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KD is an acute illness of infancy and early childhood characterized by prolonged fever, mucosal inflammation, polymorphous skin rash, erythema of the peripheral extremities, and nonsuppurative lymphadenopathy(1). Coronary artery lesions develop in 10-15% of patients with KD(2). The exact pathogenesis of KD has not yet been established. However, various immunologic abnormalities have been observed in patients with KD, including increased serum levels of cytokines, such as IL-1, IL-2, IL-6, tumor necrosis factor-α, interferon-γ, and soluble IL-2 receptors(3–8). Recent studies have suggested that SAs are involved in the pathogenesis of KD. The selective TCR Vβ2 family is increased in peripheral blood lymphocytes of acute KD patients(9–11). Leung et al.(12,13) reported that TSST-1 is directly involved in the pathogenesis of KD. However, the pathogenic role of SA in KD remains controversial(14–16). SAs stimulate very large number of T cells in a TCR Vβ-dependent manner, leading to large amounts of cytokines(19). SA-stimulated T cells do not respond to subsequent stimulation by the same SA, because SAs induce anergy or peripheral apoptosis(20,21). SAs have been established as the cause of specific staphylococcal and streptococcal infections, such as toxic shock syndrome(17,18), toxic shock-like syndrome, and scarlet fever(19).

KD and scarlet fever are both associated with a skin rash, strawberry tongue, and desquamation of the peripheral extremities during the convalescent period. Therefore, the T cell response to streptococcal SA was investigated in KD patients by comparison with staphylococcal SA to determine whether streptococcal SA is involved in the pathogenesis of KD.

METHODS

Subjects

Forty-five Japanese KD patients who fulfilled the revised criteria of the KD Research Committee of Japan(22) were enrolled in this study. They were 23 male and 22 female patients, their mean age of onset was 22 ± 15 mo, ranging from 3 mo to 5 y and 10 mo. All patients received high dose γ-globulin and aspirin therapy within 7 d of the onset of disease. Coronary artery lesions were present in 13.3% of patients. None of them had an elevated antistreptolysin O titer. The onset of KD was defined as the day on which the patient developed fever. A total of 77 blood samples were obtained from these KD patients in the acute and convalescent stages of the disease. The acute stage was defined as before the initiation of the therapy. The convalescent stage was divided into following three stages: the VEC stage was defined as within 1 mo, the EC stage was defined as from 1 to 2 mo, and the LC stage was defined from 2 mo to 1 y from the onset of illness (Table 1).

Table 1 Characteristics of patients with KD
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Twenty-four age-matched healthy children were studied as normal control subjects. A disease control group, consisting of another 13 children with various diseases in convalescent stage within 1 mo from onset, was also included in this study. The disease control group diagnoses were: AP in five, SSSS in four, and AGN in four patients. Informed consent was obtained from the parents of all patients before blood collection.

SAs. SPE-A and SPE-C were used as streptococcal SAs, and TSST-1 was used as a staphylococcal SA. All SAs were purchased from Toxin Technology (Sarasota, FL).

Lymphoid cell proliferation assay. Heparinized blood from patients and control subjects was centrifuged in Lymphoprep (Nycomed Pharma AS, Oslo, Norway) to obtain peripheral blood mononuclear cells. Purified mononuclear cells (1 × 105/well) were cultured at 37°C in 5% carbon dioxide in a round-bottomed 96-well plate (Corning, New York) with SAs (SPE-A and SPE-C, 1 ng/mL, TSST-1, 0.1 ng/mL) in 0.2 mL of RPMI 1640 containing 50 U/mL penicillin-streptomycin (ICN Biomedicals Inc., Costa Mesa, CA) and 5% heat-inactivated fetal bovine serum for 3 d. Cultures were then pulsed with 37 kBq of [3H] thymidine/well. After 6 h, cells were harvested onto glass fiber filters, and [3H]thymidine incorporation was determined by direct β counting. For proliferation assays, data represent the mean of duplicate determinations.

IL-2 assay. To reconfirm T cell activation by SAs, IL-2 production of cultured T cells obtained from KD patients (n = 26) and normal control subjects (n = 5) were examined. IL-2-dependent CTLL cells (1 × 104/well) were cultured for 20 h with 20% of the supernatant from T cell proliferation assay after 24 h in 96-well plates and then pulsed with 37 kBq of [3H]thymidine. Cells were harvested after 4 h, and [3H]thymidine incorporation was measured to estimate the IL-2 production. The IL-2 concentration (U/mL) in each sample was calculated by comparing the titrations in test samples with a standard curve for recombinant human IL-2 (Collaborative Research, Inc., Bedford, MA)(23).

Statistical analysis. T cell proliferation by each SA was analyzed by ANOVA, multiple comparison test (Bonferroni/Dunn test for post hoc test). The recovery of T cell response to SA with disease duration was tested by regression analysis. Fisher's exact probability test was used to compare the number of patients with low response to SPE-C and to other SAs. Stringent statistical evaluation was applied in analyzing data with a multitude of comparisons. When the Mann-Whitney U test was used to analyze eight clinical data between two groups of patients according to T cell response to SPE-C, the significant p value was defined as <0.006. Each comparison test was performed using Statview 4.0®(Abacus Concepts, Inc., Berkeley, CA), and a p value of <0.05 was defined as significant except when the Mann-Whitney U test was used.

RESULTS

T cell proliferation by SAs. Transient low T cell proliferation was observed in most of patients with KD in the early phase when T cells were stimulated by SPE-C (Fig. 1) (ANOVA p = 0.0003). The Bonferroni/Dunn test revealed that T cells of KD patients in the acute stage (p = 0.0001), in the VEC stage(p = 0.0005), and in the EC stage (p = 0.0031) showed statistically lower proliferation to SPE-C than those from healthy control subjects. In the LC stage, T cell responses to SPE-C recovered to the same level as that of normal control subjects. Regression analysis also indicated that the low T cell proliferation by SPE-C recovered with disease duration(r2 = 0.070, p = 0.0199). Such observations, however, were not found when T cells were stimulated by SPE-A or TSST-1.

Figure 1
figure 1

SA stimulation of T cell proliferation,(a) SPE-A, (b) SPE-C, and (c) TSST-1. The dotted line indicates the 10th percentile in normal healthy control subjects (SPE-A, 2996 cpm; SPE-C, 2343 cpm; and TSST-1, 5268 cpm).

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The incidence of low T cell responders to SAs in KD patients is shown in Figure 2. A low T cell responder to each SA was defined as a patient whose T cell proliferation (counts/min) was less than the 10th percentile of healthy control subjects. The incidence of low responders to SPE-C was statistically higher in the acute, VEC, and EC stages of KD than that of low responders to TSST-1. In the LC stage, on the other hand, there was no statistical difference in the incidence of low responders between SPE-C and other SAs.

Figure 2
figure 2

Low T cell response to SAs in patients with KD. The cut-off value was defined as the 10th percentile of T cell proliferation in normal healthy control subjects. *p < 0.05,**p < 0.01 compared with SPE-C (Fisher's exact probability test).

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T cells from patients with AGN showed decreased levels of T cell proliferation when elicited by SPE-C, but not by other SAs(Fig. 3). On the other hand, T cells from SSSS or AP patients responded normally to SPE-A, SPE-C, and TSST-1. However, the number of patients were too low to draw conclusions regarding responses in specific diseases.

Figure 3
figure 3

T cell proliferation in patients with AP(n = 5), SSSS (n = 4), and AGN (n = 4). The dotted line indicates the 10th percentile of T cell proliferation in normal healthy control subjects.

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IL-2 production by SAs. SPE-C-stimulated T cells from almost of all KD patients in the acute, VEC, and EC stages failed to produce IL-2(Fig. 4). In the LC stage, however, the ability to produce IL-2 returned to the same level as that of normal control subjects. Some patients failed to produce IL-2 stimulated by SPE-A in the acute and EC stage. On the contrary, T cells from KD patients in all stages of disease cultured with TSST-1 produced levels of IL-2 similar to normal control subjects. The numbers of samples were too low to statistical analysis.

Figure 4
figure 4

IL-2 production by SA-stimulated T cells in peripheral blood from patients with KD, (a) SPE-A, (b) SPE-C, and (c) TSST-1.

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Relationship between the T cell response to SPE-C and clinical features of KD. A higher peak platelet count and longer C-reactive protein-positive period were observed in the low responder group to SPE-C (n= 13) in the acute stage of KD when compared with those in the normal responder group (n = 8), although p value did not reach a significant level (Fig. 5). There were no significant differences between the two groups in the duration of fever, white blood cell count, erythrocyte sedimentation rate, serum level of albumin, or in the dose of γ-globulin used. The presence of coronary artery lesions did not correlate with the T cell response to SPE-C.

Figure 5
figure 5

Relationship between T cell response to SPE-C and the clinical and laboratory findings in patients with acute KD. Low responders (n = 13), <10th percentile of T cell proliferation in normal healthy control subjects. (a) Peak platelet count and(b) the duration of the C-reactive protein-positive period.

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All of five patients with a recurrent or persistent fever after the first high dose γ-globulin therapy were low responders to SPE-C but normal responders to SPE-A and TSST-1 when examined at the acute stage of KD. Statistical analysis was not carried out because of the small number of patients with recurrent or persistent fever (Fig. 6).

Figure 6
figure 6

Relationship between the T cell response to SAs and recurrent or persistent fever in patients with acute KD. (+) Patients with recurrent or persistent fever in KD after high doseγ-globulin therapy (n = 5). (-) Patients without recurrent or persistent fever (n = 16). The dotted line indicates the 10th percentile of T cell proliferation in normal healthy control subjects.

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DISCUSSION

T cells obtained from peripheral blood of KD patients showed a transient low response to streptococcal SAs, especially to SPE-C, as demonstrated by decreased proliferation and IL-2 production. This low T cell response to SPE-C was not due to the influence of γ-globulin therapy, because it was observed before the initiation of the γ-globulin therapy in these KD patients. In addition, T cell responses to SPE-C were normal afterγ-globulin therapy in four patients without KD; one patient with sepsis due to Staphylococcus aureus, one with SSSS, one with idiopathic thrombocytopenic purpura, and one with Guillain-Barré syndrome (data not shown).

The mechanism of the transient low T cell response observed in KD patients is of interest for the understanding of the pathogenesis of KD. There are two possible mechanisms for the specific T cell response to SPE-C in KD: a migration of SPE-C-reactive T cells from the peripheral circulation to inflammatory lesions and anergy of SPE-C-reactive T cells.

The first mechanism, T cell migration, is supported by some previous reports. Furukawa et al.(24–26) noted the absence of T cell activation in peripheral blood of acute KD patients, although there have been observed increased levels of cytokines in this setting(3–8). It has been reported that activated T cells were found in the bacillus Calmette-Guérin vaccination inoculation(27,28), and an expansion of Vβ2+ T cells were in the myocardium, coronary arteries, and small intestinal mucosa in patients with acute KD(13,29). These findings suggest that in patients with KD the low T cell response to SPE-C may be due to migration of SPE-C-reactive T cells from the peripheral circulation to inflammatory lesions.

The other possible mechanism for the transient low T cell response to SPE-C in KD patients is T cell anergy, which can be defined as a nonproliferative state in which cell division is prevented by the inhibition of IL-2 production(30,31). SAs are known to induce transient anergy in T cells(20,21), followed by expansion of T cells with specific TCR Vβ repertoires(19). However, studies examining the specific TCR Vβ repertoire in KD patients have produced conflicting results(9–11,14–16). Ohnishi et al.(32) have reported that characteristic Vβ depletion was observed in patients with streptococcal toxic shocklike syndrome. They suggested that the amount of SA may be an important factor in determining whether specific TCR Vβ expansion or deletion was seen(32). Miethke et al.(33) reported that the number of Vβ-specific T cells increased markedly for 3 d after mice were injected with a high concentration of SEB, with the number of Vβ-specific T cells falling below baseline levels after 5 d. They also detected T cell anergy within 24 h of injection of SEB. Low dose SEB caused no change in the number of Vβ-specific T cells but did trigger transient anergy to SEB(33). Migita and Ochi(34) reported that anergic T cells recovered 4 mo after injection of SEB. Therefore, the dose of the SA and the timing of examinations may influence the evaluations of the immunologic effects of SAs on T cells. The transient low T cell response to SPE-C in the present study may have been caused by anergy, regardless of whether the number of Vβ-specific T cells changed in acute KD.

KD is usually diagnosed after approximately 5 d from onset, which may be a sufficient period to allow the induction of anergy on T cells. If the anergy is caused by SPE-C, the amount of SPE-C may be too low to allow the T cells to release much cytokine, but sufficient to induce a state of anergy, resulting in no expansion of the specific TCR Vβ repertoire.

We did not examine any evidence of invasion of SPE-C or the presence of streptococci producing SPE-C in patients with KD and thus did not confirm a direct role for SPE-C in the etiology of KD in the present study. The failure of production of IL-2 stimulated by SPE-A found in some patients may be caused by the antigenic similarity between SPE-A and SPE-C(19). However, the present results suggest that SAs, especially SPE-C, may be involved in the pathogenesis of KD. Further investigations are needed to clarify the mechanism for the low response of T cells to SPE-C in KD patients.