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.
Subject(s)
Chemokines, CC/genetics , Conjunctiva/metabolism , Epithelium, Corneal/metabolism , Gene Expression/physiology , Macrophage Inflammatory Proteins/genetics , Thymosin/genetics , beta-Defensins/genetics , Adult , Cell Culture Techniques , Chemokine CCL20 , Chemokines, CC/metabolism , Chemokines, CC/pharmacology , Colony Count, Microbial , Conjunctiva/drug effects , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelium, Corneal/drug effects , Female , Humans , Interleukin-1beta/pharmacology , Macrophage Inflammatory Proteins/metabolism , Macrophage Inflammatory Proteins/pharmacology , Male , Middle Aged , Pseudomonas aeruginosa/drug effects , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sodium Chloride/pharmacology , Staphylococcus aureus/drug effects , Staphylococcus epidermidis/drug effects , Tears/physiology , Thymosin/metabolism , Thymosin/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , beta-Defensins/metabolism , beta-Defensins/pharmacologyABSTRACT
PURPOSE: Human beta-defensin-2 (hBD-2) and cathelicidin LL-37 are salt-sensitive cationic antimicrobial peptides expressed by ocular surface epithelia. The goal of this study was to investigate the effect of preservative-free artificial tears on hBD-2 and LL-37 antimicrobial activity against Pseudomonas aeruginosa. METHODS: P. aeruginosa was incubated with hBD-2 or LL-37 in the absence or presence (70% vol/vol) of different preservative-free artificial tears--Visine Tears (300 mOsm/kg), Tears Naturale Free (261 mOsm/kg), TheraTears (185 mOsm/kg), and Refresh Plus (325 mOsm/kg)--for 2 hours at 37 degrees C. In some experiments, P. aeruginosa was incubated with hBD-2 or LL-37 and Visine Tears or Tears Naturale Free with or without carboxymethylcellulose (0.5% vol/vol final concentration). Plates were inoculated with samples of each reaction mixture and then incubated for 24 hours at 37 degrees C. RESULTS: Visine Tears and Tears Naturale Free had little or no effect on the antimicrobial activity of 100 microg/mL hBD-2 or LL-37. In the presence of Refresh Plus and TheraTears, the activity of 100 microg/mL hBD-2 or LL-37 was reduced by 90% to 100%. Carboxymethylcellulose, at a concentration comparable to that present in Refresh Plus, reduced the effectiveness of hBD-2 or LL-37 by 40% to 90% in the presence of Tears Naturale Free and Visine Tears. CONCLUSION: Human beta-defensin-2 and cathelicidin LL-37 inhibit the growth of P. aeruginosa in vitro, but this activity is markedly reduced in the presence of Refresh Plus and TheraTears. These results suggest that carboxymethylcellulose-containing artificial tears may reduce the activity of the endogenously produced antimicrobial peptides.
