CCR1- and CCR5-mediated inactivation of leukocytes by a nonglycosaminoglycan (non-GAG)-binding variant of n-nonanoyl-CCL14 (NNY-CCL14).
Intervention on chemokine receptors to prevent directional leukocyte migration is a potential therapeutic strategy. NNY-CCL14 is a CD26-resistant lead molecule, which exerts its effects on multiple chemokine receptors (CCR1, CCR2, CCR3, and CCR5). The inhibitory effects of NNY-CCL14 in murine models of allergic airway inflammation have been assigned to its interaction with CCR1 and CCR5. In this study, a non-GAG-binding variant of NNY-CCL14 was generated by mutating basic amino acids within the identified GAG-binding 49BBXB52 motif. This CD26-resistant, non-GAG binding variant, NNY-CCL14(G,A), does not promote CCR1-dependent cell arrest on modeled endothelium. Its biological activity tested on human and murine chemokine receptors revealed distinguishing properties to NNY-CCL14. As suggested by EC50 values for intracellular calcium mobilization, NNY-CCL14(G,A) demonstrated a reduced ability to activate hCCR1, but internalization and desensitization of hCCR1 were unperturbed. Surprisingly, its activity on hCCR3 was strongly reduced, and it did not internalize mCCR3. A significantly reduced chemotactic activity of eosinophils and monocytes was observed. All biological effects mediated by NNY-CCL14(G,A) via hCCR5 and mCCR5 showed no difference to NNY-CCL14. In mice treated i.v. with NNY-CCL14(G,A), a sustained in vivo down-modulation of CCR5 was achieved over 3 h. Therefore, NNY-CCL14(G,A) inactivates leukocytes by desensitizing and internalizing multiple chemokine receptors, thus rendering them unresponsive to further stimulation by natural ligands. When administered systemically, NNY-CCL14(G,A) may modulate leukocyte functions prior to their interaction with other endothelium-bound chemokines expressed under pathophysiological conditions, such as allergic inflammation.
Gupta
Shipra
S
Center of Pharmacology and Toxicology, Clinic of Immunology and Rheumatology, Hannover Medical School, Feodor-Lynen-Strasse 31, 30625 Hannover, Germany. sgupta3@bidmc.harvard.edu
Rieder
Sebastian
S
Richter
Rudolf
R
Schulz-Maronde
Sandra
S
Manns
Johanna
J
Escher
Sylvia E
SE
Heitland
Aleksandra
A
Mack
Matthias
M
Forssmann
Wolf-Georg
WG
Elsner
Jörn
J
Forssmann
Ulf
U
D000818:Animals; D020013:Calcium Signaling; D019742:Chemokines, CC / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007962:Leukocytes / Q000378:metabolism*; D051379:Mice; D016296:Mutagenesis; D009154:Mutation; D011485:Protein Binding; D054389:Receptors, CCR1 / Q000378:metabolism*; D019713:Receptors, CCR5 / Q000378:metabolism*; D019707:Receptors, Chemokine / Q000378:metabolism
C515301:Ccr1 protein, mouse; D019742:Chemokines, CC; C551922:NNY-CCL14 protein, mouse; D054389:Receptors, CCR1; D019713:Receptors, CCR5; D019707:Receptors, Chemokine