Netrin-1 inhibits leukocyte migration in vitro and in vivo - PubMed (original) (raw)
Netrin-1 inhibits leukocyte migration in vitro and in vivo
Ngoc P Ly et al. Proc Natl Acad Sci U S A. 2005.
Abstract
Cell migration plays important roles in embryonic development and inflammation, and this process is highly regulated to ensure tissue homeostasis. A number of barriers exist to prevent the inappropriate migration of leukocytes into healthy peripheral tissues, including retention of these cells in the inactive state and maintenance of the integrity and charge of the vascular endothelium. However, active signals also are likely to exist that can repulse cells or abolish existing cell migration. One such paradigm exists in the developing nervous system, where neuronal migration is mediated by a balance between chemoattractive and chemorepulsive signals. The ability of the guidance molecule netrin-1 to repulse or abolish attraction of neuronal cells expressing the UNC5b receptor makes it an attractive candidate for the regulation of inflammatory cell migration. Here, we show that netrin-1 is expressed on vascular endothelium, where it is regulated by infection and inflammatory cytokines. The netrin-1 receptor UNC5b is strongly expressed by leukocytes, upon which netrin-1 acts as a potent inhibitor of migration to different chemotactic stimuli both in vivo and in vitro. These data suggest that endothelial expression of netrin-1 may inhibit basal cell migration into tissues and that its down-regulation with the onset of sepsis/inflammation may facilitate leukocyte recruitment.
Figures
Fig. 1.
Netrin-1 tissue expression and cellular localization. (a) Netrin-1 mRNA expression in mouse tissues was quantified by quantitative real-time RT-PCR. Each sample was analyzed in triplicate, and the amount of netrin-1 was calculated from a standard curve of known template. Results represent the average of three samples, normalized to GAPDH. (b) Immunofluorescent staining of the lung demonstrates high netrin-1 (green) expression in this tissue, particularly in the endothelium of large blood vessels and capillaries. Colocalization of netrin-1 (green, Center) with the endothelial cell marker PECAM-1 (red, Center) is seen in the merged image (yellow). Primary antibodies were omitted in control staining. (c) Immunoperoxidase staining for netrin-1 in postcapillary venules of the small intestine. Serial sections of the small intestine were stained with silver stain to show venules (V) and arterioles (A) (Top), anti-netrin-1 antibody (Middle), and an isotype control antibody (Bottom). Netrin-1 (red, arrows) appeared to be expressed on the luminal surface of postcapillary venules and, to a lesser extent, by arteriole endothelial cells. No staining was detected when netrin-1 was replaced with an isotype control antibody (Bottom).
Fig. 2.
Modulation of netrin-1 expression in the lung during infection. (a) Expression of netrin-1, IFN-γ, and TNF-α mRNA in the lung was measured by quantitative real-time RT-PCR in mice infected with S. aureus. Netrin-1 is rapidly down-regulated. Regulation of netrin-1 mRNA showed an inverse relationship to the expression of the inflammatory cytokines IFN-γ and TNF-α. (_b_-c) Treatment of human umbilical vein epithelial cells with 10 ng/ml TNF-α or IFN-γ for 6 h reduced netrin-1 expression. (b) Netrin-1 mRNA was measured by quantitative RT-PCR and normalized to β-actin. (c) Netrin-1 protein was detected by Western blotting and normalized to levels of tubulin.
Fig. 3.
Leukocytes express the netrin-1 receptor UNC5b but not DCC. Immunohistochemical staining demonstrates robust expression of UNC5b (green, Top) on the cell surface of monocytes, lymphocytes, and granulocytes that colocalizes with the leukocyte cell-surface marker CD45 (red). By contrast, DCC (green, Middle) expression is not detected on CD45-positive leukocytes. UNC5b- and DCC-transfected 293-T cells served as positive controls and show high expression of these receptors (Bottom). Negative controls included replacing primary antibodies with rabbit (UNC5b) or goat (DCC) IgG. Nuclei were stained with Hoechst 33342 stain (blue).
Fig. 4.
Netrin-1 inhibits active migration of monocytes, granulocytes, and lymphocytes in vitro. (a) Netrin-1 dose-dependently inhibits monocyte migration in response to 10 nM fMLP. Control represents monocyte migration in the absence of fMLP and netrin-1 (*, P < 0.05). (b) Netrin-1 inhibits fMLP-induced monocyte migration when present in either the lower or upper chamber, or both, in a Boyden migration plate (*, P < 0.05). Monocytes were placed in the top chamber, and migration to 10 nM fMLP in the lower chamber was measured in the presence and absence of netrin-1 (500 ng/ml) in the lower chamber or both. Netrin-1 alone did not alter baseline cell migration. (c) Netrin-1 pretreatment inhibits monocyte migration to fMLP. Monocytes were untreated (filled bars) or pretreated with netrin-1 (500 ng/ml) for 90 min and washed extensively (open bars), and migration to 10 nM fMLP was measured. Both netrin-1 pretreatment of cells and the presence of netrin-1 in the lower chamber inhibited monocyte migration to fMLP to a similar extent (*, P = 0.002). (d) Treatment of monocytes with anti-UNC5b antibody, but not control antibody, dose-dependently reverses the inhibitory effect of netrin-1 on monocyte migration (*, P < 0.05).
Fig. 5.
Netrin-1 is a broad inhibitor of leukocyte chemotaxis. (a and b) Netrin-1 inhibits both IL-8 (50 ng/ml)-induced granulocyte migration and stromal cell-derived factor 1α (10 ng/ml)-induced lymphocyte migration when placed in the lower well of a Boyden chamber (*, P ≤ 0.005). (c) Netrin-1 does not significantly affect superoxide production by neutrophils treated with 10 nM fMLP (*, P < 0.05 significantly different from control).
Fig. 6.
Netrin-1 inhibits leukocyte recruitment in vivo in a model of mouse peritonitis. (a) Mice were injected i.p. with 1 ml of PBS/1% BSA, fMLP (10 nM), netrin-1 (500 ng), or both fMLP plus netrin-1, and recruited leukocytes were harvested. fMLP induced a 2-fold increase in leukocytes in the peritoneum that was abrogated in the presence of netrin-1 (*, P < 0.005). Injection of netrin-1 alone was similar to that of the PBS control (data not shown). (b) Analysis of the leukocyte subpopulations in the peritoneal lavage revealed that 4 h after fMLP injection, a predominance of granulocytes was recruited. Netrin-1 inhibited the recruitment of all leukocyte subpopulations.
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