Lactoferrin Expression in Human and Murine Ocular Tissue

Abstract

Purpose: Lactoferrin (LF) is a multifunctional protein known to provide innate defense due to its antimicrobial and anti-inflammatory properties. In the eye, LF has been identified in the tears and vitreous humor. Its presence in other ocular tissues has not been determined. Our aim is to assess the presence of LF in the cornea, iris, retina and retinal pigment epithelium (RPE) of humans and mice.

Methods: To test for the endogenous production of LF, reverse transcription polymerase chain reaction was performed in cultured human cells from the cornea and RPE and in murine tissues. To confirm LF localization in specific ocular tissue, immunohistochemistry was performed on flat mounts of cornea, retina and RPE in human donor eyes. The presence of LF was assessed by western blotting in human and mouse ocular tissue and human culture cells (cornea and RPE). To verify antibody specificity, purified human LF and transferrin (TF) were used on 1D and 2D western blots.

Results: LF gene expression was confirmed in the cornea and RPE cell cultures from humans, suggesting that LF is an endogenously produced protein. PCR results from mouse ocular tissue showed LF expression in cornea, iris, RPE, but not in retina. These results were also consistent with immunohistochemical localization of LF in human donor tissue. Antibody reaction for human LF was specific and western blotting showed its presence in the cornea, iris and RPE tissues. A faint reaction for the retina was observed but was likely due to contamination from other ocular tissues. Multiple commercially available antibodies for murine LF cross-reacted with TF, so no reliable results were obtained for murine western blot.

Conclusion: LF is expressed in multiple eye tissues of humans and mice. This widespread expression and multifunctional activity of LF suggests that it may play an important role in protecting eye tissues from inflammation-associated diseases.

Keywords: Cornea; iris; lactoferrin; retina; retinal pigment epithelium; transferrin.

Figure 1

Expression of LF in murine tissue and human cultured cells. Image is an agarose gel resolving PCR products corresponding to LF that were amplified from the RPE, iris and cornea of C57/BL6J mice (879 bp). Human cultured cells from the RPE and corneal epithelium also produced a PCR product for LF (600 bp). Cornea E., corneal epithelium, NTC, no template control; bp, 1 kb base pair ladder.

Figure 2

Immunohistochemistry of flat mounts of cornea, RPE and retina from a human donor eye. (A) LF (ab166803) staining (green) in human cornea with DAPI-stained nuclei (blue). Arrows point to cells with nuclear and cytoplasmic staining. Box shows enlarged image of cells. (B) LF staining in human RPE. (C) LF staining in human retinal tissue counterstained with DAPI to mark nuclei. There is a positive LF staining present only in cells within blood vessels. (D) Secondary only staining of corneal epithelium cells. (E) Secondary only staining of RPE cells. Background fluorescence is caused by the lipofuscin autofluorescence. (F) Secondary only staining of retina containing a retinal blood vessel. *Green, LF; blue, DAPI; arrow, blood cells in C and F.

Figure 3

Western blot of murine and human ocular tissue. (A) Human eye tissue (retina, RPE, iris, corneal epithelium and endothelium) were probed with anti-LF antibody (ab38155). Mouse retina was included to check cross reactivity with this antibody. HLF and HTF were positive and negative controls, respectively. *H, human, M, mouse; RET, retina; RPE, Retinal pigmented epithelium, IRI, iris; COE, corneal epithelium; CON, corneal endothelium; LF, lactoferrin; TF, transferrin. (B) C57/BL6J mice eye tissue processed for protein and analyzed using anti-Lactoferrin antibody (ab77705). Human purified lactoferrin (HLF) and transferrin (HTF) were included as positive and negative controls, respectively.

Figure 4

2D gel separations of purified LF and TF. Purified proteins (LF and TF) were resolved by isoelectric focusing on an immobilized strip with a nonlinear pH gradient (3–10) in the first dimension, followed by SDS–PAGE (12%) in the second dimension. Top: Image is the Coomasie blue stained gel. Middle: PVDF membrane stained with the anti-TF antibody (ab82411) reacts with the TF protein (pI 6.8) but not LF. Bottom: PVDF membrane stained with anti-LF antibody (ab77705) reacts with LF protein (pI 8.7) but not TF. Arrows show a cross reaction with both anti-TF (B) and anti-LF (C) for an acidic protein. Ladder at the bottom of the figure shows the nonlinear pH gradient for proteins separation in the first dimensions. MW, Molecular weight marker. Numbers indicate protein size.