title: Netrin-1 Reduces Monocyte and Macrophage Chemotaxis towards the Complement Component C5a.
abstract: Netrin-1, acting at its cognate receptor UNC5b, has been previously demonstrated to inhibit CC chemokine-induced immune cell migration. In line with this, we found that netrin-1 was able to inhibit CCL2-induced migration of bone marrow derived macrophages (BMDMs). However, whether netrin-1 is capable of inhibiting chemotaxis to a broader range of chemoattractants remains largely unexplored. As our initial experiments demonstrated that RAW264.7 and BMDMs expressed high levels of C5a receptor 1 (C5aR1) on their surface, we aimed to determine the effect of netrin-1 exposure on monocyte/macrophage cell migration induced by C5a, a complement peptide that plays a major role in multiple inflammatory pathologies. Treatment of RAW264.7 macrophages, BMDMs and human monocytes with netrin-1 inhibited their chemotaxis towards C5a, as measured using two different real-time methods. This inhibitory effect was found to be dependent on netrin-1 receptor signalling, as an UNC5b blocking antibody was able to reverse netrin-1 inhibition of C5a induced BMDM migration. Treatment of BMDMs with netrin-1 had no effect on C5aR1 proximal signalling events, as surface C5aR1 expression, internalisation and intracellular Ca2+ release following C5aR1 ligation remained unaffected after netrin-1 exposure. We next examined receptor distal events that occur following C5aR1 activation, but found that netrin-1 was unable to inhibit C5a induced phosphorylation of ERK1/2, Akt and p38, pathways important for cellular migration. Furthermore, netrin-1 treatment had no effect on BMDM cytoskeletal rearrangement following C5a stimulation as determined by microscopy and real-time electrical impedance sensing. Taken together these data highlight that netrin-1 inhibits monocyte and macrophage cell migration, but that the mechanism behind this effect remains unresolved. Nevertheless, netrin-1 and its cognate receptors warrant further investigation as they may represent a potential avenue for the development of novel anti-inflammatory therapeutics.
authors: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
authors: Lewis
authors: http://orcid.org/0000-0003-4622-9890
authors: L
authors: Taylor
authors: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
authors: Maximillian Hugo
authors: MH
authors: Brodermann
authors: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
authors: David
authors: D
authors: McCaffary
authors: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
authors: Asif Jilani
authors: AJ
authors: Iqbal
authors: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
authors: David R
authors: DR
authors: Greaves
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D018932:Chemokine CCL2 / Q000235:genetics / Q000378:metabolism; D002633:Chemotaxis / Q000187:drug effects / Q000502:physiology*; D015936:Complement C5a / Q000378:metabolism*; D006801:Humans; D007155:Immunologic Factors / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000502:physiology*; D008297:Male; D008810:Mice, Inbred C57BL; D009000:Monocytes / Q000187:drug effects / Q000502:physiology; D009414:Nerve Growth Factors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D000075388:Netrin-1; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D044087:Receptor, Anaphylatoxin C5a / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*
chemical_list: C000591759:C5ar1 protein, mouse; C491768:Ccl2 protein, mouse; D018932:Chemokine CCL2; D007155:Immunologic Factors; C000617351:NTN1 protein, human; D009414:Nerve Growth Factors; C000617353:Ntn1 protein, mouse; D044087:Receptor, Anaphylatoxin C5a; D025521:Tumor Suppressor Proteins; D000075388:Netrin-1; D015936:Complement C5a; D019869:Phosphatidylinositol 3-Kinases; D002118:Calcium