Cross-regulation of TNF and IFN-alpha in autoimmune diseases

Abstract

Cytokines, most particularly TNF and type I IFN (IFN-alphabeta), have been long considered essential elements in the development of autoimmunity. Identification of TNF in the pathogenesis of rheumatoid arthritis and TNF antagonist therapy represent successes of immunology. IFN-alphabeta plays a major role in systemic lupus erythematosus (SLE), a prototype autoimmune disease characterized by a break of tolerance to nuclear components. Here, we show that TNF regulates IFN-alpha production in vitro at two levels. First, it inhibits the generation of plasmacytoid dendritic cells (pDCs), a major producer of IFN-alphabeta, from CD34+ hematopoietic progenitors. Second, it inhibits IFN-alpha release by immature pDCs exposed to influenza virus. Neutralization of endogenous TNF sustains IFN-alpha secretion by pDCs. These findings are clinically relevant, as five of five patients with systemic juvenile arthritis treated with TNF antagonists display overexpression of IFN-alpha-regulated genes in their blood leukocytes. These results, therefore, might provide a mechanistic explanation for the development of anti-dsDNA antibodies and lupus-like syndrome in patients undergoing anti-TNF therapy.

Figures

Fig. 1.

PBMCs from SOJIA patients treated with anti-TNF therapy display increased transcription of IFN-α-regulated genes. Gene expression was analyzed by using U95Av2 and U133 Affymetrix chips and was normalized to the median of healthy controls. A set of IFN-α-regulated genes, identified by the analysis of SLE patients as described in ref. , was compared in patients with or without anti-TNF therapy.

Fig. 2.

Exogenous TNF inhibits IFN-α release from PBMCs exposed to influenza virus. PBMCs obtained from healthy volunteers (a) or SLE patients (b) were cultured overnight with live influenza virus with or without TNF. IFN-α release to supernatants (y axis) was measured by ELISA. Average ± SD of IFN-α release in cultures with or without addition of TNF. n = 5, healthy volunteer samples; n = 9, SLE patient samples.

Fig. 3.

Exogenous TNF inhibits IFN-α release from pDCs exposed to influenza virus. pDCs were generated from CD34+ HPCs and sorted based on HLA-DR+CD123+CD11c- phenotype. (a) IFN-α release (y axis) after overnight culture with influenza virus with or without exogenous TNF. Paired t test was used. (b) Flow cytometry analysis of pDC phenotype after sorting or overnight culture with medium (these two conditions are from one experiment) or influenza virus with or without TNF (these two conditions are from another experiment). Four log decade scales are shown. (c) Giemsa staining of pDCs just after sorting, 24-h culture with influenza virus, or with TNF. Note the presence of prominent cytoplasmic protrusions (dendrites) on cells cultured with TNF or virus.

Fig. 4.

Blocking endogenous TNF sustains IFN-α release from pDCs exposed to influenza virus. pDCs were generated and purified as described in Fig. 3 and cultured with influenza virus. (a) Primary cultures were carried out in the presence or absence of isotype control or TNF-neutralizing mAb. Supernatants were harvested at 24 h after centrifugation of culture plates, and IFN-α release (y axis) was measured by ELISA. (b) Cell pellets were resuspended in fresh medium (without mAbs), influenza virus was added, and cultures were carried out for an additional 24 h. Supernatants from secondary cultures were analyzed by ELISA (y axis). (c) Flow cytometry analysis of HLA-DR expression by pDCs cultured for 24 h with influenza virus with or without TNF-neutralizing mAb (Right). Fluorescence intensity is shown on the x axis. Data are representative of three experiments.

Fig. 5.

TNF blocks differentiation of pDCs from CD34+ HPCs. CD34+ HPCs isolated from granulocyte colony-stimulating factor mobilized blood of healthy volunteers were cultured with FLT3-L and TPO with or without TNF. (a) Representative experiment of three performed that demonstrate the kinetics of pDC differentiation and dose response to TNF. Absolute numbers of pDCs are shown on the y axis. (b and c) Average ± SD percentage of pDCs (b, y axis) and mDCs (c, y axis) at three time points (x axes) in CD34+ HPC cultures with FLT3-L plus TPO with (filled bars) or without (open bars) TNF at 100 ng/ml. Data shown are for three experiments. (d) Absolute numbers of pDCs in cultures of CD34+ HPCs with or without addition of anti-TNF mAb. Data are representative of three experiments.