The Regulatory Role of CD99L2 in Inflammation

The inflammatory response involves a series of steps in which inflammatory stimuli activate endothelium of local blood vessels to express adhesion molecules that recruit circulating leukocytes from the blood. Transendothelial Migration (TEM) is a crucial step in the inflammatory response as it is the process in which leukocytes leave the bloodstream to enter the inflamed tissues. PECAM and CD99 are two molecules important for the initiation and completion of TEM, respectively. CD99-like 2 (L2) is a highly glycosylated 52 kD type-1 membrane protein that is the only molecule in the genome related to CD99. Similar to CD99, L2 is expressed at the borders of endothelial cells and on the surface of leukocytes. Blocking L2 using antibodies significantly reduces the recruitment of neutrophils and monocytes to sites of inflammation in mice. Similarly, L2 knockout mice have an inherent defect in transmigration into inflammatory sites. Thus, L2 has been shown to be another important protein for TEM; however, how it regulates this process is not known. L2 is different enough from CD99 (60% larger and with only 38% sequence identity) that it is not clear whether it regulates TEM by the same mechanism as CD99. Furthermore, all of the published studies on L2 have been performed in mice. The role of L2 in TEM of human cells has yet to be shown. In order to study the mechanism used by L2 and the relevance to human inflammation, we studied the role of L2 on human cells in vitro. Using an endpoint TEM assay, we show that inhibiting L2 function using either a blocking antibody or shRNA reduces TEM of human monocytes to 30% of control levels. Like PECAM and CD99, homophilic interactions between L2 on leukocytes and L2 on the endothelium are required for this process. Previous studies have shown that PECAM regulates a step in TEM that is “upstream” of the one regulated by CD99. In order to determine where L2 functions in relation to PECAM and CD99, we used an extended version of our TEM assay, the sequential block assay. Our data show that L2 functions downstream of PECAM and upstream of CD99. Preliminary data suggest that L2 can promote TEM by engaging either the signaling pathway used by PECAM or the one used by CD99. These data are consistent with L2 functioning between the PECAM and CD99. The data we have obtained in both humans and mice identify L2 as a potential target for anti-inflammatory therapy. Supported by grants from the NIH: T32-5T32AI007476-19, R37-HL064774, and F31-HL131355-01