Human caspase-5 and -4 are the two most closely related caspases. Similar to our previous results on caspase-4,16
the present study demonstrates that caspase-5 mRNA expression, protein production, activation, and catalytic activity were all inducible by a variety of proinflammatory agents, such as IL-1β, TNF-α, LPS, and monocyte coculture in addition to the previously reported, and here reconfirmed, agents LPS and IFN-γ. Expression of caspase-5 was subject to stimulation by the ER stress inducers thapsigargin and tunicamycin. These results were similar to those we previously reported for caspase-4 in hRPE cells.16
However, the contribution to ER stress–induced hRPE apoptosis by caspase-5 is much less than that of caspase-4. Although caspase-4 inhibitor Z-LEVD-fmk blocked 59% of tunicamycin-induced hRPE cell death as detected by DNA fragmentation and 62% reduction in TUNEL positivity, capase-5 and -1 dual inhibitor Z-WEHD-fmk inhibited DNA fragmentation by 32% and TUNEL positivity by 20%, suggesting that the contribution of caspase-5 to ER stress–induced hRPE cell death is much less than that of caspase-4.
Of note, Western blot analysis of caspase-5 consistently showed an intermediate product with molecular weight of 30 kDa, consistent with previous observations.3,12
The nature of the intermediate product is unclear. It could be the pro-large subunit as described for caspase-5 closely related caspase-13.25
In addition, a 42-kDa band immediately below the 47-kDa pro-caspase-5 band was observed, as previously reported.3
This protein is likely produced from pro-caspase-5 variant b, which has an expected molecular weight of 5 kDa less than pro-caspase variant a. Variants a and b are both predominant isoforms of human caspase-5.4
By using inhibitors and shRNA knockdown, we further confirmed that caspase-5 was functionally involved in the regulation of hRPE IL-8 and MCP-1 chemokine expression. LPS-induced IL-8 secretion and MCP-1 secretion were sensitive to the inhibition by Z-WEHD-fmk, an inhibitor for caspase-5 and -1. When caspase-5 was knocked down by shRNA, TNF-α–induced IL-8 and MCP-1 production was markedly reduced by 56% and 35%, respectively. hRPE IL-8 and MCP-1 expression were also inhibited by caspase-1 inhibitor Z-YVAD-fmk and caspase pan inhibitor Z-VAD-fmk, although the inhibition by Z-YVAD-fmk was more effective in LPS than TNF-α induction, suggesting that the signaling pathways induced by TNF-α are differentially regulated by caspase-5 and -1. We have shown previously that the induction of hRPE IL-8 and MCP-1 production in hRPE cells is mediated by multiple signaling pathways, including the activation of NF-κB.22,26
Activation of NF-κB has been reported by caspase-1 by a receptor interacting protein 2–dependent pathway8
or by interaction with the TRAIL-DR5 system.27
Therefore, one possible pathway for caspase-5– and -1–mediated upregulation of IL-8 and MCP-1 expression could be by the activation of NF-κB.
We demonstrated that caspase-5 and caspase-1 mutually regulated each other in their protein expression and activation. Regulating caspase-1 activation by the caspase-5 murine homolog caspase-11 has been well documented,5,28
but there has been no report on the regulation of human caspase-1 expression and activation by caspase-5. In this study, we showed that shRNA knockdown of caspase-5 reduced not only caspase-5 but also caspase-1 expression and activation. Conversely, the caspase-1 inhibitor Z-YVAD-fmk also inhibited the expression and activation of caspase-5. The latter finding was consistent with a previous study that showed caspase-1 to be required for the complete maturation of caspase-5.12
Our results suggest that caspase-5 and caspase-1 may work in concert in the inflammasome to modulate inflammation and immunity in hRPE cells.
There have been no reports about the effects of anti-inflammatory medicines on caspase-5 expression and activation. As shown in this study, proinflammatory agent-induced caspase-5 expression was mitigated by adding anti-inflammatory agents. Dex has numerous anti-inflammatory effects, which include the suppression of cytokine-mediated responses. Intraocular Dex concentrations achieved clinically through systemic and topical delivery range from 10−8
A few reports30–32
demonstrate that Dex reduces caspase-1 activation in a variety of cell types. In addition to Dex, IL-10 has been reported to relieve inflammation, improve cell survival, and inhibit caspase-1 activation in human monocytes.33
Another study showed that IL-10 reduced caspase-1 expression in rat cortical astrocytes.34
The results presented here showed that both Dex and IL-10 strongly reduce the simulated expression and activation of caspase-5; thus, IL-10 could also be a valuable therapeutic agent in ocular inflammation. In our previous study,16
neither Dex (10 μM) nor IL-10 (100 U/mL) induced noticeable apoptosis under the same conditions. Our results may indicate a new potential role of these drugs in ocular therapy. TA is a synthetic crystalline corticosteroid with potent anti-inflammatory properties. Recently, TA was delivered by intravitreal injection for the treatment of posterior ocular diseases, such as AMD,35
retinal vein occlusion,37
diabetic macular edema,38
and proliferative vitreoretinopathy.39
After intraocular injections of 20 to 25 mg TA in patients, the average concentrations of TA in aqueous humor are lower than 0.01 mg/mL during the first 12 months of injections, while the measured levels of serum TA are negligible within 4 to 92 days.40,41
In contrast to more water-soluble Dex, which is only weakly cytotoxic in hRPE cells, some studies indicate that significant TA toxicity of ARPE19 cells can be observed at a 1.0-mg/mL concentration,42
possibly because of oxidative injury.43–45
However, TA at 0.1 mg/mL, the maximal amount used in this study, has been shown to have minimal toxicity.43
Furthermore, TA at a concentration 10 times lower (0.01 mg/mL) already exhibited a significant inhibitory effect on caspase-5 activation. Thus, our results support the clinical use of TA, which has the advantage of being delivered in a sustained-release crystalline form.
The data presented here demonstrated that in LPS-primed hRPE cells, ATP and its analog, BzATP, caused time-dependent, transient increases in caspase-5 and -1 activation. ATP is present in millimolar concentrations in the cytosol of all eukaryotic cell types, and the extracellular levels are maintained at extremely low levels by ubiquitous ecto-ATPases and ecto-phosphatases in physiologic conditions.46
Extracellular ATP ubiquitously functions as an important mediator for cell-cell communication. Under pathologic conditions, such as inflammation, ATP concentrations may increase substantially by its release from damaged cells. RPE cells have a functional ATP P2X7 receptor that directly induces apoptosis and releases ATP into the subretinal space in response to chemical ischemia, cell swelling, osmotic stress, growth factors, glutamate, and other stimuli.47,48
Human RPE cells are also capable of degrading extracellular ATP, which may be released by leukocytes and RPE cells in retinal diseases. Recent evidence suggests that extracellular ATP accumulation at the sites of inflammation is considered to be a danger signal that alerts the immune system by binding to the P2X7 purinoreceptor, thereby activating NALP3 and caspase-1.24
Therefore, the current finding of involvement of caspase-5 and -1 in hRPE response to ATP may be clinically relevant.
The pathologic significance of caspase-5 has become more evident by a series of pathogenetic investigations.49–52
A polyadenosine repeat A(10)
in the caspase-5 coding sequence is often mutated, causing a frame shift in various microsatellite instability–positive cancers, including leukemia and gastrointestinal, endometrial, breast, and lung carcinomas. In addition, a polymorphism of caspase-5 has also been linked to ovarian cancer53
repeat does not exist in caspase-4. These studies suggest the clinical importance of caspase-5, for example, in tumorigenesis, perhaps related to a proapoptotic function. Our data show that caspase-5 is not as potent as caspase-4 in ER stress-induced apoptosis. Whether the proinflammatory role of caspase-5 is related to these reduced function mutations is unknown.
Human RPE cells, located at the blood-retina burrier, are putative important immunoregulatory cells that play important key roles in innate and adaptive immunity in a variety of retinal pathologic processes. Many reports have shown RPE cells to be ideal targets for infectious agents.55–58
Pathogen replication and elaboration of toxin by these agents can induce RPE cell death,55,58
which remains a potential risk factor for AMD pathogenesis.59
Inflammatory processes are also implicated in many diseases in which innate immunity contributes to pathogenesis. For example, in diabetic retinopathy, upregulation of IL-1β and caspase-1 activity occurs in retinal capillary cells.60,61
Research results up to now are just beginning to reveal how caspase-5 and caspase-1 may be involved in retinal diseases. The functional roles of caspase-5 remain poorly characterized in hRPE cells. In other cell types, NOD2, NLR protein NALP1, and caspase-1 form a complex mediating innate immune responses.62
Because caspase-5 is known to be associated with caspase-1 in NALP1 inflammasome,5
it is reasonable to propose that caspase-5 is also a downstream effector of the hRPE NLR–mediated innate immune response. The involvement of caspase-5 and the NLR signal system in retinal diseases warrants further investigation.