ABSTRACT
PURPOSE: Apoptosis was examined after Pseudomonas aeruginosa corneal infection in C57BL/6 (B6, susceptible) and BALB/c (resistant) mice. METHODS: TUNEL staining, real-time RT-PCR, polymorphonuclear neutrophils (PMNs) and macrophage (Mphi) depletion, and immunostaining were used. RESULTS: Intense TUNEL staining was seen in BALB/c versus B6 cornea at 1 versus 3 days after infection (PI) and correlated with mRNA levels for caspase-3. TUNEL staining (with or without PMN depletion) and PMN immunostaining revealed the PMN as the major apoptotic cell for both groups. Next, B6 mice with high corneal levels of the antiapoptosis neuropeptide, substance P (SP), were treated with the SP antagonist, Spantide I (with/without Mphi depletion), resulting in earlier apoptosis and diminished disease only when M(phi)s were present. SP interactions with M(phi)s were explored further by eliciting cells from both groups and stimulating them with lipopolysaccharide (LPS), with or without SP. LPS with SP treatment decreased the number of apoptotic M(phi)s in B6 but not BALB/c mice and correlated with reduced mRNA expression of NK-1R (major SP receptor) on BALB/c cells. In addition, mRNA expression for IL-12 was upregulated in LPS-stimulated B6 M(phi)s, although cells from BALB/c mice expressed more IL-10. CONCLUSIONS: These studies provide evidence that PMN apoptosis is delayed in the cornea of B6 versus BALB/c mice after bacterial infection; that in B6 mice, blocking SP interaction with the NK-1R promotes earlier apoptosis and improves disease outcome; that M(phi)s regulate PMN apoptosis; and that M(phi)s from B6 versus BALB/c mice differ in expression of the NK-1R and cytokines produced after LPS challenge.
Subject(s)
Apoptosis , Corneal Ulcer/microbiology , Eye Infections, Bacterial/microbiology , Neutrophils/pathology , Pseudomonas Infections/microbiology , Substance P/physiology , Animals , Caspase 3/genetics , Corneal Ulcer/immunology , Eye Infections, Bacterial/immunology , Female , In Situ Nick-End Labeling , Interleukin-12/genetics , Lymphocyte Depletion , Macrophages, Peritoneal/physiology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Microscopy, Confocal , Neurokinin-1 Receptor Antagonists , Neutrophil Activation , Neutrophils/metabolism , Pseudomonas Infections/immunology , RNA, Messenger/metabolism , Receptors, Neurokinin-1/genetics , Reverse Transcriptase Polymerase Chain Reaction , Substance P/analogs & derivatives , Substance P/antagonists & inhibitors , Substance P/therapeutic useABSTRACT
Corneal infection with Pseudomonas aeruginosa perforates the cornea in susceptible C57BL/6 (B6), but not resistant BALB/c, mice. To determine whether vasoactive intestinal peptide (VIP) played a role in development of the resistant response, protein expression levels were tested by immunocytochemistry and enzyme immunoassay in BALB/c and B6 corneas. Both mouse strains showed constitutive expression of corneal VIP protein and nerve fiber distribution. However, disparate expression patterns were detected in the cornea after infection. VIP protein was elevated significantly in BALB/c over B6 mice at 5 and 7 days postinfection. Therefore, B6 mice were injected with rVIP and subsequently demonstrated decreased corneal opacity and resistance to corneal perforation compared with PBS controls. rVIP- vs PBS-treated B6 mice also demonstrated down-regulation of corneal mRNA and/or protein levels for proinflammatory cytokines/chemokines: IFN-gamma, IL-1beta, MIP-2, and TNF-alpha, whereas anti-inflammatory mediators, IL-10 and TGF-beta1, were up-regulated. Treatment with rVIP decreased NO levels and polymorphonuclear neutrophil (PMN) number. To further define the role of VIP, peritoneal macrophages (Mphi) and PMN from BALB/c and B6 mice were stimulated with LPS and treated with rVIP. Treatment of LPS-stimulated Mphi from both mouse strains resulted in decreased IL-1beta and MIP-2 protein levels; PMN responded similarly. Both cell types also displayed a strain-dependent differential response to rVIP, whereby B6 Mphi/PMN responded only to a higher concentration of VIP compared with cells from BALB/c mice. These data provide evidence that neuroimmune regulation of the cytokine network and host inflammatory cells functions to promote resistance against P. aeruginosa corneal infection.
Subject(s)
Corneal Diseases/metabolism , Corneal Diseases/microbiology , Cytokines/metabolism , Pseudomonas aeruginosa/physiology , Retinal Perforations/metabolism , Vasoactive Intestinal Peptide/metabolism , Animals , Corneal Diseases/drug therapy , Cytokines/genetics , Female , Macrophages/drug effects , Mice , Neutrophils/drug effects , RNA, Messenger/genetics , Receptors, Vasoactive Intestinal Polypeptide, Type I/metabolism , Vasoactive Intestinal Peptide/therapeutic useABSTRACT
PURPOSE: To develop a rat model to experimentally monitor the potential inflammatory effects of soft contact lens (CL) usage on the cornea during extended wear (EW). METHODS: Lewis rats were fitted with EW lotrafilcon A (CIBA Vision, Duluth, GA) hydrogel lenses (Dk/t 175 barrers/cm) in the left eye, the right eye serving as a control. After 12 days (n = 5 rats) and 30 days (n = 8 rats) of continuous extended wear, corneas were removed and total RNA was extracted from both CL-wearing and non-lens-wearing eyes. Multiprobe ribonuclease protection assays (RPA) were used to detect and compare cytokine and chemokine gene expression in corneas from both groups. RESULTS: Cytokine-chemokine mRNA expression levels were similar for interleukin (IL)-1alpha, IL-1 receptor antagonist (RA), IL- 18, transforming growth factor (TGF)-beta1, TGF-beta2, TGF-beta3, and macrophage migration inhibitory factor (MIF) levels in CL-wearing and non-lens-wearing corneas after 12 or 30 days of EW. CONCLUSION: This in vivo rat model for extended contact lens wear allows analysis and comparison of mRNA levels of cytokines and chemokines in the cornea with and without EW soft CL use. Remarkably, after 12 or 30 days of continuous CL wear, there was no significant up-regulation in lens wearing corneas for any of the cytokines-chemokines tested.
Subject(s)
Contact Lenses, Extended-Wear , Models, Animal , Animals , Chemokines/genetics , Chemokines/metabolism , Cornea/metabolism , Cytokines/genetics , Cytokines/metabolism , Female , Hydrogel, Polyethylene Glycol Dimethacrylate , In Situ Hybridization , RNA, Messenger/metabolism , Rats , Rats, Inbred LewABSTRACT
Previously, thymosin beta 4 (Tbeta(4)) was found to promote wound healing in full thickness skin wounds and heptanol debrided corneas. Here, the effect of Tbeta(4) was examined treatment on corneal wound healing and inflammation in vivo after alkali injury, a more severe wound of the eye. Corneas from 129 Sv mice were chemically burned with a 2 mm disc soaked in 1 N NaOH for 30 sec. Eyes were irrigated copiously with phosphate buffered saline (PBS) and then treated topically with either Tbeta(4) (5 microg/5 microl PBS) or 5 microl PBS twice daily. Animals were killed, the eyes were enucleated, fixed and embedded in plastic resin or prepared for mRNA analysis. Mouse corneas topically treated with 5 microg of Tbeta(4) twice daily after alkali injury demonstrated accelerated re-epithelialization at all time points and decreased polymorphonuclear leukocyte (PMN) infiltration at 7 days post injury (p.i.) when compared to PBS-treated controls. mRNA transcript levels were decreased several fold for interleukin (IL)-lbeta, and the chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2 and monocyte chemoattractant protein (MCP)-1 from 1 to 7 days after injury in the Tbeta(4)- vs. PBS-treated corneas. Thus, Tbeta(4) may provide a new clinical treatment for severe traumatic corneal wound disorders by promoting rapid corneal wound healing and decreasing both PMN infiltration and inflammatory cytokine and chemokine mRNA levels.
