Figure 5.

Macrophages are not involved in HSV-1–induced lymphangiogenesis. (A) Mice expressing GFP under the proximal human VEGF-A promoter were infected with HSV-1 and examined for expression of GFP and the pan-leukocyte marker CD45. No colocalization between GFP reporter and CD45 was observed, despite the observation of numerous CD45 cells as well as GFP⁺ cells, indicating that leukocytes, including macrophages, are not an appreciable source of VEGF-A during HSV-1 infection. Shown is an image of a cornea at day 5 PI representative of two experiments. n = 6. The images were acquired with a 400× objective. Bars, 50 µm. (B) Macrophages were depleted by subconjunctival injection with either clodronate or control PBS containing liposomes. At day 5 PI, corneal F4/80⁺ macrophages were quantitated by flow cytometry in control and clodronate-treated groups. Clodronate treatment significantly reduced the number of corneal F480⁺ cells (*, P < 0.05). (C) Corneas were examined for expression of the lymphatic vessel antigen LYVE-1 and quantified as a measure of lymphatic vessel area. No significant differences in lymphatic vessel area or structure (not depicted) were observed between control and clodronate-treated groups. B and C are summaries of two experiments. Error bars represent the SEM based on the results of each cornea sample summarized for both experiments. n = 6. (D) Chimeric mice expressing GFP in bone marrow–derived cells were infected with HSV-1. Corneas were examined for expression of GFP (green) and LYVE-1 (red) to detect possible structural contributions from bone marrow–derived cells. However, no appreciable structural contribution was observed. Data are representative of two experiments. n = 8 animals. Bars, 100 µm.