Abnormal production of pro- and anti-inflammatory cytokines by lupus monocytes in response to apoptotic cells

Abstract

Monocytes are a key component of the innate immune system involved in the regulation of the adaptive immune response. Previous studies have focused on apoptotic cell clearance abnormalities in systemic lupus erythematosus (SLE) monocytes. However, whether SLE monocytes might express unique patterns of cytokine secretion in response to apoptotic cells is still unknown. Here, we used monocytes from healthy controls and SLE patients to evaluate the production of TNF-α and TGF-β in response to apoptotic cells. Upon recognition of apoptotic material, monocytes from healthy controls showed prominent TGF-β secretion (mean ± SD: 824.6±144.3 pg/ml) and minimal TNF-α production (mean ± SD: 32.6±2.1 pg/ml). In contrast, monocytes from SLE patients had prominent TNF-α production (mean ± SD: 302.2±337.5 pg/ml) and diminished TGF-β secretion (mean ± SD: 685.9±615.9 pg/ml), a difference that was statistically significant compared to normal monocytes (p≤10(-6) for TNF-α secretion, and p = 0.0031 for TGF-β, respectively). Interestingly, the unique cytokine response by SLE monocytes was independent of their phagocytic clearance efficiency, opsonizing autoantibodies and disease activity. We further showed that nucleic acids from apoptotic cells play important role in the induction of TNF-α by lupus monocytes. Together, these observations suggest that, in addition to potential clearance defects, monocytes from SLE patients have an abnormal balance in the secretion of anti- and pro-inflammatory cytokines in response to apoptotic cells. Since the abnormal cytokine response to apoptotic material in SLE is not related to disease activity and opsonizing autoantibodies, it is possible that this response might be an intrinsic property of lupus monocytes. The studies focus attention on toll-like receptors (TLRs) and their downstream pathways as mediators of this response.

Figure 1. Monocytes from patients with SLE have prominent production of TNF-α, but decreased production of TGF-β.

Monocytes obtained from healthy donors (n = 13) or patients with SLE (n = 47) were co-incubated in the presence of apoptotic Jurkat cells. After overnight incubation, production of TGF-β (A) and TNF-α (B) were determined in the supernatants by ELISA. Data is shown as boxplots. Comparison of mean cytokine secretion between groups was performed by Students t-test.

Figure 2. Production of TGF-β and TNF-α by live or apoptotic Jurkat cells, non-stimulated monocytes and monocytes plus live Jurkat cells.

Monocytes from healthy donors (A & B) or patients with SLE (C & D) were incubated alone or in the presence of live Jurkat cells. In addition, live or apoptotic Jurkat cells were incubated alone. After overnight incubation, production of TGF-β (A & C) and TNF-α (B & D) were determined in the supernatants by ELISA.

Figure 3. Pearson's correlation between phagocytosis of apoptotic cells and TNF-α production by SLE monocytes.

Control (Figure S1) or SLE monocytes were co-incubated with CFSE-labeled apoptotic Jurkat cells. The percentage of cells positive for both CD14 and CFSE was used to quantify phagocytosis of apoptotic Jurkat cells by monocytes. Only data from SLE monocytes is shown. Statistical analysis was performed with Stata 10 software (Stata Corporation, College Station, TX).

Figure 4. TNF-α production by lupus monocytes in response to apoptotic cells is sensitive to RNase and DNase treatment.

Monocytes from six consecutive SLE patients were co-incubated with apoptotic Jurkat cells (UVJ) in the absence or presence of 5 µg/ml of RNase, 16 u/ml of DNase, or both nucleases; and TNF-α production was determined by ELISA in the supernatants.