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(Investigative Ophthalmology and Visual Science. 2003;44:2683-2688.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.02-0829
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Strong In Vivo Activation of NF-{kappa}B in Mouse Lenses by Classic Stressors

George Alexander,1,2 Harald Carlsen,1,2 and Rune Blomhoff1

1From the Institute for Nutrition Research, University of Oslo, Oslo, Norway.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To examine the in vivo activation of nuclear factor (NF)-{kappa}B in mouse lens epithelia by using bacterial lipopolysaccharide (LPS), tumor necrosis factor (TNF)-{alpha}, and UV-B radiation.

METHODS. Transgenic mice containing the NF-{kappa}B-luciferase reporter were injected with LPS, TNF-{alpha} or, exposed to UV-B. After various exposure times, the mice were killed, and ocular, liver, lung, kidney, spleen, and skin tissue were obtained. Tissue homogenates were examined for luciferase activity with a luminometer. Groups of mice were also imaged in vivo through a light-intensified camera system to assess NF-{kappa}B activity.

RESULTS. LPS- and TNF-{alpha} injected NF-{kappa}B-luciferase transgenic mice yielded 20- to 40-fold increases in lens NF-{kappa}B activity, similar to other LPS- and TNF-{alpha}–responsive organs. Peak NF-{kappa}B activity occurred 6 hours after injection of TNF-{alpha} and 12 hours after injection of LPS. Peak activities were, respectively, 3 and 6 hours later than that in other tissues. Mice exposed to 360 J/m2 of UV-B exhibited a 16-fold increase in NF-{kappa}B activity 6 hours after exposure, which are characteristics similar to TNF-{alpha}–exposed mice. In vivo imaging of transgenic mice exposed to LPS, TNF-{alpha}, and UV-B radiation demonstrated a similarity between in vitro and in vivo measurements of NF-{kappa}B activity.

CONCLUSIONS. In NF-{kappa}B-luciferase transgenic mice, NF-{kappa}B activity occurs in lens epithelial tissue and is activated when the intact mouse is exposed to bacterial LPS, TNF-{alpha}, or UV-B. Lens epithelial NF-{kappa}B kinetics were comparable to those of other tissues, indicating that NF-{kappa}B may play a role in progression or arrest of lens disorders.

NF-{kappa}B is a family of transcription factors comprising p65 (RelA) and p50 subunits. It is localized in the cytoplasm and is sequestered by the inhibitory protein I{kappa}B, which renders it inactive. Five I{kappa}B forms exist, with two, I{kappa}B{alpha} and I{kappa}Bß, showing phosphorylation in response to different extracellular stimuli, ultimately modulating the activity of NF-{kappa}B. Injury-, immune-, or stress-responsive mediators, such as the cytokines IL-1, IL-6, and TNF-{alpha}, activate NF-{kappa}B by initiating the dissociation of I{kappa}B, permitting the translocation of the unbound NF-{kappa}B dimer into the nucleus. The now-activated NF-{kappa}B dimer binds to the regulatory NF-{kappa}B elements in the target genes—activating genes involved in their respective immune, tissue repair, or apoptotic processes.1

The eye is exposed to various cytokines and factors that are released as a result of injury or disease processes,2 3 and the lens itself can be exposed to inflammatory factors that are subsequently present in the aqueous humor.4 Even though the lens is located within the very center of the eye, it would be expected to exhibit cytokine-related NF-{kappa}B dynamics, because the lens epithelium has been shown to produce cytokines.5 6 Only recently has the first indication of a role for NF-{kappa}B in alteration of lens epithelial function been suggested. Dudek et al.,7 using TNF-{alpha} as an excitatory cytokine on a human lens epithelium cell line, showed strong activation of NF-{kappa}B and the degradation of both I{kappa}B-{alpha} and -ß. In addition, they demonstrated an H2O2-mediated activation of NF-{kappa}B, although, in this case, both I{kappa}Bs were not degraded, which may be related to the transformation state.8

The lens is subject to injury and disease processes that often show NF-{kappa}B activation in many other tissues. Lens damage manifests itself as an increase in opacity up to the point of the lens being completely opaque (e.g., cataractogenesis). Diabetes, for example, is strongly associated with development of cataract, and loss of lens clarity may be related to the oxidative stress associated with the presence of diabetes-induced advanced glycation end products (AGEs). Cataract is also associated with a direct and rapid injury to the eye due to trauma, or, is the result of a slow, ongoing injurious process, such as prolonged exposure to ultraviolet radiation (UV-R). When the lens epithelium is assaulted as just described, its injury response generally does not prevent the onset of cataract. A large body of research has demonstrated the various caspases and cytokines associated with the irrecoverable decline in lens clarity,9 especially in whole-lens experiments.10 Aside from the in vitro experiments of Dudek et al.,7 the role of NF-{kappa}B in lens damage is quite undefined and begs the question of whether NF-{kappa}B is under-, over-, or misresponding to the various stimuli. They suggest that an appropriate NF-{kappa}B activation pathway may be necessary to effect an appropriate NF-{kappa}B–directed gene expression that is relevant to the nature of the original stimulus. It may well be that cataractogenesis is the result of the NF-{kappa}B activation pathway’s being mismatched to the original stimulus, resulting in an inappropriate NF-{kappa}B–directed gene expression.

Although the importance of in vitro experiments is recognized, a demonstration of NF-{kappa}B dynamics in an in vivo context would be valuable for a better understanding of this transcription factor in the lens. Specifically, modulating presumed NF-{kappa}B activity through the use of "classic" stressors would provide a foundation for understanding how the lens may respond to disease and injury processes. In the present study, we exposed in vivo the eyes of transgenic NF-{kappa}B-luciferase (NF-{kappa}B-luc) mice to three kinds of classic stressors and examined the kinetics of NF-{kappa}B response. We induced externally mediated stress by exposing the eye to UV-B radiation, whereas we mimicked systemic septicemia-associated stress by treating NF-{kappa}B-luc mice with LPS.11 Finally, we administered the injury-response cytokine TNF-{alpha} to mimic a trauma-related response12 and examined subsequent NF-{kappa}B dynamics in intact native lens epithelia.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Materials
The anesthetics fentanyl citrate and midazolam were purchased from Janssen (Beerse, Belgium) and Roche (Basel, Switzerland), respectively. TNF-{alpha} was procured from R&D Systems (Minneapolis, MN), and LPS was purchased from Sigma (St. Louis, MO). Lysis buffer was obtained from Promega (Madison, WI), and luciferin substrate was purchased from Biothema (Dalaro, Sweden).

Transgenic Mouse Development
The transgenic mice used in this experiment were the same as those used by this laboratory in previous experiments.13 Briefly, superovulated female (C57 BL/6J x CBA/J)F1 mice were mated overnight with F1 males. The pronuclei of the resultant zygotes were injected with the 3x-{kappa}B-luc plasmid linearized with HindIII and BglI. Pseudopregnant CD-1–recipient mice were impregnated with the surviving zygotes. Offspring (F0) were tested for 3x-{kappa}B-luc by PCR genotyping. The founder mouse was crossed with wild-type F1 mice, and all subsequent transgenic offspring were also crossed with wild-type F1 to produce heterozygous 3x-{kappa}B-luc mice with the (C57BL/6J x CBA/J) genetic background. The mice were housed in a controlled light (12-hour light-dark), humidity, and temperature environment and were provided food and hydration ad libitum. All experiments were conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and followed the directives of the Norwegian Animal Welfare Authority.

Injections of LPS and TNF-{alpha}
Before the introduction of stimuli, the NF-{kappa}B-luciferase mice were anesthetized with a 1.25 mg/mL midazolam, 2.5 mg/mL fluanisone, and 0.079 mg/mL fentanyl citrate preparation. TNF-{alpha} (4–8 µg/kg) was introduced by an intravenous injection in the tail vein. LPS (Escherichia coli serotype 055:B5, 2 mg/kg) was similarly administered.

UV Radiation Protocol
Before exposure to UV-B, wild and NF-{kappa}B-luciferase mice were anesthetized with Hypnorm-Dormicum. For the skin-exposure experiments, we exposed the shorn ventral portion of a number of transgenic mice to 1 minimal erythema dose (MED; 360 J/m2) of UV-B radiation. From 2 to 24 hours after UV-B exposure, we recorded the photons emitted, after suitable injection of luciferin substrate, through an image-intensifying high-resolution camera. For the ocular experiments, the irises of both eyes were dilated with tropicamide (0.5%), and the right eye of each mouse received UV-B radiation ({lambda}max 295 nm) during a 15-minute period with exposure doses ranging from 0.4 to 8 kJ/m2; the left eye served as the control. The radiation apparatus consisted of an air-cooled high-pressure 100-W mercury-xenon lamp (PTI) providing light, which was collimated and directed through a water-cooled water filter, redirected through a dichroic mirror (reflecting wavelengths, <400 nm), and directed upward through an interference filter (297 FS 10-50, {lambda} max= 295 nm, {lambda} 0.5= 8.8 nm; Andover Corp., Salem, NH). The UV-B output (fluence rate, µW/cm2) was measured by a UV-B detector (model 7104; Oriel, Stratford, CT) that had been calibrated by the manufacturer within the previous 2 months. The light’s intensity was measured before and after each exposure.

Sampling Protocol for LPS, TNF-{alpha}, and UV-B Treatments
After exposure to the various regimens, the mice were humanely killed. Their eyes were immediately removed and placed in an in vitro fertilization Petri dish containing artificial aqueous humor (AAH) and the lens extracted by separation of the cornea and posterior portion of the globe along the ora serrata. Intact lenses were placed in a 1.5-mL microcentrifuge tube and quick frozen in liquid nitrogen. The luciferase assay was performed either immediately or the next day.

Luciferase Assay: In Vitro
The intact lenses were thawed, and 200 µL of lysis buffer was pipetted into the 1.5-mL microcentrifuge tube containing the lens. The microcentrifuge tube containing the tissue and lysis buffer were flash frozen in liquid nitrogen and subsequently allowed to thaw. After a 15-minute reaction period at room temperature, the tubes were placed on ice for a further 15 minutes. Subsequently, the tubes were centrifuged at 12,500g for 5 minutes at 4°C. A 100-µL aliquot of supernatant was removed for the luciferase assay and a 10-µL aliquot was used for protein determination (Bradford). Luciferase activity was determined by the introduction of 100 µL of luciferin substrate (D-luciferin) to the 10-µL aliquot of supernatant, followed immediately by brief vortexing and measurement in a luminometer (Turner Designs, Mountain View, CA). Measurements of lens weight and amount of protein liberated by freeze-thaw in lysis buffer was consistent between the eyes of different mice; therefore, the NF-{kappa}B activity in the lens, as reported by luciferase in these transgenic mice, is expressed as luminescence per eye. The measurement of NF-{kappa}B activity in other tissues were accomplished by mincing tissue in a homogenizer (Ultra Turrax; Janke & Kunkel, Staufen, Germany) in lysis buffer and subsequently freeze thawing the tissue-lysis buffer homogenate. NF-{kappa}B activity from these tissues was reported as luminescence per microgram of tissue protein.

Luciferase Assay: In Vivo
Mice that had their shorn ventral epidermis exposed to UV-B radiation were anesthetized as described and injected in the tail vein with D-luciferin (120 mg/kg) dissolved in 200 mM PBS (pH 7.8). The mice were then immediately placed in a light-sealed imaging chamber fitted with an image intensifier coupled to a charge-coupled device (CCD) camera (C2400-47; Hamamatsu, Stockholm, Sweden) fitted with a 25-mm macro lens (Schneider Optics, Hauppauge, NY). The luminescence emitting from the mouse was integrated for 10 minutes, starting 2 minutes after the injection of luciferin. Images were analyzed on computer (Image-Pro Plus 4.0 software; Media Cybernetics, Silver Spring, MD), to determine the relative flux of photons emitting from the transgenic mice.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
In Vivo Measurement of NF-{kappa}B Activity in Various Tissues
In a previous study, we have used NF-{kappa}B-luc transgenic mice to assess NF-{kappa}B activity in an in vivo setting with an intensified camera system.13 The false-color image of the control mice in Figure 1A indicates that there is general absence of intrinsic NF-{kappa}B activity, except for a few discreet regions: the neck (lymph nodes) and thoracic (thymus) and abdominal (Peyer’s patches) regions. When treated with LPS, increases in NF-{kappa}B activity can be visualized (Fig. 1B) . TNF-{alpha}–injected mice present a similar but less intense overall activation of NF-{kappa}B (Fig. 1C) . Similarly, an increase in NF-{kappa}B activity in a discreet region of the integument, which was exposed to UV-B radiation, was clearly visible (Fig. 1D) . Initial in vivo camera-based experiments in our laboratory indicated that NF-{kappa}B-luc photon fluxes from ocular tissues were not sufficient for intensified camera use (data not shown), necessitating the use of tissue homogenates and a luminometer for an accurate assessment of NF-{kappa}B activity.



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  		FIGURE 1. In vivo imaging of NF{kappa}B-luciferase activity in NF-{kappa}B-luc transgenic mice exposed to inflammatory agents. (A) Control mouse, positioned dorsally, imaged 4 hours after tail vein injection with saline. The false-color image in this and all panels represents a range of NF-{kappa}B activity reporting, with blue being the lowest, spectrally progressing through red and finally, white as the highest measurement of activity. Intrinsic NF-{kappa}B activity was present in the neck and thoracic regions. (B) Image of LPS-injected mouse 4 hours after injection. Greatest NF{kappa}B activity reported at the thyroid, lung, liver, and paw regions. (C) Image of TNF-{alpha}–injected mouse 4 hours after injection. Greatest NF-{kappa}B activity occurred in the thyroid, thymus, lung, and liver region. (D) False-color with superimposed gray-scale nonintensified image of transgenic mouse exposed to 1 MED UV-B radiation in the ventral region and imaged 24 hours after exposure. Concentric circles of color from blue to white represent increasing signal of NF-{kappa}B activity.

 
Effects of Known NF-{kappa}B Inducers on the Murine Lens
Because biological responses to inflammatory agents usually involve upregulation that occurs within a few hours, we assessed the time course of NF-{kappa}B activity regulation in the murine lens by injecting groups of transgenic mice with LPS and TNF-{alpha} and then exposing them to UV-B (Fig. 2) . LPS injection showed essentially no increase in NF-{kappa}B activity upregulation over the first 2 hours, whereas, during the next 4 hours there was a marked increase in NF-{kappa}B activity with a peak activity occurring 12 hours after injection and evidence of decline by 24 hours after injection. TNF-{alpha}, in contrast, acted in a more rapid manner (Fig. 2) with a level of NF-{kappa}B activity 2 hours after injection equal to 4 hours after injection in LPS-injected mice. The peak level of NF-{kappa}B activity did peak, however, at 6 hours after injection, with induction levels approximately half that of the peak levels of LPS-injected mice. Over the following 6 hours, NF-{kappa}B activity declined to almost basal levels with a clear basal level of activity at 24 hours after injection. The effect of the administration of 4 kJ/m2 UV-B radiation on lens NF-{kappa}B activity is also shown in Figure 2 . A steady increase in NF-{kappa}B activity occurred during the initial 6 hours after exposure, with the level of induction at 6 hours comparable to those in TNF-{alpha} injected mice. Levels of NF-{kappa}B activity significantly decreased by 24 hours after UV-B exposure.



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  		FIGURE 2. 3 Time course of NF-êB induction after injection or exposure to inflammatory agents. Lens homogenates were assessed for luciferase activity as a measure of NF-{kappa}B activity. Data represent the magnitude of induction compared with intrinsic NF-{kappa}B activity in control mice for LPS and TNF-{alpha} or compared with contralateral, nonexposed eyes for UV-B–exposed mice. Peak levels of TNF-{alpha} and LPS were significantly higher (P < 0.05, Student-Newman-Keuls test) than pre- and postpeak measurements. Peak measurement for UVB was significantly higher than initial and 2-hour measurements (P < 0.05, Student-Newman-Keuls). n = 6–8 for all measurements.

 
Time Course of NF-{kappa}B Induction by LPS and TNF-{alpha} Measured in Tissue Homogenates
To compare the kinetics of lens epithelium NF-{kappa}B response to that of other organs, we injected LPS and TNF-{alpha} and removed the organs at different intervals (Figs. 3A 3B) . The kinetics of LPS-inducted NF-{kappa}B activity in liver, spleen, and lung homogenates clearly showed a trend of maximum activation occurring 4 to 6 hours after stimulation with kidney and skin tissues responding maximally 6 hours after stimulation (Fig. 3A) . TNF-{alpha}–induced NF-{kappa}B activity (Fig. 3B) appeared to peak consistently at 3 hours after stimulation in all tissues except skin, which exhibited maximum NF-{kappa}B activation at approximately 12 hours (data not shown).



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  		FIGURE 3. 3 Time course and comparison of NF-êB induction in tissue homogenates after prior injection of LPS or TNF-{alpha}. (A) Luminometer measurements of tissue homogenates after IV injection of LPS. Data represent the percentage inducti3 on compared with peak NF-êB activation for each particular tissue. Liver, spleen, and lung tissue had first significant maximum at 4 hours, kidney and skin had first significant maxima at 6 hours. (B) Luminometer measurements of tissue homogenates after IV injection of TNF-{alpha}3. Data represent the percentage induction compared with peak NF-êB activation for each type of tissue. The peak NF-êB activity at 3 hours for all tissue except skin was significantly higher than previous measurements. Spleen tissue showed no significant reduction in NF-êB activity after initial peak. Skin tissue showed a first significant peak (compared with time 0) at 3 hours, with a significant decrease at 4 hours and significant increase at 6 hours. (C) Luminometer measurements of tiss3 ue homogenates after IV injection of LPS. Data represent manifold induction measured at peak NF-êB activation time for each tissue. Liver and lung tissue were not significantly different in induction; however, they were significantly different from all other tissues (P < 0.01). Kidney, skin, and lens tissue did not significantly differ, whereas spleen was significantly the lowest. (D) Luminometer measurements of tissue homogenates after IV injection of TNF-{alpha}. Data represent manifold induction measured at3 peak NF-êB activation time for each tissue. Liver and lung tissue were not significantly different in induction, but they were significantly different from all other tissues (P < 0.01). Kidney and lens tissue were not significantly different, but their induction levels were significantly higher than those of spleen and skin tissue. n = 6–8 for all time points and unless otherwise indicated, significance is P < 0.05 based on Student-Newman-Keuls test.

 
Relative Levels of LPS- and TNF-{alpha}–Induced NF-{kappa}B Activity in Tissue Homogenates
A comparison of the relative peak levels NF-{kappa}B activity in liver, spleen, lung, kidney, skin, and ocular tissue, as induced by LPS and TNF-{alpha}, are shown in Figures 3C and 3D , respectively. LPS-induced lens homogenates exhibited a 40-fold increase in NF-{kappa}B activity, higher than the 20-fold increase in activity in spleen tissue and not significantly different from the 50-fold increase in skin tissue homogenates. Induced NF-{kappa}B activity in kidney, liver, and lung was somewhat higher (80–210-fold) than in the lens; however, the former also exhibited a greater variation of response, compared with the consistent response of lens tissue.

TNF-{alpha}–induced NF-{kappa}B activity exhibited a remarkably similar trend, although overall, induction levels were 50% of LPS-induced activity. Lens homogenates exhibited a 20-fold increase in NF-{kappa}B activity, comparable to kidney tissue and significantly higher than the 8-fold increase in activity in spleen tissue. Skin tissue had only a threefold increase in NF-{kappa}B activity, whereas, lung and liver tissue again exhibited relatively high induction values (65–90-fold increase). Lung and liver tissue retained their high variation of response, whereas lens homogenate responses exhibited the same level of consistency seen in the LPS-induced lenses.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
We have been able to demonstrate that, under in vivo conditions, NF-{kappa}B activity is present in the murine lens epithelium, can be stimulated with known NF-{kappa}B inducers, and demonstrates kinetics consistent with a cytokine response. The NF-{kappa}B-luciferase transgenic mice used in our experiments have already demonstrated their utility in the assessment of NF-{kappa}B activity in virtually all tissues in the mouse in both an in vitro and in vivo manner.13 Contag et al.,14 using transgene luciferase-reporting, demonstrated that transgene-produced luciferase stays within the cell and that the injected luciferin enters all cells, with the photon-emitting luciferin-luciferase activity taking place within luciferase-producing cells only. Before the present report, there have been few reports of NF-{kappa}B activity in ocular tissue, with the only evidence heretofore of NF-{kappa}B activity in the lens being presented by Dudek et al.,7 who used hydrogen peroxide as an inducer on a lens epithelium culture preparation.

To assess the biological relevance of NF-{kappa}B activity in the lens, we initially assessed inflammatory agent–induced NF-{kappa}B activity in other selected tissues that already had well-established NF-{kappa}B responses. Measuring in vivo and using a light-intensified camera, we demonstrated that a number of internal tissues (Figs. 1A 1B 1C) had NF-{kappa}B responses consistent with a inflammation response when induced by bacterial LPS and the cytokine TNF-{alpha}. Similarly, the ventral integument, when shorn and exposed to UV-B radiation, also exhibited appropriate NF-{kappa}B responses (Fig. 1D) . Previous DNA-binding experiments confirmed that there was a strong association between luciferase induction and p65 DNA binding,13 indicating that our transgenic mouse model was demonstrating NF-{kappa}B activity. Clearly, our transgenic mice showed changes in NF-{kappa}B activities that were activated through separate initial pathways.

The induction of septicemia (by LPS) and inflammatory (through TNF-{alpha} and UV-B radiation) stress in the intact mouse indicated that these circulating or external factors modulate NF-{kappa}B activity in the lens epithelial tissue in a manner similar to other tissues. As illustrated in Figure 2 , lens epithelia showed a relatively rapid response to the direct administration of TNF-{alpha}, as would be expected, because TNF-{alpha} acts directly on inhibitory I{kappa}B kinases (IKKs), liberating the p50 and p65 Rel A subunits that dimerize to form NF-{kappa}B.15 The lens epithelial response to LPS indicated an initial 2-hour lag with a maximum response 6 hours later than that of TNF-{alpha}–induced NF-{kappa}B activity. Because LPS itself is believed to initiate a TNF-{alpha} chain of events leading to NF-{kappa}B activation,16 this lag period is consistent with inflammation response kinetics.17 When compared with the in vitro NF-{kappa}B responses of other tissues to LPS and TNF-{alpha}, (compare Figs. 2 3A 3B ), the NF-{kappa}B activation of lens epithelia clearly occurred 2 to 8 hours later. This may be due to the avascular nature of the intraocular compartment. The administration of any agent would first take some time to reach sufficient levels in the ocular humors to have an effect on the lens epithelial tissue and would account for some of the time lags that occurred in this experiment.4 Second, the anterior chamber of the eye is considered to be an immune-privileged region containing immunosuppressive factors that may require first the production of IL-6 to compromise this immunosuppression18 and permit measurable increases in NF-{kappa}B.

When induced by LPS the extent of NF-{kappa}B activation in the lens epithelia appears to be comparable to the skin’s response to LPS (Fig. 3C) . This is perhaps not surprising, because both tissues are epithelial in nature, and LPS may in part be promoting the production of TNF-{alpha}, which itself is promoting the activity of NF-{kappa}B.19 It must be noted, however, that the skin in our experiment exhibited a highly variable LPS-induced NF-{kappa}B activation.

As septicemia is associated with various ocular disorders including cataract, the NF-{kappa}B activity presently demonstrated in lens epithelium may have a significant role to play in these disorders. LPS has been shown to increase plasma concentrations of TNF-{alpha}20 with a subsequent stimulation of further cytokine production in immune and skeletal tissues. LPS may then be acting both directly and indirectly (through TNF-{alpha} induction) on NF-{kappa}B activity. Perhaps suppression of LPS-induced cytokine production by amiloride21 or pyrrolidine dithiocarbamate (PDTC)22 with an ensuing alteration of NF-{kappa}B dynamics may have a beneficial effect on the progression or regression of various ocular disorders. Measuring pharmacological and phytochemical alterations of NF-{kappa}B dynamics in lens and other tissues would be possible with the use of our in vivo–ex vivo NF-{kappa}B-luc mouse protocol.

A similar argument could be made for TNF-{alpha}–induced NF-{kappa}B activation of lens epithelia; however, this cytokine elicits a response in lens epithelia that compares favorably to the response in kidney tissue (Fig. 3D) . This comparison indicates that the lens epithelium has biologically relevant levels of NF-{kappa}B activity. This observation is of interest because the development of cataracts is, in part, shown to be associated with the presence of TNF-{alpha} and indicate that the action or inaction of NF-{kappa}B has a role in the development of cataracts. As seen in septicemia, a general inflammation due to disease or injury may have a deleterious effect on the lens, even though it may have been spared the initial disease or injury. For example, liver inflammation or injury is known to activate liver Kupffer cells, which produce TNF-{alpha} as a result.23 This cytokine may then enter the circulatory system, ultimately reaching the eye. It may be worthwhile to consider that NF-{kappa}B–altering agents such as dexamethasone or phytochemicals10 24 may have a (one hopes) positive influence on cataract development because corticosteroids, for example, appear to lower TNF-{alpha} levels in the aqueous humor of patients who have undergone cataract surgery25 ; however, a caveat must be introduced with regard to the use of glucocorticoids, because even though they have the effect of lowering NF-{kappa}B activity26 27 the use of these anti-inflammatory agents have been shown to induce cataract formation in chick embryos.28 29

The in vivo administration of UV-B radiation in doses shown to initiate scattering in lenses30 also demonstrated a timely increase and decrease in NF-{kappa}B activity in lens epithelial tissue (Fig. 2) . The kinetics and extent of NF-{kappa}B activation are somewhat similar to TNF-{alpha}–induced activity with UV-B–induced NF-{kappa}B activity maintaining higher levels after 24 hours, in that NF-{kappa}B activation by UV-B in human skin fibroblasts exhibit a late mechanism at 15 to 20 hours after UV exposure and results in the maintenance of NF-{kappa}B activity through a DNA-induced release of IL-1{alpha}.31 Presumably, as the UV-B radiation has a direct intracellular effect, the initial dynamics of a stress response would be similar to a TNF-{alpha}–directed response,32 although some of the dynamics of the NF-{kappa}B response would be altered by the redox state of the cell.33 Once stimulated, the UV-B–induced increase in NF-{kappa}B levels would play a proapoptotic role such has been seen in UV-exposed U20S cells.34 As seen in Figure 2 and described earlier, 24 hours after UV-B exposure NF-{kappa}B levels dropped to 50% of peak levels. Daily exposure to UV-B radiation may well cause long-term elevation of NF-{kappa}B levels, possibly through a TNF-{alpha} activation pathway, and drive the cellular apoptosis–anti-apoptosis balance, possibly modifying cellular processes in the lens epithelium sufficiently to permit cataractogenesis. This process may be altered through the use of an anticancer agent, inositol hexaphosphate (InsP6), which has been shown to alter UV-B–induced signal transduction.35 In addition, using phytochemicals that have the property of altering signal transduction pathways36 would be of value in determining ways to manipulate NF-{kappa}B levels to alter the inexorable process of lens damage as the result of UV-B exposure.

We present evidence that the murine lens exhibits NF-{kappa}B responses to classic inflammatory agents and, accounting for the physiological factors associated with the intraocular environment, these responses are comparable to those in other NF-{kappa}B–inducible tissues. Future research into various conditions that have an effect on the lens should take NF-{kappa}B dynamics into account, including the possibility that alteration of NF-{kappa}B dynamics through various agents may sufficiently mitigate lens physiology alterations and maintain lens clarity.


   Acknowledgements
 
The authors thank our technician Kari Holte and the Skin Research Department at the Ullevål University Hospital in Olso, Norway, for the use of the UV illumination apparatus.


   Footnotes
 
2 Contributed equally to the work and therefore should be considered equivalent authors. Back

Supported by The Norwegian Research Council, the Norwegian Cancer Society, and The Johan Throne Holst Foundation

Submitted for publication August 15, 2002; revised December 26, 2002; accepted January 29, 2003.

Disclosure: G. Alexander, None; H. Carlsen, None; R. Blomhoff, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: George Alexander, Johan Throne Holst Institute for Nutrition Research, University of Oslo, PO Box 1046, Blindern, 0316 Oslo, Norway; george.alexander{at}basalmed.uio.no.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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  7. Dudek, EJ, Shang, F, Taylor, A. (2001) H(2)O(2)-mediated oxidative stress activates NF-kappaB in lens epithelial cells Free Radic Biol Med 31,651-658[CrossRef][Medline][Order article via Infotrieve]
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