IL-6 promotes an increase in human mast cell numbers and reactivity through suppression of suppressor of cytokine signaling 3

Abstract

Background IL-6, which is reported to be elevated in association with mastocytosis, asthma and urticaria, is used in conjunction with stem cell factor (SCF) to generate human MCs (HuMCs) from progenitor (CD34+) cells. Despite these associations, the effects on, and mechanisms by which prolonged exposure to IL-6 alters HuMC number and function are not well understood. Objectives To study the effect of IL-6 on HuMC function, the mechanisms by which IL-6 exerts its effects, and the relationship of these findings to mastocytosis. Methods HuMCs were cultured in SCF with or without IL-6. The responses to FcεRI aggregation, and the expression of proteases and receptors including the soluble IL-6 receptor (sIL-6R) were then quantitated. Epigenetic changes in SOCS3 were determined using methylation specific PCR. Serum samples from healthy controls and patients with mastocytosis were assayed for IL-6, tryptase, and sIL-6R. Results IL-6 enhanced MC proliferation, maturation, and reactivity following FcεRI aggregation. IL-6 reduced expression of SOCS3, which correlated with methylation of the SOCS3 promoter, and increased expression and activation of STAT3. IL-6 also suppressed constitutive production of sIL-6R and serum levels of sIL-6R were similarly reduced in patients with mastocytosis. Conclusion IL-6 increases mast cell proliferation and formation of a more reactive phenotype enabled by suppressing proteolytic cleavage of sIL-6R from IL-6R and down regulation of the SOCS3 auto-inhibitory pathway. We suggest IL-6 blockade might ameliorate MC related symptoms and pathology in MC-related diseases associated with elevated IL-6 including mastocytosis.

An optimized protocol for the generation and functional analysis of human mast cells from CD34 + enriched cell populations

“…Similarly, IL-6 is known to promote huMC proliferation in vitro and thus also routinely used in the culture on huMCs [9]. In this study, IL-3 was added only once into the MC medium on the first day to some cultures.…”

“…The enriched progenitor cells were suspended at 5×10 5 cells/ml with mast cell culture medium (MC medium) [StemPro-34 Medium (Invitrogen, Grand Island, NY) supplemented with 100 ng/ml human recombinant SCF, 100 ng/ml human rIL-6 (R&D Systems, Minneapolis, MN), 100 μg/ml penicillin/streptomycin and 2 mM L-glutamine (Invitrogen, Grand Island, NY)][9]. Then 0.5 ml of the cell suspension was added to each of the 8 inner side wells of a 24-well plate (Corning, NY) (2.5 × 10 5 cell/well) and 1 ml of PBS was added to each of the outer side wells to maintain humidity and prevent evaporation of the inner wells containing cells.…”

“…For analysis of mast cell activation, cultured cells were sensitized overnight with 200 ng/ml human myeloma IgE (Millipore, MA) and then stimulated with 200 ng/ml anti-IgE (KPL, Gaithersburg, MD) for 1 or 2 hours at 37 °C, under 5% CO 2 [9]. Cells were harvested, washed once with PBS and resuspended in 200 μl aqua staining buffer (live/dead fixable aqua dead cell stain kit, Invitrogen, CA) (1 μl aqua in 2000 μl of PBS) at room temp in the dark for 20 min.…”

Influence of IL‐6, IL‐33, and TNF‐α on human mast cell activation: Lessons from single cell analysis by flow cytometry

“…Studies in rodent MCs have revealed that MCs biology is influenced by proinflammatory cytokines, such as interleukin-6 (IL-6), IL-33, and tumor necrosis factor-alpha (TNF-a) (9)(10)(11)(12). In contrast, data about the effects of these cytokines on human MCs are scarce and have mainly been gathered using mediator release assays in the supernatant of cultured human MCs (17)(18)(19)(20)(21)(22)(23). Besides, IL-33 has been shown to increase cytokine and chemokine production after IgE-independent stimulation via IgG immune complexes (16).…”

IL‐6 and IL‐1β expression is increased in autologous serum skin test of patients with chronic spontaneous urticaria

“…12,13 This observation led to the successful use of anti-IL-6 therapy to treat mast cell-related pathologies, such as mastocytosis, associated with increased IL-6 levels. Several lines of evidence support a stimulatory role of IL-6 and IL-1β on mast cells.…”