Clin. exp. Immunol. (1991) 86, 134- 139
Serum soluble CD4 and CD8 levels in Kawasaki disease S. FURUKAWA*, T. MATSUBARA*, K. TSUJI*, T. MOTOHASHI*, K. OKUMURAt & K. YABUTA* Departments of * Pediatrics and t Immunology, Juntendo University School of Medicine, Tokyo, Japan (Acceptedfor publication 9 May 1991)
SUMMARY
The levels of soluble CD4 (sCD4) and sCD8 in serum correlate with the T cell subset activation and may be important in monitoring and characterizing disease processes during immunological diseases. We compared acute Kawasaki disease (KD) with anaphylactoid purpura (AP) and acute febrile viral infections, such as measles and infectious mononucleosis (IM), in terms of serum sCD4 and sCD8 levels. The levels of serum sCD4 and sCD8 were measured by a sandwich enzyme immunoassay. In addition, peripheral blood mononuclear cell subsets were analysed by single and two-colour flowcytometric analyses in KD and IM patients. The levels of serum sCD4 and sCD8 were significantly elevated in patients during acute stages of KD, measles and IM, but not AP. Peripheral blood CD4+, CD8+ and also HLA-DR+ T cells count did not increase during the acute stage of KD; however, peripheral blood CD8+ and HLA-DR+ T cell counts were increased during the acute stage of IM. Our results suggest that there is a low level of activation of peripheral blood T cells during acute KD, or that infiltrated T cells in some local tissues of KD patients contribute to the elevated levels of serum sCD4 and sCD8. Keywords Kawasaki disease anaphylactoid soluble sCD4 and sCD8
purpura
INTRODUCTION Fujimoto, Levy & Levy (1983) showed that CD8 antigen was spontaneously released from CD8 + leukaemic cells. Subsequently the amount of soluble CD8 (sCD8) has been shown to increase upon activation of CD8+ T cells in vitro. It has been suggested that quantitative determination of sCD8 in serum may serve as an index of suppressor/cytotoxic T cell activity (Tomkinson et al., 1989). The concentration of sCD8 found in serum has been shown to be elevated in human immunodeficiency virus (HIV) infection (Reddy, Lange & Grieco, 1989), rheumatoid arthritis (Symons et al., 1990) and bronchial asthma (Hsieh, 1989). Soluble CD4 (sCD4) has been investigated in vitro with regard to the role of potent inhibitors of HIV infection (Smith et al., 1987; Deen et al., 1988; Fisher et al., 1988; Hussey et al., 1988; Traunecker, Luke & Karjalaimen, 1988). Regarding sCD4 in vivo studies, preliminary data indicate that there is a baseline level of natural sCD4 in normal sera (Kline et al., 1989), and that serum sCD4 levels are elevated in HIV infection (Reddy, Vodian & Grieco, 1990). The level of sCD4 and sCD8 in serum may be important in monitoring or characterizing disease processes and may provide insight into the immunoregulation of cell growth and cell differentiation.
measles infectious mononucleosis
Recently, it has been reported that the levels of serum cytokines, such as tumour necrosis factor-x (TN F-x) (Furukawa et al., 1988; Maury, Salo & Pelkonen, 1989), interleukin 1 (IL- 1) (Maury, Salo & Pelkonen, 1988), IL-2 receptor (IL-2R) and gamma interferon (IFN-,') levels (Matsubara, Furukawa & Yabuta, 1990), and IL-6 activity (Ueno et al., 1989) were increased during the acute stage of Kawasaki disease (KD) (Kawasaki et al., 1974). The results of these studies suggest that the immunological abnormalities observed in the patients with KD are related to hyperreactive responses in macrophages/ monocytes as well as T cells. Although peripheral blood CD14+ macrophage/monocyte and FcER2/CD23-positive macrophage/monocyte counts were increased (Furukawa et al., 1988, 1990a, 1990b), peripheral blood T cell counts did not change during the acute stage of KD (Furukawa et al., 1990a). In the present study, we investigated whether changes in sCD4 and/or sCD8 antigen levels in serum correlated with vasculitis, acute KD and anaphylactoid purpura (AP), or correlated with acute febrile viral infections, such as measles and infectious mononucleosis (IM).
MATERIALS AND METHODS The subjects of the study consisted of 33 patients with acute KD, five with AP, 14 with measles, and nine with IM, who were seen at our hospital between March 1986 and June 1990.
Correspondence: Dr Susumu Furukawa, Department of Pediatrics, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
134
Soluble CD4 and CD8 in Kawasaki disease Kawasaki disease The patients in this study met specific diagnostic criteria for KD (Kawasaki, 1985). We included 19 males and 14 females, aged from 2 months to 5 years and I month (the mean being 2 years and 8 months). The day of onset of fever was recognized as the first day of illness. Blood samples were taken for analysis of sCD4 and sCD8 levels in serum and peripheral blood mononuclear cell subsets on the third to the ninth days (the mean being 5 8 + 1 9) of illness before treatment (acute stage) and on the 21st to the 64th days (the mean being 34-4 + 15 6) of illness (convalescent stage). Two-dimensional echocardiography was used to detect the presence of coronary-artery lesions (CAL). In accordance with the KD cardiovascular lesion diagnostic criteria of the Research Committee on Kawasaki Disease, Ministry of Health and Welfare, Japan, coronary arteries with diameters of 4 mm or greater were regarded as showing the presence of CAL (Research Committee on Kawasaki Disease, 1984). During a 30-day period after the onset of the disease, nine KD patients in this study had CAL. Twelve of 33 KD patients were analysed for serum sCD4 and sCD8 levels in acute and convalescent sera. Seventeen of 33 KD patients were analysed for peripheral blood mononuclear cell subsets obtained by single-colour flow-cytometric analysis in the acute and convalescent bloods. In addition, six of 17 KD patients were analysed for peripheral blood mononuclear cell subsets obtained by twocolour flow-cytometric analysis in the acute and convalescent bloods.
Anaphylactoid purpura This study included four males and one female aged from 3 years and 7 months to 14 years and 6 months (the mean being 8 years and 4 months). The diagnosis was based on common clinical symptoms, such as mild fever, purpura, arthritis and colicky abdominal pain. All of these patients had purpura. From the onset of purpura to sampling, an average of 5 2 + 3-7 days had elapsed. Anaphylactoid purpura patients in this study had no renal involvements. Measles This study included two males and 12 females aged from I year and 6 months to 10 years and 4 months (the mean being 3 years and 6 months). From the onset of fever to sampling, an average of 5-5 + 1 6 days had elapsed. Four of the 14 patients with measles had sera taken during the convalescent stage, the 20th to the 32nd day of the illness (the mean being 26 + 8 5).
Infectious mononucleosis This study included five males and four females aged from 1 year and 3 months to 13 years and 11 months (the mean being 5 years and 10 months). The diagnosis was based on common clinical symptoms of acute IM and atypical lymphocytes in the peripheral blood. All patients were positive for heterophile antibody and IgM antibody to Epstein-Barr virus (EBV) capsid antigen as determined by indirect fluorescence assay (Henle, Henle & Horwitz, 1974). From the onset of fever to sampling, an average of 8-3 + 2 5 days had elapsed. These patients had values on peripheral blood mononuclear cell subsets obtained by single- and two-colour flow-cytometric analyses in the acute blood. Eight of the nine patients with IM had sera taken during the convalescent stage, which tested positive for antibody to
135
EBV nuclear antigen (Reedman & Klein, 1973) during the 102nd to 200th day of the illness (the mean being 1513 + 39-9).
Healthy children The control subjects were 12 healthy children, seven males and five females aged from 2 months to 4 years and 7 months (the mean being 2 years and 4 months). The control samples were tested in parallel with the patients' samples.
Quantification of CD8 concentration in serum The CD8 concentration in serum was determined using a CELLFREE T8 test kit (T cell Sciences, Cambridge, MA). The principle of the method was a sandwich enzyme immunoassay as described by Tomkinson et al. (1989). The assay employs two murine anti-CD8 monoclonal antibodies (MoAb) recognizing different epitopes of the CD8 molecule. The first anti-CD8 MoAb was absorbed onto a polystyrene 96-well microtitre plate. CD8 present in the samples or standards bound to the antibody-coated wells, while unreacted sample components were removed by washing. The horseradish peroxidase (HRP)conjugated second anti-CD8 MoAb was then added, which bound to the sCD8 captured by the first antibody and thus completed the sandwich format of the assay. After the removal of unbound HRP-conjugated anti-CD8 by washing, O-phenylenediamine was added to the wells. A coloured product was formed in proportion to the amount of CD8 present in the sample. The reaction was terminated with stop solution (2 N H2SO4) and absorbance at 490 nm was measured. A standard curve was constructed using standards of a known concentration supplied by the manufacturer, and unknown samples were determined from the standard curve. The lower limit of the test kit was about 50 U/ml. Quantification of CD4 concentration in serum The CD4 concentration in serum was determined using a CELLFREE CD4 test kit (T cell Sciences, Cambridge, MA). The principle of the method was a sandwich enzyme immunoassay as described by Kline et al. (1989) using two murine antiCD4 MoAbs. The practical procedure is similar to that of the CELLFREE T8 test kit. The lower limit of the test kit was 12 U/ ml. Measurement of the peripheral blood CD3+, CD4+ and CD8+ T cell counts Using a method previously described (Furukawa et al., 1988, 1990a, 1990b), the percentage of the peripheral blood T cell subsets among mononuclear cells was assayed with a fluorescence-activated cell sorter (FACS IV, Becton Dickinson ICS, Mountain View, CA). For fluorescence procedures the MoAbs used were isothiocyanate-conjugated MoAb of Leu 4/CD3 (Becton Dickinson Monoclonal Center, Inc, San Jose, CA), Leu 3a/CD4 and Leu 2a/CD8, and phycoerythrin-conjugated MoAb of HLA-DR, in single- and two-colour flow-cytometric analyses. From the ratio of peripheral blood mononuclear cells to the leucocyte count, the absolute count of CD3+, CD4+, CD8+, HLA-DR+CD4+ or HLA-DR+CD8+ T cells was calculated. Statistical analysis Statistical analyses were performed using Student's t-test and paired t-test.
136
S. Furukawa et al. Table 1. Serum soluble CD4 (sCD4) and soluble CD8 (sCD8) levels of patients with Kawasaki disease (KD), anaphylactoid measles and infectious mononucleosis (IM), and of control subjects
purpura
(AP),
KD
n Mean day from onset sCD4 (U/ml) sCD8 (U/ml)
Acute
Convalescent
AP
33
12 34 4+ 15 6 30 6+5 9 393 8 + 84 3 0 08+0 03
5 52 + 37 20 0+2-5 317 0 + 78 8 0 07+0 03
58 + 1 9 42 9+ 18 5t 532 4+ 171 4t 0 09+0 05
sCD4/sCD8
IM
Control subjects
14
9
12
55 + 1 6
83 +2 5 65 6+ 12.0*
Measles
62 4+25-9* 924 3 + 505 6* 0 10+0 06
13566 +7463*
0.004+0.001*
28 1 +4 4 391 7 + 71 9 0 07+0 02
Values are expressed as mean + s.d. * Significant at P < 0-01 versus control subjects. t Significant at P < 0-01 Lversus convalescent stage of KD and control subjects. 100l0
0
80
0o
0 E
0 D
,-
60 Ccn
0
a) 40
0
E
0
Os8
*8
0 -0 0
(n
*0 *0
20
0
,-1
n
3-4 n=10 l
5-6
7-8
9-10
n/=7 n=12 Acute stage
n=4
21 -64 n=12 Convalescent stage
Time from onset of fever (days)
Fig. 1. The correlation between the day of onset of fever and Normal range; 0, case with coronary-artery lesion; , mean;
serum 0,
RESIU LTS Table shows serum sCD4 and sCD8 levels during the acute and convalescent stages of KD, the acute stages of AP, measles and IM, and in control subjects. Serum sCD4 and sCD8 levels found in patients with KD were high during the acute stage of the disease, as compared with those found at the convalescent stage and in control subjects. In addition, elevated levels of serum sCD4 and sCD8 were found in patients with measles and IM during the acute stage, as compared with those in control subjects. These increased sCD4 and sCD8 levels, found in patients with measles and IM, were seen to return to the normal range during the convalescent stage (measles, 30 9 + 4 6 (sCD4), 404 5 + 27-6 (sCD8); IM, 31 + 6 6 (sCD4), 432-4 + 140 2 (sCD8), mean + s.d. respectively). It was noted that serum sCD8 levels in patients with IM were markedly elevated during the acute stage
soluble CD4 (sCD4) levels during acute Kawasaki disease. l, without coronary-artery lesion.
case
of the disease. In contrast, serum sCD4 and sCD8 levels in patients with AP did not increase during the acute stage of the disease. Figures and 2 show the correlation between the days of onset of fever and serum sCD4 and sCD8 levels during acute KD. There were no significant differences in serum sCD4 and sCD8 levels between KD patients with and without CAL. Table 2 shows the absolute counts of leucocytes, mononuclear cells, CD3+, CD4+, CD8+, HLA-DR-CD4+ and HLA-DR+CD8+ T cells during the acute and convalescent stages of KD, the acute stage of IM and in control subjects. Absolute counts of CD3 +, CD4+ and CD8 ' T cells found in patients with KD did not increase during the acute stage of the disease, in comparison with those found at the convalescent stage and in control subjects. In addition, there were no significant differences in the absolute counts of HLA-
137
Soluble CD4 and CD8 in Kawasaki disease
I000
0 0
-
800 _
0
a
0 0
0 D
600 _
-
0
.J
0
U,
0
0
0
0
E
0
000
1
*I
0
400 _
000
:-
0
(17
200
0
3-4 n=10
21 -64 9-10 7-8 5-6 n=12 n=7 n=4 n=12 Convalescent stage Acute stage Time from onset of fever (days)
Fig. 2. The correlation between the day of onset of fever and serum soluble CD8 (sCD8) levels during acute Kawasaki disease. U, Normal range; 0, case with coronary-artery lesion; -, mean; 0, case without coronary-artery lesion. Table 2. Peripheral blood T cell subsets in patients with Kawasaki disease (KD) and infectious mononucleosis (IM) KD
n Leucocytes
Mononuclear cell CD3+ Tcell CD4+ Tcell CD8+ Tcell CD4+ cell/CD8+ cell n
HLA-DR+CD4+ T cell HLA-DR+CD8+ T cell
Acute
Convalescent
IM acute
Control subjects
17 17 618 + 6682* 4832+2517 2270+ 1450 1693+1023 771-1+475-2 2-3+0-8 6 78-1 + 59-8 33-3 + 17-8
17 8627 +2424 4672+1345 2855+1149 2051+757-4 1063+447-1 2-1 +0-9 6 52 3 +20-8 44 5 + 7-8
9 16 633 + 6377* 13 664 + 6009* 10625+5012* 2817+1497 7219+3319* 0-4+0-2* 9 837+380* 6152 + 2776*
12 8356+2204 4826+ 1201 3117+1096 2212+744-5 1037+379-9 2-3+09 6 57-4+ 15-3 55 2 + 29-0
Values are expressed as mean + s.d./mm3. * Significant at P < 0 01 versus control subjects.
DR+CD4+ T cells and HLA-DR+CD8+ T cells during the
were also measured in patients' sera during the acute stage of AP
acute stage of KD and in control subjects. In contrast, absolute counts of CD3+, CD8+, HLA-DR+CD4+ and HLA-
and the acute febrile stages of measles and IM. Although AP shows clinical symptoms which differ from those of KD, it shares some pathological findings with KD, namely vasculitis of small vessels; therefore the levels of sCD4 and sCD8 in sera of both groups of patients were measured to determine the role of T cell subsets in the pathogenesis of vasculitis. We also quantified sCD4 and sCD8 in sera of patients with measles and IM, which represent acute febrile viral infections, to which KD may also belong. The activation of the immune system with measles has been previously reported (Griffin et al., 1989, 1990).
DR+CD8+ T cells found in patients with IM increased during the acute stage of the disease, in comparison with those in control subjects.
DISCUSSION Our study represents the first report on the simultaneous determination of sCD4 and sCD8 in the sera of patients with acute KD. In parallel studies, the levels of both soluble antigens
138
S. Furukawa et al.
Peripheral blood T cells are the main target cells of measles virus infection. In contrast, acute IM is a self-limiting lymphoproliferative disease caused by EBV (Henle, Henle & Deihil, 1968). Although B lymphocytes are the target cells of EBV infection, the expansion of T lymphocytes, primarily those of the CD8 cytotoxic/suppressor subset, is responsible for the lymphocytosis evident during acute IM (De Waele, Thielemans & Van Camp, 1981). In the present study, KD, measles and IM patients had increased levels of serum sCD4 and sCD8 during the acute stage of the disease (Table 1). We confirmed the results of previous studies showing raised serum sCD8 levels in patients with measles (Griffin et al., 1989) and IM (Tomkinson et al., 1989). However, serum sCD4 and sCD8 levels in patients with AP did not increase during the acute stage. We reported that patients with AP had increased serum TNF-a levels, but not IL-2R and IFN-y levels in serum during the acute stage (Furukawa et al., 1989). Although peripheral blood CD4+ T cells, CD8+ T cell and also HLA-DR+ T cell counts were not increased during the acute stage of KD in the present study, serum sCD4 and sCD8 levels were elevated during the acute stage of KD, suggesting that T cells were activated under these conditions. These findings agree with our previous study which showed that serum IFN-y and IL-2R levels increased during the acute stage of KD (Matsubara, Furukawa & Yabuta, 1990). Since the expression of HLA-DR on peripheral blood T cells in KD patients was not increased during the acute stage, it is likely that there is a mild activation of peripheral blood T cells. Whether or not tissue lymphocytes were activated was not determined in the present study, although we have previously reported that skin lesions in biopsy specimens of KD were infiltrated with activated T cells as well as macrophages/monocytes (Sugawara et al., 1987). The contribution oflocalized T cells in affected tissues to the elevated serum levels of IL-2R was observed in patients with organ transplant rejections (Colvin et al., 1989). In the present study, peripheral blood CD8+, HLADR+CD8+, and also HLA-DR+CD4+ T cell counts increased during the acute stage of IM. It is noted that markedly increased CD8+ and CD8+HLA-DR+ T cell counts as well as sCD8 levels were seen during the acute stage of IM. We confirmed earlier studies that serum sCD8 levels were seen to increase markedly during the acute stage of IM patients, which correlated with the observation of an increased number of peripheral blood T cells expressing HLA-DR, a phenotypic characteristic of T cell activation in IM patients (Tomkinson et al., 1989). With regard to the sCD4/sCD8 ratio, KD was similar to measles, but differed from IM. These results were also supported by our previous report that the changes in natural killer cell and CD8+ T cell subsets in acute KD were in contrast to those of IM (Furukawa et al., 1991). Although we failed to prove significance in statistical analysis, serum sCD4 levels in patients with KD were seen to increase during the early part of the acute stage and sCD8 levels were seen to increase during the latter part of the acute stage. It remains unclear whether the reciprocal changes in serum sCD4 and sCD8 levels during the acute stage are related to the pathogenesis of KD.
ACKNOWLEDGMENTS This work was partially supported by a Research Grant (from 1988 to
1990) for Kawasaki disease from the Ministry of Health and Welfare, Japan. We wish to thank T. Obara for his support.
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Soluble CD4 and CD8 in Kawasaki disease Abstract, 4th Annual Conference on Clinical Immunology, Arlington, VA. MATSUBARA, T., FURUKAWA, S. & YABUTA, K. (1990) Serum levels of tumor necrosis factor, interleukin 2 receptor and gamma interferon in Kawasaki disease involved coronary-artery lesion. Clin. Immunol. Immunopathol. 56, 29. MAURY, C.P.J., SALO, E. & PELKONEN, P. (1988) Circulating interleukinI# in patients with Kawasaki disease. N. Engl. J. Med. 319, 1670. MAURY, C.P.J., SALO, E. & PELKONEN, P. (1989) Elevated circulating tumor necrosis factor-a in patients with Kawasaki disease. J. lab. clin. Med. 113, 651. REDDY, M.M., LANGE, M. & GRIECO, M.H. (1989) Elevated soluble CD8 levels in sera of human immune deficiency virus-infected populations. J. cdin. Microbiol. 27, 257. REDDY, M.M., VODIAN, M. & GRIECO, M.H. (1990) Elevated levels of CD4 antigen in sera of human immunodeficiency virus-infected populations. J. cdin. Microbiol. 28, 1744. REEDMAN, B.M. & KLEIN, G. (1973) Cellular localization of an EpsteinBarr virus (EBV)-activated complement-fixing antigen in producer and non producer lymphoblastoid cell lines. Int. J. Cancer, 11, 499. RESEARCH COMMITTEE ON KAWASAKI DISEASE (1984) Report ofSubcommittee on Standardization of Diagnostic Criteria and Reporting of
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Coronary Artery Lesions in Kawasaki Disease p. 56. Ministry of Health and Welfare, Tokyo, Japan. SMITH, D.H., BYRN, R.A., MARSTERS, S.A., GREGORY, T., GROOPMAN, J.E. & CAPON, D.J. (1987) Blocking of HIV-l infectivity by a soluble, secreted form of the CD4 antigen. Science, 238, 1704. SUGAWARA, T., HATTORI, S., HIROSE, S., FURUKAWA, S., YABUTA, K. & SHIRAI, T. (1987) Immunopathology of the skin lesions of Kawasaki disease. In Kawasaki Disease (ed. by S.T. Schulman) p. 185. Alan R. Liss, New York. SYMONS, J.A., WOOD, N.C., DIGIOVINE, F.S. & DUFF, G.W. (1990) Soluble CD8 in patients with rheumatic disease. Clin. exp. Immunol. 80, 354. TOMKINSON, B.E., BROWN, M.C., IP, S.H., CARRABIS, S. & SULLIVAN, J.L. (1989) Soluble CD8 during T cell activation. J. Immunol. 142, 2230. TRAUNECKER, A., LUKE, W. & KARJALAIMEN, K. (1988) Soluble CD4 molecules neutralize human immunodeficiency virus type 1. Nature, 331, 84. UENO, Y., TAKANO, N., KANEGANE, H., YOKOI, T., YACHIE, A., MIYAWAKI, T. & TANIGUCHI, N. (1989) The acute phase nature of interleukin 6: studies in Kawasaki disease and other febrile illnesses. Clin. exp. Immunol. 76, 337.
Serum soluble CD4 and CD8 levels in Kawasaki disease S. FURUKAWA*, T. MATSUBARA*, K. TSUJI*, T. MOTOHASHI*, K. OKUMURAt & K. YABUTA* Departments of * Pediatrics and t Immunology, Juntendo University School of Medicine, Tokyo, Japan (Acceptedfor publication 9 May 1991)
SUMMARY
The levels of soluble CD4 (sCD4) and sCD8 in serum correlate with the T cell subset activation and may be important in monitoring and characterizing disease processes during immunological diseases. We compared acute Kawasaki disease (KD) with anaphylactoid purpura (AP) and acute febrile viral infections, such as measles and infectious mononucleosis (IM), in terms of serum sCD4 and sCD8 levels. The levels of serum sCD4 and sCD8 were measured by a sandwich enzyme immunoassay. In addition, peripheral blood mononuclear cell subsets were analysed by single and two-colour flowcytometric analyses in KD and IM patients. The levels of serum sCD4 and sCD8 were significantly elevated in patients during acute stages of KD, measles and IM, but not AP. Peripheral blood CD4+, CD8+ and also HLA-DR+ T cells count did not increase during the acute stage of KD; however, peripheral blood CD8+ and HLA-DR+ T cell counts were increased during the acute stage of IM. Our results suggest that there is a low level of activation of peripheral blood T cells during acute KD, or that infiltrated T cells in some local tissues of KD patients contribute to the elevated levels of serum sCD4 and sCD8. Keywords Kawasaki disease anaphylactoid soluble sCD4 and sCD8
purpura
INTRODUCTION Fujimoto, Levy & Levy (1983) showed that CD8 antigen was spontaneously released from CD8 + leukaemic cells. Subsequently the amount of soluble CD8 (sCD8) has been shown to increase upon activation of CD8+ T cells in vitro. It has been suggested that quantitative determination of sCD8 in serum may serve as an index of suppressor/cytotoxic T cell activity (Tomkinson et al., 1989). The concentration of sCD8 found in serum has been shown to be elevated in human immunodeficiency virus (HIV) infection (Reddy, Lange & Grieco, 1989), rheumatoid arthritis (Symons et al., 1990) and bronchial asthma (Hsieh, 1989). Soluble CD4 (sCD4) has been investigated in vitro with regard to the role of potent inhibitors of HIV infection (Smith et al., 1987; Deen et al., 1988; Fisher et al., 1988; Hussey et al., 1988; Traunecker, Luke & Karjalaimen, 1988). Regarding sCD4 in vivo studies, preliminary data indicate that there is a baseline level of natural sCD4 in normal sera (Kline et al., 1989), and that serum sCD4 levels are elevated in HIV infection (Reddy, Vodian & Grieco, 1990). The level of sCD4 and sCD8 in serum may be important in monitoring or characterizing disease processes and may provide insight into the immunoregulation of cell growth and cell differentiation.
measles infectious mononucleosis
Recently, it has been reported that the levels of serum cytokines, such as tumour necrosis factor-x (TN F-x) (Furukawa et al., 1988; Maury, Salo & Pelkonen, 1989), interleukin 1 (IL- 1) (Maury, Salo & Pelkonen, 1988), IL-2 receptor (IL-2R) and gamma interferon (IFN-,') levels (Matsubara, Furukawa & Yabuta, 1990), and IL-6 activity (Ueno et al., 1989) were increased during the acute stage of Kawasaki disease (KD) (Kawasaki et al., 1974). The results of these studies suggest that the immunological abnormalities observed in the patients with KD are related to hyperreactive responses in macrophages/ monocytes as well as T cells. Although peripheral blood CD14+ macrophage/monocyte and FcER2/CD23-positive macrophage/monocyte counts were increased (Furukawa et al., 1988, 1990a, 1990b), peripheral blood T cell counts did not change during the acute stage of KD (Furukawa et al., 1990a). In the present study, we investigated whether changes in sCD4 and/or sCD8 antigen levels in serum correlated with vasculitis, acute KD and anaphylactoid purpura (AP), or correlated with acute febrile viral infections, such as measles and infectious mononucleosis (IM).
MATERIALS AND METHODS The subjects of the study consisted of 33 patients with acute KD, five with AP, 14 with measles, and nine with IM, who were seen at our hospital between March 1986 and June 1990.
Correspondence: Dr Susumu Furukawa, Department of Pediatrics, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
134
Soluble CD4 and CD8 in Kawasaki disease Kawasaki disease The patients in this study met specific diagnostic criteria for KD (Kawasaki, 1985). We included 19 males and 14 females, aged from 2 months to 5 years and I month (the mean being 2 years and 8 months). The day of onset of fever was recognized as the first day of illness. Blood samples were taken for analysis of sCD4 and sCD8 levels in serum and peripheral blood mononuclear cell subsets on the third to the ninth days (the mean being 5 8 + 1 9) of illness before treatment (acute stage) and on the 21st to the 64th days (the mean being 34-4 + 15 6) of illness (convalescent stage). Two-dimensional echocardiography was used to detect the presence of coronary-artery lesions (CAL). In accordance with the KD cardiovascular lesion diagnostic criteria of the Research Committee on Kawasaki Disease, Ministry of Health and Welfare, Japan, coronary arteries with diameters of 4 mm or greater were regarded as showing the presence of CAL (Research Committee on Kawasaki Disease, 1984). During a 30-day period after the onset of the disease, nine KD patients in this study had CAL. Twelve of 33 KD patients were analysed for serum sCD4 and sCD8 levels in acute and convalescent sera. Seventeen of 33 KD patients were analysed for peripheral blood mononuclear cell subsets obtained by single-colour flow-cytometric analysis in the acute and convalescent bloods. In addition, six of 17 KD patients were analysed for peripheral blood mononuclear cell subsets obtained by twocolour flow-cytometric analysis in the acute and convalescent bloods.
Anaphylactoid purpura This study included four males and one female aged from 3 years and 7 months to 14 years and 6 months (the mean being 8 years and 4 months). The diagnosis was based on common clinical symptoms, such as mild fever, purpura, arthritis and colicky abdominal pain. All of these patients had purpura. From the onset of purpura to sampling, an average of 5 2 + 3-7 days had elapsed. Anaphylactoid purpura patients in this study had no renal involvements. Measles This study included two males and 12 females aged from I year and 6 months to 10 years and 4 months (the mean being 3 years and 6 months). From the onset of fever to sampling, an average of 5-5 + 1 6 days had elapsed. Four of the 14 patients with measles had sera taken during the convalescent stage, the 20th to the 32nd day of the illness (the mean being 26 + 8 5).
Infectious mononucleosis This study included five males and four females aged from 1 year and 3 months to 13 years and 11 months (the mean being 5 years and 10 months). The diagnosis was based on common clinical symptoms of acute IM and atypical lymphocytes in the peripheral blood. All patients were positive for heterophile antibody and IgM antibody to Epstein-Barr virus (EBV) capsid antigen as determined by indirect fluorescence assay (Henle, Henle & Horwitz, 1974). From the onset of fever to sampling, an average of 8-3 + 2 5 days had elapsed. These patients had values on peripheral blood mononuclear cell subsets obtained by single- and two-colour flow-cytometric analyses in the acute blood. Eight of the nine patients with IM had sera taken during the convalescent stage, which tested positive for antibody to
135
EBV nuclear antigen (Reedman & Klein, 1973) during the 102nd to 200th day of the illness (the mean being 1513 + 39-9).
Healthy children The control subjects were 12 healthy children, seven males and five females aged from 2 months to 4 years and 7 months (the mean being 2 years and 4 months). The control samples were tested in parallel with the patients' samples.
Quantification of CD8 concentration in serum The CD8 concentration in serum was determined using a CELLFREE T8 test kit (T cell Sciences, Cambridge, MA). The principle of the method was a sandwich enzyme immunoassay as described by Tomkinson et al. (1989). The assay employs two murine anti-CD8 monoclonal antibodies (MoAb) recognizing different epitopes of the CD8 molecule. The first anti-CD8 MoAb was absorbed onto a polystyrene 96-well microtitre plate. CD8 present in the samples or standards bound to the antibody-coated wells, while unreacted sample components were removed by washing. The horseradish peroxidase (HRP)conjugated second anti-CD8 MoAb was then added, which bound to the sCD8 captured by the first antibody and thus completed the sandwich format of the assay. After the removal of unbound HRP-conjugated anti-CD8 by washing, O-phenylenediamine was added to the wells. A coloured product was formed in proportion to the amount of CD8 present in the sample. The reaction was terminated with stop solution (2 N H2SO4) and absorbance at 490 nm was measured. A standard curve was constructed using standards of a known concentration supplied by the manufacturer, and unknown samples were determined from the standard curve. The lower limit of the test kit was about 50 U/ml. Quantification of CD4 concentration in serum The CD4 concentration in serum was determined using a CELLFREE CD4 test kit (T cell Sciences, Cambridge, MA). The principle of the method was a sandwich enzyme immunoassay as described by Kline et al. (1989) using two murine antiCD4 MoAbs. The practical procedure is similar to that of the CELLFREE T8 test kit. The lower limit of the test kit was 12 U/ ml. Measurement of the peripheral blood CD3+, CD4+ and CD8+ T cell counts Using a method previously described (Furukawa et al., 1988, 1990a, 1990b), the percentage of the peripheral blood T cell subsets among mononuclear cells was assayed with a fluorescence-activated cell sorter (FACS IV, Becton Dickinson ICS, Mountain View, CA). For fluorescence procedures the MoAbs used were isothiocyanate-conjugated MoAb of Leu 4/CD3 (Becton Dickinson Monoclonal Center, Inc, San Jose, CA), Leu 3a/CD4 and Leu 2a/CD8, and phycoerythrin-conjugated MoAb of HLA-DR, in single- and two-colour flow-cytometric analyses. From the ratio of peripheral blood mononuclear cells to the leucocyte count, the absolute count of CD3+, CD4+, CD8+, HLA-DR+CD4+ or HLA-DR+CD8+ T cells was calculated. Statistical analysis Statistical analyses were performed using Student's t-test and paired t-test.
136
S. Furukawa et al. Table 1. Serum soluble CD4 (sCD4) and soluble CD8 (sCD8) levels of patients with Kawasaki disease (KD), anaphylactoid measles and infectious mononucleosis (IM), and of control subjects
purpura
(AP),
KD
n Mean day from onset sCD4 (U/ml) sCD8 (U/ml)
Acute
Convalescent
AP
33
12 34 4+ 15 6 30 6+5 9 393 8 + 84 3 0 08+0 03
5 52 + 37 20 0+2-5 317 0 + 78 8 0 07+0 03
58 + 1 9 42 9+ 18 5t 532 4+ 171 4t 0 09+0 05
sCD4/sCD8
IM
Control subjects
14
9
12
55 + 1 6
83 +2 5 65 6+ 12.0*
Measles
62 4+25-9* 924 3 + 505 6* 0 10+0 06
13566 +7463*
0.004+0.001*
28 1 +4 4 391 7 + 71 9 0 07+0 02
Values are expressed as mean + s.d. * Significant at P < 0-01 versus control subjects. t Significant at P < 0-01 Lversus convalescent stage of KD and control subjects. 100l0
0
80
0o
0 E
0 D
,-
60 Ccn
0
a) 40
0
E
0
Os8
*8
0 -0 0
(n
*0 *0
20
0
,-1
n
3-4 n=10 l
5-6
7-8
9-10
n/=7 n=12 Acute stage
n=4
21 -64 n=12 Convalescent stage
Time from onset of fever (days)
Fig. 1. The correlation between the day of onset of fever and Normal range; 0, case with coronary-artery lesion; , mean;
serum 0,
RESIU LTS Table shows serum sCD4 and sCD8 levels during the acute and convalescent stages of KD, the acute stages of AP, measles and IM, and in control subjects. Serum sCD4 and sCD8 levels found in patients with KD were high during the acute stage of the disease, as compared with those found at the convalescent stage and in control subjects. In addition, elevated levels of serum sCD4 and sCD8 were found in patients with measles and IM during the acute stage, as compared with those in control subjects. These increased sCD4 and sCD8 levels, found in patients with measles and IM, were seen to return to the normal range during the convalescent stage (measles, 30 9 + 4 6 (sCD4), 404 5 + 27-6 (sCD8); IM, 31 + 6 6 (sCD4), 432-4 + 140 2 (sCD8), mean + s.d. respectively). It was noted that serum sCD8 levels in patients with IM were markedly elevated during the acute stage
soluble CD4 (sCD4) levels during acute Kawasaki disease. l, without coronary-artery lesion.
case
of the disease. In contrast, serum sCD4 and sCD8 levels in patients with AP did not increase during the acute stage of the disease. Figures and 2 show the correlation between the days of onset of fever and serum sCD4 and sCD8 levels during acute KD. There were no significant differences in serum sCD4 and sCD8 levels between KD patients with and without CAL. Table 2 shows the absolute counts of leucocytes, mononuclear cells, CD3+, CD4+, CD8+, HLA-DR-CD4+ and HLA-DR+CD8+ T cells during the acute and convalescent stages of KD, the acute stage of IM and in control subjects. Absolute counts of CD3 +, CD4+ and CD8 ' T cells found in patients with KD did not increase during the acute stage of the disease, in comparison with those found at the convalescent stage and in control subjects. In addition, there were no significant differences in the absolute counts of HLA-
137
Soluble CD4 and CD8 in Kawasaki disease
I000
0 0
-
800 _
0
a
0 0
0 D
600 _
-
0
.J
0
U,
0
0
0
0
E
0
000
1
*I
0
400 _
000
:-
0
(17
200
0
3-4 n=10
21 -64 9-10 7-8 5-6 n=12 n=7 n=4 n=12 Convalescent stage Acute stage Time from onset of fever (days)
Fig. 2. The correlation between the day of onset of fever and serum soluble CD8 (sCD8) levels during acute Kawasaki disease. U, Normal range; 0, case with coronary-artery lesion; -, mean; 0, case without coronary-artery lesion. Table 2. Peripheral blood T cell subsets in patients with Kawasaki disease (KD) and infectious mononucleosis (IM) KD
n Leucocytes
Mononuclear cell CD3+ Tcell CD4+ Tcell CD8+ Tcell CD4+ cell/CD8+ cell n
HLA-DR+CD4+ T cell HLA-DR+CD8+ T cell
Acute
Convalescent
IM acute
Control subjects
17 17 618 + 6682* 4832+2517 2270+ 1450 1693+1023 771-1+475-2 2-3+0-8 6 78-1 + 59-8 33-3 + 17-8
17 8627 +2424 4672+1345 2855+1149 2051+757-4 1063+447-1 2-1 +0-9 6 52 3 +20-8 44 5 + 7-8
9 16 633 + 6377* 13 664 + 6009* 10625+5012* 2817+1497 7219+3319* 0-4+0-2* 9 837+380* 6152 + 2776*
12 8356+2204 4826+ 1201 3117+1096 2212+744-5 1037+379-9 2-3+09 6 57-4+ 15-3 55 2 + 29-0
Values are expressed as mean + s.d./mm3. * Significant at P < 0 01 versus control subjects.
DR+CD4+ T cells and HLA-DR+CD8+ T cells during the
were also measured in patients' sera during the acute stage of AP
acute stage of KD and in control subjects. In contrast, absolute counts of CD3+, CD8+, HLA-DR+CD4+ and HLA-
and the acute febrile stages of measles and IM. Although AP shows clinical symptoms which differ from those of KD, it shares some pathological findings with KD, namely vasculitis of small vessels; therefore the levels of sCD4 and sCD8 in sera of both groups of patients were measured to determine the role of T cell subsets in the pathogenesis of vasculitis. We also quantified sCD4 and sCD8 in sera of patients with measles and IM, which represent acute febrile viral infections, to which KD may also belong. The activation of the immune system with measles has been previously reported (Griffin et al., 1989, 1990).
DR+CD8+ T cells found in patients with IM increased during the acute stage of the disease, in comparison with those in control subjects.
DISCUSSION Our study represents the first report on the simultaneous determination of sCD4 and sCD8 in the sera of patients with acute KD. In parallel studies, the levels of both soluble antigens
138
S. Furukawa et al.
Peripheral blood T cells are the main target cells of measles virus infection. In contrast, acute IM is a self-limiting lymphoproliferative disease caused by EBV (Henle, Henle & Deihil, 1968). Although B lymphocytes are the target cells of EBV infection, the expansion of T lymphocytes, primarily those of the CD8 cytotoxic/suppressor subset, is responsible for the lymphocytosis evident during acute IM (De Waele, Thielemans & Van Camp, 1981). In the present study, KD, measles and IM patients had increased levels of serum sCD4 and sCD8 during the acute stage of the disease (Table 1). We confirmed the results of previous studies showing raised serum sCD8 levels in patients with measles (Griffin et al., 1989) and IM (Tomkinson et al., 1989). However, serum sCD4 and sCD8 levels in patients with AP did not increase during the acute stage. We reported that patients with AP had increased serum TNF-a levels, but not IL-2R and IFN-y levels in serum during the acute stage (Furukawa et al., 1989). Although peripheral blood CD4+ T cells, CD8+ T cell and also HLA-DR+ T cell counts were not increased during the acute stage of KD in the present study, serum sCD4 and sCD8 levels were elevated during the acute stage of KD, suggesting that T cells were activated under these conditions. These findings agree with our previous study which showed that serum IFN-y and IL-2R levels increased during the acute stage of KD (Matsubara, Furukawa & Yabuta, 1990). Since the expression of HLA-DR on peripheral blood T cells in KD patients was not increased during the acute stage, it is likely that there is a mild activation of peripheral blood T cells. Whether or not tissue lymphocytes were activated was not determined in the present study, although we have previously reported that skin lesions in biopsy specimens of KD were infiltrated with activated T cells as well as macrophages/monocytes (Sugawara et al., 1987). The contribution oflocalized T cells in affected tissues to the elevated serum levels of IL-2R was observed in patients with organ transplant rejections (Colvin et al., 1989). In the present study, peripheral blood CD8+, HLADR+CD8+, and also HLA-DR+CD4+ T cell counts increased during the acute stage of IM. It is noted that markedly increased CD8+ and CD8+HLA-DR+ T cell counts as well as sCD8 levels were seen during the acute stage of IM. We confirmed earlier studies that serum sCD8 levels were seen to increase markedly during the acute stage of IM patients, which correlated with the observation of an increased number of peripheral blood T cells expressing HLA-DR, a phenotypic characteristic of T cell activation in IM patients (Tomkinson et al., 1989). With regard to the sCD4/sCD8 ratio, KD was similar to measles, but differed from IM. These results were also supported by our previous report that the changes in natural killer cell and CD8+ T cell subsets in acute KD were in contrast to those of IM (Furukawa et al., 1991). Although we failed to prove significance in statistical analysis, serum sCD4 levels in patients with KD were seen to increase during the early part of the acute stage and sCD8 levels were seen to increase during the latter part of the acute stage. It remains unclear whether the reciprocal changes in serum sCD4 and sCD8 levels during the acute stage are related to the pathogenesis of KD.
ACKNOWLEDGMENTS This work was partially supported by a Research Grant (from 1988 to
1990) for Kawasaki disease from the Ministry of Health and Welfare, Japan. We wish to thank T. Obara for his support.
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