Role of osteopontin in neutrophil function

Abstract

Osteopontin (OPN) is important for the function of fibroblasts, macrophages and lymphocytes during inflammation and wound healing. In recent studies of experimental colitis we demonstrated exacerbated tissue destruction in OPN-null mice, associated with reduced tumour necrosis factor-alpha expression and increased myeloperoxidase activity. The objective of this investigation therefore was to determine the importance of OPN expression in neutrophil function. Although, in contrast to macrophages, neutrophils expressed low levels of OPN with little or no association with the CD44 receptor, intraperitoneal recruitment of neutrophils in OPN-null mice was impaired in response to sodium periodate. The importance of exogenous OPN for neutrophil recruitment was demonstrated by a robust increase in peritoneal infiltration of PMNs in response to injections of native or recombinant OPN. In vitro, OPN(-/-) neutrophils exhibited reduced chemokinesis and chemotaxis towards N-formyl methionyl leucyl phenylalanine (fMLP), reflecting a reduction in migration speed and polarization. Exogenous OPN, which was chemotactic for the neutrophils, rescued the defects in polarization and migration speed of the OPN(-/-) neutrophils. In contrast, the defensive and cytocidal activities of OPN(-/-) neutrophils, measured by assays for phagocytosis, generation of reactive oxygen species, cytokine production and matrix metalloproteinase-9, were not impaired. These studies demonstrate that, while exogenous OPN may be important for the recruitment and migration of neutrophils, expression of OPN by neutrophils is not required for their destructive capabilities.

Figure 1

Expression of OPN in neutrophils. (a) OPN mRNA levels in neutrophils (PMNs) were measured by RT-PCR and compared with OPN expression by RAW 264.7 macrophages, using β-actin as a control. A single 408-bp amplicon was amplified with much lower amounts in the neutrophils (+ RT). Only a trace of product was observed in the absence of reverse transcriptase (– RT). Quantitative PCR revealed that OPN mRNA in the neutrophils was 75-fold less than in the RAW 264.7 cells. (b) Confocal immunofluorescence analysis of polarized neutrophils stained for cell-surface surface (non-permeablized cells) CD44 (red) and cellular (permeabilized cells) OPN (green). No significant colocalization of the punctate OPN with the CD44, which is concentrated in the uropod, was evident. (c) Immunofluorescence staining of neutrophils with TRITC-phalloidin to examine F-actin distribution showed no differences between the OPN^–/– null and WT cells regardless of their polarization state.

Figure 2

Migration of neutrophils in transwell chambers. Neutrophils were analysed for migration in response to 10⁻⁶ mol/l fMLP stimulation and OPN using BSA as a control. The number of neutrophils migrating through the 3·0-μm pores after 30 min in Transwell chambers was determined and the means ± SD were calculated for triplicate samples. (a) Migration toward fMLP, which was consistently decreased in OPN^–/– neutrophils, could be partially rescued by low concentrations of exogenous macrophage OPN added to both sides (BS) of the membrane, but decreased migration in both WT cells and in OPN^–/– cells at higher concentrations. (b) A dose-dependent increase in migration towards OPN added to the lower chamber (LC) was observed for both WT and OPN^–/– cells, the OPN^–/– neutrophils responding more strongly. The results from one of three replicate experiments are shown.

Figure 3

Polarization and migration of neutrophils in a Zigmond chamber. (a) The proportion of neutrophils that polarized randomly and in the direction of fMLP was determined after 15 min. Fewer cells were seen to display directed migration for both WT and OPN^–/– neutrophils. Addition of exogenous macrophage OPN to OPN^–/– cells increased both random and directed migration reproducibly, but not significantly (P > 0·1). (b) Analysis of neutrophils over the 15-min time interval consistently showed a lower directed polarization for the OPN^–/– cells, but the differences were not statistically significant. Exogenous OPN increased both random and directed polarization to similar levels observed in WT neutrophils but the increases were only significant (*P < 0·05) for the random polarization. (c) The average migration speed of OPN^–/– neutrophils was lower than the WT cells (*P < 0·05). While fMLP stimulation increased the average speed of both WT and OPN^–/– neutrophils, the effect was less in the OPN^–/– neutrophils. Addition of exogenous OPN increased migration of OPN^–/– cells approximately three-fold (***P < 0·001). Results are expressed as means ± SD.

Figure 4

Recruitment of neutrophils in vivo. The total number of immune cells in peritoneal exudates following intraperitoneal injections of 5 mm periodate or 20 μg/ml OPN was determined in Wright–Giemsa-stained slides using a haemocytometer. (a) Recruitment of immune cells by periodate was more than three-fold lower (open bars) in OPN^–/– null mice with a disproportionately lower recruitment of neutrophils (black bars). (b) In response to administration of full-length native or recombinant OPN an infiltrate containing predominantly neutrophils was obtained, whereas PBS vehicle control did not recruit any significant numbers of neutrophils. Results are expressed as mean ± SD.

Figure 5

Phagocytosis assays. The ability of LPS-stimulated neutrophils to phagocytose polystyrene beads was assessed by incubating cells for 25 min at 37° with fluorescent beads and the number of beads internalized by neutrophils was determined by flow cytometry. No differences between cells derived from OPN^–/– null mice or WT controls in the phagocytosis of beads were observed. Specific phagocytosis mediated through Fc or complement receptors (C5) was analysed using red blood cells labelled with Texas Red–sulphonyl chloride dye. No significant differences were observed between cells derived from OPN^–/– mice or WT controls in the phagocytosis of beads. Samples from three separate experiments were analysed by fluorescence microscopy and flow cytometry and the results are expressed as the percentage of cells containing one or more beads (mean ± SD).

Figure 6

Neutrophil production of H₂O₂ was assessed from the mean fluorescence intensity (MFI) of rhodamine-123 and used as a measure of the oxidative burst. Samples were analysed by fluorescence microscopy and flow cytometry (mean ± SD). (a) No differences were observed between the WT and OPN^–/– neutrophils stimulated with either fMLP or LPS. (b) The ratio of fluorescence between stimulated and unstimulated PMNs confirmed the lack of significant differences in oxidative burst ability.

Figure 7

Cytokine release and gelatin enzymography. Neutrophils were incubated with 0·1 µm PMA to stimulate neutrophil degranulation. (a) Cytokines released into PBS were analysed on a cytokine blot. The key to duplicate spots for cytokines and both positive (PC) and negative (NC) controls is shown in the right panel. No differences in the release of cytokines by WT and OPN^–/– cells were evident in these assays. (b) Gelatinolytic enzymes in cell extracts and in conditioned PBS with and without stimulation with PMA were analysed by enzymography. Only pro-MMP-9 and activated MMP-9 were detected, together with an unidentified slower migrating protein with gelatinase activity. Most of the activity corresponded to the pro-MMP-9, which was effectively released into the PBS by the PMA treatment. No differences in MMP-9 expression between the WT and OPN^–/– neutrophils were evident in replicate experiments.