vasoactive-intestinal-peptide and Corneal-Ulcer

Vasoactive intestinal peptide (VIP) is an anti-inflammatory neuropeptide that downregulates proinflammatory cytokines and promotes healing in a susceptible model of P. aeruginosa keratitis. Growth factors also play a role in corneal healing and restoration of tissue homeostasis after wounding. However, whether VIP treatment modulates growth factors to promote healing in the infected cornea remains untested and is the purpose of this study.. C57BL/6 (B6) mice were injected with VIP and mRNA and protein levels, and immunostaining for EGF, FGF, HGF, and VEGF-A were done. Exogenous treatment with a mixture of the growth factors also was tested and levels of cytokines, defensins, and bacterial counts were determined.. Real-time RT-PCR, immunostaining, and ELISA data demonstrated that treatment with VIP enhanced levels of EGF, FGF, and HGF during disease, and that VEGF-A, and associated angiogenic molecules also were increased by VIP. Moreover, immunohistochemical studies confirmed that both epithelial and stromal cells participated in growth factor production. Most notably, treatment with a mixture of EGF, FGF, and HGF after disease onset, prevented corneal perforation when compared with controls. This outcome was associated with downregulation of proinflammatory cytokines such as macrophage inflammatory protein-2 (MIP-2), upregulation of anti-inflammatory cytokines such as TGF-β, and antimicrobials β-defensins 2 and 3, as well as decreased plate counts at 1 day postinfection (p.i.) (P = 0.0001).. Collectively, the data provide evidence that VIP treatment modulates growth factors, angiogenic molecules, and defensins in the infected cornea and that this in turn promotes healing and restoration of tissue homeostasis.

Topics: Animals; Colony Count, Microbial; Cornea; Corneal Neovascularization; Corneal Perforation; Corneal Ulcer; Cytokines; Defensins; Enzyme-Linked Immunosorbent Assay; Eye Infections, Bacterial; Female; Gene Expression; Intercellular Signaling Peptides and Proteins; Mice; Mice, Inbred C57BL; Pseudomonas Infections; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Vascular Endothelial Growth Factor A; Vasoactive Intestinal Peptide

This study tested the hypothesis that the neuropeptide vasoactive intestinal peptide (VIP) regulates adhesion molecule expression, reduces inflammatory cell migration and infiltration into the Pseudomonas aeruginosa-infected cornea of susceptible B6 mice, and promotes corneal healing and resistance.. B6 mice received daily intraperitoneal (IP) injections of VIP from -1 through 5 days after infection. Control mice were similarly injected with sterile phosphate-buffered saline (PBS). Transcript levels of adhesion molecules were determined by PCR array, then select molecules were tested individually by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and confirmed at the protein level by enzyme-linked immunosorbent assay (ELISA) or immunofluorescent staining with confocal laser scanning microscopy at various time points after infection to assess the effects of VIP treatment in the regulation of adhesion molecule expression.. Injection of B6 mice with VIP compared with PBS resulted in significant downregulation of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, platelet-endothelial cell adhesion molecule-1, and P-selectin and L-selectin mRNA expression. Protein levels for ICAM-1 and VCAM-1, detected by ELISA, supported the mRNA data at similar time points. Immunofluorescence staining further confirmed the effects of VIP treatment, showing reduced corneal expression of ICAM-1/leukocyte function-associated antigen (LFA-1) and VCAM-1/very late antigen-4 (VLA-4) at select time points compared with PBS-treated animals.. VIP treatment downregulates the production of adhesion molecules integral to the transmigration process of host inflammatory cells (polymorphonuclear neutrophils, macrophages) into the infected cornea. This results directly in reduced cellular infiltration, less stromal destruction, and better disease outcome.

Topics: Animals; Cell Adhesion Molecules; Cell Movement; Corneal Ulcer; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Eye Infections, Bacterial; Female; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation; Immunity; Injections, Intraperitoneal; Macrophages; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Neuroprotective Agents; Neutrophils; Prednisolone; Pseudomonas Infections; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Vasoactive Intestinal Peptide; Wound Healing

Studies have shown that blocking substance P (SP) binding to neurokinin 1 receptor with spantide I prevents Pseudomonas aeruginosa-induced corneal perforation in susceptible C57BL/6 mice. This study tested the effect of SP injection on the resistance response (cornea heals) of BALB/c mice.. The day before infection, mice were injected intraperitoneally with SP or PBS. Disease was graded by clinical score, slit lamp, plate count, real-time RT-PCR, and ELISA assays, and polymorphonuclear neutrophils (PMNs) were quantitated using a myeloperoxidase assay. In additional experiments, BALB/c mice were injected intraperitoneally with vasoactive intestinal peptide (VIP) antagonist and similarly analyzed.. Mice injected with SP exhibited worsened disease on days 1 to 7 after infection compared with controls. SP injection resulted in elevated PMN levels and viable bacterial counts in the cornea 3 and 5 days after infection. mRNA expression for NFkappaB and type 1 cytokines (e.g., IFN-gamma), as well as for TNF-alpha, MIP-2, IL-18, IL-6, and IL-1beta, were significantly elevated, whereas VIP and cytokines TGF-beta and IL-10 were significantly reduced. Differences in mRNA expression were selectively confirmed at the protein level by ELISA for NFkappaB, IL-1beta, and IL-10. VIP antagonist treatment also resulted in exacerbated disease scores, elevated proinflammatory mediators, and reduced anti-inflammatory mediators.. These data provide evidence that the neuropeptide SP, among its broad systemic effects, is a potent neuroimmunoregulator that promotes susceptibility in the resistant BALB/c mouse by overcoming the anti-inflammatory effects of VIP and IL-10 and that a balance between SP and VIP levels may be critical in disease resolution.

Topics: Animals; CD4-Positive T-Lymphocytes; Corneal Ulcer; Cytokines; Disease Susceptibility; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Eye Infections, Bacterial; Female; Inflammation Mediators; Killer Cells, Natural; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Neutrophils; NF-kappa B; Pseudomonas aeruginosa; Pseudomonas Infections; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Substance P; Th1 Cells; Vasoactive Intestinal Peptide