Antigen presentation by macrophages is enhanced by the uptake of necrotic, but not apoptotic, cells

Abstract

The aim of this study was to determine whether phagocytosis of necrotic or apoptotic cells affects antigen presentation by murine bone marrow-derived macrophages. After uptake of necrotic neutrophils, macrophages were able to stimulate significantly higher T cell proliferation in vitro against both the recall antigen albumin and the mitogen concanavalin A. No such effect was seen following phagocytosis of apoptotic neutrophils. Flow cytometry revealed that, within 4h of ingestion, macrophages that had taken up the necrotic cells expressed higher levels of CD40 than those that had phagocytosed apoptotic cells. Macrophage cultures pulsed with apoptotic, but not necrotic, neutrophils contained higher levels of transforming growth factor beta1, but lower concentrations of tumour necrosis factor alpha, compared to untreated controls. Our interpretation of these results is that macrophages that have taken up necrotic neutrophils co-stimulate T cells with greater efficiency due to rapid CD40 up-regulation, whereas those that have ingested apoptotic cells are not only ineffective in co-stimulation, but also secrete inhibitory cytokine.

Fig. 1

The effect of pulsing macrophages with necrotic or apoptotic neutrophils on the ability to induce splenic T cell proliferation against the mitogen Con A. Macrophages were allowed to take up dying neutrophils for 30 min and the non-ingested cells washed off before addition of the T cells and stimulation with Con A. The results in replicate cultures (n = 3) are summarized, and significant differences (P < 0·05) between proliferative responses are bracketed. Proliferation in wells containing T cells or macrophages alone was minimal (cpm <375). Stimulation: ▪, Con A; □, none.

Fig. 2

Representative experiment (n = 4) showing the effect of pulsing macrophages with necrotic or apoptotic neutrophils on the ability to induce splenic T cell proliferation by presenting the recall antigen ovalbumin. Macrophages were allowed to take up dying neutrophils for 30 min and the uningested cells washed off. The macrophages were then pulsed with ovalbumin for 1h, washed and the T cells added. Significant differences (P < 0·05) between proliferative responses are bracketed. Stimulation ▪, Ovalbumin; □, none.

Fig. 3

The differential effects of necrotic or apoptotic neutrophil uptake on presentation of ovalbumin by macrophages are not dependent on the concentration of macrophages or antigen in cultures. Splenic T cell proliferation was stimulated by macrophages that had been plated out at 0·5 × 10⁶ (a) and (b) or 10⁶ (c) cells/ml and pulsed with the recall antigen ovalbumin at 10 (a) or 100 (b and c) μg/ml. The percentage changes in proliferation are relative to the corresponding responses elicited by macrophages that have not been incubated with dying neutrophils and are calculated from the stimulation indices of three replicate cultures.

Fig. 4

The effect of pulsing macrophages with necrotic or apoptotic neutrophils on the expression after 4h of MHC class II and the co-stimulatory molecules CD40, CD80 and CD86. The mean results of repeat experiments are summarized (MHC class II n = 2, CD40 n = 6, CD80 n = 2, CD86 n = 6). Significant differences (P < 0·05) between staining levels are bracketed. Staining: ▪, MHC class II; □, CD40; , CD80;, CD86.

Fig. 5

The effect of pulsing macrophages with necrotic or apoptotic neutrophils on the production of TGF-β1 (a) or TNF-α (b). For each cytokine, the duplicate results of at least three repeat experiments are summarized and significant differences (P < 0·05) between responses are bracketed.