Autoperfused Micro Flow Chamber Reveals Functional Upregulation of VLA–4 on Circulating Leukocytes During Endotoxin–Induced Uveitis

Abstract

Purpose: : To investigate functional upregulation of specific leukocyte adhesion molecules, such as the Very Late Antigen 4 (VLA–4), during endotoxin–induced uveitis (EIU) using our recently introduced Autoperfused Micro Flow Chamber system.

Methods: : To construct the micro flow chamber, microslides (0.4 x 0.04mm in dimension) were coated with recombinant P–selectin (5µg/ml), vascular cell adhesion molecule–1 (VCAM–1) (5µg/ml) or both molecules (5µg/ml each). The microslides were connected to biocompatible polyester tubing at both ends and the tubing ends were cannulated into the right carotid artery and the left jugular vein of EIU and control rats under anesthesia. EIU was induced 24h prior to the experiments by injection of 100µg of LPS into the footpad of Lewis rats. During the circulation of the animal’s native blood cells through the chamber, leukocyte interaction with the immobilized proteins was visualized and recorded using an upright intravital microscope. After 5 minutes of blood flow through the chamber, the microslides were removed and gently perfused with saline to remove red blood cells and unbound leukocytes. Leukocytes adhering firmly to the chamber surfaces were quantified and compared between EIU animals and normal controls (n=5 each).

Results: : Firm leukocyte adhesion to immobilized P–selectin was similar between EIU and control animals (43.3±9.7/mm² and 37.2±15.5/mm², respectively, p=0.37). However, leukocyte adhesion to VCAM–1 was significantly higher in EIU animals compared to controls (13.1±5.3/mm² and 2.3±0.8/mm², respectively, p=0.04). Furthermore, in the P–selectin and VCAM–1 double–coated chambers, firm leukocyte adhesion was dramatically increased in EIU rats compared with normal controls (342.8±44/mm² and 137±41.3/mm², respectively, p=0.001).

Conclusions: : The increased adhesion of EIU leukocytes to immobilized VCAM–1 under flow conditions suggests an important role for the leukocyte integrin VLA–4, the known binding partner of VCAM–1, during this process. Furthermore, the increased adhesion of EIU leukocytes on P–selectin and VCAM–1 doubly coated chambers suggests an important synergistic effect between these molecules during uveitis. These data provide novel evidence for a functional upregulation of VLA–4 during EIU. The VLA–4 interaction with VCAM–1 may be an important target for limiting leukocyte recruitment during uveitis.

Keywords: uveitis-clinical/animal model • inflammation