Fig. 2.

Tregs suppress the LPS-induced proinflammatory response of monocytes/macrophages. Monocytes were cultured as described in the legend to Fig. 1. After 40 h of coculture, LPS (50 ng/ml) was added and 24 h later cytokine/chemokine production was measured by ELISA (IL-6, TNF-α, and IL-10) or Luminex (IL-1β, IL-8, MIP-1α, MCP-1, and IL-1Ra). The average production of eight independent experiments ± SEM of proinflammatory (A) and antiinflammatory (B) cytokines/chemokines are shown for the three culture conditions. Significant differences (P < 0.03; Θ, no T vs. CD25⁺; Δ, no T vs. CD25⁻; #, CD25⁻ vs. CD25⁺) are shown in the graphs. Neither GM-CSF nor IL-12p70 was not detected (data not shown). (C) After 40 h, the cocultures were depleted of T cells, and the purified monocytes were either unstimulated (control) or stimulated with LPS for 15 min before total cell lysates were generated. The mean NF-κB p50 activation ± SEM of four independent experiments is shown. (D) Ten micrograms of total cell lysates from monocytes cultured in the absence (lanes 1 and 4) or presence of CD25⁻ (lanes 2 and 5) or CD25⁺ (lanes 3 and 6) T cells that were untreated (lanes 1–3) or treated with LPS (lanes 4–6) was separated on 10% SDS gels and immunoblotted with an antiphosphotyrosine antibody, pY (Upper) or a control anti-β-actin antibody (Lower). A representative example of four independent experiments is shown.