Direct effects of HP Acthar Gel on human B lymphocyte activation in vitro

Abstract

Introduction: Both clinical experience and experimental evidence have suggested that Adrenocorticotropic hormone (ACTH) might directly exert immunomodulatory effects not dependent on adrenal steroidogenesis.

Methods: The direct effects of H.P. Acthar Gel (Acthar), a repository preparation containing a porcine ACTH analogue, on human B lymphocyte function were studied in vitro using peripheral blood B cells isolated using anti-CD19 coated magnetic beads and activated by interleukin 4 (IL-4) and CD40 ligand (CD40L). Analysis of expression of messenger RNA (mRNA) encoding activation-induced cytidine deaminase (AICDA) was carried out by quantitative real-time polymerase chain reaction (PCR). Cellular proliferation was assessed by a flow cytometric technique using intracellular staining with carboxyfluorescein succinimidyl ester (CFSE). Immunoglobulin G (IgG) production was measured in cell supernatants using an immunoassay.

Results: Acthar was found to exert acute, dose-dependent inhibitory effects on IL-4/CD40L-mediated induction of the expression of activation-induced cytidine deaminase (AICDA) after 24 hours, as well as sustained inhibition of B cell proliferation and IgG production during five more days of culture, without deleterious effects on B cell viability.

Conclusions: These experiments demonstrate that Acthar can exert direct effects on the humoral immune system independent of any role in the regulation of adrenal steroidogenesis. Although the impact of these findings on clinical disease was not evaluated in this study, these data support the therapeutic potential of Acthar for the management of autoimmune diseases characterized by B cell activation and aberrant humoral immune function.

Figures

Fig 1

Immunoglobulin production by activated human B lymphocytes in vitro is suppressed by Acthar. IgG (top panel) and IgM (bottom panel) were measured in supernatants from human peripheral B cells cultured for 6 days under basal conditions, with added IL-4/CD40L alone, or with IL-4/CD40L plus added Acthar at ACTH analog concentrations of approximately 0.124, 1.24, or 2.49 μM (stepped bars). Parallel cultures contained IL-4/CD40L plus matched volumes of placebo. Supernatants were harvested and assayed for IgG or IgM concentration and results analyzed by ANOVA (Kruskal–Wallis test, p <0.0001 for IgG; p = 0.0551 for IgM). **Significantly different from IL-4/CD40L-stimulated samples by Dunn’s post test at p <0.01. *Significantly different from IL-4/CD40L-stimulated samples by Dunn’s post test at p <0.05. Results shown are mean ± SEM for eight independent experiments. Acthar H.P. Acthar Gel®, CD40L CD40 ligand, Ig immunoglobulin, IL interleukin

Fig 2

Proliferation of activated human B lymphocytes in vitro is suppressed by Acthar. a The percentage of cells that divided, b the average number of divisions of all cells (division index), and c the fold-expansion of all cells (expansion index) were assessed in CSFE-loaded human peripheral B cells cultured for 6 days under basal conditions, with added IL-4/CD40L alone, or with IL-4/CD40L plus added Acthar at ACTH analog concentrations of approximately 0.124, 1.24, or 2.49 μM. Parallel cultures contained IL-4/CD40L plus matched volumes of placebo. Cells were examined by flow cytometry and data analyzed by ANOVA (Kruskal–Wallis test, p <0.0001 for percentage of cells divided, p <0.0001 for division index, and p <0.0001 for expansion index). **Significantly different from IL-4/CD40L-stimulated samples by Dunn’s post test at p <0.01. *Significantly different from IL-4/CD40L-stimulated samples at p <0.05. Results shown are mean ± SEM for six independent experiments. Acthar H.P. Acthar Gel®, CD40L CD40 ligand, IL interleukin

Fig. 3

Viability of B lymphocytes in vitro is unaffected by Acthar. The percentage of live cells (negative for staining) was assessed in human peripheral B cells cultured for 6 days under basal conditions, with added IL-4/CD40L alone, or with IL-4/CD40L plus added Acthar at ACTH analog concentrations of approximately 0.124, 1.24, or 2.49 μM. Parallel cultures contained IL-4/CD40L plus volumes of placebo at equal volumes. At the end of the culture period cells were stained with Live/Dead stain as described in Methods. Cells were analyzed by flow cytometry. Statistical analysis (ANOVA) revealed none of the treatment values to be significantly different from IL-4/CD40L-stimulated sample. Results shown are mean ± SEM for six independent experiments. Acthar H.P. Acthar Gel®, CD40L CD40 ligand, FITC fluorescein isothiocyanate, IL interleukin

Fig. 4

Expression of AICDA mRNA in activated human B lymphocytes in vitro is suppressed by Acthar. Expression of AICDA mRNA was assessed in human peripheral B cells cultured a for 1 day or b for 6 days under basal conditions, with added IL-4/CD40L alone, or with IL-4/CD40L plus added Acthar at ACTH analog concentrations of approximately 0.124, 1.24, or 2.49 μM. Parallel cultures contained IL-4/CD40L plus placebo at matched volumes. mRNA was isolated and quantitated by RT/PCR as described in Methods. Data were analyzed by ANOVA (p = 0.0002 on day 1 and p = 0.9973 on day 6). *Significantly different from IL-4/CD40L-stimulated samples by Tukey’s post test at p <0.05; **Significantly different from IL-4/CD40L-stimulated samples by Tukey’s post test at p <0.01. Results shown are mean ± SEM of six independent experiments at 1 day and of seven independent experiments at 6 days. Acthar H.P. Acthar Gel®, AICDA activation-induced cytidine deaminase, CD40L CD40 ligand, IL interleukin