Nerve Growth Factor Induces Migration of Murine Monocytes Into the Cornea

Abstract

Purpose: : Previously we showed that NGF stimulated murine monocyte migration in vitro. However our cell population was only 60% pure based on flow cytometry. Here we confirmed this observation with highly purified cells and examined the effect of NGF on migration of monocyte lineage cells in to the cornea in vivo.

Methods: : Monocytes were purified from peripheral blood with a continuous Percoll gradient (67%, 58%, 53%, 50%), and negative immunomagnetic selection against B220, CD4 and CD8. Purity was determined by flow cytometry for CD3 and CD115. The effect of NGF (0.5 - 200ng/ml) on migration of monocytes in vitro was tested using blind-well chambers. NGF (2µg/ml, 2µl) or PBS was injected intrastromally into corneas of C57BL/6 mice. The percentage of CD11c, Ly6C, and CD115 positive cells in the epithelium and in the stroma was determined by flow cytometry 4 hours after injection.

Results: : With the current isolation method, 92% of the isolated cells were CD115+CD3-, i.e. monocyte lineage cells. In vitro NGF induced concentration-dependent migration of these monocytes, with the maximum effect at 50ng/ml (146.1±26.3 cells/4 fields), which was significantly (t-test p<0.01, n=3) higher than untreated control (4.3±1.5 cells/4 fields). With NGF application in vivo, the percentage of monocyte lineage cells(7% CD11c+, CD115+, Ly6C^low and 1.3% CD11c+, CD115+, Ly6C^high ) in the corneal epithelium (n=2) increased compared with the PBS control (2.4% CD11c+, CD115+, Ly6C^low and 0.6% CD11c+, CD115+, Ly6C^high). Similarly in the stroma (n=2), the percentage of monocyte lineage cells was higher with NGF(4.8% CD11c+, CD115+, Ly6C^low and 4.0% CD11c+, CD115+, Ly6C^high ), than with PBS (1.1% CD11c+, CD115+, Ly6C^low and 1.8% CD11c+, CD115+, Ly6C^high ).

Conclusions: : The present study using highly purified cells confirmed that NGF stimulated monocyte migration. Further, NGF induced migration of monocyte lineage cells into the cornea in vivo. The results indicate that NGF may facilitate the recruitment of monocyte lineage cells and modulate immune responses initiated by those cells in the cornea.

Keywords: cornea: basic science • flow cytometry • immunomodulation/immunoregulation