HoxB8 neutrophils replicate Fcγ receptor and integrin-induced neutrophil signaling and functions

Abstract

Neutrophils are short-lived, terminally differentiated leukocytes that form an essential part of host immunity and play a key role in acute and chronic inflammation. The analysis of these important cells is hindered by the fact that neutrophils are not amenable to culture, transfection, or transduction. Conditionally HoxB8-immortalized mouse hematopoietic progenitors are suitable for in vitro differentiation of a range of myeloid cells, including neutrophils. Integrins and FcγRs are cell surface receptors, the ligation of which is required for a range of neutrophil functions that are important in health and disease. We show here that HoxB8 neutrophils express major neutrophil integrins and FcγRs. They respond to FcγR and integrin stimulation in a manner that is comparable with primary neutrophils, in terms of intracellular signaling. HoxB8 neutrophils also perform a range of FcγR/integrin-dependent neutrophil functions, including, generation of reactive oxygen species, degranulation, and chemotaxis. Our findings suggest that HoxB8 neutrophils represent a faithful experimental model system for the analysis of Fc and integrin receptor-dependent neutrophil functions.

Keywords: Fc Receptors; adhesion molecules; granulocytes; human cell lines; intracellular signaling; monocytes/macrophages; neutrophils; transgenic/knockout mice.

Figure 1

HoxB8 neutrophils express neutrophil integrins and FcγRs. (A) A representative cytocentrifuge preparations of Quick‐Diff stained HoxB8 neutrophils after 5 d of differentiation. Scale bar, 5 μm. (B, C) HoxB8 neutrophil surface FcγRs (B) and integrins (C) as indicated were analyzed by flow cytometry. Representative FACS plots are shown from a minimum of 3 separate experiments performed with HoxB8 neutrophils that had been enriched by separation over a discontinuous percoll gradient. Black traces, BMNs; red/grey traces, HoxB8 neutrophils; broken lines, isotype controls; for FcγRIV, broken line represents secondary antibody only control

Figure 2

Integrin and FcγR stimulation‐induced intracellular signaling events in BMNs and HoxB8 neutrophils. BMNs and HoxB8 neutrophils (from 3 different bone marrow donors) were stimulated (A) for 1 min with 1 μM fMLF in suspension, or by being plated onto (B) pRGD or (C) immobilized ICs for 12 min, or (D) for 10 min with 10 μg/ml insoluble ICs in suspension. Signaling events were analyzed by Western blotting. Hsp90 served as a loading control. Representative blots are shown. Images were put into greyscale for ease of viewing after densitometry. Densitometric analysis was carried out to calculate the fold activations obtained in individual experiments. Mean fold activations (±sem) obtained with HoxB8 neutrophils and BMNs under all the stimulation conditions of at least 4 separate experiments are plotted. Statistical analysis was by unpaired t test

Figure 3

HoxB8 neutrophils generate ROS upon integrin/FcγR stimulation. (A) HoxB8 neutrophils were or were not subjected to purification by percoll gradient before being stimulated with 100 nM PMA or its vehicle for ROS assays. A representative example is shown. Red symbols, no percoll gradient; blue symbols, with percoll gradient (B–G). ROS generation of percoll‐enriched HoxB8 cells that had or had not been pre‐incubated with DPI prior to stimulation with PMA (B, E), being plated onto pRGD (C, F), or immobilized ICs (D, G). Representative examples (mean ± range) from a minimum of 4 separate experiments are plotted (B–D). Integrated results are plotted normalized to the activated condition (E–G). *P < 0.05; **P < 0.01; statistical analysis was by t test. Results shown in this figure were obtained with HoxB8 neutrophils from a single donor, and reflect those obtained with cells obtained from two other donors on a different plate reader

Figure 4

HoxB8 neutrophils degranulate upon integrin/FcγR stimulation. HoxB8 neutrophils were stimulated by being plated onto pRGD or immobilized ICs, or with 1 μM fMLF in the presence of 10 μM cytochalasin B for 1 h. Released gelatinase (A) and lactoferrin (B) in the supernatant was determined by in gel‐zymography (A) and ELISA (B). A broken line in (B) indicates that the readings obtained with fMLF and cytochalasin B were obtained with more dilute supernatants. Bars show mean ± sem from at least 3 separate experiments performed with HoxB8 neutrophils obtained from 2 different bone marrow donors. *P < 0.05; **P < 0.01; ***P < 0.001; statistical analysis was by paired t test (A) and by unpaired t test (B)

Figure 5

HoxB8 neutrophil chemotaxis. HoxB8 neutrophils were allowed to chemotax toward fMLF. (A) Tracks of individual cells from experiments performed on three different days are combined in this spider plot. * indicates the source of chemoattractant. (B) Total and Euclidian distances travelled by the cells shown in (A) are plotted. Error bars show sem. (C) Cropped still from a time‐lapse movie showing 3 HoxB8 neutrophils with characteristic migrating morphology comprising leading edge (arrowhead) and trailing end (asterisk)