Induction of angiogenesis and inflammatory cell influx by purified heterophils. (A) Collagen onplants supplemented with buffer alone (control) or 5 × 104 heterophils isolated from peripheral blood were engrafted on the CAM of 10-day-old embryos incubated ex ovo. Onplants were harvested and fixed in formalin or frozen in OCT compound at 72 hours after the angiogenic response was scored. Paraffin sections stained with hematoxylin and eosin (H&E) indicate similar tissue composition of the onplants (top panels). Arrows point to some of newly formed angiogenic vessels visualized more frequently, especially between the grids, in the onplants supplemented with heterophils. Cryosections were immunohistochemically stained with chMMP-9–specific antibody to visualize heterophils (arrows, middle panels). In addition, paraffin sections were immunohistochemically stained with anti–chMMP-13 antibody identifying brown rounded cells of monocyte/macrophage lineage (bottom panels). (B) Control onplants with and without exogenously added heterophils were scored for angiogenesis (top graph; bar indicates mean), heterophil influx (middle graph), and infiltration by monocytes (bottom graph) as described in Figure 7. Data are presented as means ± SEM. One of 3 independently performed experiments is shown. A statistically significant difference was confirmed (P < .01) for the 2 experimental groups depicted in the scatter and bar graphs.
