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. 2008 Dec;125(4):591-600.
doi: 10.1111/j.1365-2567.2008.02873.x.

Tumour necrosis factor (TNF)-alpha primes murine neutrophils when triggered via formyl peptide receptor-related sequence 2, the murine orthologue of human formyl peptide receptor-like 1, through a process involving the type I TNF receptor and subcellular granule mobilization

Karin Onnheim  1 , Johan Bylund, Francois Boulay, Claes Dahlgren, Huamei Forsman
Affiliations

Tumour necrosis factor (TNF)-alpha primes murine neutrophils when triggered via formyl peptide receptor-related sequence 2, the murine orthologue of human formyl peptide receptor-like 1, through a process involving the type I TNF receptor and subcellular granule mobilization

Karin Onnheim et al. Immunology. 2008 Dec.

Abstract

Neutrophil granulocytes play an important role in innate host defence against microbial invasions and they are also the key effector cells in mediating host tissue damage. These functions often rely on the production of reactive oxygen species (ROS) from the membrane-bound NADPH-oxidase system. The magnitude of ROS production varies depending on the state of the cells, i.e. resting or primed. Many priming agents as well as potent NADPH-oxidase activators have been identified and characterized for human neutrophils. The cytokine tumour necrosis factor (TNF)-alpha is one prominent example of a priming agent and the synthetic hexapeptide WKYMVm is an agonist that triggers an activation of the NADPH-oxidase of human neutrophils through two members of the formyl peptide family of receptors, formyl peptide receptor (FPR) and FPR-like 1 (FPRL1). This peptide also activates murine neutrophils but the precise receptor involved has not been previously characterized. We show in this study that WKYMVm activates stably transfected HL60 cells expressing murine formyl peptide receptor-related sequence 2 (Fpr-rs2) and that activation of murine neutrophils with WKYMVm is blocked by an FPRL1-specific antagonist. WKYMVm is thus an agonist for Fpr-rs2 and we suggest that this receptor is in fact the mouse orthologue of FPRL1. In addition, we show that the WKYMVm response in murine neutrophils can be primed by TNF-alpha and this priming process involves mobilization of subcellular granules. The results obtained using neutrophils derived from TNF receptor type I (TNFRI)-deficient animals suggest that TNF-alpha exerts its priming effect via the TNFRI.

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Figures

Figure 1
Figure 1
WKYMVm induced superoxide release from murine neutrophils. Murine neutrophils (5 × 105 cells) were isolated from bone marrow and incubated with chemiluminescence (CL) reagents, including isoluminol and horseradish peroxidase, at 37° for 2 hr either alone (control) or with pertussis toxin (PTX) (500 ng/ml final concentration), after which reactive oxygen species (ROS) production was triggered by the addition of WKYMVm (100 nm) or phorbol 12-myristate 13-acetate (PMA) (50 nm; inset). The extracellular superoxide release was measured using an isoluminol-amplified CL technique. Representative kinetics of the CL response from three experiments are shown. Abscissa, time of CL recording (min); ordinate, the CL response (arbitrary units).
Figure 2
Figure 2
WKYMVm induces an increase in calcium in murine formyl peptide receptor-related sequence 2 (Fpr-rs2) over-expressing HL60 cells. Undifferentiated HL60 cells transfected with Fpr-rs2 were loaded with Fura-2, and an intracellular calcium increase was triggered by the addition of WKYMVm at various final concentrations. (a) The percentage of the maximum calcium response as calculated from the peak fluorescence (340 nm) for each concentration of WKYMVm and the maximum response induced by 1 μm WKYMVm. (b) A representative calcium response induced by the L-form of WKYMVm, WKYMVM (100 nm). (c) The specific FPR antagonist cyclosporin H (1 μm; dotted line), the FPRL1 antagonist WRW4 (0·25 μm; dashed line) or Krebs–Ringer phosphate buffer (KRG) (control; solid line) was preincubated with cells during the 5-min pre-warming period before the addition of WKYMVm (10 nm). Changes in the level of cytosolic calcium were determined by measurement of the light emitted at 509 nm during excitation at 340 and 380 nm. The results are presented as a ratio between the fluorescence intensities at 340 and 380 nm from one representative experiment of six independent experiments.
Figure 3
Figure 3
Effects of WRW4 and cyclosporine H on WKYMVm-induced superoxide anion production. Murine neutrophils (5 × 105 cells) were isolated from bone marrow and primed with tumour necrosis factor (TNF)-α at 37° for 30 min before stimulation with WKYMVm (5 nm). The indicated concentrations of the specific formyl peptide receptor (FPR) antagonist cyclosporine H (open boxes) or the FPRL1 antagonist WRW4 (closed boxes) were included in the 5-min pre-warming period before the addition of WKYMVm. Superoxide release upon WKYMVm stimulation was continuously recorded using the chemiluminescence (CL) technique, and the results are expressed as percentage inhibition compared with samples without any antagonist. The data presented are from one representative experiment of three.
Figure 4
Figure 4
Time-dependent priming effect of the WKYMVm response by tumour necrosis factor (TNF)-α. (a) Mouse neutrophils were either left untreated (solid line) or primed with mouse recombinant TNF-α (50 ng/ml; dashed line) at 37° for 30 min and the superoxide release triggered by WKYMVm (100 nm) was recorded. (b) The same type of experiment was performed but the time for priming with TNF-α (50 ng/ml) was varied from 10 to 30 min (closed boxes), and the peak values of the response (superoxide release) were determined by chemiluminescence (CL) technique and compared with the corresponding control values obtained with cells that received no cytokine (control; open boxes). The data represent mean values (± standard deviation) from six independent experiments and a P value < 0·05 (*) was regarded as significant.
Figure 5
Figure 5
No priming induced by tumour necrosis factor (TNF)-α in TNF receptor type I (TNFRI)-deficient (TNFRI−/−) cells. Bone marrow neutrophils were isolated from wild-type (WT) animals (strain C57B/6; open bars) or mice deficient in TNFRI on a pure C57B/6 background (TNFRI−/−, filled bars). The cells were primed with TNF-α (50 ng/ml) or keratinocyte-derived chemokine (KC) (50 ng/ml) at 37° for 30 min, and then activated with WKYMVm (100 nm final concentration). Superoxide release was measured and the peak values from primed and non-primed animals were compared and expressed as a fold increase. The data are expressed as mean ± standard deviation (n = 6) and a P value < 0·05 (*) was regarded as significant.
Figure 6
Figure 6
No change in surface complement receptor 3 (CR3) expression upon tumour necrosis factor (TNF)-α priming in TNF receptor type I (TNFRI)-deficient (TNFRI−/−) cells. Neutrophils were isolated from a bone marrow TNFRI−/− mouse and primed with either TNF-α (dotted line) or keratinocyte-derived chemokine (KC) (dashed line), or left untreated (control; solid line), followed by fixation in 2% paraformaldehyde. The CR3 expression was examined by flow cytometry with a fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse CR3 antibody. A representative histogram from six independent experiments is shown. The inset shows the percentage of CR3 up-regulation in wild-type (WT) cells and TNFRI−/− cells upon TNF-α and KC priming in comparison to the non-primed cells. Data are expressed as mean values ± standard deviation (n = 6).
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