Globotriaosylceramide (Gb 3/CD77) is synthesized and surface expressed by bovine lymphocytes upon activation in vitro

Abstract

Neutral glycosphingolipids (GSLs) are considered activation markers on human lymphocytes, which are fundamental for studying the immune system. For cattle, only a limited number of activation markers has yet been identified. We recently showed that Shiga toxin 1, known to use globotriaosylceramide (Gb 3 syn. CD77) as a cellular receptor, depresses proliferation of activated bovine lymphocytes [Infect. Immunol. 67 (1999b) 2209]. In order to confirm the expression of Gb 3/CD77 on bovine lymphocytes, we flowcytometrically examined a bovine B-lymphoma cell line (BL-3) and bovine peripheral blood mononuclear cells (PBMC) before and after mitogenic stimulation and biochemically characterized neutral GSLs extracted from PBMC. CD77 was detected on the surface of BL-3 cells and cultured PBMC essentially after mitogenic stimulation. Although expressed by all PBMC subpopulations identified, the portion of CD77 + cells was highest for BoCD8 + cells, followed by B-cells and BoCD4 + cells at day 4 of cultivation. Ceramide trihexoside of stimulated PBMC was structurally determined as Gal(α1-4)Gal(1-4)Glc(1-1)ceramide (Gb 3). Biochemically, Gb 3 was also detected within unstimulated PBMC which contained ceramide monohexoside (CMH) and Gb 3 in a ratio of about 4:1. However, stimulation induced an increase of CMH and Gb 3 by a factor of 2.5 and 10, respectively, implicating that bovine lymphocytes regulate surface expression of Gb 3/CD77 predominantly by quantitative changes in the Gb 3 metabolism. This report presents Gb 3/CD77 as the first GSL identified on bovine immune cells and highly recommends this activation dependent antigen as a useful tool to investigate lymphocyte activation within the bovine immune system.