Page 136 - 2020_01-Haematologica-web

A. Tobio et al.
intensity (MFI) was associated with KIT+ cells. As SCF in the media may induce IL-6 directly or through autocrine/paracrine signals in clonal and non-clonal cells in these cultures, we examined the expression of IL-6 in BM biopsies by immunofluorescence (IF). IF images of BM of patients with SM indicated that IL-6 intracellular content was mostly associated with mast cells (Online Supplementary Figure S1). Thus, the combination of ex vivo and in situ experiments suggests mast cells as the predom- inant producers of IL-6, with variable participation of other cell lineages. Data are also consistent with an asso- ciation between increased IL-6 expression by BM mast cells and D816V-KIT frequency.
Expression of D816V-KIT causes IL-6 upregulation and secretion
Given these associations and that mast cells and their progenitors often carry somatic D816V-KIT variants in SM, we investigated the hypothesis that D816V-KIT
P=0.025 AB
(Figure 1B), suggesting a contributory role for mast cells in
IL-6 production. In addition, we analyzed intracellular IL-
6 staining in single BM mast cells by flow cytometry from
three separate patients (see Online Supplementary Table S1
for patients' characteristics). Patient 1 had idiopathic ana-
phylaxis and did not meet criteria for SM and thus was
used as a control. This patient had no detectable D816V-
KIT, 0.098% of BM cells were CD3–/CD34–/KIT+/FcεRI+
(mast cells) and a minor percentage of these were IL-6 pos-
itive (0.063%) (Figure 1C, left panel). However, CD3-
/CD34–/KIT+/FcεRI+ cells from Patients 2 and 3, with BM
D816V frequencies of 2.7% and 5.5%, were 77% and
99% positive for IL-6, respectively (Figure 1C, middle and
right panels, respectively). In this ex vivo experiment,
where BM cells were cultured up to 4 days (d) in the pres-
(KIT /FcεRI , KIT /FcεRI and KIT /FcεRI ) also showed positive intracellular IL-6 staining (Online Supplementary Table S2). However, the highest IL-6 mean fluorescence
ence of SCF, cell lineages other than mast cells +––+––
C
Figure 1. Production of IL-6 by bone marrow cells and mast cells is enhanced in patients with systemic mastocytosis (SM) in association with the D816V-KIT allelic burden. (A) IL-6 release from bone marrow mononuclear cells isolated from five patients with SM with D816V-KIT bone marrow allelic frequencies of <5 (Patients 4 and 5 in Online Supplementary Table S1) or >5 (Patients 6, 7, and 10). BM aspirates were cultured for 2-4 days and IL-6 released into the culture media was meas- ured by ELISA. Data are the mean±Standard Error of Mean (SEM). (B) Correlation between IL-6 released into the media by BM cells and the percentage of mast cells in those cultures which was determined by flow cytometry. (C) Flow cytometry histograms showing intracellular IL-6 staining in BM mast cells. Mast cells were gated as CD3–/CD34–/KIT3/FcεRI+ within the BM cells of three patients (Patients 1-3 in Online Supplementary Table S1), with D816V-KIT allelic frequencies in the bone marrow also indicated in the figure. Patient 1 had idiopathic anaphylaxis and did not meet criteria for SM but was used as a control. The percentage of IL-6 positive cells within the mast cell population is indicated in the histograms. ISM: indolent SM.
126
haematologica | 2020; 105(1)