Suppressive Effects of Cyclosporin A and FK-506 on Superoxide Generation in Human Polymorphonuclear Leukocytes Primed by Tumor Necrosis Factor α Yasushi.

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Suppressive Effects of Cyclosporin A and FK-506 on Superoxide Generation in Human Polymorphonuclear Leukocytes Primed by Tumor Necrosis Factor α Yasushi Goto, Takeshi Kono, Masamitsu Ishii Journal of Investigative Dermatology Volume 115, Issue 6, Pages 986-989 (December 2000) DOI: 10.1046/j.1523-1747.2000.00181.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Concentration-response function for fMLP-induced O2 generation in unprimed and TNF-α-primed human PMNL: effects of CsA and FK-506. (A, B) Unprimed human PMNL; (A) TNF-α-primed human PMNL. For unprimed PMNL, CsA (○), FK-506 (•) (final concentration 0.01 nM-1 μM), or solvent (control) was added to the reaction mixture 12 min before fMLP stimulation (A, B). For primed PMNL, CsA (□), FK-506 (▪) (final concentration 0.01 nM-1 μM), or solvent (control) was added to the reaction mixture 2 min before addition of 10 units per ml of TNF-α or solvent alone. After 10 min priming with TNF-α, fMLP was added (A). To precisely determine the effects in unprimed PMNL, the graphs for unprimed cells were magnified in scale for the vertical axis (B). Values are the means of assays (n = 5 or 6) of an experiment performed with six preparations of PMNL. Bars, SD; *p <0.05; **p <0.01. Journal of Investigative Dermatology 2000 115, 986-989DOI: (10.1046/j.1523-1747.2000.00181.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Effect of FK-506 on tyrosyl phosphorylation of PMNL protein. After incubation of PMNL (2 × 106 cells) with FK-506 or solvent for 2 min and TNF-α 10 units per ml for 10 min, neutrophil proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by transfer to PVDF membrane. Tyrosine residues in phosphorylated proteins were detected by immunoblotting with antiphosphotyrosine antibody. Lane 1, without TNF-α or FK-506; lanes 2–7, with 10 units per ml of TNF-α lanes 3–7, with 10 units per ml of TNF-α and FK-506; lane 3, 10-9 M of FK-506; lane 4, 10-8 M; lane 5, 10 -7 M; lane 6, 10-6 M; lane 7, 10-5 M. Journal of Investigative Dermatology 2000 115, 986-989DOI: (10.1046/j.1523-1747.2000.00181.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Concentration-response function for tyrosyl phosphorylation of 115 kDa protein in human PMNL primed with TNF-α: effects of CsA and FK-506. CsA (○) (final concentration 1 nM-10 μM), FK-506 (•) (final concentration 1 nM-10 μM), or solvent (control) was added to the reaction mixture 2 min before addition of 10 units per ml of TNF-α or solvent alone. After 10 min priming with TNF-α, cells were harvested and tyrosyl phosphorylation of 115 kDa protein was assayed as described in Materials and Methods. Values are the means of assays (n = 6) of an experiment performed with five preparations of PMNL. Bars, SD; *p <0.01. Journal of Investigative Dermatology 2000 115, 986-989DOI: (10.1046/j.1523-1747.2000.00181.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions