Cytokine production by bone marrow mononuclear cells in inherited bone marrow failure syndromes - PubMed (original) (raw)

Cytokine production by bone marrow mononuclear cells in inherited bone marrow failure syndromes

Ken Matsui et al. Br J Haematol. 2013 Oct.

Abstract

Fanconi anaemia (FA), dyskeratosis congenita (DC), Diamond-Blackfan anaemia (DBA), and Shwachman-Diamond syndrome (SDS) are characterized by the progressive development of bone marrow failure. Overproduction of tumour necrosis factor-α (TNF-α) from activated bone marrow T-cells has been proposed as a mechanism of FA-related aplasia. Whether such overproduction occurs in the other syndromes is unknown. We conducted a comparative study on bone marrow mononuclear cells to examine the cellular subset composition and cytokine production. We found lower proportions of haematopoietic stem cells in FA, DC, and SDS, and a lower proportion of monocytes in FA, DC, and DBA compared with controls. The T- and B-lymphocyte proportions were similar to controls, except for low B-cells in DC. We did not observe overproduction of TNF-α or IFN-γ by T-cells in any patients. Induction levels of TNF-α, interleukin (IL)-6, IL-1β, IL-10, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor in monocytes stimulated with high-dose lipopolysaccharide (LPS) were similar at 4 h but lower at 24 h when compared to controls. Unexpectedly, patient samples showed a trend toward higher cytokine level in response to low-dose (0·001 μg/ml) LPS. Increased sensitivity to LPS may have clinical implications and could contribute to the development of pancytopenia by creating a chronic subclinical inflammatory micro-environment in the bone marrow.

Keywords: bone marrow; cytokines; immunology; inherited bone marrow failure syndromes; monocytes.

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Figures

Fig 1

Patients with IBMFS have lower percentages of HSCs and monocytes than the controls. Bone marrow mononuclear cells from patients with FA, DC, DBA, or SDS, and healthy adult controls were analysed by flow cytometry. The cells were surface stained for the pan-leucocyte marker CD45 and for markers of leucocyte subsets. The plots show percentages of double-positive cells in the total bone marrow mononuclear cells found in a live cell-gate, which was based on the forward and side scatter characteristics. (A) The percentages of CD34+ (HSCs) cells were lower than the controls in FA, DC, and SDS. (B) The percentages of CD14+ (monocytes) cells were lower in FA, DC, and DBA than in the controls. (C) The percentages of CD3+(T-lymphocytes) cells were similar to the controls in all IBMFS. (D) The percentage of CD19+ (B-lymphocytes) cells was lower in DC than in the controls. Each symbol represents an individual, and the horizontal bars show the means. Asterisks indicate statistical significance (p < 0.05). IBMFS, inherited bone marrow failure syndrome; FA, Fanconi anaemia; DC, dyskeratosis congenita; DBA, Diamond-Blackfan anaemia, SDS, Shwachman-Diamond syndrome; Ctrl, control; HSCs, haematopoietic stem cells.

Fig 2

The percentages of IFN-γ– or TNF-α–producing CD3+ T cells in patients with IBMFS are not higher than the controls. Bone marrow mononuclear cells were incubated with or without P+I for 4 h, surface stained for CD3, and then for intracellular IFN-γ and TNF-α. (A) Representative dot plots from single individuals in each group showed that stimulated T cells produced IFN-γ and TNF-α in all groups. (B-C) The production of IFN-γ and TNF-α was not increased in any of the syndromes; it was decreased in DBA. IBMFS, inherited bone marrow failure syndrome; FA, Fanconi anaemia; DC, dyskeratosis congenita; DBA, Diamond-Blackfan anaemia, SDS, Shwachman-Diamond syndrome; Ctrl, control; IFN-γ, γ-interferon; TNF-α, tumour necrosis factor-α; P+I, Phorbol 12-myristate 13-acetate + ionomycin

Fig 3

The proportions of TNF-α–producing CD14+ monocytes are lower than in the controls in FA and DBA. Bone marrow mononuclear cells were stimulated with 1 μg/ml of LPS for 4 h and stained for CD19 and CD14, and then for intracellular TNF-α. (A) Representative dot plots from single individuals showed LPS-stimulated CD19+ B cells do not produce TNF-α. (B) CD14+ monocytes in the same samples shown in (A) produced TNF-α in all groups. (C) Lower proportions of monocytes in FA and DBA produced TNF-α. IBMFS, inherited bone marrow failure syndrome; FA, Fanconi anaemia; DC, dyskeratosis congenita; DBA, Diamond-Blackfan anaemia, SDS, Shwachman-Diamond syndrome; Ctrl, control; IFN-γ, γ-interferon; TNF-α, tumour necrosis factor-α; LPS, lipopolysaccharide.

Fig 4

CD14+ monocytes from the majority of IBMFS patients are hypersensitive to low-dose LPS. Bone marrow mononuclear cells were stimulated with 1.0, 0.1, or 0.001 μg/ML of LPS for 4 h and stained for CD14, then for intracellular TNF-α. All 14 samples in the control group were tested for each stimulation condition. For the syndrome groups, the following numbers of samples were tested: no-stimulation, 0.001 and 1.0 μg/ml LPS: FA (N = 9/9); DC (N = 7/12); DBA (N = 12/12); and SDS (N = 7/7); 0.1 μg/ml LPS: FA (N = 7/9); DC (N = 7/12); DBA (N = 11/12); and SDS (N = 4/7). (A) Dot plots of single individuals from each group are shown. All samples responded to 1 and 0.1 μg/ml of LPS, but only IBMFS patients responded to 0.001 μg/ml. Individuals who responded to the lowest dose of LPS are presented in this panel. (B) Higher proportions of CD14+ monocytes produced TNF-α in FA, DBA, and SDS in response to 0.001 μg/mL of LPS. (C) The median fluorescent intensity of TLR4 on CD14+ cells was comparable among the groups. IBMFS, inherited bone marrow failure syndrome; FA, Fanconi anaemia; DC, dyskeratosis congenita; DBA, Diamond-Blackfan anaemia, SDS, Shwachman-Diamond syndrome; Ctrl, control; IFN-γ, γ-interferon; TNF-α, tumour necrosis factor-α; LPS, lipopolysaccharide.

Fig 5

Cells from patients show a trend toward cytokine induction in response to low-dose LPS. Determination of cytokine concentrations in the LPS-stimulated culture supernatants was performed as described in Methods. To normalize for the difference in the number of CD14+ monocytes, we calculated stimulation indexes to determine the fold-increase over the no-stimulation controls. The levels of cytokines other than TNF-α in the no-stimulation controls were below the limit of detection. For these cytokines, an average number (12 assays) of the lowest concentration detected for each standard was used as the value for the no-stimulation control to calculate the stimulation index: IL-6 (3.1 pg/ml); IL-1β (3.18 pg/ml); IL-10 (2.89 pg/ml); G-CSF (3.00 pg/ml); and GMCSF (3.19 pg/ml). (A) The mean fold-increase in TNF-α after 4 and 24 h of stimulation showed a trend toward higher induction in FA, DC, and SDS at the lowest dose of LPS. (B) Inductions of IL-6 and IL-1β were similar between the groups, but SDS was higher at 4 h. (C) The induction levels of IL-6, IL-1β, IL-10, G-CSF, and GM-CSF after 24 h of LPS stimulation were generally lower in IBMFS than in the controls at the high doses, but greater at the lowest dose in FA, DC, and SDS. IBMFS, inherited bone marrow failure syndrome; FA, Fanconi anaemia; DC, dyskeratosis congenita; DBA, Diamond-Blackfan anaemia, SDS, Shwachman-Diamond syndrome; Ctrl, control; IFN-γ, γ-interferon; TNF-α, tumour necrosis factor-α; LPS, lipopolysaccharide; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor.

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Cytokine production by bone marrow mononuclear cells in inherited bone marrow failure syndromes - PubMed (original) (raw)