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. 2008 Jan;151(1):139-45.
doi: 10.1111/j.1365-2249.2007.03532.x. Epub 2007 Dec 7.

Neutrophil secretion products regulate anti-bacterial activity in monocytes and macrophages

O Soehnlein  1 , E Kenne, P Rotzius, E E Eriksson, L Lindbom
Affiliations

Neutrophil secretion products regulate anti-bacterial activity in monocytes and macrophages

O Soehnlein et al. Clin Exp Immunol. 2008 Jan.

Abstract

Macrophages represent a multi-functional cell type in innate immunity that contributes to bacterial clearance by recognition, phagocytosis and killing. In acute inflammation, infiltrating neutrophils release a wide array of preformed granule proteins which interfere functionally with their environment. Here, we present a novel role for neutrophil-derived granule proteins in the anti-microbial activity of macrophages. Neutrophil secretion obtained by antibody cross-linking of the integrin subunit CD18 (X-link secretion) or by treatment with N-Formyl-Met-Leu-Phe (fMLP secretion) induced a several-fold increase in bacterial phagocytosis by monocytes and macrophages. This response was associated with a rapid activation of the monocytes and macrophages as depicted by an increase in cytosolic free Ca(2+). Interestingly, fMLP secretion had a more pronounced effect on monocytes than the X-link secretion, while the opposite was observed for macrophages. In addition, polymorphonuclear cells (PMN) secretion caused a strong enhancement of intracellular reactive oxygen species (ROS) formation compared to incubation with bacteria. Thus, secretion of neutrophil granule proteins activates macrophages to increase the phagocytosis of bacteria and to enhance intracellular ROS formation, indicating pronounced intracellular bacterial killing. Both mechanisms attribute novel microbicidal properties to PMN granule proteins, suggesting their potential use in anti-microbial therapy.

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Figures

Fig. 1
Fig. 1
(a–e) Polymorphonuclear cell (PMN) secretion products induce phagocytosis in human and murine monocytes and macrophages. (a–d) Human THP-1 (a), human macrophages (b), murine WEHI-3B (c) or RAW 264·7 (d) were treated with X-link secretion, N-Formyl-Met-Leu-Phe (fMLP secretion) or fMLP alone (10 nM) for 24 h. Control (ctrl) indicates treatment with cell culture medium only. After stimulation, Alexa Fluor 488-labelled Staphylococcus aureus were added and incubated with the cells for 1 h. The number of incorporated bacteria per cell was quantified by fluorescence microscopy. Data are expressed as mean ± standard deviation; n = 6–8 for each bar. *Significant difference compared to ctrl; **significant difference compared to ctrl and X-link secretion in (a) and (c) or compared to ctrl and fMLP secretion in (b) and (d). (e) Composition of PMN secretion after CD18 cross-linking and fMLP activation. Proteins in X-link secretion and fMLP secretion were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Western blotting and antibody staining for marker proteins of the PMN granules were used to identify release of the different granule subsets. Each Western blot is representative of at least three independent experiments.
Fig. 2
Fig. 2
(a–b) Monocytes and macrophages are activated by polymorphonuclear cell (PMN) secretion. Dynamic change in fluorescence intensity of human THP-1 (a), human macrophages (b), murine WEHI-3B (c) or murine RAW 264·7 (d) loaded with fluo4/AM after stimulation with X-link secretion, N-Formyl-Met-Leu-Phe (fMLP secretion) or cell culture medium [control (ctrl)]. Fluorescence intensity depicting intracellular Ca2+ mobilization was measured before stimulation, 30 s, 90 s and 150 s after stimulation in a fluorescence plate reader. Values are expressed as percentage of mean fluorescence intensity (MFI) before treatment. *Significant increase in MFI compared to ctrl treatment; **significant difference compared to ctrl and X-link secretion in (a) and (c) or compared to ctrl and fMLP secretion in (b) and (d). Values are given as mean ± standard deviation; n = 6–8 for each point.
Fig. 3
Fig. 3
(a–b) Intracellular reactive oxygen species (ROS) formation in macrophages in response to polymorphonuclear cell (PMN) secretion. Measurement of intracellular ROS formation by assessing the fluorescence intensity of human macrophages labelled with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA). (a) Macrophages were pretreated with X-link secretion for 24 h and the ROS formation was measured after addition of Staphylococcus aureus for 80 min (S. aureus + X-link secretion). Treatment with culture medium [control (ctrl)] or S. aureus without prior incubation with X-link secretion (S. aureus) were used as controls. (b) Macrophages were grown in a 96-well plate, labelled with H2DCFDA and the fluorescence was measured over 80 min after stimulation with medium (ctrl), X-link secretion, S. aureus or X-link secretion and S. aureus. All data points indicate mean values of at least eight measurements. Note the different scales of the y-axis in (a) and (b).
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