Therapy with Nonglycosaminoglycan‐Binding Mutant CCL7: A Novel Strategy to Limit Allograft Inflammation

Abstract

Chemokines are immobilized by binding to glycosaminoglycans (GAGs). A non-GAG-binding mutant CCL7 (mtCCL7) was developed that retained its affinity for chemokine receptors. This mtCCL7 induced leukocyte chemotaxis in diffusion gradients but did not stimulate trans-endothelial migration (p < 0.01). Unlike wild-type CCL7, mtCCL7 persisted in the circulation of BALB/c mice for more than 6 h and prevented leukocyte infiltration of skin isografts (p < 0.05). Treatment with mtCCL7 marginally increased the survival of C57BL/6 to BALB/c skin allografts and reduced graft infiltration by CD3 + cells (p < 0.05). Importantly, mtCCL7 promoted long-term (>40 day) graft survival following minor histocompatibility (HY) antigen mismatched C57BL/6 skin transplantation; control grafts were rejected by day 24. Treatment with mtCCL7 produced a significant decrease in the frequency of IFN-c producing donor-reactive splenic T cells, reduced CCR2 expression by circulating leukocytes for 6 h (p < 0.01) and blocked the normal increase in affinity of a 4b 1 integrins for VCAM-1 following transient chemokine stimulation. These data suggest that mtCCL7 persists in the circulation and reduces both specific T-cell priming and the capacity of circulating immune cells to respond to GAG-bound chemokine at sites of developing inflammation.

CCL2 nitration is a negative regulator of chemokine-mediated inflammation

“…It was found that unlike WT-CCL2, the NO 2 -CCL2 was not capable of inducing transendothelial leukocyte migration in vitro . A similar failure of cell migration has been reported previously for leukocyte transmigration assays performed using non-GAG binding chemokines152930.…”

Chemokine Oligomerization in Cell Signaling and Migration

“…That these interactions are important has been convincingly demonstrated by several studies which showed that chemokine mutants devoid of their ability to oligomerize or interact with GAGS are functionally deficient in vivo. 22,45,96 …”

Chemokines: established and novel targets in atherosclerosis

“…This is exemplified by a mutation of proline 8 into alanine in CCL2, which resulted in an obligate monomeric form that retained in vitro activity towards CCR2 but nevertheless acted as an antagonist in vivo , possibly by displacing endogenous CCL2 from GAGs on the vessel wall (Handel et al, 2008). Likewise, mutation of the GAG‐binding sites in CCL5, CCL7 and CXCL12 yielded chemokine mutants that were competent in receptor activation in vitro but possessed anti‐inflammatory dominant‐negative properties in animal models of inflammation, including atherosclerosis (Ali et al, 2010; Braunersreuther et al, 2008; O'Boyle et al, 2009). It is intriguing how chemokine mutants with reduced oligomerization or binding to GAGs might nevertheless act as inhibitors for their wild‐type counterparts.…”