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Abnormal Natural Killer Cell Function in Systemic Sclerosis: Altered Cytokine Production and Defective Killing Activity  Mayuka Horikawa, Minoru Hasegawa,

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Presentation on theme: "Abnormal Natural Killer Cell Function in Systemic Sclerosis: Altered Cytokine Production and Defective Killing Activity  Mayuka Horikawa, Minoru Hasegawa,"— Presentation transcript:

1 Abnormal Natural Killer Cell Function in Systemic Sclerosis: Altered Cytokine Production and Defective Killing Activity  Mayuka Horikawa, Minoru Hasegawa, Kazuhiro Komura, Ikuko Hayakawa, Koichi Yanaba, Takashi Matsushita, Kazuhiko Takehara, Shinichi Sato  Journal of Investigative Dermatology  Volume 125, Issue 4, Pages (October 2005) DOI: /j X x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Frequency and absolute number of peripheral blood natural killer (NK) cells in diffuse cutaneous systemic sclerosis patients, limited cutaneous systemic sclerosis patients, and normal controls (CTL). The frequency of NK cells was determined as CD56+ cells by immunofluorescence staining with flow cytometric analysis (A). Absolute numbers of NK cells were calculated from the relative frequency of NK cells and the absolute leukocyte (B). The horizontal bars represent mean values with statistically significant differences between groups indicated. Journal of Investigative Dermatology  , DOI: ( /j X x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 CD16, CD69, and CD62L expression on CD56+ natural killer (NK) cells in diffuse cutaneous systemic sclerosis (dcSSc) patients, limited cutaneous systemic sclerosis (lcSSc) patients, and normal controls (CTL). For CD16 expression levels on NK cells, two-color analysis was performed using fluorescein isothiocyanate (FITC)-conjugated anti-CD16 and phycoerythrin (PE)-conjugated anti-CD56 monoclonal Abs (mAb). For the expression of CD69 and CD62L, two-color analysis was conducted using a combination of FITC-conjugated anti-CD56 and PE-conjugated either anti-CD69 or anti-L-selectin mAb. All samples were analyzed by flow cytometry with identical instrument settings. Horizontal dashed lines are provided for reference. The number in each quadrant represents a percentage of all lymphocytes. The numbers beside the square represent the number of cells within each square as a percentage of all CD56+ NK cells. Representative histograms of dcSSc patients, lcSSc patients, and CTL are shown. Journal of Investigative Dermatology  , DOI: ( /j X x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 CD16 expression level, CD69, and CD62L frequency on natural killer (NK) cells from diffuse cutaneous systemic sclerosis patients, limited cutaneous systemic sclerosis patients, and normal controls (CTL). CD16 expression levels on CD56+ NK cells (A) and frequency of CD69+ cells (B) and CD62L (C) in CD56+ NK cells were determined by flow cytometric analysis as described in Figure 2. The horizontal bars represent mean values with statistically significant differences between groups indicated. Journal of Investigative Dermatology  , DOI: ( /j X x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Cytokine production by natural killer (NK) cells from diffuse cutaneous systemic sclerosis patients, limited cutaneous systemic sclerosis patients, and normal controls (CTL). Purified NK cells (0.5 × 105) were stimulated with either medium alone (non-stimulated) or phorbol 12-myristate 13-acetate and ionomycin (stimulated) for 24 h. Cell supernatants were analyzed by ELISA to determine the levels of interferon-γ, interleukin (IL)-5, IL-6, and IL-10. The horizontal bars represent mean values with statistically significant differences between groups indicated. Journal of Investigative Dermatology  , DOI: ( /j X x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Natural cytotoxicity by natural killer (NK) cells. Cytotoxicity activity of NK cells from diffuse cutaneous systemic sclerosis patients, limited cutaneous systemic sclerosis patients, and normal controls (CTL) was assessed by flow cytometry. The K562 target cells stained with DiOC18 were mixed with isolated NK cells at effector:target (E:T) ratios of 5:1, 10:1, 20:1, and 40:1. Propidium iodide was then added to the mixture and incubated at 37°C for 2 h. Two-color flow cytometric analysis was performed to assess NK lytic activity. Percent cytotoxicity was calculated by the frequency of dead propidium iodide-positive target cells divided by the total target cells stained with DiOC18. Journal of Investigative Dermatology  , DOI: ( /j X x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 Granzyme B secretion by natural killer (NK) cells from diffuse cutaneous systemic sclerosis patients, limited cutaneous systemic sclerosis patients, and normal controls (CTL). Purified NK cells (0.5 × 105) were stimulated with either medium alone (non-stimulated) or phorbol 12-myristate 13-acetate and ionomycin (stimulated) for 24 h. Cell supernatants were analyzed by ELISA to determine granzyme B levels. The horizontal bars represent mean values with statistically significant differences between groups indicated. Journal of Investigative Dermatology  , DOI: ( /j X x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

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