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Production and expression of Gro-alpha and RANTES by peripheral blood mononuclear cells isolated from patients with Kawasaki disease and measles.

We investigated whether the production and gene expression of Gro-alpha and RANTES in Kawasaki disease differ in measles. Forty-two samples from 14 patients in different clinical stages of Kawasaki disease, eight samples from 8 patients in the acute stage of measles and seven samples from 7 healthy children were collected. The present study was performed using ELISA and RT-PCR for the productions and gene expression of the chemokines. The production of Gro-alpha was markedly elevated during the acute stage of measles compared with Kawasaki disease. Moreover, the expression of Gro-alpha was increased in every case of measles, but not in Kawasaki disease. The production of RANTES was elevated in the acute stage of both diseases when compared to the healthy control. However, the plasma RANTES level did not change significantly according to the clinical stages of Kawasaki disease. A correlation between the production and gene expression of RANTES and Gro-alpha was not found in Kawasaki disease. These results suggest that Kawasaki disease differs from measles with regard to Gro-alpha production and expression, but not RANTES. Gro-alpha might play an important role in the acute stage of measles, however not in Kawasaki disease. Further studies are needed to clarify the efficacy of Gro-alpha as a marker in measles.

The study cohort were composed of three groups. The first group included 14 consecutive patients (10 males, 4 females; mean age, 35.6 months, range 11 to 83 months) who met the diagnostic criteria for Kawasaki disease as described elsewhere (1). All patients received high dose single intravenous gamma globulin (IVIG) infusion (Liv-gamma 2 g/kg in 10 to 12 hr) in combination with oral aspirin (Rhonal 60-100 mg/kg divided into 3 equal doses) treatment within the first 10 days of illness (acute phase) and were prospectively evaluated at Yeungnam University Hospital, Dae-Gu, Korea. All patients underwent serial blood sampling and echocardiographic evaluation. The blood sampling and echocardiographic study was done according to the disease course as follows; acute stage, before treatment with IVIG and aspirin, subacute stage, 7 to 10 days after intravenous gamma globulin treatment, and convalescent stage, 30 to 40 days after the onset of the Kawasaki disease. Normal ranges for coronary artery size defined according to diagnostic criteria of the Research Committee on Kawasaki disease, Ministry of Health and Welfare, Japan (17). In younger than 5 yr, internal lumen diameters of 3 mm or more, and in aged 5 yr or older, internal lumen diameter of 4 mm or more is considered coronary artery dilatation. Three patients were found to have dilatations of the coronary arteries. In all of them, the echocardiographic findings became normal during one year of follow-up. None of the patients died.
The second group included 8 samples from eight patients (3 males, 5 females; mean age, 28.1 months, ranges from 7 to 128 months) in the acute febrile stage of measles. And 7 healthy children (4 males, 3 females; mean age, 34.3 months, ranges from 8 to 55 months) who had no apparent underlying diseases composed the third group (Table 1).
Informed consents were obtained from the parents of the patients included in this study.

Echocardiographic assessment of coronary artery
The 2-dimensional echocardiographic study was performed with a 7.5 MHz transducer (Sequoia CA, U.S.A.) to obtain the coronary artery dimension at the level of the aortic root in the parasternal short axis view. All patients received a single dose of chloral hydrate (80 mg/kg) orally for sedation.

Sera
Samples of venous blood were obtained from patients and controls, and were centrifuged at 2,000 rpm/min for 30 min. After the isolation of peripheral blood mononuclear cells (PBMCs), serum samples were frozen at -70 for the ELISA assay.

Reagents
Reverse transcriptase-polymerase chain reaction (RT-PCR) kits were purchased from Perkin-Elmer (Norwalk, CT, U.S.A.). Trizol solution for total RNA isolation and a 100 bp DNA ladder as molecular weight marker were obtained from Gibco/ BRL (Life Technologies, Gaithersburg, MD, U.S.A.).

ELISA assay
ELISAs for RANTES and Gro-were performed according to manufacturer' s instruction using kits from R&D systems (Minneapolis, MN, U.S.A.). Briefly, add 100 L of standards or patient serum was incubated in anti-human RANTES (or Gro-) coated well plates at room temperature for 2 hr. After the 200 L of prepared biotinylated antibody was added, the plates were incubated at room temperature for 1 hr, and then the 200 L of tetramethylbenzidine substrate solution was added and the plates were developed in the dark state at room temperature for 20 min. The reaction was ceased by adding 50 L of stop solution. Absorbence of serum was measured at 450 nm for RANTES and Gro-.
PBMCs isolation and total RNA isolation PBMC were isolated by density centrifugation on a Ficoll-Hypaque gradient at 2,000 rpm for 30 min, then total RNA was isolated with the use of Trizol solution, as instructed by the manufacturer. Briefly, after the addition of 1 mL of Trizol and 200 L of chloroform followed by centrifugation, the aqueous phase was combined with an equal volume of isopropanol. The precipitated pellet was washed with 70% ethanol, and suspended again in diethylpyrocarbonate (DEPC)-treated water.

Statistical Methods
Results were presented as mean SD. Statistical differences were analyzed by the Mann-Whitney test. A p-value

Plasma levels of Gro-
The plasma levels of Gro-were markedly elevated during the acute stage of measles than those of the patients with Kawasaki disease and healthy control group (p<0.01) (Fig. 1). Among the patients with Kawasaki disease, it is somewhat elevated in the subacute stage, but we could not find obvious elevation of plasma Gro-level when compared with healthy control group (Fig. 2).

Plasma levels of RANTES
The plasma levels of RANTES were more elevated in the acute stage of Kawasaki disease than measles and healthy controls (Fig. 3), but there was no statistical significance. Among the patients with Kawasaki disease, there was no obvious correlation between clinical stages of the Kawasaki disease and the plasma levels of RANTES, yet it is elevated in each stage (Fig. 4).

Expression of Gro-and RANTES mRNA in PBMCs
The expression of Gro-mRNA was increased in every case of measles, but was not in the acute stage of the Kawasaki disease and the control group. RANTES mRNA was ex-  pressed extensively in both diseases, but not in the control group (Fig. 5).
According to the clinical stages in Kawasaki disease, RAN-TES mRNA was expressed inconsistently, but Gro-mRNA was not expressed (Fig. 6).

DISCUSSION
Kawasaki disease is an acute febrile illness of early childhood (1). Although the clinical and epidemiological features suggest an infectious origin, its etiology remains unknown. Histopathological findings in Kawasaki disease indicate a vasculitis that predominantly affects the medium-sized arteries (19,20).
The differential diagnosis includes scarlet fever, toxic shock syndrome, measles, drug reaction, viral infection, and other vasculitis syndromes. Especially, Kawasaki disease shares some clinical findings with measles such as high fever, conjunctivitis, and skin rash. It is important to differentiate of these diseases during the acute febrile stage in endemic areas of measles.
The chemokines (chemotactic cytokines) are a large family of cytokines composed of small pro-inflammatory peptides that regulate trafficking, activation and sometimes the proliferation of myeloid, lymphoid, melanocytes, and endothelial cells (22). The chemokines presently comprise approximately 60 members. Based on the predicted primary amino acid structure, the superfamily is divided into four groups  1st  2nd  3rd  1st  2nd  3rd  1st  2nd  3rd  1st  2nd  3rd  1st  2nd  3rd actin Pt2 Pt3 Pt4 Pt5 (23)(24)(25): the CXC or chemokines, the CC or chemokines, the C or chemokines, and the CX3C or chemokines. The chemokine Gro-is a member of the CXC chemokine and is chemotactic for neutrophils, basophils, monocytes, and lymphocytes, and is responsible for much of the tissue damages associated with chronic infection. Gro-is over expressed, contribute to the ongoing inflammatory process and neutrophil infiltration associated with various diseases including psoriasis (26,27), rheumatoid arthritis (28), and ulcerative colitis (29). And also, it is angiogenic (30), like IL-8, and has a role in wound healing (31,32) through the stimulation of the proliferation of keratinocytes. It is also speculated, although not yet proven, Gro-performs the function of IL-8 in mice (6).
Recent study reports that IL-8 and RANTES have been suggested to be involved in the pathogenesis of Kawasaki disease (5,33). Moreover, RANTES mRNA was induced by measles virus infection (34). So we focused on RANTES and Gro-instead of IL-8.
In this study, the production of Gro-was markedly elevated during the acute stage of measles than Kawasaki disease and healthy control. And there were no remarkable changes of plasma level of Gro-according to the clinical stages in Kawasaki disease (levels were below 150 pg/mL). This was coincided with the expression patterns of Gro-mRNA. The expression of Gro-mRNA was increased in every case of measles, but not in Kawasaki disease, even in different clinical stages. Therefore, the production and gene expressions of Gro-seem to have no significant correlation on the clinical stages of Kawasaki disease. Otherwise, we assume that Gromight play an important role in the acute febrile stage of measles.
The chemokine RANTES belongs to the CC chemokines and attract mainly T lymphocytes and monocytes, and is involved in a number of inflammatory diseases, including rheumatoid arthritis (35), allergy in airway (36), and multiple sclerosis (37).
The production of RANTES was elevated in the acute stage of Kawasaki disease and measles than healthy control in this study. However, there were no significant changes of plasma RANTES levels according to the clinical stages of Kawasaki disease. The mRNA of RANTES was expressed, but was not consistent according to the clinical stages. We presume that would be the natural course of Kawasaki disease or one of the effects of the IVIG infusion. So we could not find any obvious correlation between the plasma level and gene expression of RANTES in the patients with Kawasaki disease.
And also, there was no obvious relation between clinical characteristics of the disease including the coronary artery dilatation and RANTES. These results were similar to Wong et al. (5), demonstrating that RANTES gene expression levels were significantly elevated, but there was no obvious correlation between the clinical stage of Kawasaki disease and chemokine expression level. Otherwise, they reported that persistence or increased expression of RANTES gene into convalescent stage may contribute to further risk of coronary artery dilatation. In the acute stage of measles, the expression of RANTES mRNA was strong that was similar to Noe et al. (34). The expression patterns of chemokine genes are variable according to the studies (37)(38)(39)(40). Further study for the chemokine expressions and roles are still required in Kawasaki disease and measles.
In conclusion, these results suggested that Kawasaki disease differs from measles with regard to Gro-production and expression, but not RANTES. Gro-might play an important role in the acute febrile stage of measles, but not in Kawasaki disease. Further studies are needed to clarify the efficacy of Gro-as a marker for activities of measles.