Ocular surface expression and in vitro activity of antimicrobial peptides

Abstract

Purpose: Human ocular surface epithelia express four antimicrobial peptides (APs): beta -defensin (hBD) 1-3 and LL-37. Here the expression of additional APs (hBD 4-6, HE2beta 1; histatin-1, -3; liver expressed antimicrobial peptide-1, -2; macrophage inflammatory protein (MIP)-3alpha, and thymosin (T)beta -4) was sought and activity against common ocular pathogens studied.

Methods: AP expression was determined in human corneal and conjunctival epithelial cells (HCEC, HCjEC) by RT-PCR and in corneal sections by immunostaining. Antimicrobial assays were performed to assess peptide (hBD 1-3, LL-37, MIP-3alpha, and Tbeta 4) activity against Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), and Staphylococcus epidermidis (SE) in the presence of NaCl or tears.

Results: HCEC and HCjEC expressed MIP-3alpha and Tbeta 4. hBD 1-3, MIP-3alpha, and Tbeta 4 showed activity against PA. hBD-3 had potent activity against SA and SE, whereas hBD-2, MIP-3alpha and Tbeta 4 had moderate activity and hBD-1 had none. NaCl markedly attenuated, and tears almost completely inhibited the activity of hBD 1-2 and Tbeta 4, but not that of hBD-3.

Conclusions: The ocular surface epithelia additionally express MIP-3alpha and Tbeta 4 which have moderate antimicrobial activity. The current data support a role for hBD-3 as an antimicrobial peptide in vivo, but call in to question the effectiveness of some other APs. However, further study is required to conclusively elucidate the physiological role of each AP.

Figure 1

Expression of MIP-3α and Tβ4 mRNA and protein by ocular surface epithelia. (A) RT-PCR. The figure shows representative results for cornea = scraped human corneal epithelium (n = 3); conjunctiva = primary cultured human conjunctival epithelial cells (n = 2); + ve controls = positive controls: testis (hBD 4–6, HE2β1, MIP-3α), salivary gland (Hist-1, -3), liver (LEAP 1-2), and thymus (Tβ4). (B) Immunoblotting. The figure shows representative results for: STD (standard) = 5 ng human rMIP-3α or 10 ng Tβ4 synthetic peptide; scraped = 25 μg cellular protein from two scraped human corneal epithelial samples (n = 2); cultured = 25 μg cellular protein from primary cultured human corneal epithelial cells (n = 3).

Figure 2

Immunostaining of MIP-3α and Tβ4 in the cornea. The figure shows representative images for MIP-3α (left) and Tβ4 (right) expression in normal human corneal epithelium (sections from central cornea). Specific immunolabeling for MIP-3α or Tβ4 is shown in red; DAPI: nuclear labeling (blue); MIP-3α or Tβ4+ DAPI: merged image; control: background labeling in the absence of primary antibody; phase: phase contrast image. Identical findings were seen in corneas from three different donors. (Scale bars: 40 μm).

Figure 3

Effect of pro-inflammatory cytokines on MIP-3α and Tβ4 expression in ocular surface epithelial cells. MIP-3α (A) and Tβ4 (B) mRNA expression by corneal epithelial cells treated with IL-1β(10 ng/ml) or TNF-α (10 ng/ml) for 24 hours. M = base pair marker. Real-time PCR showing relative levels of mRNA expression for MIP-3α and Tβ4 in IL-1β (C) or TNF-α (D) treated epithelial cell samples. The figure shows representative results for primary cultured HCEC (n = 3, * p < 0.05, compared to the untreated control). Media = normalized expression in media-treated samples set to one.

Figure 4

Antibacterial effect of hBD 1-3 and Tβ4 against common ocular pathogens. (A-B) 10⁷ cfu/ml PA (ATCC 27853) were incubated with hBD-1 (A) or hBD-2 (B) for 2 hours. The graphs show the average data of three experiments. The EC₅₀values were 21.4 ± 1.5 and 1.2 ± 0.2 μg/ml for hBD-1 and hBD-2, respectively. (C) 10⁷ cfu/ml PA (ATCC 27853), SA (ATCC 29213), and SE (ATCC 155) were incubated with hBD-3 for 2 hours. The graph shows the average data of three experiments against each strain. The EC₅₀values were 3.4 ± 1.3 (PA), 5.3 ± 1.5 (SA), and 0.9 ± 0.2 μg/ml (SE). (D) 10⁷ cfu/ml PA (ATCC 27853 and ATCC 19660) were incubated with Tβ4 for 2 hours. The graph shows the average data of three experiments against each strain. The EC₅₀ values were 18.4 ± 1.5 μg/ml (PA, ATCC 27853) and 28.7 ± 1.6 μg/ml (PA, ATCC 19660). (E) 10⁷ cfu/ml PA (ATCC 27853), SA (ATCC 29213), and SE (ATCC 155) were incubated with LL-37 (50 μg/ml), MIP-3α (25 μg/ml), and Tβ4 (200 μg/ml) at 37°C for 2 hours. The graph shows representative data from two experiments.

Figure 5

Antibacterial effect of hBD 1–3 and Tβ4 against Pseudomonas aeruginosa (ATCC 27853) in the presence of NaCl and Tears. 10⁷ cfu/ml PA were incubated with (A) hBD-1 (20 μg/ml or 100 μg/ml), (B) hBD-2 (1 μg/ml or 100 μg/ml), (C) hBD-3 (3 μg/ml or 100 μg/ml), or (D) Tβ4 (25 μg/ml or 1 mg/ml) in the presence or absence of 150 mM NaCl or tears (70% v/v) at 37°C for 2 hours. The graph shows representative data from one of two or three experiments. PB = phosphate buffer, control.