Neutrophils protect the retina of the injected eye from infection after anterior chamber inoculation of HSV-1 in BALB/c mice

Abstract

Purpose: To determine whether infiltrating polymorphonuclear leukocytes PMNs play a role in preventing early direct anterior-to-posterior spread of herpes simplex virus (HSV)-1 and/or in preventing the spread of HSV-1 from the brain back to the retina of the injected eye after anterior chamber (AC) inoculation.

Methods: BALB/c mice were treated with monoclonal antibody RB6-8C5 (Gr-1) against PMNs or control IgG and inoculated with HSV-1.

Results: In Gr-1-treated mice, PMNs were depleted in the peripheral blood and in the HSV-1-infected eye. More virus (2-3 logs) was recovered from the inoculated eye of Gr-1 antibody-treated mice than from control mice. Immunohistochemistry revealed disseminated virus-infected cells in the junction between the anterior and the posterior segment and also in the posterior segment of the HSV-1-inoculated eye in Gr-1-treated mice. In control IgG-treated mice, virus-infected cells were observed only within the AC. More virus (3 logs) was recovered from the contralateral suprachiasmatic nucleus (SCN), and increased virus staining was observed in the ipsilateral optic nerve of Gr-1-treated mice compared with control mice. In Gr-1-treated mice, the central retina was virus-infected in a patchy fashion beginning on day 7 post infection (pi), and the infection progressed to involve the entire retina.

Conclusions: Since both direct anterior-to-posterior spread of virus and spread via the optic nerve occurred in PMN-depleted mice, these results suggest that PMNs play an important role both in limiting intraocular spread of virus in the injected eye and in controlling spread of the virus from the brain into the optic nerve and retina of the injected eye.

Figure 1

Photomicrographs illustrating infiltration of FITC–Gr-1⁺ cells in the cornea (C), anterior chamber (AC), iris (I), and ciliary body (CB) of the HSV-1–inoculated eyes of mice treated with Gr-1 antibody or control antibody at days 3, 5, and 8 pi. Gr-1 staining of the eyes of (A, E, I) Gr-1 antibody– and (C, G, K) control antibody–treated mice. Merged images of Gr-1 staining and DAPI staining of the eyes of (B, F, J) Gr-1 antibody– and (D, H, L) control antibody–treated mice. Original magnification: ×200.

Figure 2

Flow cytometry results showing percentages of FITC–Gr-1⁺ neutrophils in the peripheral blood of Gr-1 antibody– (A) and control antibody–treated (B) mice at day 5 pi. Efficacy of systemic depletion of Gr-1⁺ neutrophils in Gr-1 antibody– and control antibody–treated mice at day 5 pi (C). The percentages of FITC–Gr-1⁺ neutrophils in cells derived from the whole eyes of HSV-1–inoculated Gr-1 antibody– (D) and control antibody–treated (E) mice at day 5 pi. The efficacy of depletion of Gr-1⁺ neutrophils in the eye-derived cells of Gr-1 antibody– and control antibody–treated mice at day 5 pi (F).

Figure 3

Photomicrographs showing that neutrophil depletion correlated with increased HSV-1 infection of the parenchyma of the anterior segment of the inoculated eye at day 5 pi. (A–D) In Gr-1–treated mice, HSV-1–infected cells were observed at day 5 pi in the ciliary body, in RPE cells and in some peripheral retinal cells; a few Gr-1⁺ neutrophils were also seen. (E–H) Diffuse HSV-1 infection of the ciliary body and sclera with massive infiltration of Gr-1⁺ neutrophils was observed in the injected eyes of control antibody–treated mice. In control mice, most of the Gr-1⁺ cells were also HSV-1⁺ (compare D with H). (A, E) HSV-1 staining; (B, F) Gr-1 staining; (C, G) DAPI; (D, H) merged images. S, sclera; J, junction of ciliary body and pars plana.

Figure 4

(A–D) Photomicrographs showing that neutrophil depletion correlated with fulminant HSV-1 infection of the posterior retina; (E–H) retinas of control antibody–treated mice shown for comparison. (A, E) HSV-1 staining; (B, F) Gr-1 staining; (C, G) DAPI; (D, H) merged images.

Figure 5

HSV-1–inoculated eyes were removed from control antibody– and Gr-1–treated mice at days 3, 5, and 8 pi. The eyes were homogenized individually, and the titer of virus was determined by plaque assay on Vero cells. Each value is the mean ± SD of infectious virus recovered from three injected eyes.

Figure 6

(A–D) Photomicrographs demonstrating that neutrophil depletion correlated with HSV-1 infection of the ipsilateral optic nerve at day 8 pi; (E–H) ipsilateral optic nerves of control antibody–treated mice shown for comparison. (A, E) HSV-1 staining; (B, F) Gr-1 staining; (C, G) DAPI; (D, H) merged images.

Figure 7

Replication of HSV-1 in cultures of neutrophils, monocytes and Vero cells at 0, 2, 4, 8, 12, 21, and 36 hours after exposure to HSV-1 at an MOI of 5 pfu/cell. Virus titers (mean ± SD) were determined by plaque assay on Vero cells.