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. Author manuscript; available in PMC: 2010 Oct 1.
Published in final edited form as: J Immunol. 2009 Jul 13;183(3):2122–2132. doi: 10.4049/jimmunol.0804187

Figure 1. Neutrophils secrete a soluble anti-inflammatory factor.

Figure 1

(a+b) HMDM were stimulated with either LPS or CD40L/IFN-γ (CI) along with added apoptotic neutrophils (LPSN or CIN) for 18 hours, prior to harvesting culture supernatants for assay of TNF-α by ELISA. In triplicate wells anti-TGFβ was added to assess the role of TGFβ in mediating the immunosuppressive effect of apoptotic neutrophils [LPSN(anti-TGFβ) or CIN(anti-TGFβ)]. In addition apoptotic neutrophils were separated from activated HMDM by a transwell [LPS(N) or CI(N)] for the duration of the culture period. Macrophages alone (M) or unstimulated macrophages cultured with apoptotic neutrophils (MN) did not secrete TNF-α.

(c+d) Neutrophils were cultured for up to 24 hours, harvested at set timepoints and ultracentrifuged to remove cell membranes and apoptotic bodies. This neutrophil conditioned medium (NCM) was added (as 25% final vol) to LPS or CD40L/IFN-γ (CI) stimulated macrophages. After 18 hours of culture HMDM culture supernatants were harvested and assayed for TNF-α by ELISA.

Representative of 10 experiments performed with different human donors. Error bars represent SEM and significance of *** p ≤0.0002, ** p ≤0.002, * p ≤0.02.