Total protein concentration and T-cell suppression activity of aqueous humour before and after penetrating keratoplasty.

PURPOSE: We wished to determine whether immune privilege parameters assayed in aqueous humour (AqH) are relevant to the fate of penetrating keratoplasty (PK) in humans. METHODS: AqH was collected in 28 patients before PK (prospective cohort), in 6 patients with no history of graft rejection undergoing cataract surgery after PK (acceptors), in another 6 patients undergoing treatment of an acute endothelial immune reaction (rejectors), and in 65 controls undergoing uncomplicated cataract extraction. AqH was tested for total protein concentration and the ability to suppress T-cell activation. RESULTS: AqH protein concentrations of acceptors and rejectors post-PK were elevated (2.7 ± 0.8 and 2.7 ± 0.7 mg/ml, respectively) compared with pre-PK AqH level and cataract controls (1.0 ± 0.1 mg/ml, P = 0.01). All AqH samples suppressed T-cell activation, irrespective of source and timing of AqH removal. CONCLUSION: Assays of immune privilege markers in AqH suggest that PK surgery may result in a sustained loss of integrity of the blood-aqueous barrier. Although trends were evident, values of immune privilege markers determined pre- and post-PK were not statistically significantly different between the study groups. However, further prospective studies determining additional immune privilege markers have to be conducted in order to find out whether these markers might serve as predictive parameters for immune reactions following PK.

JIP1 binding to RBP-Jk mediates cross-talk between the Notch1 and JIP1-JNK signaling pathway.

Notch1 signaling has a critical function in maintaining a balance among cell proliferation, differentiation, and apoptosis. Our earlier work showed that the Notch1 intracellular domain interferes with the scaffolding function of c-Jun N-terminal kinase (JNK)-interacting protein-1 (JIP1), yet the effect of JIP1 for Notch1-recombining binding protein suppressor of hairless (RBP-Jk) signaling remains unknown. Here, we show that JIP1 suppresses Notch1 activity. JIP1 was found to physically associate with either intracellular domain of Notch1 or RBP-Jk and interfere with the interaction between them. Furthermore, we ascertained that JIP1 caused the cytoplasmic retention of RBP-Jk through an interaction between the C-terminal region of JIP1 including Src homology 3 domain and the proline-rich domain of RBP-Jk. We also found that RBP-Jk inhibits JIP1-mediated activation of the JNK1 signaling cascade and cell death. Our results suggest that direct protein-protein interactions coordinate cross-talk between the Notch1-RBP-Jk and JIP1-JNK pathways.

Early Involvement of Immune/Inflammatory Response Genes in Retinal Degeneration in DBA/2J Mice.

PURPOSE: The DBA/2J (D2) mouse carries mutations in two of its genes, Tyrp1 and Gpnmb. These alterations result in the development of an immune response in the iris, leading to iris atrophy and pigment dispersion. The development of elevated intraocular pressure (IOP) in this model of glaucoma is considered to be a significant factor leading to the death of retinal ganglion cells (RGCs). Changes in gene expression in the retina have already been correlated with the appearance of elevated IOP in the D2 mouse. The purpose of the present study was to determine if any changes in gene expression occur prior to the development of IOP. METHODS: The IOP was measured monthly using a rebound tonometer in D2 and age-matched C57/BL6 (B6) mice (normal controls). D2 animals with normal IOP at 2 and 4 M were used. In addition, mice at the age of 6-7 M were included to look for any trends in gene expression that might develop during the progression of the disease. Separate RNA samples were prepared from each of three individual retinas for each age, and gene expression profiles were determined with the aid of mouse oligonucleotide arrays (Agilent). A subset of genes was examined with the aid of real-time PCR. Immunocytochemistry was used to visualize changes in the retina for some of the gene-products. RESULTS: Four hundred and thirteen oligonucleotide probes were differentially expressed in the retinas of 4 M versus 2 M old D2 mice. The most significantly up-regulated genes (181) were associated with immune responses including interferon signaling, the complement system and the antigen presentation pathway, whereas the down-regulated genes (232) were linked to pathways related to cell death and known neurological diseases/disorders. These particular changes were not revealed in the age-matched B6 mice. By 6 M, when IOP started to increase in many of the D2 mice, more robust changes of these same genes were observed. Changes in the levels of selected genes, representative of different functions/pathways, were validated with RT-PCR, and changes in glial responses were visualized in the retina with immunocytochemistry. CONCLUSIONS: The results showed that the expression of genes related to the immune response and acute stress were altered independently of the development of elevated IOP, and indicated early involvement of the immune system in the onset of the disease. The later development of elevated IOP, observed in this animal model, was coincident with continued changes in expression of genes observed at earlier time points. Further studies are warranted to identify the roles of specific genes identified here with respect to the death of the RGCs.

DJ-1 modulates UV-induced oxidative stress signaling through the suppression of MEKK1 and cell death.

DJ-1 is a multifunctional protein that performs functions in transcriptional regulation and oxidative stress, and the loss of its function is believed to result in the onset of Parkinson's disease (PD). In this study, we report that DJ-1 protects against UV-induced cell death through the suppression of the JNK1 signaling pathway. The results of both binding and kinase studies have revealed that MEKK1 is the direct target of DJ-1. The C-terminus of DJ-1 was crucial to the inhibition of the MEKK1 kinase activity. Wild-type DJ-1 sequesters MEKK1 within the cytoplasm and the L166P mutant facilitates the translocation of MEKK1 toward the nucleus without physical association. Both DJ-1 knockdown and pathogenic L166P mutant were determined to be highly susceptible to the UV-induced activation of the MEKK1-SEK1-JNK1 signaling cascade and cell death. Taken together, our findings show that missense mutation in DJ-1 sensitizes cells to stress-induced cell death through the MEKK1-SEK1-JNK1 signaling pathway, a process, which may trigger the early onset of PD.

Immune privilege persists in eyes with extreme inflammation induced by intravitreal LPS.

Since immune privilege is believed to exist in the eye in order to suppress sight-destroying inflammation, we wondered whether eyes with intraocular inflammation retain the immune privileged state. Intraocular inflammation was induced by injection of lipopolysaccharide (LPS) into the vitreous cavity of BALB/c mouse eyes, which showed a peak in intensity at approximately 9 h. At this time point, inflamed eyes were examined for their capacity to afford immune privilege to injected allogeneic tumor cells, and to promote anterior chamber-associated immune deviation (ACAID) to antigens injected locally. In addition, aqueous humor (AqH) harvested from inflamed eyes was tested for its ability to suppress T cell activation. Surprisingly, eyes with acute, intense intraocular inflammation allowed allogeneic tumor cells to form progressively growing tumors, and these same eyes promoted ACAID. Moreover, AqH harvested from inflamed eyes strongly inhibited T cell activation. We conclude that the type of extreme, intraocular inflammation evoked by intravitreally injected LPS fails to abolish immune privilege in the eye. These findings are discussed in light of the effects of other types of inflammation on the integrity of ocular immune privilege, and with respect to the capacity of the eye to maintain immune privilege by more than one mechanism.

CXC chemokine GRO is essential for neutrophil infiltration in LPS-induced uveitis in rabbits.

The purpose of this study was to investigate the role and regulation of the CXC chemokine GRO and the interaction between GRO and IL-8 in LPS-induced uveitis in rabbits. Uveitis was induced by intravitreal injection of 100 ng of LPS in rabbits. After the LPS injection, GRO was produced in aqueous humor and peaked at 24 hr. Immunohistochemistry showed that ciliary epithelial cells were responsible for production of GRO. Blocking the activity of GRO by anti-GRO serum reduced LPS-induced aqueous neutrophil counts by 80%, but did not reduce the mononuclear cell counts or protein levels or IL-8 levels. Regulation of GRO production by TNFalpha, IL-1 and IL-8 was studied. Anti-TNFalphamAb alone did not inhibit the 24 hr LPS induced GRO levels, whereas rrIL-1Ra inhibited the GRO production by 58%. The combination of anti-TNFalpha mAb and rrIL-1Ra inhibited 93% of GRO production. Although treatment with anti-IL-8 IgG inhibited the neutrophil infiltration by 66%, treatment with this antibody did not inhibit GRO production. Taken together, our results suggest that GRO is an essential mediator for neutrophil infiltration in LPS-induced uveitis in rabbits. Most of GRO production is mediated by TNFalpha and IL-1. GRO and IL-8 act in concert to mediate neutrophil infiltration.

Role and regulation of IL-8 and MCP-1 in LPS-induced uveitis in rabbits.

Intravitreal injection of lipopolysaccharide (LPS) induces leukocyte infiltration and protein leakage into the aqueous humor. In the present study, we investigated the role of IL-8 and MCP-1 and regulation of these chemokines by TNFalpha and IL-1 in LPS-induced uveitis in rabbits. After intravitreal injection of LPS, generation of IL-8 in the aqueous humor showed a biphasic pattern with the first peak at 12 hr and the second one at 24 hr, while MCP-1 was produced in a monophasic pattern and peaked at 24 hr. Immunohistochemistry showed that ciliary epithelial cells and infiltrating leukocytes were the producing cells of IL-8 and MCP-1. Administration of anti-IL-8 IgG suppressed by 66% the peak levels of LPS-induced aqueous neutrophil counts at 24 hr but did not suppress aqueous mononuclear cell counts or protein levels. anti-MCP-1 IgG inhibited aqueous mononuclear cell counts by 41% and protein levels by 28%, but did not inhibit aqueous neutrophil counts. The levels of LPS-induced aqueous IL-8 and MCP-1 at 12 hr were inhibited by anti-TNFalpha mAb but not by an IL-1 receptor antagonist (IL-1Ra), while concentrations of the two chemokines at 24 hr were inhibited by both anti-TNFalpha mAb and IL-1Ra. A combination of anti-TNFalpha mAb and rrIL-1Ra had an additive effect on the 24 hr-chemokine levels and inhibited up to 90% chemokine production. Taken together, our results show that IL-8 mediates neutrophil infiltration, while MCP-1 mediates mononuclear cell infiltration and protein leakage in LPS-induced uveitis in rabbits. Levels of aqueous IL-8 and MCP-1 at 12 hr are regulated by TNFalpha, while levels at 24 hr are regulated by TNFalpha and IL-1.

Involvement of TNF alpha, IL-1 beta and IL-1 receptor antagonist in LPS-induced rabbit uveitis.

The objective of the study was to investigate involvement of TNF alpha, IL-1 beta and IL-1 receptor antagonist (IL-1Ra) in lipopolysaccharide (LPS)-induced uveitis. Intravitreal injection of LPS (100 ng) to rabbits induced a massive leukocyte infiltration and protein leakage into the aqueous humor. Aqueous leukocyte counts and protein levels reached a peak 24 hr after this injection. The peak concentrations of aqueous TNF alpha (230 +/- 37 pg ml-1, at 9 hr) and IL-1 beta (185 +/- 80 pg ml-1, at 18 hr) preceded peak levels of aqueous leukocyte counts and protein levels. In contrast, the levels of aqueous IL-1Ra peaked at 48 hr (12,239 +/- 1964 pg ml-1) and a fairly high concentration of IL-1Ra remained when the inflammatory reactions subsided. Immunohistochemistry and leukocyte-depletion studies showed that infiltrating leukocytes were the major cellular sources of aqueous TNF alpha, IL-1 beta and IL-1Ra. Intravitreal injection of homologous TNF alpha (0.1-1.5 micrograms) or IL-1 beta (0.5-5 ng) reproduced a rapid leukocyte infiltration and protein leakage. Administration of anti-TNF alpha mAb (10 micrograms) suppressed the number of LPS-induced infiltrating neutrophils by 50%, mononuclear cells by 58%, and protein leakage by 42%. Administration of rabbit IL-1Ra (10 micrograms) also suppressed neutrophil influx by 78%, however, neither mononuclear cell influx nor protein leakage was inhibited by rabbit IL-1Ra. Co-administration of the two inhibitors enhanced inhibition of neutrophil infiltration to 88%, and protein leakage to 64%. We conclude that TNF alpha and IL-1 beta are the principal mediators of LPS-induced uveitis. Our observations also suggest that endogenous IL-1Ra may down-regulate inflammatory reactions.