INTRODUCTION

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The recognition of bronchial asthma as an inflammatory disease led to a search for soluble markers that would be useful in assessing airway inflammation. A variety of cytokines, adhesion molecules, and eosinophil-associated proteins have been evaluated in this respect (1, 2). Interleukin-6 (IL-6) is increased in bronchoaveolar lavage fluid obtained from asthmatic patients (3) and in nasal secretions after antigen challenge (4). We previously reported that patients with stable asthma exhibited an increase in circulating IL-6 and that its levels were increased to a greater extent during an asthmatic attack (5). Our previous study also indicated that the airway inflammation caused by antigen inhalation could similarly increase the circulating levels of IL-6. Wang and coworkers (6) demonstrated that serum levels of IL-6 and TNF- were each increased in normal volunteers after the inhalation of swine dust.

Gosset and colleagues (7) reported that IL-6 production from alveolar macrophages is increased in patients with asthma, especially in patients showing a dual asthmatic response. An increased expression of IL-6 gene and protein was demonstrated in bronchial epithelial cells of asthmatic patients (8). Mast cells and eosinophils, which are increased in the asthmatic airways, are reported to release IL-6 (9, 10). IL-6 exhibits a variety of immune and proinflammatory effects (11). It can activate the T-cells as well as the natural killer cells, with such activation being characteristic of asthmatic patients. IL-6 is involved in the synthesis of human IgE induced by IL-4, and it inhibits the growth of fibroblasts and bronchial epithelial cells. IL-6 can directly promote the survival of mast cells without the involvement of autocrine factors. Preincubation of mast cells with IL-6 can significantly upregulate the IgE-mediated histamine release (12). However, the epithelial cell-specific expression of IL-6 in transgenic mice leads to inflammation of the airway and a reduction in the bronchial reactivity to methacholine, suggesting that the production of IL-6 may have a beneficial effect in bronchial asthma (13). Such observations indicate the involvement of IL-6 in the pathophysiology of bronchial asthma.

IL-6 acts via specific receptors that consist of the IL-6 binding glycoprotein gp80 and the signal transducer gp130. Soluble IL-6 receptor (sIL-6R, sCD126) is made from gp80 and is excreted as a 52-kD molecule in human urine. Unlike other soluble receptors, the soluble form of the members of IL-6 receptor family such as IL-6, ciliary neurotrophic factor, leukemia inhibitory factor, and IL-12 can act as agonists (14). Soluble IL-6R can bind to IL-6. In turn, this complex can bind to cell surface gp130 to transduce the IL-6 signal. A persistently elevated serum concentration of IL-6 may downregulate gp80 and make the cells unresponsive to IL-6. Such a refractory state is overcome in vitro by the addition of sIL-6R. Thus, sIL-6R is considered to be important in the responses mediated by IL-6.

The present study evaluated serum levels of sIL-6R in patients with asthma versus control subjects and evaluated its significance in the pathophysiology of asthma, information not previously reported.

	METHODS

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We evaluated serum IL-6 levels in 20 consecutive Japanese patients with asymptomatic asthma with FEV₁ > 70%. There were six men and 14 women 22 to 76 yr of age (mean, 44.7 yr). Of this group, 12 had atopic and eight nonatopic asthma. This determination was based on the positive results of intradermal skin tests and/or of levels of specific IgE (MAST; multiple antigen simultaneous test). All of the patients had intermittently used beta-agonists given by inhalation, six patients had also received sustained-release theophylline, and 15 had also received inhaled steroid (400 µg/d). Blood was drawn when the methacholine inhalation test was carried out by using an Astograph (TCK 6100H; Chest Co., Tokyo, Japan) (15). Airway resistance (Raw) was observed until it increased to twice the Raw/control obtained by inhalation of control saline. The methacholine concentration, which starts to increase in Raw, was determined from the dose-response curve and assessed as Cmin (concentration minimum). As controls, we selected 18 normal subjects (eight men and 12 women 22 to 67 yr of age; mean, 40 yr) based on normal findings in blood chemistry tests, complete blood counts, and no evidence of inflammatory disease, including a negative result for C-reactive protein. The age and sex of the patients and control subjects did not differ significantly.

To measure the serum levels of sIL-6R in each patient in the symptomatic as well as the asymptomatic state, paired serum samples were obtained at 4-wk intervals in 10 patients. PEF was recorded using the Assess Peak Flow Meter (Healthscan Products Inc., Cedar Grove, NJ). These patients were treated with bronchodilators and/or intravenously with steroids followed by additional prednisolone given orally, 10 to 30 mg/d for 5 to 14 d. To evaluate the control changes in sIL-6R levels, we evaluated 10 asymptomatic patients with stable asthma (no fall in best PEF < 80% for at least 6 mo) over a period of 4 wk. Serial measurements of sIL-6R (Days 1, 3, and 5) were conducted in seven other patients who had been admitted to our hospital with acute severe asthma. None of these patients had infection evidenced by clinical examinations (lack of sore throat, fever, etc.) and the examination of sputum (lack of purulence with predominance of neutrophils).

We conducted inhalation tests using as the allergen a serially diluted solution of the house dust mite at the maximum concentration of 0.1 wt/vol (endotoxin-free; Torii Pharmaceutical Co., Tokyo, Japan). The methacholine inhalation test was also carried out in accordance with the standard method (not using an Astograph) recommended by the Japanese Society of Allergology. A fall in FEV₁ of 20% was regarded as an immediate response. A fall in FEV₁ 3 to 8 h after challenge by more than 20% of the prechallenge value was regarded as a late reaction. Venous blood was sampled before the inhalation test and 24 h after the final inhalation.

Venous blood was allowed to clot for 1 to 2 h, and serum was obtained and stored at 80° C until used. Levels of sIL-6R in serum were determined by a specific ELISA using a murine antihuman IL-6R mAb (MT-18; IgG_2b; 2 µg/ml) and polyclonal guinea pig anti-sIL-6R IgG fraction (1 µg/ml) (16, 17). Because the affinity of MT-18 mAb for IL-6R is not influenced by the complex formation between IL-6 and IL-6R, this assay detects both complexed and uncomplexed IL-6R (18). Recombinant sIL-6R (Tosoh Corp., Ayase, Kanagawa, Japan) was used as the standard. The limit of detection in this assay was 0.1 ng/ml. This assay is specific for human sIL-6R because of nonreactivity with murine sIL-6R, human IL-1, IL-2, IL-3, IL-4, IFN-, TNF-, or sIL-2R. The intraassay coefficient of variation was 4.7%, whereas that of the interassay variation was 2.2%.

Data are shown as means ± SD. Differences between groups were estimated by the Mann-Whitney U-test or Wilcoxon's test, as applicable. Spearman's rank sum test was used to evaluate correlations. A level of statistical significance was defined as p < 0.05.

	RESULTS

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Serum levels of sIL-6R in the patients with asthma significantly exceeded those of the normal control subjects (n = 20: 132 ± 31 ng/ml versus n = 18: 111 ± 16 ng/ml, p < 0.05) (Figure 1). However, there was no significant correlation between the serum levels of sIL-6R and bronchial hyperreactivity (Cmin) (r, p) (0.04, 0.97) or the serum levels of IgE (0.15, 0.51). Levels of sIL-6R did not differ significantly in the atopic (n = 12) versus the nonatopic (n = 8) patients (125 ± 31 versus 127 ± 31 ng/ml, respectively).

View larger version (14K): [in this window] [in a new window]   Figure 1.   Serum levels of sIL-6R in patients with bronchial asthma (n = 20) and in healthy control subjects (n = 18). The levels of patients with asthma (132 ± 31 ng/ml) were significantly higher than those of control subjects (111 ± 16 ng/ml; Mann-Whitney U-test, p < 0.05).

The serum concentration of sIL-6R obtained at an emergency visit to treat a spontaneous asthmatic attack (PEF, 294 ± 73 L/min) was significantly elevated as compared with that obtained in the asymptomatic state (4 wk later; PEF, 393 ± 103 L/min) (n = 10: 150 ± 42 versus 139 ± 38 ng/ml, respectively; p < 0.05) (Table 1). Patients with stable asthma showed no significant change in the serum concentration of sIL-6R during the same period (n = 10: 113 ± 21 versus 112 ± 22 ng/ml).

The results of serial measurements of serum concentrations of sIL-6R during an emergency admission for a severe asthmatic attack (n = 6) are shown in Figure 2. There was a significant further increase in the sIL-6R concentration on the second and third days of admission (p < 0.05). The concentration returned to the levels observed on the day of admission on the fifth day of admission.

View larger version (20K): [in this window] [in a new window]   Figure 2.   Serial measurements of sIL-6R in serum after admission for treatment of a severe asthmatic attack (n = 6). The serum levels of sIL-6R were 136 ± 40, 158 ± 46, 154 ± 35, and 143 ± 24 ng/ml on the first, second, third, and fifth days of admission, respectively.

In evaluation of the sIL-6R concentration in patients with mite sensitivity 24 h after inhalation of this allergen (n = 7), five patients showed a single early response and two patients showed a dual response. A significant increase in sIL-6R, from 123 ± 20 to 133 ± 25 ng/ml is shown in Figure 3. However, the inhalation of the nonspecific bronchoconstrictor, methacholine (n = 9), did not influence the serum concentration of sIL-6R. Soluble IL-6R concentration before methacholine inhalation tended to be higher than that before mite inhalation. However, there was no significant difference between them (p = 0.10).

View larger version (20K): [in this window] [in a new window]   Figure 3.   The levels of sIL-6R before and 24 h after bronchial challenge (left: dust mite allergen, n = 7; right: methacholine, n = 9). The inhalation of allergen induced a significant increase of sIL-6R concentration, from 123 ± 20 to 133 ± 25 ng/ml. Methacholine challenge induced no significant change in sIL-6R concentration (from 131 ± 33 to 127 ± 29 ng/ml).

	DISCUSSION

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The present study demonstrated that the serum levels of sIL-6R in patients with asthma were increased as compared with those of normal control subjects. Its levels were further increased in an exacerbation of asthma. Because sIL-6R was increased after the inhalation of allergen, the circulating levels of sIL-6R may reflect airway inflammation.

The mechanism for the increase in serum sIL-6R is unclear at present. It may result from an increased production of IL-6, as IL-6 can induce IL-6R expression in mice in vivo (19). We previously observed a patient with the Churg-Strauss syndrome in the transient vasculitic phase with an elevated sIL-6R level that paralleled that of IL-6 (20). Thus, the elevation of circulating sIL-6R in patients with asthma would be an indirect indicator of the severity of airway inflammation, i.e., airway inflammation induces IL-6 production; the increase in IL-6 would then increase sIL-6R. This concept would explain the prolonged elevation of sIL-6R observed after a severe asthmatic attack (Figure 2).

An increase in serum levels of sIL-6R have been reported in patients with infectious disease (21), benign or malignant hematologic disease (22, 23), inflammatory bowel disease (24), collagen-vascular disease (25), and lung disease (17, 26). These reports suggest that circulating and local levels of sIL-6R could reflect disease activity, and be useful in evaluation of the prognosis of a particular disease. In our experience, the increase of serum sIL-6R observed in patients with asthma was mild compared with other conditions such as rheumatoid arthritis (153.9 ± 56.9 ng/ml, n = 15; maximal value, 307 ng/ml) (unpublished observations) and interstitial pneumonia (138.7 ± 39.7 ng/ml, n = 17; maximal value, 229 ng/ml) (17).

Eosinophil cationic protein (ECP) and sIL-2R have been well studied as soluble markers of allergic inflammation. ECP could reflect an increase in the activity of the circulating eosinophil population. Its levels in serum reflect the severity of the disease, bronchial hyperreactivity, and treatment efficacy (1). sIL-2R may reflect T-cell activity, which is an underlying feature of asthmatic pathology (27). sIL-6R could be produced by IL-6R-expressing cells (16). The IL-6R is expressed on monocytes, T-cells, activated B-cells, and many other nonlymphoid cells, including epithelial cells, fibroblasts, hepatocytes, neural cells, and mesothelial cells (28, 29). This may explain why such a large amount of soluble receptor of IL-6 (around 100 ng/ml) was found in normal serum, as compared with the small amount of sIL-2R (around 1 to 2 ng/ml).

The limitations in using a sIL-6R as a soluble marker in bronchial asthma should be noted. First, these levels were not correlated with such key clinical parameters as bronchial hyperreactivity. Second, its levels varied widely so that the cutoff level could not be determined. However, when measured serially in an individual patient, this substance could serve as a marker for monitoring disease activity of bronchial asthma. It should also be noted that the sIL-6R level in other specimens such as sputum or urine could be a better marker, but we have not investigated this at present.

In conclusion, circulating concentrations of sIL-6R may reflect the severity of airway inflammation. A possible soluble marker should be evaluated by several points, as mentioned by O'Byrne and Hargreave (2), such as whether it could assess asthma control, improve the management of asthmatic patients, identify patients at risk for a severe or fatal exacerbation, and reduce severe attacks. Of particular, we need to know exactly what marker could be added during follow-up of the patients in a routine way. For this purpose, a multiparametric analysis should be done, including various indices used in the clinic to monitor asthma. The present study warrants investigations of these points to determine the benefits of measuring sIL-6R.