Tumor necrosis factor α release in peripheral blood mononuclear cells of cutaneous lupus and dermatomyositis patients

Abstract

Introduction: Several studies have reported that TNFα is substantially increased within skin lesions of patients with discoid lupus erythematosus (DLE), subacute cutaneous lupus erythematosus (SCLE) and dermatomyositis (DM) compared to controls. Elevated TNFα has been reported in the sera of some patients with systemic lupus erythematosus, DLE and SCLE, but not in the sera of patients with DM. Because of the key pathogenic role of autoimmunity in these diseases, in this study we sought to evaluate TNFα production by a readily available source of immune cells (namely, peripheral blood mononuclear cells (PBMCs)) taken from controls and from patients with cutaneous lupus or DM.

Methods: Freshly isolated PBMCs were cultured overnight, and TNFα protein accumulation in conditioned medium was determined. In addition, flow cytometry using cell-type-specific markers was performed to determine the sources of TNFα. One-way analysis of variance and Dunnett's multiple comparisons test were performed for statistical comparisons.

Results: Accumulation of TNFα protein in conditioned medium containing PBMCs from DLE patients, but not from SCLE, TLE or DM patients, was significantly greater (19-fold) than that from controls (P < 0.001). In DLE PBMCs, increased TNFα was produced by circulating monocytes and myeloid dendritic cells (mDCs). The mean TNFα fluorescence intensity, but not the total number, of both monocytes and mDCs (P < 0.01) from DLE patients was significantly greater (2.3-fold) than that of controls. There were significantly more (13.3-fold) mDCs with intracellular TNFα in blood from DLE patients (P < 0.001) and DM patients (P < 0.001) compared to controls. Most importantly, a positive correlation was seen in DLE patients between their disease activity measured using the Cutaneous Lupus Erythematosus Disease Area and Severity Index and TNFα protein secretion (r = 0.61, P < 0.08).

Conclusions: TNFα protein production by PBMCs is greater in DLE patients than in patients with other cutaneous forms of lupus and DM or in controls. Flow cytometric studies demonstrated that circulating monocytes and mDCs contributed to this increased TNFα production. Monocytes and mDCs are present in lesional skin, and the increased TNFα production by these cells and other PBMCs likely increase the number of inflammatory cells seen in DLE skin relative to other subsets of cutaneous lupus erythematosus and DM. These results provide a possible biological explanation for the denser infiltrate seen in DLE relative to DM.

Figure 1

TNFα protein production by unstimulated peripheral blood mononuclear cells from DLE, SCLE, tumid TLE and DM patients and from controls. TLE should be written in the figure Peripheral blood mononuclear cells (PBMCs) were cultured for 24 hours, followed by collection of supernatants. The concentration of TNFα in the supernatants was measured by ELISA. Bars show the means and SD. ***P < 0.001 vs control PBMCs by Dunnett's multiple comparison test. DLE = discoid lupus erythematosus, DM = dermatomyositis, TLE, tumid lupus erythematosus, SCLE, subacute cutaneous lupus erythematosus.

Figure 2

TNFα in peripheral blood mononuclear cell supernatants from discoid lupus erythematosus (DLE) patients correlated with Cutaneous Lupus Erythematosus Disease Area and Severity Index scores (r = 0.61).

Figure 3

Flow cytometric analysis of intracellular TNFα production by myeloid dendritic cells and monocytes from patients and controls. Upper panels show gating of peripheral blood mononuclear cells (PBMCs) from representative patients with discoid lupus erythematosus (DLE), dermatomyositis (DM) or controls stained with fluorochrome-conjugated appropriate antibodies (A) myeloid dendritic cells (lineage negative, HLADR⁺, CD11c⁺) or (B) monocytes (CD64⁺CD14⁺). Lower panels show intracellular expression of TNFα in (A) myeloid dendritic cells or (B) monocytes. The numbers in the upper panels state the percentage of cell subset per total population of PBMCs. The numbers in the lower panels state the percentage of myeloid dendritic cells or monocytes producing TNFα.

Figure 4

Defining the cell population responsible for TNFα production. (A) CD64⁺CD14⁺ monocytes, (B) CD11c⁺HLA-DR^hi/low myeloid dendritic cells, (C) CD123⁺ plasmacytoid dendritic cells and (D) CD3⁺ T lymphocytes from five discoid lupus erythematosus (DLE) and five dermatomyositis (DM) patients, as well as from five controls, were analyzed by flow cytometry to assess their ability to produce intracellular TNFα. Bars show the means and SD. *** P < 0.001 vs controls by Dunnett's multiple comparisons test.

Figure 5

Measurement of mean fluorescence intensity (MFI). The MFI of TNFα release from (A) CD64⁺CD14⁺ monocytes, (B) CD11c⁺HLA-DR^hi/low myeloid dendritic cells, (C) CD123⁺ plasmacytoid dendritic cells, and (D) CD3⁺ T lymphocytes from five discoid lupus erythematosus (DLE) and five dermatomyositis (DM) patients, as well as from five controls, as determined by flow cytometry. Bars show the means and SD. ** P < 0.01 vs controls by Dunnett's multiple comparison test.

Figure 6

Measurement of cell populations by flow cytometry. (A) CD64⁺CD14⁺ monocyte and (B) CD11c⁺HLA-DR^hi/low myeloid dendritic cell populations from five discoid lupus erythematosus (DLE) and five dermatomyositis (DM) patients, as well as from five controls, as determined by flow cytometry.