showing a correlation between neopterin concentrations and the formation of CAL in their patients. Plasma levels of neopterin have not been reported in KD. Since the prolonged activation of neutrophils was not accompanied by macrophage activity one may assume that the elevated levels of complexed HNE are from activated PMNs and not from monocytes or macrophages. However, we cannot exclude the possibility that more subtle macrophage activation may be present.

Several biological effects have been attributed to HNE, both beneficial and pathological. Among the beneficial effects, killing of pathogens (such as Gram-negative bacteria and fungi) has been shown in vitro and in animal studies [20]. Pathological properties include the uncontrolled degradation of extracellular matrix components, such as elastin and collagen [20]. These substrates are also part of the subendothelial basement membrane and the interstitium, which form the crucial matrix for the vascular lining. Moreover, HNE can both activate MMP9 [21] and cleave TIMPs, thus favouring enhanced MMP activity [22]. Whether this is the case in the KD plasma samples is not known, but the increased NGAL and MMP9 levels and that of HNE are suggestive of an increased MMP activity.

MMPs are thought to be involved in processes of vascular damage, such as atherosclerosis and arterial aneurysm formation [6,7]. Recently, several groups separately described the presence of elevated plasma and serum levels of circulating MMP1, -2, -3 and -9 in KD, although the study period was limited to five weeks after onset of disease. NGAL was not measured though [8,9].

Also HNE has been described to be elevated in the acute phase of KD [9,23]. Both MMPs and HNE were reported to be higher in patients who developed CAL compared to those who did not [9].

In the literature some studies report a correlation between inflammatory parameters such as CRP and CAL [24,25], whereas we and others [26,27] do not find a significant correlation. However, our cohort may be too small to find a correlation, since CAL developed in only two patients. On the other hand, vasculitis in KD may be more systemic and not limited to the inflammation of the coronary arteries. The formation of CAL therefore does not necessarily reflect the extent of the vascular inflammation. Moreover, it is not known whether differences in the individual sensitivity to a given amount of free elastase or other proteolytic activities play a role in the vascular damage detected by echocardiography.

The site of the neutrophil activation is unknown. Direct and prolonged activation within the bloodstream seems unlikely, without the perturbation of other protein cascades. In sepsis, where neutrophils are massively activated and often a neutropenia is present due to adhesion and extravasation, the strongly increased HNE levels in patients normalize within 7 days [34]. In KD the release of HNE by PMN most probably occurs in the vascular lesions or at extravascular sites. At present, we cannot discriminate between these two possibilities. With respect to the chronic PMN activation it is to be noted that their half-life in the circulation is only 8 h. In the tissues PMNs dwell for 24 h before they will undergo apoptosis. This process of apoptosis prevents the PMNs from spillage of their toxic content such as HNE and allows their recognition by tissue macrophages for rapid uptake and clearance.

Elevation of levels of HNE over a long period of time indicates that KD results in long-term neutrophil activation and -potentially in vascular damage other than acute aneurysm formation in the coronary vasculature. Indeed, several studies report long-term vascular complications in KD patients, such as an impaired endothelial function, increased intima-media thickness (IMT), restenosis and atherosclerosis, irrespective of coronary artery involvement in the acute phase of the disease [2830]. These are all conditions with an increased risk for cardiovascular morbidity and with a need for long-term follow-up.

To date, histopathological investigations of the endothelium and adventitium are scarce [31] and not directly focused on these long-term issues of the disease. Combined with morphological evaluation, such as IMT measurements, and functional tests, such as flow-mediated dilation (FMD) measurement and distensibility tests, this might lead to new additional treatment regimens. For instance, preliminary data on Ulinastatin, an inhibitor of HNE, suggest a beneficial effect on the coronary vasculature [32]. Alternatively, MMP9 production in the wall of human abdominal aortic aneurysm is suppressed by cerivastatin, a member of the cholesterol-lowering HMG-CoA reductase inhibitor-family (‘statins’) [33]. Besides the cholesterol-lowering effects, several other beneficial effects on inflammation, thrombosis and endothelial function, have been described recently for statins. Whether statins or other therapeutic interventions could have a role in the (long-term) treatment of KD has to be investigated.

Acknowledgments

MH Biezeveld is funded by the Netherlands Heart Foundation (NHS 99·189). The funding sources had no role in study design, data collection, data analysis, data interpretation or writing of the report.

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