Antimicrobial defensin peptides of the human ocular surface

Abstract

Background/aims: The antimicrobial activity of the tear film exceeds the activity of its known constituents. The authors postulate that this excess activity is the result of antimicrobial peptides called defensins, and they aimed to look for defensins in the human eye.

Methods: Evidence of defensin production was sought by reverse transcriptase polymerase chain reaction (RT-PCR). Intron spanning primers were designed for beta defensins 1 and 2, and alpha defensins 5 and 6. RT-PCR was performed on cornea, conjunctiva, and lacrimal gland samples, and reaction products were size fractionated and sequenced to confirm their identity. A monoclonal antibody was utilised for the detection of alpha defensins 1, 2, and 3 in tissue sections and in immunoblots of tears.

Results: RT-PCR revealed beta defensin 1 message in samples of conjunctiva, cornea, and lacrimal gland. beta Defensin 2 message was detected in the conjunctiva and cornea but was absent from the lacrimal gland. alpha Defensin 5 and 6 message was absent in these tissues but alpha defensins 1, 2, and 3 were detected in normal tears, lacrimal gland, and inflamed conjunctiva by immunochemistry.

Conclusion: The data suggest the human eye innately produces a spectrum of antimicrobial defensin peptides. Defensins hold therapeutic potential in ocular infections as they have a broad spectrum of antimicrobial activity (bacteria fungi and viruses ) and accelerate epithelial healing.

Figure 1

(A) Analysis of β defensin 1 expression by RT-PCR. Lane M=100 bp ladder, lane −ve = negative control sample (DEPC H₂O replacing c-DNA), lane 1= neutrophils, lane 2=retina, lane 3=conjunctiva, lane 4=cornea, lane 5=lacrimal gland. Bronchial cell positive control RT-PCR confirmed a product at 215 bp (not shown). β Defensin 1 primer sequence: 5'-CCC AGT TCC TGA AAT CCT GA-3'; 5'-CAG GTG CCT TGA ATT TTG GT-3'. Product size = 215 bp. (B) Analysis of β defensin 2 expression by RT-PCR. Lane M=100 bp ladder, lane 1=skin, lane 2=conjunctiva, lane 3=cornea, lane 4=lacrimal gland, lane −ve=negative control sample (DEPC H₂O replacing c-DNA). β Defensin 2 primer sequence: 5'-CCA GCC ATC AGC CAT GAG GGT-3'; 5'-GGA GCC CTT TCT GAA TCC GCA-3'. Product size = 255 bp.

Figure 2

Dot immunoblots of normal human tears showing positive staining for α defensins 1-3. Blot A: immunostain with anti α defensin 1-3 monoclonal antibody; dot 1=tears, dot 2=non-defensin peptide mixture (negative control). Blot B (negative control): immunostain as blot A except wash buffer replaced anti α defensin 1-3 monoclonal antibody; dot 3=tears, dot 4=non-defensin peptide mixture. APAAP stain.

Figure 3

(A) Immunohistochemistry of inflamed conjunctiva showing positive staining for α defensins 1-3 in neutrophils in the conjunctival epithelium (arrow) and substantia propria (immunoperoxidase paraffin ×100). (B) Immunohistochemistry of lacrimal gland showing positive staining for α defensins 1-3 in the ductular epithelium (arrows) (APAAP cryosection ×100).