CD99 is essential for leukocyte diapedesis in vivo

Abstract

Recruitment of leukocytes into inflamed tissue requires migration of leukocytes from the blood stream across the endothelial lining and the basement membrane of the local blood vessels. CD99 in humans is a 32-kDa highly O-glycosylated cell surface protein expressed on most leukocytes. The authors recently found CD99 to be expressed in leukocytes and at human endothelial cell contacts. Human CD99 is involved in homophilic interaction between the two cell types and participates in the transendothelial migration of monocytes and polymorphonuclear neutrophils (PMNs) in vitro. To test the role of CD99 in vivo, the authors cloned murine CD99 (muCD99), expressed it in vitro, and generated a blocking monoclonal antibody against it. We first showed that muCD99 is expressed on mouse leukocytes as well as enriched at the endothelial cell borders. Transfection of cells with muCD99 imparts on them the ability to aggregate in a CD99-dependent homophilic manner. Cells expressing muCD99 did not bind to cells expressing murine or human platelet endothelial call adhesion molecule (PECAM) or human CD99. In the thioglycollate peritonitis model of inflammation, anti-CD99 monoclonal antibody blocked the recruitment of neutrophils and monocytes by over 40% and 80%, respectively, at 18 h. Microscopy showed that this blocking occurred at the luminal surface of venules. The authors conclude that CD99 plays a major role in the emigration of leukocytes in vivo.

Figure 1. Murine CD99-EGFP is expressed at the cell surface

A) muCD99 is expressed on the plasma membrane of the cell as well as perinuclearly. B) Western blot against EGFP showing native muCD99-EGFP (left lane) and trypsin digested muCD99-EGFP (right lane). A shorter band around 33 kDa represents ~ 1/3 of the muCD99-EGFP that is present on the cell surface and susceptible to extracellular proteolysis. C and D) Flow cytometric analysis of a clone expressing muCD99-EGFP (light line) shows a uniform population expressing EGFP (C), while staining of the same clone with a monoclonal antibody against muCD99 (light line; detected with a PE-labeled secondary antibody) shows uniformity of muCD99 expression (D). In C and D filled histograms represent nontransfected cells and isotype control, respectively.

Figure 2. Murine CD99 is expressed on leukocytes and at endothelial cell borders

A-C) Mouse blood was stained with anti-muCD99 and gated by other fluorescent antibodies into leukocyte subpopulations. Expression of muCD99 on leukocytes gated for subpopulations: A) B-cells (CD19 positive). B) T-cells (CD3 positive). C) Myeloid cells (Ly6 C/G positive cells). D) Cultured murine endothelial cell monolayers were fixed and stained with anti-muCD99 mAb, which was detected with fluorescent secondary antibody. MuCD99 is restricted to cell borders.

Figure 3. Expression of muCD99 imparts on cells the ability to aggregate

A) L-cells expressing muCD99 aggregate with themselves in a time and calcium dependent manner, whereas L-cells transfected with a vector only do not aggregate (see part D). Solid line, with 1 mM Ca ⁺², dashed line, no calcium. B) A monoclonal antibody against muCD99 blocks aggregation by 50% (white bar) compared to control IgG (black bar). The same antibody does not affect aggregation of clones expressing PECAM. C & D) Two clones expressing muCD99-EGFP at different levels (solid line for lower expression, dashed line for higher expression) are shown by FACS (C). The L-cell aggregation is muCD99 concentration dependent (D), since the clone with the higher expression aggregates two times faster in the presence of calcium. High expressor (▼), low expressor (▲), transfected with EGFP vector only (■). The data shown are experiments representative of 3 – 6 independent experiments. Experimental errors were always less than 10% of the absolute value and are omitted for clarity.

Figure 4. L-cell aggregation induced by muCD99 is due to homophilic interactions

A) muCD99 transfectants mixed with an equal number of CFSE-labeled control cells aggregated only with other cells that expressed muCD99. Note only unlabeled cells in aggregates (arrowheads). B) Unlabeled muCD99 expressing cells (arrowheads) were mixed with CFSE-labeled L-cells expressing huPECAM. Both cell types aggregated, but only in a homophilic manner. (C-F) The Y-axis indicates the number of aggregates counted containing the percentage of muCD99 expressing cells (indicated on the X-axis) when muCD99 transfectants are mixed with an equal number of cells expressing empty vector (C), or transfectants expressing huPECAM (D), huCD99 (E), or muPECAM (F). The parabola shaped pattern on D-F is expected if each cell-type aggregates homophilically. Thus, most aggregates have either a very low percentage of muCD99+ cells in them or a very high percentage of muCD99+ cells. See diagram above part D, where filled circles represent muCD99-expressing cells. Data shown are representative of at least three independent experiments.

Figure 5. Anti-muCD99 blocks diapedesis in vivo

FVB mice received anti-muCD99 or control IgG i.v. one hour before they were injected i.p. with PBS or thioglycolate (Thio) and were sacrificed 18h later for peritoneal lavage. Cell count for neutrophils (A) or monocytes/macrophages (B) showed that anti-muCD99 blocked leukocyte emigration. Data are mean ± S.E.M. for three independent experiments and are representative of a total of six independent experiments. * p < 0.01.

Figure 6. The block in diapedesis is at the level of the endothelial wall

Mice were pretreated with control rat IgG (A) or anti-muCD99 (B) and injected with thioglycollate as in Figure 5. Venules from mice pretreated with control rat IgG showed few leukocytes attached the venular wall (A) whereas venules from mice treated with an anti-muCD99 (B) displayed numerous leukocytes apparently interacting with the endothelial cells (arrowheads). C) Percentage of attached leukocytes in each group. D) Percentage of venules with more than one leukocyte attached. Scale bar = 20 μm. In parts C and D control is significantly different from anti-CD99 treated p < 0.02