MyD88 Deficiency Protects Against Dry Eye-Induced Damage.

Purpose: Dry eye disease (DED) is a multifactorial disease associated with ocular surface inflammation. Toll-like receptors (TLRs) are integral in the initiation of inflammatory signaling. Therefore, we evaluated the effect of TLR-deficiency on dry eye-related ocular surface damage and inflammation using a mouse model of experimental dry eye (EDE). Methods: C57BL/6 wild-type (WT), MyD88-/-, and IL-1R-/- mice were exposed to EDE conditions for 5 days. Tear production was measured by phenol red thread test and ocular surface damage assessed with fluorescein staining. Corneal homogenates were obtained for matrix metalloproteinase (MMP) and cytokine expression analysis by Luminex assay and quantitative PCR. In addition, whole eyes and eyelids were dissected and goblet cells and Meibomian glands were imaged, respectively. Results: Following 5 days of EDE, WT mice had extensive ocular surface staining, while MyD88-/- mice had no increased staining above non-EDE conditions. Similarly, MyD88-/- mice did not have increased corneal MMP-2, 3, or 8 concentrations, as seen with WT mice. MyD88-deficiency also resulted in decreased corneal cytokine levels. In addition, MyD88-/- mice had significantly lower conjunctival goblet cell counts compared with both WT (EDE) and IL-1R-/- (non-EDE) mice. However, there was no difference in Meibomian gland morphology between WT, IL-1R-/-, and MyD88-/- mice. Conclusions: These studies demonstrate the importance of TLR signaling in dry eye development. Mice lacking TLR signaling, MyD88-/-, were protected from EDE-induced ocular surface damage and inflammatory mediator expression, warranting further investigation into TLR inhibition as a potential therapeutic for DED.

High-Mobility Group Box 1 in Dry Eye Inflammation.

Purpose: To determine high-mobility group box 1 (HMGB1) expression during experimental dry eye (EDE) and dry eye-like culture conditions and elucidate its role in corneal dry eye-related inflammation. Methods: EDE was induced in 8- to 12-week-old C57BL/6 mice. Corneal tissue sections and lysates from EDE and untreated mice were evaluated for HMGB1 expression by immunostaining and quantitative real-time PCR (qPCR). For in vitro studies, human corneal epithelial cells (HCEC) were treated with hyperosmolar media, toll-like receptor (TLR) agonists, or proinflammatory cytokines to determine HMGB1 expression. HCEC were also treated with human recombinant HMGB1 (hrHMGB1) alone or in combination with inflammatory stimuli, and TNFα, IL-6, and IL-8 expression evaluated by qPCR and ELISA. Nuclear factor-κB (NF-κB) p65 nuclear translocation was determined by immunostaining. Results: EDE mice had higher corneal HMGB1 RNA and protein expression compared to untreated animals. In HCEC, hyperosmolar stress and TNFα treatment stimulated HMGB1 production and secretion into culture supernatants. However, in vitro stimulation with hrHMGB1 did not induce secretion of TNFα, IL-6, or IL-8 or NF-κB p65 nuclear translocation. In addition, the inflammatory response elicited by TLR agonists fibroblast-stimulating lipopeptide-1 and lipopolysaccharide was not enhanced by hrHMGB1 treatment. Conclusions: HMGB1 expression was enhanced by dry eye conditions in vivo as well as in vitro, during hyperosmolar stress and cytokine exposure, suggesting an important role for HMGB1 in dry eye disease. However, no direct inflammatory effect was observed with HMGB1 treatment. Therefore, under these conditions, HMGB1 does not contribute directly to dry eye-induced inflammation and its function at the ocular surface needs to be explored further.

Designed Host Defense Peptides for the Treatment of Bacterial Keratitis.

Purpose: To limit corneal damage and potential loss of vision, bacterial keratitis must be treated aggressively. Innovation in antimicrobials is required due to the need for empirical treatment and the rapid emergence of bacterial resistance. Designed host defense peptides (dHDPs) are synthetic analogues of naturally occurring HDPs, which provide defense against invading pathogens. This study investigates the use of novel dHDPs for the treatment of bacterial keratitis. Methods: The minimum inhibitory concentrations (MICs) were determined for dHDPs on both Gram-positive and -negative bacteria. The minimum biofilm eradication concentrations (MBEC) and in vitro time-kill assays were determined. The most active dHDP, RP444, was evaluated for propensity to induce drug resistance and therapeutic benefit in a murine Pseudomonas aeruginosa keratitis model. Results: Designed HDPs were bactericidal with MICs ranging from 2 to >64 µg/mL and MBEC ranging from 6 to 750 µg/mL. In time-kill assays, dHDPs were able to rapidly reduce bacterial counts upon contact with as little as 2 µg/mL. RP444 did not induce resistance after repeated exposure of P. aeruginosa to subinhibitory concentrations. RP444 demonstrated significant efficacy in a murine model of bacterial keratitis as evidenced by a significant dose-dependent decrease in ocular clinical scores, a significantly reduced bacterial load, and substantially decreased inflammatory cell infiltrates. Conclusions: Innovative dHDPs demonstrated potent antimicrobial activity, possess a limited potential for development of resistance, and reduced the severity of murine P. aeruginosa keratitis. These studies demonstrate that a novel dHDP may have potential to treat patients with sight-threatening bacterial keratitis.

MyD88 contribution to ocular surface homeostasis.

The cornea must maintain homeostasis, enabling rapid response to injury and microbial insult, to protect the eye from insult and infection. Toll-like receptors (TLRs) are critical to this innate immune response through the recognition and response to pathogens. Myeloid differentiation primary response (MyD88) is a key signaling molecule necessary for Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R)-mediated immune defense and has been shown to be necessary for corneal defense during infection. Here, we examined the intrinsic role of TLR signaling in ocular surface tissues by determining baseline levels of inflammatory mediators, the response to mechanical stimuli, and corneal infection in MyD88-deficient mice (MyD88-/-). In addition, cytokine, chemokine, and matrix metalloproteinase (MMP) expression was determined in ocular surface cells exposed to a panel of TLR agonists. Compared to wild-type (WT) animals, MyD88-/- mice expressed lower MMP-9 levels in the cornea and conjunctiva. Corneal IL-1α, TNFα, and conjunctival IL-1α, IL-2, IL-6, and IL-9 levels were also significantly reduced. Additionally, CXCL1 and RANTES expression was lower in both MyD88-/- tissues compared to WT and IL-1R-/- mice. Interestingly, MyD88-/- mice had lower corneal sensitivities (1.01±0.31 gm/mm2) than both WT (0.59±0.16 gm/mm2) and IL-1R-/- (0.52±0.08 gm/mm2). Following Pseudomonas aeruginosa challenge, MyD88-/- mice had better clinical scores (0.5±0.0) compared to IL-1R-/- (1.5±0.6) and WT (2.3±0.3) animals, but had significantly more corneal bacterial isolates. However, no signs of infection were detected in inoculated uninjured corneas from either MyD88 or IL-1R-deficient mice. This work furthers our understanding of the importance of TLR signaling in corneal defense and immune homeostasis, showing that a lack of MyD88 may compromise the baseline innate response to insult.

Vitamin D Induces Global Gene Transcription in Human Corneal Epithelial Cells: Implications for Corneal Inflammation.

PURPOSE: Our previous studies show that human corneal epithelial cells (HCEC) have a functional vitamin D receptor (VDR) and respond to vitamin D by dampening TLR-induced inflammation. Here, we further examined the timing of the cytokine response to combined vitamin D-TLR treatment and used genome-wide microarray analysis to examine the effect of vitamin D on corneal gene expression. METHODS: Telomerase-immortalized HCEC (hTCEpi) were stimulated with polyinosinic-polycytidylic acid (poly[I:C]) and 1,25-dihydroxyvitamin D3 (1,25D3) for 2 to 24 hours and interleukin (IL)-8 expression was examined by quantitative (q)PCR and ELISA. Telomerase-immortalized HCEC and SV40-HCEC were treated with 1,25D3 and used in genome-wide microarray analysis. Expression of target genes was validated using qPCR in both cell lines and primary HCEC. For confirmation of IκBα protein, hTCEpi were treated with 1,25D3 for 24 hours and cell lysates used in an ELISA. RESULTS: Treatment with 1,25D3 increased poly(I:C)-induced IL-8 mRNA and protein expression after 2 to 6 hours. However, when cells were pretreated with 1,25D3 for 24 hours, 1,25D3 decreased cytokine expression. For microarray analysis, 308 genes were differentially expressed by 1,25D3 treatment in hTCEpi, and 69 genes in SV40s. Quantitative (q)PCR confirmed the vitamin D-mediated upregulation of target genes, including nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α (IκBα). In addition to increased transcript levels, IκBα protein was increased by 28% following 24 hours of vitamin D treatment. CONCLUSIONS: Microarray analysis demonstrates that vitamin D regulates numerous genes in HCEC and influences TLR signaling through upregulation of IκBα. These findings are important in dissecting the role of vitamin D at the ocular surface and highlight the need for further research into the functions of vitamin D and its influence on corneal gene expression.

Effects of Topically Applied Vitamin D during Corneal Wound Healing.

Vitamin D is an important regulator of immune function and largely acts to dampen chronic inflammatory events in a variety of tissues. There is also accumulating evidence that vitamin D acts to enhance initial inflammation, beneficial during both infection and wound healing, and then promotes resolution and prevention of chronic, damaging inflammation. The current study examines the effect of topical vitamin D in a mouse of model of corneal epithelial wound healing, where acute inflammation is necessary for efficient wound closure. At 12 and 18 hours post-wounding, vitamin D treatment significantly delayed wound closure by ~17% and increased infiltration of neutrophils into the central cornea. Basal epithelial cell division, corneal nerve density, and levels of VEGF, TGFß, IL-1ß, and TNFα were unchanged. However, vitamin D increased the production of the anti-microbial peptide CRAMP 12 hours after wounding. These data suggest a possible role for vitamin D in modulating corneal wound healing and have important implications for therapeutic use of vitamin D at the ocular surface.

Vitamin D: Implications for ocular disease and therapeutic potential.

Vitamin D is a multifunctional hormone that is now known to play a significant role in a variety of biological functions in addition to its traditional role in regulating calcium homeostasis. There are a large number of studies demonstrating that adequate vitamin D levels are important in maintaining health and show that vitamin D is able to be utilized at local tissue sites. In the eye, we have increasing evidence of the association between disease and vitamin D. In this narrative review, we summarize recent findings on vitamin D and its relationship to various ocular pathologies and the therapeutic potential for some of these, as well as examine the basic science studies that demonstrate that vitamin D is biologically relevant in the eye.

Toll-like receptor activation modulates antimicrobial peptide expression by ocular surface cells.

The ability of the ocular surface to respond to pathogens is in part attributed to toll-like receptors (TLRs) that recognize conserved motifs on various microbes. This study examines TLR expression on various ocular surface cells, if TLR agonists can modulate the expression of antimicrobial peptides (AMPs), human beta defensins (hBD) and cathelicidin (hCAP-18/LL-37) which maybe functionally active against Pseudomonas aeruginosa (PA) and if TLR agonists or AMPs can modulate TLR mRNA expression. TLR1-10 mRNA expression was examined in corneal epithelial, corneal stromal cells and conjunctival epithelial cells by RT-PCR. To confirm protein expression flow cytometry or immunostaining was performed for selected TLRs on some cell cultures. Ocular surface cells were cultured with a range of TLR agonists and then hBD-1, 2, 3, or hCAP-18 mRNA and protein expression was determined by RT-PCR and immunoblotting. In some experiments, cells were cultured with a cocktail of agonists for TLR3, 5 and 6/2 and the antimicrobial activity of the culture media was tested against PA. TLR mRNA expression was also examined in primary human corneal epithelial cells (HCEC) treated with either 3 µg/ml of hBD-2, 5 µg/ml of LL-37 or TLR4, 5 and 9 agonists. Overall, the ocular surface cells expressed mRNA for most of the TLRs but some differences were found. TLR2 was not detected in corneal fibroblasts, TLR4 was not detected in primary cultured or freshly isolated HCEC, TLR5 was not detected in conjunctival epithelial cells (IOBA-NHC) and corneal fibroblasts, TLR7 was not detected in freshly isolated HCEC and TLR10 was not detected in HCEC and IOBA-NHC. TLR8 mRNA was not expressed by any of the samples tested. Immunostaining of cadaver corneas revealed TLR5 and 9 expression throughout the cornea while TLR3 was significantly expressed only in the epithelium. Flow cytometry and immunostaining revealed cultured fibroblasts expressed TLR9 but had no significant TLR3 expression. hBD-2 expression was upregulated by TLR1/2, 3, 4, 5 and 6/2 agonists depending on the cell type, whereas only the TLR3 agonist upregulated the expression of hCAP-18 in primary HCEC. The combination of TLR3, 5 and 6/2 agonists in primary HCEC, upregulated hBD-2 and hCAP-18 mRNA and peptide expression and secretion into the culture media, which significantly killed PA. This antimicrobial activity was primarily attributed to LL-37. TLR agonists did not modulate TLR expression itself, however, LL-37 or hBD-2 downregulated TLR5, 7 and/or 9 mRNA depending on the cell type. TLRs are expressed on the ocular surface and TLR agonists trigger the production of LL-37 and hBD-2, with LL-37 being particularly important for protecting the ocular surface against PA infection.

Antibacterial activity and cytotoxicity of PEGylated poly(amidoamine) dendrimers.

We have investigated the antibacterial activity and cytotoxicity of a series of amino-terminated poly(amidoamine) (PAMAM) dendrimers modified with poly(ethylene glycol) (PEG) groups. The antibacterial activity of the PAMAM dendrimers and their derivatives against the common ocular pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, was evaluated by their minimum inhibitory concentrations (MICs). For the unmodified third and fifth generation (G3 and G5) amino-terminated dendrimers, the MICs against both P. aeruginosa and S. aureus were in the range of 6.3-12.5 microg mL(-1), comparable to that of the antimicrobial peptide LL-37 (1.3-12.5 microg mL(-1)) and within the wide range of 0.047-128 microg mL(-1) for the fluoroquinolone antibiotics. PEGylation of the dendrimers decreased their antibacterial activities, especially for the Gram-positive bacteria (S. aureus). The reduction in potency is likely due to the decrease in the number of protonated amino groups and shielding of the positive charges by the PEG chains, thus decreasing the electrostatic interactions of the dendrimers with the negatively-charged bacterial surface. Interestingly, localization of a greater number of amino groups on G5 vs. G3 dendrimers did not improve the potency. Significantly, even a low degree of PEGylation, e.g. 6% with EG(11) on G3 dendrimer, greatly reduced the cytotoxicity towards human corneal epithelial cells while maintaining a high potency against P. aeruginosa. The cytotoxicity of the PEGylated dendrimers to host cells is much lower than that reported for antimicrobial peptides. Furthermore, the MICs of these dendrimers against P. aeruginosa are more than two orders of magnitude lower than other antimicrobial polymers reported to date. These results motivate further exploration of the potential of cationic dendrimers as a new class of antimicrobial agents that may be less likely to induce bacterial resistance than standard antibiotics.

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.

Cathelicidin-deficient (Cnlp -/- ) mice show increased susceptibility to Pseudomonas aeruginosa keratitis.

PURPOSE: To examine the clinical progression and innate immune responses during Pseudomonas aeruginosa (PA) keratitis in cathelicidin-deficient (KO) mice. METHODS: PA (ATCC 19660) keratitis was induced in KO mice and wild-type (WT) littermates generated on a 129/SVJ background. Clinical score and histopathology were used to monitor the progression of infection at postinfection (PI) days 1, 3, 7, 14, and 21. Mouse corneas were harvested for viable bacteria quantitation, and myeloperoxidase (MPO) assays were performed to determine the number of infiltrating neutrophils. ELISA was used to quantitate interleukin (IL)-1beta, IL-6, macrophage inflammatory peptide (MIP)-2, keratinocyte-derived chemokine (KC), tumor necrosis factor (TNF)-alpha, and vascular endothelial growth factor (VEGF) levels in the corneas. RESULTS: WT mice were resistant (cornea healed), whereas KO mice showed increased susceptibility (corneas failed to recover by 21 days or perforated) to PA infection. Clinical scores were significantly elevated in the infected corneas of KO mice versus WT mice at 7, 14, and 21 days PI. Absence of cathelicidin resulted in significantly delayed clearance of PA in the cornea and an increased number of infiltrating neutrophils at 1, 3, 7, and 14 days PI. KO mice also exhibited differential expression of protein levels for IL-1beta, IL-6, MIP-2, KC, TNF-alpha, and VEGF up to day 21 PI compared with the WT mice. CONCLUSIONS: Cathelicidin-deficient mice showed considerable susceptibility to PA keratitis. The present study demonstrates direct in vivo evidence that endogenous expression of cathelicidin provides defense against corneal PA infection indicating its importance in host innate immunity at the ocular surface.

In vitro activity of human beta-defensin 2 against Pseudomonas aeruginosa in the presence of tear fluid.

Pseudomonas aeruginosa causes vision-threatening keratitis and is difficult to treat due to emerging resistance. Human beta-defensin 2 (hBD-2) is an antimicrobial peptide expressed by ocular surface epithelia with broad-spectrum activity against various pathogens, including P. aeruginosa. The activity of hBD-2 against P. aeruginosa in the presence of human tears or NaCl was studied. In some experiments, tears were heat-inactivated, filtered, and separated into cationic/anionic fractions or mucin MUC5AC was removed by immunoprecipitation before use. Immunoprecipitation was performed to study the interaction between hBD-2 and MUC5AC. hBD-2 activity was reduced by 40 to 90% in the presence of 17.5 to 70% (vol/vol) tears. NaCl reduced hBD-2 activity, but at most it could account for only 36% of the inhibitory effect of tears. Heat inactivation and filtration attenuated the ability of tears to inhibit hBD-2 activity by 65 and 68%, respectively. Anionic tear fractions significantly reduced (86%) the activity of hBD-2, whereas only a 22% reduction was observed with the cationic fractions. In the absence of MUC5AC, the activity of hBD-2 was restored by 64%. Immunoprecipitation studies suggested that the loss of hBD-2 activity in tears is due to a direct binding interaction with MUC5AC. Our data showed that the antimicrobial activity of hBD-2 is sensitive to the presence of human tears and that this is partly due to the salt content and also the presence of MUC5AC. These data cast doubt on the effectiveness of hBD-2 as an antimicrobial peptide, and additional studies are required to conclusively elucidate its role in innate immunity at the ocular surface in vivo.

Aldehyde dehydrogenase (ALDH) 3A1 expression by the human keratocyte and its repair phenotypes.

Transparency is essential for normal corneal function. Recent studies suggest that corneal cells express high levels of so-called corneal crystallins, such as aldehyde dehydrogenase (ALDH) and transketolase (TKT) that contribute to maintaining cellular transparency. Stromal injury leads to the appearance of repair phenotype keratocytes, the corneal fibroblast and myofibroblast. Previous studies on keratocytes from species such as bovine and rabbit indicate that the transformation from the normal to repair phenotype is accompanied by a loss of corneal crystallin expression, which may be associated with loss of cellular transparency. Here we investigated if a similar loss occurs with human keratocyte repair phenotypes. Human corneal epithelial cells were collected by scraping and keratocytes were isolated by collagenase digestion from cadaveric corneas. The cells were either processed immediately (freshly isolated keratocytes) or were cultured in the presence of 10% fetal bovine serum or transforming growth factor-beta to induce transformation to the corneal fibroblast and myofibroblast phenotypes, respectively. RT-PCR, western blotting and immunolabeling were used to detect mRNA and protein expression of ALDH isozymes and TKT. ALDH enzyme activity was also quantitated and immunolabeling was performed to determine the expression of ALDH3A1 in human corneal tissue sections from normal and diseased corneas. Human corneal keratocytes isolated from three donors expressed ALDH1A1 and ALDH3A1 mRNA, and one donor also expressed ALDH2 and TKT. Corneal epithelial cells expressed ALDH1A1, ALDH2, ALDH3A1 and TKT. Compared to normal keratocytes, corneal fibroblast expression of ALDH3A1 mRNA was reduced by 27% (n=5). ALDH3A1 protein expression as detected by western blotting was markedly reduced in passage zero fibroblasts and undetectable in higher passages (n=3). TKT protein expression was reduced in fibroblasts compared to keratocytes (n=2). ALDH3A1 enzyme activity was not detectable in corneal fibroblasts (n=6) but was readily detected in corneal epithelial cells (0.29+/-0.1U/mg protein, n=4) and keratocytes (0.05+/-0.009U/mg protein, n=7). ALDH3A1 expression was also reduced in corneal fibroblasts and myofibroblasts as determined by immunolabeling of the cells in culture (n=3) and in diseased corneal tissues in situ (n=2). We conclude that expression of the crystallin ALDH3A1 is decreased in repair phenotype human keratocytes, compared to normal human keratocytes. Extrapolating from studies of bovine and rabbit, the reduced expression of ALDH3A1 may contribute to the loss of corneal transparency experienced by human patients after injury and refractive surgeries.

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.

Renal expression of the C3a receptor and functional responses of primary human proximal tubular epithelial cells.

Although complement activation and deposition have been associated with a variety of glomerulopathies, the pathogenic mechanisms by which complement directly mediates renal injury remain to be fully elucidated. Renal parenchymal tissues express a limited repertoire of receptors that directly bind activated complement proteins. We report the renal expression of the receptor for the C3 cleavage product C3a, a member of the anaphylatoxin family. C3aR is highly expressed in normal human and murine kidney, as demonstrated by immunohistochemistry and in situ hybridization. Its distribution is limited to epithelial cells only, as glomerular endothelial and mesangial cells showed no evidence of C3aR expression. The C3aR is also expressed by primary renal proximal tubular epithelial cells in vitro as demonstrated by FACS, Western blot, and RT-PCR. In vitro C3aR is functional in terms of its capacity to bind 125I-labeled C3a and generate inositol triphosphate. Finally, using microarray analysis, four novel genes were identified and confirmed as transcriptionally regulated by C3aR activation in proximal tubular cells. These studies define a new pathway by which complement activation may directly modulate the renal response to immunologic injury.