Ocular surface expression and in vitro activity of antimicrobial peptides.

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.

Effect of hyperosmolality on beta-defensin gene expression by human corneal epithelial cells.

PURPOSE: As human beta-defensins (hBD) are important antimicrobial peptides at epithelial surfaces, including the ocular surface, we tested the effect of hyperosmolar conditions on the expression of these peptides by human corneal epithelial cells (HCECs). METHODS: Simian virus 40-transformed HCECs (n = 5) or primary cultured HCECs (n = 5) were treated with serum-free media or serum-free hyperosmolar (400-500 mOsm/kg) media for 24 hours or serum-free 500 mOsm/kg media for 12 to 48 hours. The effect of hyperosmolality on interleukin-1beta (IL-1beta)-induced hBD-2 expression was also tested. IL-6 expression was studied as a marker of IL-1beta function. Expression of hBD-1, -2, and -3 and IL-6 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). The levels of active IL-1beta (culture supernatants and cell lysates) and pro-IL-1beta (cell lysates) were detected by enzyme-linked immunosorbent assay. RESULTS: HCECs constitutively expressed hBD-1 and -3 but not hBD-2. Hyperosmolar media had no effect on the basal expression of hBD-1 or -3 and did not induce the expression of hBD-2. Treatment with 500 mOsm/kg media for 24 hours decreased the ability of IL-1beta to upregulate hBD-2 and IL-6 expression. Active or pro-IL-1beta was not detected in any cell culture sample. CONCLUSION: Our results suggest that the hyperosmolar environment observed in diseases such as dry eye does not alter defensin expression. However, a hyperosmolar environment may influence cytokine function in ocular surface cells and thus affect their response to injury and inflammation.

Multifunctional roles of human cathelicidin (LL-37) at the ocular surface.

PURPOSE: The goals of this study were to examine the expression of the antimicrobial peptide LL-37 in the corneal epithelium during wound healing and to investigate whether LL-37 stimulates human corneal epithelial cell (HCEC) migration, proliferation, and cytokine production. METHODS: Expression of LL-37 was determined by RT-PCR and immunostaining in tissue sections and HCECs scraped from corneas before (original) and after (regrown) re-epithelialization. The antimicrobial activity of LL-37 against Pseudomonas aeruginosa (PA) was determined in the presence of NaCl and tears. Blind-well chamber assays were performed to study the effect of LL-37 on migration. Proliferation was determined using calcein-AM, and cytotoxicity was evaluated by MTT assay. ELISA was performed to assess the ability of LL-37 to stimulate HCEC cytokine secretion. RESULTS: LL-37 peptide was present throughout the corneal epithelium (n=4). All original corneal epithelial samples expressed a low level of LL-37 (n=10). Regrown epithelial samples collected 24 (n=3 of 5) or 48 (n=4 of 5) hours after wounding showed upregulated expression of LL-37. LL-37 killed PA in the presence of NaCl (EC50=10.3+/-2.5 microg/mL) and retained its activity in tears (n=3). LL-37 induced HCEC migration (n=5) and secretion of IL-8, IL-6, IL-1beta, and TNF-alpha (2- to 23-fold, n=4-7). Inhibitor studies indicated that LL-37's effects are mediated through multiple pathways involving a G protein-coupled receptor (formyl peptide receptor-like 1 in migration) and the epidermal growth factor receptor (n=2 to 5). LL-37 did not stimulate HCEC proliferation (n=3) and high concentrations (>10 microg/mL) were cytotoxic (n=3). CONCLUSIONS: LL-37 expression is upregulated in regenerating human corneal epithelium, has antibacterial activity against ocular pathogens under physiologically relevant conditions, and stimulates HCEC migration and cytokine production. These findings suggest that LL-37 acts as a multifunctional mediator that helps protect the cornea from infection and modulates wound healing.

Human cathelicidin (LL-37), a multifunctional peptide, is expressed by ocular surface epithelia and has potent antibacterial and antiviral activity.

PURPOSE: This study determined whether LL-37 (cathelicidin) is expressed by conjunctival and corneal epithelia as part of ocular host defense. The antimicrobial activity of LL-37 was also assessed in vitro against Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), Staphylococcus epidermidis (SE), herpes simplex virus type 1 (HSV-1), and adenovirus (Ad). METHODS: Expression of LL-37/hCAP 18 mRNA and LL-37 protein was determined by reverse transcription-polymerase chain reaction (RT-PCR) and immunoblotting, respectively, in scraped human corneal epithelium and primary cultured human corneal and conjunctival epithelial cells. The EC50 values for three strains of PA and one each of SA and SE were determined for LL-37. LL-37 antiviral inhibition of HSV-1 and adenovirus was assessed by direct inactivation assays. Toxicity of LL-37 to A549 cells was evaluated by a MTT assay. RESULTS: LL-37/hCAP18 mRNA and LL-37 peptide were expressed by human corneal and conjunctival epithelial cells. Antibacterial activity for LL-37 was demonstrated (EC50 values for the three PA strains were 2.8 +/- 1.3, 1.9 +/- 0.3, and 3.6 +/- 2.1; for SA: 1.6 +/- 1.5; for SE: 1.3 +/- 1.9 microg/ml). LL-37 produced a significant reduction (p < 0.001 ANOVA) in HSV-1 and Ad19 viral titers with distinctly different time-kill curves (p < 0.001). LL-37 (up to 111 microM) produced no toxicity in A549 cells. CONCLUSIONS: Corneal and conjunctival epithelia express LL-37 as part of mucosal innate immunity to protect against bacterial and viral ocular infections.

Defensin expression by the cornea: multiple signalling pathways mediate IL-1beta stimulation of hBD-2 expression by human corneal epithelial cells.

PURPOSE: To investigate the expression of human beta-defensins (hBDs) by human corneal epithelium and determine the effects of proinflammatory cytokines on expression of human beta-defensin (hBD)-2 by human corneal epithelial cells (HCECs) in culture. METHODS: RNA was extracted from corneal epithelial cells scraped from cadaveric corneas and from cultured HCECs, and RT-PCR was performed to detect hBD-1, -2, and -3 mRNA. To study the effects of proinflammatory cytokines on expression of defensin, HCECs were cultured and then exposed to interleukin (IL)-1beta or tumor necrosis factor (TNF)-alpha for up to 36 hours, with a range of concentrations (0.01-100 ng/mL). In some experiments, cells were pretreated with various cell signaling pathway inhibitors before the addition of IL-1beta. At the end of the incubations, the cells were harvested for RT-PCR and the culture media collected for the detection by immunoblot analysis of secreted defensin peptide. RESULTS: All epithelial tissue collected from cadaveric corneas expressed mRNA for hBD-1. hBD-2 was detectable in two of eight donors corneas, whereas hBD-3 was detected in five. All primary cultures of HCECs expressed hBD-1 and -3. A faint band for hBD-2 was detectable in three of eight cultures. Cultures of simian virus (SV)40-transformed HCECs always expressed hBD-1 and -3, but did not express hBD-2 under control conditions. IL-1beta and TNFalpha each stimulated the expression of hBD-2 in HCECs and were more effective in combination than alone. The effects of IL-1beta were concentration- (maximal at 10 ng/mL) and time-dependent (maximal at 12 hours and 24 hours for hBD-2 mRNA expression and protein secretion, respectively). The upregulation of hBD-2 mRNA persisted for at least 24 hours after removal of IL-1beta. The NFkappaB inhibitors pyrrolidinedithiocarbamate (PDTC; 100 microM), caffeic acid phenethyl ester (CAPE; 90 microM), and MG-132 (25 microM), blocked IL-1beta-stimulated expression of hBD-2. The p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 (5 microM) and the c-Jun NH2-terminal kinase (JNK) inhibitor SP600125 (25 microM) partially blocked (by 47% and 59%, respectively) the effect of IL-1beta. However, PD98059, an ERK inhibitor, had no effect. Genistein (50 microM) and dexamethasone (1 microM) also partially blocked (by 26% and 28%, respectively) the effect of IL-1beta. CONCLUSIONS: Human corneal epithelium expresses hBD-1 and -3. hBD-2 is not typically present, but its expression can be stimulated by proinflammatory cytokines such as IL-1beta, acting through mitogen-activated protein (MAP) kinase and nuclear factor (NF)-kappaB pathways. Because IL-1 is known to be increased at the ocular surface after injury, the current observations provide a mechanism to explain the previous finding that hBD-2 is upregulated in regenerating corneal epithelium. Cytokine stimulation of hBD-2 expression most likely provides additional protection against infection and raises the possibility that this defensin in particular may be involved in the wound-healing response, per se.

Identification, cloning, and functional characterization of a murine lipoxin A4 receptor homologue gene.

To identify additional members of the murine N-formyl-Met-Leu-Phe peptide receptor family (fMLF-R), a mouse macrophage cDNA library was screened using the open reading frame of murine N-formyl peptide receptor. Four individual hybridizing cDNA clones were maintained through tertiary screening. One cDNA clone was a truncated, polyadenylated version of the previously described murine-fMLF-R. The other three cDNA clones varied in length, but contained identical open reading frame sequences. One clone, 8C10, was selected for further study and shared 70% sequence identity with murine-fMLF-R and 89% sequence identity with murine lipoxin A4 receptor cDNA. When placed into the pcDNA-3 expression vector and cotransfected with Galpha16 cDNA into COS-1 cells, 8C10 cDNA induced the production of inositol-1,4,5-triphosphate when concentrations of 1-1600 nM lipoxin A4 (LXA4) were tested as ligands. Northern blot analysis of murine organs indicated that the 8C10 message is present in lung, spleen, and adipose tissue. Moreover, mice treated with LPS demonstrated increased expression of 8C10 message in spleen and adipose tissue, while showing a slight reduction in lung. We have also characterized the 8C10 structural gene from a 129Sv/J genomic library and have determined its size to be >6.1 kb in length and comprised of two exons separated by a 4.8-kb intron. Collectively, these data indicate that this homologue receptor is closely related to the murine LXA4 receptor and functionally responds to LXA4 as a ligand.