CD99-like 2 (CD99L2)-deficient mice are defective in the acute inflammatory response

Abstract

CD99-Like 2 (CD99L2) is a Type I glycoprotein expressed on leukocytes and endothelial cells as well as other cell types. It is related to CD99, although it shows only 38% sequence identity. CD99L2 has been shown to play a role in leukocyte extravasation in mice under various inflammatory conditions using anti-CD99L2 antibodies and, in one case by targeted deletion of CD99L2. We report here studies on an independently made CD99L2 "knockout mouse" that extend our knowledge of the role of CD99L2 in inflammation. CD99L2 deficiency did not affect the total or relative numbers of circulating leukocyte subsets, red blood cells, or platelets. Neither did CD99L2 deficiency affect the expression of ICAM-1, PECAM, or CD99 on endothelial cells. Mice lacking CD99L2 had a defective inflammatory response in the thioglycollate peritonitis model with a greater than 80% block in neutrophil infiltration and a nearly complete block in monocyte emigration into the peritoneal cavity measured 16h after the inflammatory challenge. The mice will be a useful resource to study the role of CD99L2 in various acute and chronic inflammatory diseases.

Keywords: Adhesion molecule; CD99L2; Endothelium; Inflammation; Knockout mouse; Leukocyte.

Figure 1. Targeting scheme for and verification of deletion of CD99L2 by homologous recombination

See Materials and Methods for details. A. Two forward primers and a common reverse primer were designed for a multiplex PCR system. Black boxes with numbers denote exons. PGK-NEO cassette in the inverse orientation is shown in the knockout allele. B. PCR results from wild-type, heterozygous, and CD99L2-deficient mice. Numbers at the right indicate the molecular size in base pairs.

Figure 2. Expression of relevant endothelial cell adhesion molecules by CD99L2-deficient mice

Frozen sections of mouse kidney from wild-type (WT) or CD99-deficient (KO) mice were incubated with antibodies against ICAM-1, PECAM-1, CD99L2 (L2), or CD99 then with HRP-conjugated secondary antibody or Secondary antibody alone (negative control). After development of peroxidase reaction product, slides were counterstained with hematoxylin. Brown reaction product is seen on endothelial cells of all levels: arterioles (a), glomerular capillaries (g), venules (v), and peritubular capillaries. Scale bar = 80 μm.

Figure 3. Expression of PECAM and CD99 is similar on leukocytes from wild-type and CD99L2^−/− mice

Flow cytometric profiles of PECAM and CD99 on leukocytes gated by forward and side scatter as well as cell-specific markers. At least 100,000 events were recorded per sample. Details of the full surface marker panel can be found in Materials and Methods. Black histogram = unstained negative control; blue histogram = wild-type; red histogram = CD99L2^−/−.

Figure 4. CD99L2^−/− mice have defective acute inflammatory response

Age-and sex-matched wild-type (WT) or CD99L2 knockout (L2 KO) mice were left untreated for baseline measurements (− THIO) or received an intraperitoneal injection of thioglycollate (+ THIO) 16 hours prior to sacrifice and harvest of peritoneal cells. Very few PMN were present in the unstimulated peritoneal cavity of unstimulated mice. Black bars = PMN; grey bars = Monocytes. Mean ± SEM of 3 independent experiments *** p<0.001; **** p< 0.0001 by two-way ANOVA.