The pan-chemokine inhibitor NR58-3.14.3 abolishes tumour necrosis factor-alpha accumulation and leucocyte recruitment induced by lipopolysaccharide in vivo

Chemokines participate in the regulation of leucocyte recruitment in a wide variety of inflammatory processes, including host defence and diseases suc h as asthma, atherosclerosis and autoimmune disorders. We have previously d escribed the properties of Peptide 3, the first broad-specificity chemokine inhibitor in vitro. Here, we report the properties of NR58-3.14.3, a retro inverso analogue of Peptide 3. NR58-3.14.3 inhibited leucocyte migration in duced by a range of chemokines, including monocyte chemoattractant protein- 1 (MCP-1) (2.5 nM), macrophage inflammatory protein-1 alpha (MIP-1 alpha) ( 5 nM), regulated on activation, normal T-cell expressed and presumably secr eted (RANTES) (20 nM), stromal cell-derived factor-1 alpha (SDF-1 alpha) (2 5 nM) and interleukin-8 (IL-8) (30 nM), but did not affect migration induce d by N-formyl-methionyl-leucyl-phenylalanine (FMLP) or complement C5a (>100 muM). NR58-3.14.3 is therefore approximate to 1000-fold more potent than P eptide 3 but retains the broad-spectrum chemokine inhibitory activity of th e parent peptide. In vivo, pretreatment with a systemic dose of 10 mg of NR 58-3.14.3, but not the inactive derivative NR58-3.14.4. abolished leucocyte recruitment in response to intradermal injection of 500 ng of MCP-1 into r at skin. This suggests that NR58-3.14.3 is a functional chemokine inhibitor in vivo as well as in vitro. We utilized NR58-3.14.3 as a tool to investig ate the role of chemokine activity during leucocyte recruitment in response to lipopolysaccharide (LPS) in vivo. NR58-3.14.3, but not NR58-3.14.4. abo lished leucocyte recruitment in response to intradermal injection of 50 ng of LPS into rat skin. Furthermore, NR58-3.14.3 completely inhibited LPS-ind uced accumulation of tumour necrosis factor-alpha (TNF-alpha). This data is consistent with a model in which multiple chemokines act in parallel upstr eam of TNF-alpha. NR58-3.14.3 is therefore a powerful anti-inflammatory age nt in vivo, suppressing proinflammatory cytokine production and leucocyte r ecruitment in response to endotoxin stimulus in rat skin.