| Abstract No.: | A-C1103 |
| Country: | Canada |
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| Title: | MECHANISM FOR ANTI-INFLAMMATORY EFFECTS OF ANTI-α4 MONOCLONAL ANTIBODY TREATMENT AFTER SPINAL CORD INJURY INCLUDES INTEGRIN INTERNALIZATION |
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| Authors/Affiliations: | 1 Jennifer Fleming*; 1 Lynee Weaver;
1 University of Western Ontario, Robarts Research Institute, London, ON, Canada
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| Content: | Objectives: Rolling and firm adhesion of leukocytes to endothelial cells is mediated, in part, by an α4β1 integrin/vascular cell adhesion molecule-1 (VCAM1) interaction. Blockade of this interaction with an anti-α4 monoclonal antibody (α4 mAb) after spinal cord injury decreases intraspinal influx of neutrophils and monocytes, thereby reducing inflammation and oxidative damage, and improving neurological recovery. The purpose of this study was to determine the response of neutrophils to α4 mAb binding, and to determine the mechanism responsible for α4 mAb-mediated decreases in neutrophil influx into the injured cord.
Methods and Materials: Neutrophil oxidative and phagocytic activity in response to α4 mAb binding were assessed by flow cytometry using dihydrorhodamine1,2,3 and fluorescent microspheres as probes, respectively. Blood samples assessed for oxidative activity were either untreated, cytokine-primed or cytokine-primed and chemically stimulated in vitro. Neutrophil viability was determined using 7AAD, a cell death marker and AnnexinV, an apoptosis marker. To assess internalisation of the integrin/mAb complex, surface α4 mAb was detected by indirect labelling using a secondary antibody conjugated to AlexaFluor488. Loss of fluorescence connoted internalization of the complex. Lysosomal degradation of the internalized complex was assessed by direct immunolabeling (detecting intracellular and extracellular fluorescence) and concanamycinA, an lysosomal inhibitor. Neutrophil transmigration across TNFα-stimulated rat cerebrovascular endothelial monolayers was assessed after mAb treatment.
Results: Neither the α4 mAb nor the control mAb had any effect on phagocytic activity, or survival. Compared to control mAb treatment, α4 mAb treatment (<1h) had no effect on oxidative activity in resting, primed or activated neutrophils. However, incubation of blood samples with α4 mAb for 1h or 2h caused small increases in oxidative activity in resting neutrophils. To assess internalization, neutrophils were incubated with the α4 mAb at 37°C for 2h, 4h and 6h. AlexaFluor488 fluorescence decreased significantly by ~44%, ~48%, and ~65%, respectively, indicating that the complex was no longer on the surface of the neutrophils. Incubation at 4°C, to prevent endocytosis, verified that the integrin/mAb complex was internalized rather than shed from the surface, as fluorescence intensity did not change with time. When direct immunofluorecent staining was used, fluorescence intensity increased with time, again suggesting that the integrin/mAb complexes were accumulating intracellularly and verifying internalization rather than shedding of the complexes. In this experiment, treatment of neutrophils with concanamycinA had no effect on fluorescence intensity, demonstrating that the complexes were not degraded by the lysosome. Finally when examined in the transwell assay, the α4 mAb treatment decreased neutrophil transendothelial migration by 34.9±0.06% compared to the control mAb treatment.
Conclusions: The reduced intraspinal neutrophil influx in α4 mAb-treated animals after SCI and the reduced transendothelial migration in vitro may be caused, in part, by internalisation of the integrin/mAb complex, making the integrin unavailable for VCAM1 binding. Binding of the mAb to neutrophils activated them after long exposure. Despite this, the mAb treatment leads to significantly improved outcomes. The integrin-induced activation may have minimal overall impact. |
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