CHAPTER 38

CYTOKINE-INDUCED RETINAL DEGENERATION:ROLE OF SUPPRESSORS OF CYTOKINE SIGNALING

(SOCS) PROTEINS IN PROTECTION OF THE NEURORETINA

Charles E. Egwuagu, Cheng-Hong Yu, Rashid M. Mahdi, Marie Mameza,Chikezie Eseonu, Hiroshi Takase, and Samuel Ebong*

1. INTRODUCTION

The vertebrate retina is comprised of a collection of highly specialized cell types, witheach subtype playing unique roles and functions in the reception, transduction and con-version of incident light rays into visual images. The photo-transduction mechanism isextremely sensitive and minute alterations in the relative abundance of any retinal cell typecan severely compromise the quality of the visual image. Because ganglion cells and otherretinal neurons are terminally differentiated cells, it has been argued that evolutionarily con-served mechanisms must exist to protect retinal neurons from injury or death caused byexposure to environmental toxins or toxic bi-products of intermediary metabolism. Forexample, neuroretinal cells require protection from infectious agents that occasionally col-onize and kill them, leading to permanent loss of such cells. Although intraocular infectionsis rapidly cleared by inflammatory cells, prolonged secretion of inflammatory cytokines inthe retina may induce cytopathic effects that can produce retinal degenerative changes andpossibly retinal degeneration. Although much effort has been made in characterizing chro-mosomal mutations and other biochemical lesions that may underlie the development ofretinal degeneration, few studies have addressed the role of inflammation or inflammatorymediators in pathogenic mechanisms of retinal degenerative diseases. In fact, inflammationand dysregulation of activities of proinflammatory cytokines have been implicated in patho-genesis of other human degenerative diseases including Alzheimer’s disease and multiplesclerosis.

In this study, expression of the proinflammatory cytokine, interferon gamma (IFNg),was targeted to the lens of transgenic (TR) rats and the lens was used to serve as a depot

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*National Eye Institute, National Institutes of Health, MD, U.S.A.

for releasing IFNg into retina. This TR rat model allows us to directly test the hypothesisthat prolonged exposure of retinal cells to pro-inflammatory cytokines, as may occur duringpersistent chronic infection of the retina, can induce retinal disease or retinal degenerativechanges. We have also investigated mechanisms that may underlie protection of retinal cellsfrom hypoxia. Because suppressors of cytokine signaling (SOCS) proteins play importantrole in regulating the activation, intensity and duration of cytokine- and stress-inducedsignals,1-3 we examined whether retinal cells respond to cytokines and oxidative stress byinducing SOCS expression.

2. RESULTS

2.1. IFNg Induces Retinal Degenerative Changes and Apoptosis of Ganglion Cells

The chimeric construct used for generating the TR rats with constitutive expression ofIFNg in the eye consists of the aA-Crystallin promoter fused to the mouse IFNg codingsequence.4 Because of lens-specificity of the aA-Crystallin promoter,5,6 transgene expres-sion occurs preferentially in the lens and its effects are initially confined to this tissue.However, after the first month of postnatal life, the lens capsule begins to disintegrate, releas-ing lenticular material into the posterior chamber and vitreous cavity and this coincides temporally with appearance of the effects of IFNg on the retina. Appearance of retinal in-foldings is observed in adult TR rats after three months of age (Fig. 38.1A) and number andsize of these folds increase with time (data not shown). In addition, growth of the ganglioncell layer is significantly inhibited and its thickness approximates one-half of that seen inWT eye (data not shown).

The inhibition of ganglion cell growth is of particular importance in view of the criti-cal functions of these cells in the visual process. It is therefore interesting to note that Insitu detection of apoptotic cells by the TUNEL assay revealed presence of apoptotic cellsin TR but not in WT rat retina (Fig. 38.1B). It is even more remarkable that the apoptoticresponse is restricted to the ganglion cell layer, suggesting that ganglion cells are more sen-sitive to the effects of IFNg. In addition, the morphological changes seen in the TR rat retinacorrelates with upregulated expression of interferon regulatory factors 1 (IRF-1), interferon

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Figure 38.1. Chronic exposure of retinal cells can induce retinal degenerative changes.

consensus binding protein (ICSBP), RT1-Ba (equivalent to mouse MHC class II), ICAM-1 and TNFa genes (data not shown), suggesting that pathogenic effects of IFNg are medi-ated, in part, by altering normal patterns of gene expression in the eye.

2.2. Retinal Response to Inflammatory Cytokines is Under Feedback-Regulation by SOCS

We next examined whether cytokine activities in the retina are under feedback regula-tion by SOCS proteins. To establish that SOCS proteins are expressed in the retina, we iso-lated total RNA from human or murine retina, prepared cDNAs and subjected them to 30cycles of PCR amplification as reported previously.7 We found that CIS, SOCS4, SOCS5,SOCS6, SOCS7 are constitutively expressed at very high levels in human retina whileSOCS3 is not detectable even after 35 cycles of RT-PCR amplification. Although SOCS1 isalso detected, it is at very low levels as detection required 35 cycles.

To examine whether SOCS expression is induced in retinal cells by proinflammatorycytokines it was necessary to establish that these cytokines do indeed activate gene transcription in the retina. Human retinal pigment epithelial (hRPE) or Müller cell line wasstimulated with either interleukin 4 (IL-4) (10ng/ml) or IFNg (100u/ml) for 15min and transcriptional activation was assessed by gel-shift assay. Activation by IL-4 or IFNg ismediate through STAT6 or STAT1, respectively.8 As shown in Fig. 38.3, a retarded band isinduced by the STAT6 probe in nuclear extracts from cells stimulated with IL-4 while IFNg-

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Figure 38.2. SOCS mRNA transcripts are constitutivelyexpressed in the retina.

Figure 38.3. Inflammatory cytokines activate JAK/STAT signaling pathways in retinal cells.

stimulated cells induce a STAT1 band-shift. Presence of STAT1 or STAT6 in the retardedband is confirmed by super-shift analysis indicated by SS.

We then examined whether SOCS proteins are induced in human retinal cells by pro-inflammatory cytokines. Müller or hRPE cells was washed, starved for 2h before stimula-tion with IFNg or IL-4 and then analyzed for induction of SOCS expression by real-timeRT-PCR. As indicated in Fig. 38.4, expression of SOCS1 or SOCS3 is induced by bothcytokines, although intensity or kinetics of induction is different. Although SOCS6 orSOCS7 are constitutively expressed in these cells, they are not induced in response to theseinflammatory cytokines.

We next examined whether retina cells respond to hypoxia by inducing SOCS expres-sion. Mouse retina explants were propagated for varying amounts of time under hypoxiacondition. Induction of vascular endothelial growth factor (VEGF) or hypoxia-inducingfactor 1 (HIF-1a) expression, two markers of hypoxia, was used to verify that the cells wereindeed exposed to significant hypoxia. Induction of SOCS expression was analyzed by real-time RT-PCR and as indicated in Fig. 38.5, only SOCS3 is induced.

3. DISCUSSION AND CONCLUSION

Inflammatory cells that mediate host immunity to intraocular pathogens producecopious amounts of pro-inflammatory cytokines, IFNg and IL-4. In this study we show thatprolonged secretion of IFNg in the neuroretina promotes formation of retinal in-foldings inthe photoreceptor layer and induces apoptotic death of retinal ganglion cells. However, theseresults appear to be at variance with the fact that humans are constantly infected with avariety of pathogens that induce expression of this proinflammatory with no evidence of

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Figure 38.4. Retinal cell response to proinflammatory cytokines is under feedback regulation by SOCS proteins.

similar clinical or histological manifestation. It is however of note that these symptoms occuronly in older rats and is therefore consistent with age-dependent occurrence of retinal degen-erative diseases in older humans. The data suggests the possibility that retinal degenerationis a slow and progressive pathogenic condition that is initiated or amplified, in part, by pro-inflammatory cytokines produced by inflammatory cells elicited by low-grade persistentinfections. Thus, retinal degenerative diseases may occur only in individuals that are notable to adequately control activities of these inflammatory mediators.

Neurotrophic factors and neuregulatory cytokines, such as, CNTF, OSM, CT1, LIF,IGFs and FGFs are also produced during inflammation and have been shown to counteractdeleterious effects of proinflammatory cytokines. Thus, retinal degenerative diseases mayoccur in individuals that are not able to adequately control activities of inflammatory medi-ators or activate protective mechanism that confer protection of retinal neuronal cells fromcytokine- or stress-induced retinal damage. In the developing CNS and during spinal cordor brain injury, the steady-state levels of neuregulatory cytokines determines whether theneural progenitors would differentiate to neurons or astrocyte pathway and is therefore animportant determinant of whether healing, repair or regeneration would occur.9-11 It is ofnote that most proinflammatory cytokines and neurotrophic/neuregulatory factors mediatetheir effects through activation Janus kinase (JAK)/signal transducers and activators of tran-scription (STAT) pathway.12,13 Homeostatic regulation of activities of these competingfactors is essential to normal physiology of the retina and under stringent control by endoge-nous feedback regulators of the JAK/STAT signal transduction pathway.14

Recent studies on cellular mechanisms that switch off signals induced by growth factors and cytokines have uncovered existence of a family of endogenous negative feed-back regulators, generically called suppressors of cytokine signaling (SOCS).1-3 The bestcharacterized members of the 8-member SOCS family are SOCS1, SOCS3, SOCS5 andCIS (cytokine induced SH2-domain protein) and expression one or more of these proteinsis transiently induced by a wide variety of inflammatory and anti-inflammatory cytokines,including interferon IFNg, IL-3, IL-4, IL-6, IL-12, IL-13, leukemia-inhibitory factor, stemcell factor, CNTF, GM-CSF and leptin.1-3 Growth factors such as IGF-1, PDGF, FGFs, EGF,prolactin, growth hormone and erythropoietin also induce their expression Inhibitory effectsof SOCS proteins derive from direct interactions with cytokine receptors and/or JAKkinases, thereby preventing recruitment of STATs to the signaling complex.1-3 In addition

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Figure 38.5. Retinal cell response to hypoxia is under feedback regulation by SOCS proteins.

to functioning in a classical feedback regulatory loop, SOCS proteins can also inhibitresponses to cytokines that are different from those that induce their expression. Interest inSOCS proteins stems from the belief that SOCS may serve to integrate multiple extracel-lular signals that converge on a target cell or tissue. We show here that CIS, SOCS5, SOCS6,SOCS7 are constitutively expressed at very high levels in human and murine retinas andalthough SOCS1, SOCS2 or SOCS3 is not detectable in the normal retina, expression ofthese SOCS members is significantly upregulated by proinflammatory cytokines in retinalcells. We further show that retinas maintained under hypoxic conditions express elevatedlevels of HIF-1a and VEGF mRNAs and expression of these genes results in significantinduction of SOCS expression. However, in contrast to induction of SOCS1, SOCS2 andSOCS3 by retinal cells in response of to proinflammatory cytokines, response to hypoxia isunder feedback regulation by only SOCS3, suggesting a remarkable specificity of SOCS-mediated regulation in the retina.

In summary, we have shown in this study that similar to other neurodegenerative dis-eases, apoptotic death of retinal ganglion cells and retinal degeneration may result, in part,from chronic exposure of ocular cells to pro-inflammatory cytokines, as may occur inchronic inflammatory diseases of the eye. We also show that SOCS proteins are constitu-tively expressed in the retina and that retinal cells respond to cytotoxic cytokines or tohypoxic conditions by upregulating SOCS expression. These results are remarkable becauseSOCS proteins generally have a short half-life and are not detectable in many tissues.Demonstration that retinal cells respond to exposure to cytotoxic cytokines and hypoxia byupregulating SOCS expression, suggests that SOCS proteins may mitigate injurious effectsof environmental, chemical or oxidative stress and should be exploited as neuroprotectiveagents of the mammalian retina.

4. REFERENCES

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