Reactive oxygen species (ROS) generated during inflammation are believed to play critical roles in various ocular diseases. However, the underlying mechanisms remain poorly understood. We investigated if pro-inflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin-1β (IL-1β), and interferon-γ (IFN-γ), induce ROS in human retinal pigment epithelial (RPE) cells. TNF-α, IL-1β and IFN-γ increased both intracellular and extracellular ROS production in a time-and dosedependent manner. Thenoyltrifluoroacetone (TTFA), an inhibitor of mitochondrial respiratory chain, blocked TNF-α-and IFN-γ-, but not IL-1β-induced ROS, whereas other two mitochondrial respiratory chain inhibitors, rotenone and antimycin A, had no effect. NADPH oxidase inhibitor (diphenylene iodinium) abolished the ROS production induced by IL-1β or IFN-γ, but not by TNF-α, whereas 6-aminonicotinamide (6AN), an inhibitor of the hexose monophosphate shunt (HMS), had no significant effects on the ROS induced by all three cytokines. ROS scavengers, pyrrolidinedithiocarbamate (PDTC) and N-acetyl-cysteine (NAC), reduced the levels of ROS induced by TNF-α, IL-1β and IFN-γ (P < 0.05). Collectively, these results demonstrate that TNF-α, IL-1β and IFN-γ increase mitochondrial-and NADPH oxidase-generated ROS in human RPE cells.

CD14, a GPI-linked protein, plays a pivotal role in LPS-mediated signaling by potentiating leukocyte adherence, activation, and cytokine production. Recent studies have identified the Toll-like receptor 4 (TLR4) as a membrane cofactor in LPS-mediated transmembrane signaling in cytokine induction, although the mechanism responsible for this cooperation is unknown. Using fluorescence resonance energy transfer (RET) techniques, we demonstrate that LPS triggers a physical association between CD14 and TLR4. Because LPS stimulation up-regulates CD14 and TLR4 expression, it was necessary to control for the possibility that these newly expressed molecules were associated with one another independent of LPS stimulation. Although the calcium ionophore A23187 increased the expression of CD14 and TLR4, they did not exhibit energy transfer. However, following A23187 treatment, LPS promoted physical proximity between CD14 and TLR4. Therefore, we suggest that a close interaction between CD14 and TLR4 participates in LPS signaling, leading to nuclear translocation of NF-κB.

Age-related macular degeneration (AMD) is a genetically heterogeneous disease that leads to progressive and irreversible vision loss among the elderly. Inflammation, oxidative damage, cholesterol metabolism and/or impaired function of retinal pigment epithelium (RPE) have been implicated in AMD pathogenesis. We examined toll-like receptor 4 (TLR4) as a candidate gene for AMD susceptibility because: (i) the TLR4 gene is located on chromosome 9q32-33, a region exhibiting evidence of linkage to AMD in three independent reports; (ii) the TLR4-D299G variant is associated with reduced risk of atherosclerosis, a chronic inflammatory disease with subendothelial accumulation; (iii) the TLR4 is not only a key mediator of proinflammatory signaling pathways but also linked to regulation of cholesterol efflux and (iv) the TLR4 participates in phagocytosis of photoreceptor outer segments by the RPE. We examined D299G and T399I variants of TLR4 in a sample of 667 unrelated AMD patients and 439 unrelated controls, all of Caucasian ancestry. Multiple logistic regression demonstrated an increased risk of AMD in carriers of the G allele at TLR4 residue 299 (odds ratio=2.65, P=0.025), but lack of an independent effect by T399I variant. TLR4-D299G showed an additive effect on AMD risk (odds ratio=4.13, P=0.002) with allelic variants of apolipoprotein E (APOE) and ATP-binding cassette transporter-1 (ABCA1), two genes involved in cholesterol efflux. Interestingly, the effect of TLR4, APOE and ABCA1 variants on AMD susceptibility was opposite to that of association with atherosclerosis risk. Our data provide evidence of a link between multiple diverse mechanisms underlying AMD pathogenesis.

Having previously associated metabolic oscillations with cell locomotion, we hypothesized that patients with abnormalities in neutrophil trafficking may display aberrant intracellular oscillations. A pyoderma gangrenosum patient exhibiting aberrant leukocyte trafficking in vivo and skin ulceration without infection was identified. This patient's neutrophils constitutively overexpressed and clustered the leukocyte integrins CR3 and CR4 and failed to display appropriate integrin-to-GPI receptor interactions. Increased levels of tyrosine phosphorylation were observed. NAD(P)H oscillations, which are sinusoidal in normals, were chaotic with multiple frequency components in this patient's neutrophils. Normal cell shape and sinusoidal NAD(P)H oscillations were restored by providing a pulsed electric field to drive metabolic oscillations and by temperature reduction. N-acetyl-D-glucosamine disrupted CR3 clusters and sinusoidal NAD(P)H oscillations returned. Anecdotal reports suggest that local hypothermia is clinically useful for this patient. These data define the first metabolic oscillation-associated disease and suggest that pyoderma gangrenosum can be classified as a dynamical disease at the cellular level.