Impaired Responses of Peripheral Blood Mononuclear Cells to Staphylococcal Superantigen in Patients with Severe Atopic Dermatitis: A Role of T Cell Apoptosis

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Impaired Responses of Peripheral Blood Mononuclear Cells to Staphylococcal Superantigen in Patients with Severe Atopic Dermatitis: A Role of T Cell Apoptosis Takashi Yoshino, Hideo Asada, Shigetoshi Sano, Toshiaki Nakamura, Satoshi Itami, Kunihiko Yoshikawa Journal of Investigative Dermatology Volume 114, Issue 2, Pages 281-288 (February 2000) DOI: 10.1046/j.1523-1747.2000.00878.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Proliferation of PBMC in response to SEB. PBMC were isolated from healthy controls (C, n = 19) and AD patients divided into three groups: L, low clinical score group, score 27–48 (n = 27); M, medium clinical score group, score 49–59 (n = 27); H, high clinical score group, score 60–83 (n = 27). The PBMC were cultured with SEB for 3 d and [3H]thymidine incorporation was measured. Bars represent mean counts ± SEM. Journal of Investigative Dermatology 2000 114, 281-288DOI: (10.1046/j.1523-1747.2000.00878.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Clinical course study of proliferative response of PBMC from AD patients in comparison with clinical conditions. The samples were taken sequentially from the patients with various clinical scores and assessed for proliferative response against SEB. Four representatives of AD patients are shown. Error bars represent SEM. Journal of Investigative Dermatology 2000 114, 281-288DOI: (10.1046/j.1523-1747.2000.00878.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Proliferative response against SEB in autologous combination culture of TC and APC populations. APC and TC were prepared from good or poor proliferative PBMC, i.e., good APC, poor APC, good TC, poor TC. Four kinds of combination culture and only APC and TC populations were assessed for [3H]thymidine incorporation under SEB stimulation. Error bars represent mean counts ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005. Journal of Investigative Dermatology 2000 114, 281-288DOI: (10.1046/j.1523-1747.2000.00878.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Cytokine production by PBMC stimulated with SEB. PBMC were isolated from healthy controls (C, n = 9) and two groups of AD patients: low clinical score group (L, n = 13) and high clinical score group (H, n = 10). The levels of tumor necrosis factor α, IFN-γ, IL-2, IL-4, IL-6, IL-10, and IL-12 in each culture supernatant of SEB-stimulated PBMC were measured with ELISA, described in Materials and Methods. Bars represent mean values ± SEM. *p < 0.05, **p < 0.005, ***p < 0.0005. Journal of Investigative Dermatology 2000 114, 281-288DOI: (10.1046/j.1523-1747.2000.00878.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 TC viability under SEB stimulation. PBMC were isolated from the group of controls (C, n = 6) and two groups of AD patients: low clinical score group (L, n = 6) and high clinical score group (H, n = 6). The cells were cultured in triplicate under SEB stimulation and counted twice on days 1, 3 and 5. Vertical lines represent SEM. *p < 0.001, compared with group C. Journal of Investigative Dermatology 2000 114, 281-288DOI: (10.1046/j.1523-1747.2000.00878.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Expression of APO2.7 antigen in CD3+ cells after SEB stimulation. PBMC were derived from healthy controls (C#1, C#2, C#3, and C#4) and AD patients (p#12, p#24, and p#39) at mild and severe stages. Non-treated cells (Non-treated) and 24 h SEB-treated cells (Treated) were stained with anti-CD3 MoAb, followed by staining with anti-APO2.7 (solid line) and isotype control MoAb (dotted line). CD3+ cells are gated and plotted in histograms. Numbers under horizontal bars indicate the rate of cells with intense expression of APO2.7 in CD3+ cells (%). PBMC from healthy controls were treated with 10−4 M dexamethasone for 48 h, and were used as a positive control for apoptosis of TC. Journal of Investigative Dermatology 2000 114, 281-288DOI: (10.1046/j.1523-1747.2000.00878.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Expression of APO2.7 antigen in CD3+ cells after SEB stimulation. PBMC were derived from healthy controls (C#1, C#2, C#3, and C#4) and AD patients (p#12, p#24, and p#39) at mild and severe stages. Non-treated cells (Non-treated) and 24 h SEB-treated cells (Treated) were stained with anti-CD3 MoAb, followed by staining with anti-APO2.7 (solid line) and isotype control MoAb (dotted line). CD3+ cells are gated and plotted in histograms. Numbers under horizontal bars indicate the rate of cells with intense expression of APO2.7 in CD3+ cells (%). PBMC from healthy controls were treated with 10−4 M dexamethasone for 48 h, and were used as a positive control for apoptosis of TC. Journal of Investigative Dermatology 2000 114, 281-288DOI: (10.1046/j.1523-1747.2000.00878.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Detection of apoptotic CD16−CD19−PBMC by staining with Hoechst 33258 following SEB stimulation. PBMC were derived from healthy controls and AD patients at mild and severe stages. CD16−CD19−PBMC were prepared by negative selection with anti-CD19 and anti-CD16 MoAb, and magnetic beads coated with sheep antimouse IgG antibodies. CD16−CD19−PBMC were incubated with SEB for 3 d. The cells were fixed in 1% glutaraldehyde, and were stained with Hoechst 33258 (0.2 mM) in PBS. Two representatives of AD patients (p#24 and p#39) at mild and severe stages, and one representative of healthy control (C#1), are shown. Scale bar: 20 μm. Journal of Investigative Dermatology 2000 114, 281-288DOI: (10.1046/j.1523-1747.2000.00878.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Inhibition of Fas-FasL mediated apoptosis with blocking antibody to FasL. PBMC derived from AD patients (p#24, p#39, and p#43) at severe stages were cultured with SEB and with a neutralizing anti-FasL MoAb or an isotype control MoAb for 24 h. Expression of APO2.7 in CD3+ cells was assessed by FACS analysis. Journal of Investigative Dermatology 2000 114, 281-288DOI: (10.1046/j.1523-1747.2000.00878.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions