The inhibitory effects of topically active glucocorticoids on IL-4, IL-5, and interferon-γ production by cultured primary CD4+ T cells Shelby P. Umland,

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Presentation transcript:

The inhibitory effects of topically active glucocorticoids on IL-4, IL-5, and interferon-γ production by cultured primary CD4+ T cells Shelby P. Umland, PhD, D.Kyle Nahrebne, MS, Shad Razac, BS, Andrew Beavis, BS, Kenneth J. Pennline, PhD, Robert W. Egan, PhD, M.Motasim Billah, PhD Journal of Allergy and Clinical Immunology Volume 100, Issue 4, Pages 511-519 (October 1997) DOI: 10.1016/S0091-6749(97)70144-1 Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 1 Analysis of frequency of memory and naive CD4+ T cells and cell sorting parameters for isolation of total CD4+, naive CD4+, and memory CD4+ T cells. A, Single-parameter histogram of CD4 expression on anti-CD4-Tricolor (TC)–stained PBMCs, illustrating a defined peak for CD4+ cells. Sort region (R4) was used to exclude all CD4- cells from further analysis. B, Two-parameter contour plot showing analysis of CD4+ cells for the expression of CD45RO-phycoerythrin and CD45RA-fluorescein isothiocyanate. Sort gates R2 and R3 were defined, and cells were sorted simultaneously for naive (CD4+CD45RAhiCD45ROlo) and memory (CD4+CD45RAloCD45ROhi) cells, respectively. The population of CD4+ T cells contained approximately 45% naive cells (CD4+CD45RAhiCD45ROlo, lower right quadrant) and 40% memory cells (CD4+CD45RAloCD45ROhi, upper left quadrant). C and D, Contour plots showing reanalysis of sorted cells. Cells defined by sort gates R2 and R3 appeared within their respective gate on reanalysis and were mutually exclusive. Sorted cells were greater than 98% pure. FITC, Fluorescein isothiocyanate. Journal of Allergy and Clinical Immunology 1997 100, 511-519DOI: (10.1016/S0091-6749(97)70144-1) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 1 Analysis of frequency of memory and naive CD4+ T cells and cell sorting parameters for isolation of total CD4+, naive CD4+, and memory CD4+ T cells. A, Single-parameter histogram of CD4 expression on anti-CD4-Tricolor (TC)–stained PBMCs, illustrating a defined peak for CD4+ cells. Sort region (R4) was used to exclude all CD4- cells from further analysis. B, Two-parameter contour plot showing analysis of CD4+ cells for the expression of CD45RO-phycoerythrin and CD45RA-fluorescein isothiocyanate. Sort gates R2 and R3 were defined, and cells were sorted simultaneously for naive (CD4+CD45RAhiCD45ROlo) and memory (CD4+CD45RAloCD45ROhi) cells, respectively. The population of CD4+ T cells contained approximately 45% naive cells (CD4+CD45RAhiCD45ROlo, lower right quadrant) and 40% memory cells (CD4+CD45RAloCD45ROhi, upper left quadrant). C and D, Contour plots showing reanalysis of sorted cells. Cells defined by sort gates R2 and R3 appeared within their respective gate on reanalysis and were mutually exclusive. Sorted cells were greater than 98% pure. FITC, Fluorescein isothiocyanate. Journal of Allergy and Clinical Immunology 1997 100, 511-519DOI: (10.1016/S0091-6749(97)70144-1) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 1 Analysis of frequency of memory and naive CD4+ T cells and cell sorting parameters for isolation of total CD4+, naive CD4+, and memory CD4+ T cells. A, Single-parameter histogram of CD4 expression on anti-CD4-Tricolor (TC)–stained PBMCs, illustrating a defined peak for CD4+ cells. Sort region (R4) was used to exclude all CD4- cells from further analysis. B, Two-parameter contour plot showing analysis of CD4+ cells for the expression of CD45RO-phycoerythrin and CD45RA-fluorescein isothiocyanate. Sort gates R2 and R3 were defined, and cells were sorted simultaneously for naive (CD4+CD45RAhiCD45ROlo) and memory (CD4+CD45RAloCD45ROhi) cells, respectively. The population of CD4+ T cells contained approximately 45% naive cells (CD4+CD45RAhiCD45ROlo, lower right quadrant) and 40% memory cells (CD4+CD45RAloCD45ROhi, upper left quadrant). C and D, Contour plots showing reanalysis of sorted cells. Cells defined by sort gates R2 and R3 appeared within their respective gate on reanalysis and were mutually exclusive. Sorted cells were greater than 98% pure. FITC, Fluorescein isothiocyanate. Journal of Allergy and Clinical Immunology 1997 100, 511-519DOI: (10.1016/S0091-6749(97)70144-1) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 1 Analysis of frequency of memory and naive CD4+ T cells and cell sorting parameters for isolation of total CD4+, naive CD4+, and memory CD4+ T cells. A, Single-parameter histogram of CD4 expression on anti-CD4-Tricolor (TC)–stained PBMCs, illustrating a defined peak for CD4+ cells. Sort region (R4) was used to exclude all CD4- cells from further analysis. B, Two-parameter contour plot showing analysis of CD4+ cells for the expression of CD45RO-phycoerythrin and CD45RA-fluorescein isothiocyanate. Sort gates R2 and R3 were defined, and cells were sorted simultaneously for naive (CD4+CD45RAhiCD45ROlo) and memory (CD4+CD45RAloCD45ROhi) cells, respectively. The population of CD4+ T cells contained approximately 45% naive cells (CD4+CD45RAhiCD45ROlo, lower right quadrant) and 40% memory cells (CD4+CD45RAloCD45ROhi, upper left quadrant). C and D, Contour plots showing reanalysis of sorted cells. Cells defined by sort gates R2 and R3 appeared within their respective gate on reanalysis and were mutually exclusive. Sorted cells were greater than 98% pure. FITC, Fluorescein isothiocyanate. Journal of Allergy and Clinical Immunology 1997 100, 511-519DOI: (10.1016/S0091-6749(97)70144-1) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 2 IL-5 and IFN-γ secretion by cultured naive and memory CD4+ T cells. Sorted total CD4+, naive CD4+, and memory CD4+ T cells were cultured for 6 days and stimulated for cytokine production as described in the Methods. Two experiments representing two different donors (A, C = donor 1 and B, D = donor 2) are shown. Immobilized anti-CD3 and soluble anti-CD28 were used at 10 and 1 μg/ml, respectively. Phorbol myristate acetate was used at a final concentration of 1 ng/ml. Sort parameters shown in Fig. 1 were derived from donor 2. Values shown represent mean ± SEM of triplicate cultures. FACS analysis after 6-day culturing indicated that the cells were 96.8% CD4+. A third experiment provided similar results. PMA, Phorbol myristate acetate; UD, below the limits of detection. Journal of Allergy and Clinical Immunology 1997 100, 511-519DOI: (10.1016/S0091-6749(97)70144-1) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 2 IL-5 and IFN-γ secretion by cultured naive and memory CD4+ T cells. Sorted total CD4+, naive CD4+, and memory CD4+ T cells were cultured for 6 days and stimulated for cytokine production as described in the Methods. Two experiments representing two different donors (A, C = donor 1 and B, D = donor 2) are shown. Immobilized anti-CD3 and soluble anti-CD28 were used at 10 and 1 μg/ml, respectively. Phorbol myristate acetate was used at a final concentration of 1 ng/ml. Sort parameters shown in Fig. 1 were derived from donor 2. Values shown represent mean ± SEM of triplicate cultures. FACS analysis after 6-day culturing indicated that the cells were 96.8% CD4+. A third experiment provided similar results. PMA, Phorbol myristate acetate; UD, below the limits of detection. Journal of Allergy and Clinical Immunology 1997 100, 511-519DOI: (10.1016/S0091-6749(97)70144-1) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 2 IL-5 and IFN-γ secretion by cultured naive and memory CD4+ T cells. Sorted total CD4+, naive CD4+, and memory CD4+ T cells were cultured for 6 days and stimulated for cytokine production as described in the Methods. Two experiments representing two different donors (A, C = donor 1 and B, D = donor 2) are shown. Immobilized anti-CD3 and soluble anti-CD28 were used at 10 and 1 μg/ml, respectively. Phorbol myristate acetate was used at a final concentration of 1 ng/ml. Sort parameters shown in Fig. 1 were derived from donor 2. Values shown represent mean ± SEM of triplicate cultures. FACS analysis after 6-day culturing indicated that the cells were 96.8% CD4+. A third experiment provided similar results. PMA, Phorbol myristate acetate; UD, below the limits of detection. Journal of Allergy and Clinical Immunology 1997 100, 511-519DOI: (10.1016/S0091-6749(97)70144-1) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 2 IL-5 and IFN-γ secretion by cultured naive and memory CD4+ T cells. Sorted total CD4+, naive CD4+, and memory CD4+ T cells were cultured for 6 days and stimulated for cytokine production as described in the Methods. Two experiments representing two different donors (A, C = donor 1 and B, D = donor 2) are shown. Immobilized anti-CD3 and soluble anti-CD28 were used at 10 and 1 μg/ml, respectively. Phorbol myristate acetate was used at a final concentration of 1 ng/ml. Sort parameters shown in Fig. 1 were derived from donor 2. Values shown represent mean ± SEM of triplicate cultures. FACS analysis after 6-day culturing indicated that the cells were 96.8% CD4+. A third experiment provided similar results. PMA, Phorbol myristate acetate; UD, below the limits of detection. Journal of Allergy and Clinical Immunology 1997 100, 511-519DOI: (10.1016/S0091-6749(97)70144-1) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 3 GC inhibition of IL-5 (A), IFN-γ (B), and IL-4 (C) secretion by CD4+ T cells from donor 1. Purified CD4+ T cells were cultured for 6 days as described in Methods. Cells were stimulated with immobilized anti-CD3 plus soluble anti-CD28 mAb in the presence or absence of the indicated GC for 48 hours, at which time culture supernatants were collected. Results are shown as percent inhibition (see Methods) compared with DMSO control cultures versus GC (in nanomoles per liter) tested. Control cytokine levels (mean ± SEM pg/ml, in quadruplicate cultures) in immobilized anti-CD3 and soluble anti-CD28–stimulated cultures for this donor were: IL-5, 4046 ± 205; IL-4, 2650 ± 131; and IFN-γ, 9897 ± 594. BUD, Budesonide. Journal of Allergy and Clinical Immunology 1997 100, 511-519DOI: (10.1016/S0091-6749(97)70144-1) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 3 GC inhibition of IL-5 (A), IFN-γ (B), and IL-4 (C) secretion by CD4+ T cells from donor 1. Purified CD4+ T cells were cultured for 6 days as described in Methods. Cells were stimulated with immobilized anti-CD3 plus soluble anti-CD28 mAb in the presence or absence of the indicated GC for 48 hours, at which time culture supernatants were collected. Results are shown as percent inhibition (see Methods) compared with DMSO control cultures versus GC (in nanomoles per liter) tested. Control cytokine levels (mean ± SEM pg/ml, in quadruplicate cultures) in immobilized anti-CD3 and soluble anti-CD28–stimulated cultures for this donor were: IL-5, 4046 ± 205; IL-4, 2650 ± 131; and IFN-γ, 9897 ± 594. BUD, Budesonide. Journal of Allergy and Clinical Immunology 1997 100, 511-519DOI: (10.1016/S0091-6749(97)70144-1) Copyright © 1997 Mosby, Inc. Terms and Conditions

Fig. 3 GC inhibition of IL-5 (A), IFN-γ (B), and IL-4 (C) secretion by CD4+ T cells from donor 1. Purified CD4+ T cells were cultured for 6 days as described in Methods. Cells were stimulated with immobilized anti-CD3 plus soluble anti-CD28 mAb in the presence or absence of the indicated GC for 48 hours, at which time culture supernatants were collected. Results are shown as percent inhibition (see Methods) compared with DMSO control cultures versus GC (in nanomoles per liter) tested. Control cytokine levels (mean ± SEM pg/ml, in quadruplicate cultures) in immobilized anti-CD3 and soluble anti-CD28–stimulated cultures for this donor were: IL-5, 4046 ± 205; IL-4, 2650 ± 131; and IFN-γ, 9897 ± 594. BUD, Budesonide. Journal of Allergy and Clinical Immunology 1997 100, 511-519DOI: (10.1016/S0091-6749(97)70144-1) Copyright © 1997 Mosby, Inc. Terms and Conditions