October 2001
Volume 42, Issue 11
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Anatomy and Pathology/Oncology  |   October 2001
Expression of Serine Protease Inhibitor 3 in Ocular Tissues in Endotoxin-Induced Uveitis in Rat
Author Affiliations
  • Akira Takamiya
    From the Departments of Anatomy and
    Ophthalmology, Asahikawa Medical College, Asahikawa; and
  • Masumi Takeda
    Ophthalmology, Asahikawa Medical College, Asahikawa; and
  • Akitoshi Yoshida
    Ophthalmology, Asahikawa Medical College, Asahikawa; and
  • Hiroshi Kiyama
    From the Departments of Anatomy and
    Department of Anatomy, Osaka City University Graduate School of Medicine, Abeno-ku Osaka, Japan.
Investigative Ophthalmology & Visual Science October 2001, Vol.42, 2427-2433. doi:
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      Akira Takamiya, Masumi Takeda, Akitoshi Yoshida, Hiroshi Kiyama; Expression of Serine Protease Inhibitor 3 in Ocular Tissues in Endotoxin-Induced Uveitis in Rat. Invest. Ophthalmol. Vis. Sci. 2001;42(11):2427-2433.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To ascribe the serine protease inhibitor 3 (SPI-3) as an ocular acute inflammatory molecule and to clarify its producing cells in an endotoxin-induced uveitis (EIU) model.

methods. Male Wistar rats were injected with lipopolysaccharide (LPS), and the expression of SPI-3 mRNA in the ocular tissues was examined by in situ hybridization (ISH) and Northern blot analysis. A combination of ISH and immunohistochemistry (IHC) were performed to prove the colocalization of SPI-3 mRNA and either glial fibrillary acidic protein (GFAP) or OX-42. The expression of phosphorylated STAT3 (pSTAT3) was demonstrated by IHC and Western blot after LPS injection. The colocalization of SPI-3 mRNA and pSTAT3 was finally examined by the double labeling of ISH and IHC.

results. After LPS injection, the expression of SPI-3 mRNA in ocular tissues was quickly upregulated and reached a peak between 12 and 24 hours after injection. An intense mRNA signal was observed in epithelial cells of the iris and ciliary body and the innermost retinal layer. In the retina, SPI-3 mRNA was colocalized with GFAP, demonstrating that the cells expressing SPI-3 mRNA were astrocytes. After LPS treatment, SPI-3 mRNA and pSTAT3 were colocalized in retinal astrocytes, and pSTAT3 expression appeared slightly earlier than that of SPI-3 mRNA.

conclusions. Ocular inflammation induced the transient expression of SPI-3 mRNA in retinal astrocytes and epithelial cells in the iris and ciliary body, particularly during early phase of the inflammation. Simultaneously, the activation of STAT3 (phosphorylation of STAT3) occurred slightly earlier in astrocytes. This supports the previous in vitro results that SPI-3 expression is induced in a STAT3-mediated manner. SPI-3 may have some crucial roles in preventing some degenerative proteolysis, which is induced by inflammatory stimuli.

Endotoxin, the lipopolysaccharide (LPS)-containing component of Gram-negative bacterial cell walls, 1 causes various inflammatory responses when administered systemically to animals and human volunteers. 2 3 Systemic injection of LPS produces a model for human uveitis such as Reiter’s syndrome and ankylosing spondylarthritis. 4 5 Evidence of uveitis, such as cells and flare appears at 4 hours, achieves a maximum level at 16 to 24 hours, and subsides gradually until day 7 after LPS injection. 6 In LPS-treated rats, infiltration of inflammatory cells into the anterior segments, 7 8 9 and the posterior segments of the eye is clearly observed. 6 10 11 The inflammatory cells produce various proinflammatory cytokines such as interleukin (IL) 1β, IL-6, interferon gamma, and tumor necrosis factor alpha, 12 13 14 15 that are supposed to elicit various inflammatory conditions, including inflammatory degenerative proteolysis. 
The serine protease inhibitor (SPI) family includes genes in both plants and animals that display a large diversity in their gene structure and regulation as well as the function of their products. In rat liver, three members of this family, SPI-1, SPI-2, and SPI-3, have been cloned and their nucleotide sequences determined. 16 17 18 Amino acid sequences of SPI-3 has approximately 70% homology to that of SPI-1 and SPI-2. 18 Despite the great similarity among the sequence proteins, mRNA localization and expression responses to the physiologic status of the animal vary. For instance, SPI-1 and SPI-2 genes are expressed in normal rat livers, but SPI-3 is virtually silent in normal rats and becomes transiently active during acute inflammation in the rat liver 16 18 19 and also after transient ischemia in rat brain. 19 20 21 Among these family members, SPI-3 is unique and intriguing, because SPI-3 may function specifically under inflammatory conditions to inhibit some proteolytic activity. This inflammatory response of SPI-3 is demonstrated in the liver, pancreas, and ischemic brain; however, there are no reports of SPI-3 expression in ocular tissue in response to inflammatory stimuli. In this study, we investigated whether this inflammatory response molecule is expressed in response to inflammatory stimulation in ocular tissue as well as in the liver and pancreas. In addition, a line of recent in vitro reports demonstrated that the expression of the SPI-3 gene was upregulated by IL-6, 19 20 22 23 and the signal transducers and activators of transcription 3 (STAT3) binding element, which is indispensable for the SPI-3 induction by IL-6 20 was located in the promoter region of the SPI-3 gene. Thus, we also investigated whether STAT3 activation occurs simultaneously in the SPI-3–expressing cells in the uveitis rat model. 
Materials and Methods
Animals
Inbred male Wistar rats, weighing 180 to 200 g, were used in this study. All animals were examined by slit-lamp and indirect ophthalmoscopy to avoid abnormalities before the experiments were performed. All animal procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Induction of EIU
LPS (Salmonella typhimurium; Sigma Chemical, St. Louis, MO) was dissolved in sterile pyrogen-free saline 0.9% at a concentration of 1 mg/ml. Rats were injected with 150 μg of LPS solution in the right footpad 9 24 after they were anesthetized by intraperitoneal injection of pentobarbital (0.3 mg/kg). LPS treatments were performed from 7 to 9 PM to regulate the effects of LPS by their circadian rhythm. 25  
Section Preparation
For eyeball section preparation, the rats were killed with the overdose of pentobarbital, and perfusion was performed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB). The eyes were enucleated and postfixed overnight in the same solution at 4°C. The eyes then were dehydrated and embedded in paraffin wax (Tissuprep; Fisher Scientific, Pittsburgh, PA). Eyeball sections of 7-μm thickness were mounted on 3-aminopropyltriethoxysilane–coated slides for in situ hybridization (ISH) or immunohistochemistry (IHC). When double labeling of ISH for SPI-3 and IHC pSTAT3 was performed, 8-μm-thick sections were used to prevent from decreased immunoreactivity. The sections were stored under dry conditions until histologic analysis. 
In Situ Hybridization
Using digoxigenin (DIG)-UTP–labeled cRNA probe, ISH (DIG-ISH) was performed. For SPI-3 mRNA detection, rat cDNA fragments for SPI-3 (GenBank X16359, nt 1429–1817, 389 bp) were isolated from inflamed rat liver using RT-PCR. These fragments, which were not similar to SPI-1 and SPI-2, were subcloned into pBluescript II KS + vector (Stratagene, La Jolla, CA). This template was linearized, and the DIG-labeled cRNA probe was prepared by in vitro transcription using T7 RNA polymerase (Roche Molecular Biochemicals, Indianapolis, IN). Forty nanograms of probe were used per slide. 
All prehybridization procedures were performed under RNase-free conditions at room temperature as previously reported. 26 The sections were deparaffinized, treated with proteinase K (10μ g/ml) for 3.5 minutes, washed in 0.1 M PB, fixed in 4% paraformaldehyde/0.1 M PB for 10 minutes, and washed in 0.1 M PB again. After being treated with 0.2 M HCl for 10 minutes and washed in 0.1 M PB, acetylation was performed with 0.1 M triethanolamine/0.25% acetic anhydride for 10 minutes, and the sections were washed in 0.1 M PB, dehydrated in ascending ethanol series, incubated in chloroform for 10 minutes, and dried. Hybridization was carried out for approximately 12 hours at 58°C in hybridization buffer (50% deionized formamide, 20 mM Tris-HCl [pH 8.0], 5 mM EDTA [pH 8.0], 0.3 M NaCl, 10 mM PB, 10% dextran sulfate, 0.2% sarcocyl, 1× Denhardt’s solution, 0.5 mg/ml yeast tRNA, and denatured 0.2 mg/ml salmon sperm DNA). After hybridization, the slides were washed in 50% formamide/2× SSC for 30 minutes at 65°C, immersed in RNase buffer containing 1 mg/ml RNase A for 30 minutes at 37°C, and immersed again in RNase buffer. They were then washed in 50% formamide/2× SSC for 30 minutes at 65°C and rinsed with 2× SSC for 10 minutes at 65°C and 0.1× SSC for 10 minutes at room temperature. After equilibration in buffer 1 (100 mM Tris-HCl [pH 7.6] and 150 mM NaCl) for 5 minutes, blocking was performed with 20% sheep serum in buffer 2 (buffer 1 with 0.5% skim milk and 0.1% Tween 20) for 2 hours at room temperature. The slides were incubated over night at 4°C with alkaline phosphatase–conjugated Fab fragments against DIG antibody (diluted 1:2000 in 5% sheep serum in buffer 2; Roche). For colorization, the slides were washed for 30 minutes three times in buffer 1, equilibrated in buffer 3 (100 mM Tris-HCl [pH 9.5], 100 mM NaCl, and 50 mM MgCl2) for 10 minutes, and stained with NBT/BCIP Stock Solution (Roche) in buffer 3 at room temperature for 6 to 12 hours with finding staining. The reaction was stopped with 10 mM Tris-HCl [pH 7.6]/1 mM EDTA, and the slides were mounted or subsequently processed for IHC as described previously for double staining. 
Northern Blot Analysis
Analysis of retinal SPI-3 mRNA expression by Northern hybridization was performed. First, animals were killed as described previously. The eyes were immediately enucleated, and retinas were dissected from the scleral wall 26 at 0, 6, 12, 24, and, 72 hours after LPS injection. The retinas were homogenized in guanidine thiocynate, and total RNA was extracted from the tissue samples using a phenol-chloroform procedure. 27 Ten micrograms of total RNA was heated to 65°C for 15 minutes in 50% formamide, 20 mM morpholinopropanesulphonic acid, 5 mM sodium acetate, 1 mM EDTA, 2.2 M formaldehyde. The total RNA was electrophoresed in formaldehyde-agarose gels. Total RNA was transferred to nylon transfer membranes (Amersham Pharmacia Biotechnology, Amersham, UK) according to standard procedures. 28 Hybridization was performed at 65°C with a 32P-labeled rat SPI-3 cDNA probe. The membrane was washed for 30 minutes at room temperature in 2× SSC, 0.1% SDS; 30 minutes at 65°C in 1× SSC, 0.1% SDS; and 30 minutes at room temperature in 0.1× SSC, 0.1% SDS and subjected to autoradiography at 80°C. 
Immunohistochemistry
The sections were deparaffined, rinsed in phosphate-buffered saline (PBS), treated with proteinase K (10 μg/ml) for 3 minutes, rinsed in PBS three times, and incubated in blocking solution containing 0.5% Triton X-100/3% bovine serum albumin/0.02% sodium azide in PBS for 30 minutes at room temperature. These pretreated sections were incubated with the first primary antibody for Tyr705-phosphorylated STAT3 (pSTAT3; New England Biolabs, Beverly, MA) at a dilution of 1:200 overnight at 4°C. The sections then were rinsed three times in PBS, incubated with secondary antibody (goat biotinylated anti-rabbit IgG diluted 1:500; Vector Laboratories, Burlingame, CA) for 2 hours at room temperature, rinsed three times in PBS, and incubated in avidin/biotin-peroxidase complex (Vector Laboratories) in PBS for 1 hour at room temperature. They were rinsed in PBS and immersed in 50 mM Tris-HCl (pH 7.6). Coloration was performed in Tris-HCl containing diaminobenzidine (DAB) and hydrogen peroxide. 
For IHC after DIG-ISH of both paraffin sections and flatmounted retinas, after staining with NBT/BCIP stock solution, IHC was performed from incubation in blocking solution for 30 minutes at room temperature as described previously. 
Flat-Mount Preparation
After perfusion with 4% paraformaldehyde in 0.1 M PB, incisions were made at the ora seratta to remove the anterior segment of the eyes, lens, and vitreous body. After the retina was carefully detached from the scleral wall and postfixed in the same perfusion solution overnight at 4°C, ISH was started from prehybridization as described previously. 
To attempt double labeling of DIG-ISH, after staining with NBT/BCIP stock solution, subsequent IHC for GFAP (at a dilution of 1:3000; Sigma Chemical) or OX-42 (at a dilution of 1:800; Serotec, Raleigh, NC) was performed as previously. Goat biotinylated anti-mouse IgG (diluted 1:500; Vector Laboratories) was used for the secondary antibody. 
Retinal Protein Extraction and Western Blot Analysis
After the animals were killed as described previously, the eyes were immediately enucleated, and the retinas were dissected from the scleral wall 26 at 0, 3, 6, 12, 24, and 72 hours after LPS injection. The total protein in the retina was prepared according to previously reported methods. 26 29 The extracted retinas were solubilized in 3% SDS buffer (1 mM orthovanadium, 0.19 μl/ml aprotinin, and 0.1 μg/ml PMSF), and boiled for 10 minutes. The lysates were added to same volume of 0.3 M sucrose, homogenized, and centrifuged at 14,000 rpm for 20 minutes at 4°C. Protein levels were quantified using BCA protein assay (Pierce, Rutherford, IL). The lysates were stored at −80°C until Western blot analysis. 
For Western blot analysis, 50 μg total protein in SDS sample buffer was applied to each lane. The samples were electrophoresed in 10% SDS-polyacrylamide gel. After blotting onto PVDF membranes, the membranes were washed in TBST (Tris-buffered saline containing 0.1% Twen-20), blocked in 5% skim milk TBST, rinsed in TBS for 15 minutes at room temperature, and incubated with pSTAT3 antibody (at a dilution of 1:1000) overnight at 4°C. The membrane was incubated with the secondary antibody (donkey horseradish peroxidase–linked anti-rabbit antibody [Amersham; diluted 1:3000 in 5% skim milk TBST]) for 1 hour at room temperature, and the ECL Western blot analysis system (Amersham) was used for detection. To analyze the expression level of STAT3 protein with the same membrane, the membrane was submerged in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62 mM Tris-HCl[ pH 6.8]) and incubated at 50°C for 30 minutes with occasional agitation, and the primary and secondary antibodies were removed from the membrane completely. The membrane was washed in TBST, blocked in 5% skim milk TBST, rinsed in TBS for 15 minutes at room temperature, and incubated with STAT3 antibody (Santa Cruz Biotechnology, Santa Cruz, CA; at a dilution of 1:2000) overnight at 4°C. We analyzed the expression of β-actin as a suitable internal control using the same membrane after complete removing the STAT3 reaction as above. A prestained molecular weight standard was purchased from New England Biolabs. 
Results
Expression of SPI-3 mRNA in Ocular Tissues after LPS Treatment
Under normal conditions, no expression of SPI-3 was observed in the iris, ciliary body, choroid, and retina (Figs. 1 and 2) . However, the SPI-3 mRNA hybridization signal appeared 6 hours after LPS treatment, and the signal intensity became strengthened from 12 to 24 hours after LPS injection. The SPI-3 mRNA-positive cells were localized in the epithelial cells of the iris and ciliary body, and cells in the retinal superficial layer (Figs. 1 and 2) . The cells expressing SPI-3 mRNA in the retina were oval and flat (Fig. 2) . The expression level decreased thereafter and returned to the control level, which was below the detection level, at 72 hours after LPS injection (Figs. 1 and 2)
Quantitative Analysis of SPI-3 mRNA in Retina after LPS Treatment
To examine the expression level of SPI-3 mRNA quantitatively, we performed Northern blot analysis at the same time points after LPS treatment. The expression of SPI-3 mRNA was first detected at 6 hours after treatment as a specific single band at approximately 2.2 kb (Fig. 3) , and the expression level peaked at 24 hours after LPS treatment. The expression decreased thereafter and could not be detected at 72 hours after LPS treatment. These results were in good agreement with the histologic observation mentioned previously (Fig. 2)
Identification of Cell Species Expressing SPI-3 mRNA in the Retina
DIG-ISH using flat-mount preparation of the retinas was also attempted to obtain further information about the positive cell species. Several positive cells were spread on the innermost retinal surface, numerous positive cells were along retinal vessels (Fig. 4A 4B) . To identify the cell species, double labeling of SPI-3 mRNA and either GFAP or OX42 were carried out using flat-mounted retinas. SPI-3 mRNA signals were colocalized with GFAP immunoreactivity (Fig. 4C) , but not with OX-42 (Fig. 4D) . According to this double-labeling result and the restricted layer localization of the positive cells, the cells in the retina SPI-3 mRNA expressing were assumed to be astrocytes, and not microglia, macrophages, or Müller cells. 
Quantitative Protein Analysis for STAT3 and pSTAT3
Previous reports showed that the promoter of the SPI-3 gene had an acute-phase response factor (APRF/STAT3) binding site and stimulation of IL-6 upregulated SPI-3 expression in vitro. 20 30 Thus, we also examined quantitatively a change in the expression levels of STAT3 and pSTAT3 after LPS treatment (Fig. 5) . We performed Western blot analysis in whole retinas at different time points (0, 3, 6, 12, 24, and 72 hours after LPS treatment). No significant change in STAT expression in the retina after LPS stimulation was observed; however, a substantial increase in pSTAT3 expression was found after LPS injection. In the control retinas no apparent band for pSTAT3 was seen, but a slight increase in the positive band appeared 3 hours after the injection. A substantial increase in pSTAT3 immunoreactivity was observed from 6 to 24 hours after the injection. This upregulation of pSTAT3 expression decreased thereafter, and no positive band was detected 72 hours after the injection. This indicates that LPS stimulation does not induce additional STAT3 expression, but does induce substantial phosphorylation of STAT3, suggesting that a STAT3-mediated pathway is activated by LPS in the retina. 
pSTAT3 Immunoreactivity in SPI-3 mRNA-Positive Astrocytes after LPS Injection
IHC using anti-pSTAT3 antibody revealed a clear increase in pSTAT3 staining in cells located in the innermost retinal layer after LPS injection (Fig. 6) . Because the localization of pSTAT3-positive cells was very similar to that of SPI-3-positive cells in the retina, we examined double labeling of SPI-3 mRNA and pSTAT3 immunoreactivity. The SPI-3 mRNA signal and pSTAT3 immunoreactivity were colocalized in astrocytes in the nerve fiber layer (Fig. 7) . These results indicate that LPS-induced phosphorylation of STAT3 occurred simultaneously within the SPI-3-positive astrocytes. 
Discussion
In this study, we demonstrated that expression of endogenous SPI-3 is induced in specific cells of the ocular tissues in response to inflammatory stimulation. The SPI-3 mRNA positive signal is found in epithelial cells of the iris and ciliary body, and astrocytes in the retina. Previous studies showed that SPI-3 is almost entirely silent in normal (non-inflammatory) rats but is transiently activated in response to inflammatory stimuli in the liver 16 18 and pancreas. 31 The present study revealed a similar inflammatory response of SPI-3 also exists in ocular tissues. This transient expression is marked from 6 to 24 hours after LPS injection, indicating that the expression occurred relatively early in the inflammatory response. Thus, SPI-3 may inhibit proteolysis activity, which occurs during the early phase of inflammation. Although the function of this protease is still obscure, SPI-3 could be a marker for the early phase of the inflammatory response. In the retina, the inflammation elicits activation of some cell species, in particular, microglia, macrophage, and Müller cells, and among these cells little is known about the role of astrocytes under these conditions. In this study, we demonstrate that among these reactive cell species, only astrocytes can synthesize SPI-3, which may be one of their important functions during inflammation. In addition, because SPI-3 has a putative signal peptide sequence to be released and glycosylation is evident, 18 this molecule is supposed to be released and function in the extracellular space. In fact, all the SPI-3-mRNA positive cells in the innermost layer of each tissue suggest that the released SPI-3 can readily spread into the intraocular space. Because there are no available antibodies to measure the released SPI-3, it is impossible to confirm the release. 
Previous studies speculated that because SPI-3 is expressed only under inflammatory conditions, it might have protective effects against inflammatory damage. 16 21 31 In fact, many serine protease inhibitors have potent protective activities such as wound-healing repairs. For instance, a recent paper reported that the secretory leukocyte protease inhibitor (SLPI) is a pivotal endogenous factor necessary for optimal wound healing. 32 SLPI is a multi-potent serine protease inhibitor with anti-inflammatory, anti-viral, anti-fungal and anti-bacterial properties. In addition, SLPI antagonizes LPS-induced pro-inflammatory mediator synthesis by monocytes and macrophages. 33 34 Although the functional consequences of SPI-3 are yet unknown, it is likely that SPI-3 may have similar anti-inflammatory properties under local inflammatory conditions. Another intriguing aspect of inflammation concerns nerve regeneration. Recently, Benowitz et al. 35 reported that macrophage activation led to greatly increased regeneration of injured optic nerves. They observed that the lens puncture somehow caused massive infiltration of macrophage into the eye, which caused marked activation of Müller cells. Under this inflammatory condition, crushed optic nerve regeneration was significantly upregulated. Although the molecular mechanism underlying how inflammation accelerates nerve regeneration is unknown, an inflammation-activated protease inhibitor such as SPI-3 might contribute to nerve regeneration to some extent. Our unpublished data suggest that optic nerve injury does not induce expression of SPI-3, whereas rat motor nerve transection dramatically induced SPI-3 expression in the injured motor neurons. Retinal ganglion cells with injured nerves cannot regenerate, even though motor neurons with damaged nerves can survive and regenerate, and the difference in SPI-3 expression may affect their fate. Recently, the protein inhibitor 6 (PI-6; the human orthologue of SPI-3) is proved as an inhibitor of the cathepsin G, and the cathepsin G activates a proapoptotic protease, caspase-7. 36 37 Thus PI-6 (or SPI-3) could be a potent inhibitor of caspase-7–mediated apoptosis. In this respect SPI-3 may prevent a caspase-7–mediated apoptosis, which is caused by damage such as nerve injury or inflammatory stimulation. More detailed studies are need to determine the functional significance of SPI-3. 
In vitro studies have demonstrated that the expression of the SPI-3 gene is upregulated by IL-6. 19 20 22 23 This induction of SPI-3 mRNA was observed approximately 1 hour after IL-6 stimulation in rat-cultured primary hepatocytes, and the expression level peaked 24 hours after stimulation. 19 Coincidentally, it was reported that expression of IL-6 mRNA significantly increases in the iris, ciliary body, and retina in the early inflammatory phase of the rat EIU model. 12 13 In addition, activation of the Janus kinase (JAK)–STAT3 pathway in response to stress stimuli and ciliary neurotrophic factor (CNTF) was also reported in retinal astrocytes. 38 Because the STAT3 is rapidly activated by phosphorylation at the tyrosine residue by JAKs after the IL-6 stimulation as well as CNTF, leukemia inhibitory factor, oncostatin M, and IL-11, 39 40 41 we examined the STAT3 activation in the retina after LPS injection using Western blot analysis and IHC. LPS stimulation elicited STAT3 activation in retinal astrocytes. In addition, the double-labeling study clearly demonstrated the colocalization of pSTAT3 and SPI-3 in the astrocytes. An intriguing aspect of this experiment is that the pSTAT3 appears slightly earlier than SPI-3. Moreover, it was reported that the possible STAT3 binding elements were included in the promoter region of the rat SPI-3 gene, and the binding site was shown to be indispensable for SPI-3 induction by IL-6. 20 These would strongly suggest that SPI-3 expression is induced after STAT3 activation in the retina of EIU model. This in vivo result may support the in vitro evidence that SPI-3 expression occurs in a STAT3-mediated manner. Therefore, it might be concluded that LPS elicits release of IL-6 and/or some inflammation-associated cytokines from inflammatory cells such as a macrophage or other cells and activates thereby the JAK-STAT pathways in retinal astrocytes. This STAT3 activation might induce further SPI-3 expression to prevent a proteolytic insult caused by excessively activated serine proteases. 
 
Figure 1.
 
Expression of SPI-3 mRNA was demonstrated by ISH using a DIG-labeled rat SPI-3 specific antisense cRNA probe in the iris (A and B), ciliary body (C and D), and choroid (E and F) before LPS treatment (A, C, and E) and 12 hours after LPS treatment (B, D, and F). In the iris and ciliary body, although no signals are observed before LPS treatment, the hybridization signal increases substantially in the epithelial cells of the iris and ciliary body after LPS treatment. In the choroid, no SPI-3 mRNA signal was found before or after LPS treatment. CHO, choroid; RPE, retinal pigment epithelium. Original magnification, ×400.
Figure 1.
 
Expression of SPI-3 mRNA was demonstrated by ISH using a DIG-labeled rat SPI-3 specific antisense cRNA probe in the iris (A and B), ciliary body (C and D), and choroid (E and F) before LPS treatment (A, C, and E) and 12 hours after LPS treatment (B, D, and F). In the iris and ciliary body, although no signals are observed before LPS treatment, the hybridization signal increases substantially in the epithelial cells of the iris and ciliary body after LPS treatment. In the choroid, no SPI-3 mRNA signal was found before or after LPS treatment. CHO, choroid; RPE, retinal pigment epithelium. Original magnification, ×400.
Figure 2.
 
Expression of SPI-3 mRNA was demonstrated by ISH using a DIG-labeled SPI-3 cRNA probe in the retina at different time points after LPS treatment (A through E). Photographs show that the signal is upregulated until 24 hours after the LPS injection. The signals return to the preinjection level 72 hours after LPS treatment. Expression of SPI-3 mRNA is found in the flat cells of the retinal superficial layer (B through D, arrowheads). NFL, nerve fiber layer; GCL, ganglion cell layer. Original magnification, ×400.
Figure 2.
 
Expression of SPI-3 mRNA was demonstrated by ISH using a DIG-labeled SPI-3 cRNA probe in the retina at different time points after LPS treatment (A through E). Photographs show that the signal is upregulated until 24 hours after the LPS injection. The signals return to the preinjection level 72 hours after LPS treatment. Expression of SPI-3 mRNA is found in the flat cells of the retinal superficial layer (B through D, arrowheads). NFL, nerve fiber layer; GCL, ganglion cell layer. Original magnification, ×400.
Figure 3.
 
Northern blot analysis for SPI-3 mRNA in retinas after LPS treatment. (A) Expression of SPI-3 mRNA reached a detectable level at 6 hours and peaked at 24 hours after LPS injection. At 72 hours after LPS injection, no expression was detected. (B) Expression of GAPDH mRNA was used as an internal control for the amount of total RNA. Numbers on the right side indicate RNA size in kb.
Figure 3.
 
Northern blot analysis for SPI-3 mRNA in retinas after LPS treatment. (A) Expression of SPI-3 mRNA reached a detectable level at 6 hours and peaked at 24 hours after LPS injection. At 72 hours after LPS injection, no expression was detected. (B) Expression of GAPDH mRNA was used as an internal control for the amount of total RNA. Numbers on the right side indicate RNA size in kb.
Figure 4.
 
Cells expressing SPI-3 mRNA in a flatmounted retina. (A and B) SPI-3 mRNA expression is demonstrated by ISH using a flatmounted preparation of retina at 12 hours after LPS treatment. SPI-3 mRNA expression is seen in oval cells in the retinal superficial layer (blue cytosolic staining). Numerous cells expressing SPI-3 mRNA are found along retinal vessels. Original magnification, ×400. (B) Higher magnification of (A); original magnification, ×200. (C) Double labeling of SPI-3 mRNA by ISH and GFAP by IHC. Cells expressing SPI-3 mRNA (blue cytosolic staining) were colocalized in GFAP immunoreactive cells (brown cytosolic staining). The end feet of Müller cells are also stained by GFAP antibody, but are negative for SPA-3 mRNA. Original magnification, ×400. (D) Double labeling of SPI-3 mRNA by ISH and OX42 by IHC. The SPI-3 mRNA positive cells (arrowheads) and OX42 immunoreactive cells (arrows) are distinct. Original magnification,× 400.
Figure 4.
 
Cells expressing SPI-3 mRNA in a flatmounted retina. (A and B) SPI-3 mRNA expression is demonstrated by ISH using a flatmounted preparation of retina at 12 hours after LPS treatment. SPI-3 mRNA expression is seen in oval cells in the retinal superficial layer (blue cytosolic staining). Numerous cells expressing SPI-3 mRNA are found along retinal vessels. Original magnification, ×400. (B) Higher magnification of (A); original magnification, ×200. (C) Double labeling of SPI-3 mRNA by ISH and GFAP by IHC. Cells expressing SPI-3 mRNA (blue cytosolic staining) were colocalized in GFAP immunoreactive cells (brown cytosolic staining). The end feet of Müller cells are also stained by GFAP antibody, but are negative for SPA-3 mRNA. Original magnification, ×400. (D) Double labeling of SPI-3 mRNA by ISH and OX42 by IHC. The SPI-3 mRNA positive cells (arrowheads) and OX42 immunoreactive cells (arrows) are distinct. Original magnification,× 400.
Figure 5.
 
Western blot analysis for STAT3 (A) and phosphorylated STAT3 (B). Each lane was loaded with 50 μg of total protein extracted from a normal retina or samples taken at various time points after LPS treatment. Western blot analysis of β-actin was used as an internal control for the amount of total protein (C). Numbers on the right side indicate the protein size in kDa.
Figure 5.
 
Western blot analysis for STAT3 (A) and phosphorylated STAT3 (B). Each lane was loaded with 50 μg of total protein extracted from a normal retina or samples taken at various time points after LPS treatment. Western blot analysis of β-actin was used as an internal control for the amount of total protein (C). Numbers on the right side indicate the protein size in kDa.
Figure 6.
 
IHC for phosphorylated STAT3 in the retina. (A) Control; (B) 12 hours after LPS treatment. In the control retina, no immunopositive staining is observed in astrocytes (arrows; A), whereas intense nuclear immunostaining is apparent (brown nuclear staining) in the astrocytes (arrows; B). Original magnification,× 400.
Figure 6.
 
IHC for phosphorylated STAT3 in the retina. (A) Control; (B) 12 hours after LPS treatment. In the control retina, no immunopositive staining is observed in astrocytes (arrows; A), whereas intense nuclear immunostaining is apparent (brown nuclear staining) in the astrocytes (arrows; B). Original magnification,× 400.
Figure 7.
 
Colocalization of SPI-3 mRNA and pSTAT3 in the retinal astrocytes. SPI-3 mRNA positive staining (blue cytosolic staining) and pSTAT3 immunoreactive staining (brown nuclear staining) are seen simultaneously in the retinal astrocytes. NFL, nerve fiber layer; GCL, ganglion cell layer. Original magnification,× 400.
Figure 7.
 
Colocalization of SPI-3 mRNA and pSTAT3 in the retinal astrocytes. SPI-3 mRNA positive staining (blue cytosolic staining) and pSTAT3 immunoreactive staining (brown nuclear staining) are seen simultaneously in the retinal astrocytes. NFL, nerve fiber layer; GCL, ganglion cell layer. Original magnification,× 400.
Ulevitch RJ, Tobias PS. Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Annu Rev Immunol. 1995;13:437–457. [CrossRef] [PubMed]
Morrison DC, Ulevitch RJ. The effects of bacterial endotoxins on host mediation systems. A review. Am J Pathol. 1978;93:526–617. [PubMed]
van Deventer SJ, Buller HR, ten Cate JW, et al. Experimental endotoxemia in humans: analysis of cytokine release and coagulation, fibrinolytic, and complement pathways. Blood. 1990;76:2520–2526. [PubMed]
Rosenbaum JT, McDevitt HO, Guss RB, Egbert PR. Endotoxin-induced uveitis in rats as a model for human disease. Nature. 1980;286:611–613. [CrossRef] [PubMed]
De Vos AF, Hoekzema R, Kijlstra A. Cytokines and uveitis: a review. Curr Eye Res. 1992;11:581–597. [CrossRef] [PubMed]
Yang P, de Vos AF, Kijlstra A. Macrophages in the retina of normal Lewis rats and their dynamics after injection of lipopolysaccharide. Invest Ophthalmol Vis Sci. 1996;37:77–85. [PubMed]
Hoekzema R, Verhagen C, van Haren M, Kijlstra A. Endotoxin-induced uveitis in the rat. The significance of intraocular interleukin-6. Invest Ophthalmol Vis Sci. 1992;33:532–539. [PubMed]
McMenamin PG, Crewe J. Endotoxin-induced uveitis. Kinetics and phenotype of the inflammatory cell infiltrate and the response of the resident tissue macrophages and dendritic cells in the iris and ciliary body. Invest Ophthalmol Vis Sci. 1995;36:1949–1959. [PubMed]
Behar-Cohen FF, Savoldelli M, Parel JM, et al. Reduction of corneal edema in endotoxin-induced uveitis after application of L-NAME as nitric oxide synthase inhibitor in rats by iontophoresis. Invest Ophthalmol Vis Sci. 1998;39:897–904. [PubMed]
Ruiz-Moreno JM, Thillaye B, de Kozak Y. Retino-choroidal changes in endotoxin-induced uveitis in the rat. Ophthalmic Res. 1992;24:162–168. [CrossRef] [PubMed]
Marie O, Thillaye-Goldenberg B, Naud MC, de Kozak Y. Inhibition of endotoxin-induced uveitis and potentiation of local TNF- alpha and interleukin-6 mRNA expression by interleukin-13. Invest Ophthalmol Vis Sci. 1999;40:2275–2282. [PubMed]
de Vos AF, van Haren MA, Verhagen C, Hoekzema R, Kijlstra A. Kinetics of intraocular tumor necrosis factor and interleukin-6 in endotoxin-induced uveitis in the rat. Invest Ophthalmol Vis Sci. 1994;35:1100–1106. [PubMed]
Planck SR, Huang XN, Robertson JE, Rosenbaum JT. Cytokine mRNA levels in rat ocular tissues after systemic endotoxin treatment. Invest Ophthalmol Vis Sci. 1994;35:924–930. [PubMed]
de Vos AF, Klaren VN, Kijlstra A. Expression of multiple cytokines and IL-1RA in the uvea and retina during endotoxin-induced uveitis in the rat. Invest Ophthalmol Vis Sci. 1994;35:3873–3883. [PubMed]
Yoshida M, Yoshimura N, Hangai M, Tanihara H, Honda Y. Interleukin-1 alpha, interleukin-1 beta, and tumor necrosis factor gene expression in endotoxin-induced uveitis. Invest Ophthalmol Vis Sci. 1994;35:1107–1113. [PubMed]
Pages G, Rouayrenc JF, Le Cam G, Mariller M, Le Cam A. Molecular characterization of three rat liver serine-protease inhibitors affected by inflammation and hypophysectomy. Protein and mRNA analysis and cDNA cloning. Eur J Biochem. 1990;190:385–391. [CrossRef] [PubMed]
Pages G, Rouayrenc JF, Rossi V, et al. Primary structure and assignment to chromosome 6 of three related rat genes encoding liver serine protease inhibitors. Gene. 1990;94:273–282. [CrossRef] [PubMed]
Ohkubo K, Ogata S, Misumi Y, Takami N, Ikehara Y. Molecular cloning and characterization of rat contrapsin-like protease inhibitor and related proteins. J Biochem (Tokyo). 1991;109:243–250.
Kordula T, Bugno M, Lason W, Przewlocki R, Koj A. Rat contrapsins are the type II acute phase proteins: regulation by interleukin 6 on the mRNA level. Biochem Biophys Res Commun. 1994;201:222–227. [CrossRef] [PubMed]
Kordula T, Travis J. The role of Stat and C/EBP transcription factors in the synergistic activation of rat serine protease inhibitor-3 gene by interleukin-6 and dexamethasone. Biochem J. 1996;313:1019–1027. [PubMed]
Tsuda M, Kitagawa K, Imaizumi K, et al. Induction of SPI-3 mRNA, encoding a serine protease inhibitor, in gerbil hippocampus after transient forebrain ischemia. Mol Brain Res. 1996;35:314–318. [CrossRef] [PubMed]
Le Cam A, Legraverend C. Transcriptional repression, a novel function for 3′ untranslated regions. Eur J Biochem. 1995;231:620–627. [CrossRef] [PubMed]
Simar-Blanchet AE, Paul C, Mercier L, Le Cam A. Regulation of expression of the rat serine protease inhibitor 2.3 gene by glucocorticoids and interleukin-6. A complex and unusual interplay between positive and negative cis-acting elements. Eur J Biochem. 1996;236:638–648. [CrossRef] [PubMed]
Goureau O, Bellot J, Thillaye B, Courtois Y, de Kozak Y. Increased nitric oxide production in endotoxin-induced uveitis. Reduction of uveitis by an inhibitor of nitric oxide synthase. J Immunol. 1995;154:6518–6523. [PubMed]
Borovikova LV, Ivanova S, Zhang M, et al. Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin. Nature. 2000;405:458–462. [CrossRef] [PubMed]
Takeda M, Kato H, Takamiya A, Yoshida A, Kiyama H. Injury-specific expression of activating transcription factor-3 in retinal ganglion cells and its colocalized expression with phosphorylated c-Jun. Invest Ophthalmol Vis Sci. 2000;41:2412–2421. [PubMed]
Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156–159. [PubMed]
Sambrook J, Fritsch EF, Maniatis T. Extraction, purification, and analysis of messenger RNA from eukaryotic cells. Ford N Nolan C Ferguson M eds. Molecular Cloning: A Laboratory Manual. 1988;7:1–87. Cold Spring Harbor Laboratory Press New York.
Kenney AM, Kocsis JD. Peripheral axotomy induces long-term c-Jun amino-terminal kinase-1 activation and activator protein-1 binding activity by c-Jun and junD in adult rat dorsal root ganglia In vivo. J Neurosci. 1998;18:1318–1328. [PubMed]
Kordula T, Travis J. Activation of the rat serine proteinase inhibitor 3 gene by interferon gamma via the interleukin 6-responsive element. Biochem J. 1995;309:63–67. [PubMed]
Chen MC, Schuit F, Pipeleers DG, Eizirik DL. IL-1beta induces serine protease inhibitor 3 (SPI-3) gene expression in rat pancreatic beta-cells. Detection by differential display of messenger RNA. Cytokine. 1999;11:856–862. [CrossRef] [PubMed]
Ashcroft GS, Lei K, Jin W, et al. Secretory leukocyte protease inhibitor mediates non-redundant functions necessary for normal wound healing [In Process Citation]. Nat Med. 2000;6:1147–1153. [CrossRef] [PubMed]
Zhu J, Nathan C, Ding A. Suppression of macrophage responses to bacterial lipopolysaccharide by a non-secretory form of secretory leukocyte protease inhibitor. Biochim Biophys Acta. 1999;1451:219–223. [CrossRef] [PubMed]
Sano C, Shimizu T, Sato K, Ogasawara K, Tomioka H. Effects of secretory leukocyte protease inhibitor on the production of some cytokines and nitric oxide by murine peritoneal macrophages in response to lipopolysaccharide stimulation and Mavium complex infection. Kekkaku. 1999;74:563–570. [PubMed]
Leon S, Yin Y, Nguyen J, Irwin N, Benowitz LI. Lens injury stimulates axon regeneration in the mature rat optic nerve. J Neurosci. 2000;20:4615–4626. [PubMed]
Zhou Q, Salvesen GS. Activation of pro-caspase-7 by serine proteases includes a non-canonical specificity. Biochem J. 1997;324:361–364. [PubMed]
Scott FL, Hirst CE, Sun J, et al. The intracellular serpin proteinase inhibitor 6 is expressed in monocytes and granulocytes and is a potent inhibitor of the azurophilic granule protease, cathepsin G. Blood. 1999;93:2089–2097. [PubMed]
Peterson WM, Wang Q, Tzekova R, Wiegand SJ. Ciliary neurotrophic factor and stress stimuli activate the Jak-STAT pathway in retinal neurons and glia. J Neurosci. 2000;20:4081–4090. [PubMed]
Heim MH. The Jak-STAT pathway: cytokine signalling from the receptor to the nucleus. J Recept Signal Transduct Res. 1999;19:75–120. [CrossRef] [PubMed]
Lutticken C, Wegenka UM, Yuan J, et al. Association of transcription factor APRF and protein kinase Jak1 with the interleukin-6 signal transducer gp130. Science. 1994;263:89–92. [CrossRef] [PubMed]
Stahl N, Boulton TG, Farruggella T, et al. Association and activation of Jak-Tyk kinases by CNTF-LIF-OSM-IL-6 beta receptor components. Science. 1994;263:92–95. [CrossRef] [PubMed]
Figure 1.
 
Expression of SPI-3 mRNA was demonstrated by ISH using a DIG-labeled rat SPI-3 specific antisense cRNA probe in the iris (A and B), ciliary body (C and D), and choroid (E and F) before LPS treatment (A, C, and E) and 12 hours after LPS treatment (B, D, and F). In the iris and ciliary body, although no signals are observed before LPS treatment, the hybridization signal increases substantially in the epithelial cells of the iris and ciliary body after LPS treatment. In the choroid, no SPI-3 mRNA signal was found before or after LPS treatment. CHO, choroid; RPE, retinal pigment epithelium. Original magnification, ×400.
Figure 1.
 
Expression of SPI-3 mRNA was demonstrated by ISH using a DIG-labeled rat SPI-3 specific antisense cRNA probe in the iris (A and B), ciliary body (C and D), and choroid (E and F) before LPS treatment (A, C, and E) and 12 hours after LPS treatment (B, D, and F). In the iris and ciliary body, although no signals are observed before LPS treatment, the hybridization signal increases substantially in the epithelial cells of the iris and ciliary body after LPS treatment. In the choroid, no SPI-3 mRNA signal was found before or after LPS treatment. CHO, choroid; RPE, retinal pigment epithelium. Original magnification, ×400.
Figure 2.
 
Expression of SPI-3 mRNA was demonstrated by ISH using a DIG-labeled SPI-3 cRNA probe in the retina at different time points after LPS treatment (A through E). Photographs show that the signal is upregulated until 24 hours after the LPS injection. The signals return to the preinjection level 72 hours after LPS treatment. Expression of SPI-3 mRNA is found in the flat cells of the retinal superficial layer (B through D, arrowheads). NFL, nerve fiber layer; GCL, ganglion cell layer. Original magnification, ×400.
Figure 2.
 
Expression of SPI-3 mRNA was demonstrated by ISH using a DIG-labeled SPI-3 cRNA probe in the retina at different time points after LPS treatment (A through E). Photographs show that the signal is upregulated until 24 hours after the LPS injection. The signals return to the preinjection level 72 hours after LPS treatment. Expression of SPI-3 mRNA is found in the flat cells of the retinal superficial layer (B through D, arrowheads). NFL, nerve fiber layer; GCL, ganglion cell layer. Original magnification, ×400.
Figure 3.
 
Northern blot analysis for SPI-3 mRNA in retinas after LPS treatment. (A) Expression of SPI-3 mRNA reached a detectable level at 6 hours and peaked at 24 hours after LPS injection. At 72 hours after LPS injection, no expression was detected. (B) Expression of GAPDH mRNA was used as an internal control for the amount of total RNA. Numbers on the right side indicate RNA size in kb.
Figure 3.
 
Northern blot analysis for SPI-3 mRNA in retinas after LPS treatment. (A) Expression of SPI-3 mRNA reached a detectable level at 6 hours and peaked at 24 hours after LPS injection. At 72 hours after LPS injection, no expression was detected. (B) Expression of GAPDH mRNA was used as an internal control for the amount of total RNA. Numbers on the right side indicate RNA size in kb.
Figure 4.
 
Cells expressing SPI-3 mRNA in a flatmounted retina. (A and B) SPI-3 mRNA expression is demonstrated by ISH using a flatmounted preparation of retina at 12 hours after LPS treatment. SPI-3 mRNA expression is seen in oval cells in the retinal superficial layer (blue cytosolic staining). Numerous cells expressing SPI-3 mRNA are found along retinal vessels. Original magnification, ×400. (B) Higher magnification of (A); original magnification, ×200. (C) Double labeling of SPI-3 mRNA by ISH and GFAP by IHC. Cells expressing SPI-3 mRNA (blue cytosolic staining) were colocalized in GFAP immunoreactive cells (brown cytosolic staining). The end feet of Müller cells are also stained by GFAP antibody, but are negative for SPA-3 mRNA. Original magnification, ×400. (D) Double labeling of SPI-3 mRNA by ISH and OX42 by IHC. The SPI-3 mRNA positive cells (arrowheads) and OX42 immunoreactive cells (arrows) are distinct. Original magnification,× 400.
Figure 4.
 
Cells expressing SPI-3 mRNA in a flatmounted retina. (A and B) SPI-3 mRNA expression is demonstrated by ISH using a flatmounted preparation of retina at 12 hours after LPS treatment. SPI-3 mRNA expression is seen in oval cells in the retinal superficial layer (blue cytosolic staining). Numerous cells expressing SPI-3 mRNA are found along retinal vessels. Original magnification, ×400. (B) Higher magnification of (A); original magnification, ×200. (C) Double labeling of SPI-3 mRNA by ISH and GFAP by IHC. Cells expressing SPI-3 mRNA (blue cytosolic staining) were colocalized in GFAP immunoreactive cells (brown cytosolic staining). The end feet of Müller cells are also stained by GFAP antibody, but are negative for SPA-3 mRNA. Original magnification, ×400. (D) Double labeling of SPI-3 mRNA by ISH and OX42 by IHC. The SPI-3 mRNA positive cells (arrowheads) and OX42 immunoreactive cells (arrows) are distinct. Original magnification,× 400.
Figure 5.
 
Western blot analysis for STAT3 (A) and phosphorylated STAT3 (B). Each lane was loaded with 50 μg of total protein extracted from a normal retina or samples taken at various time points after LPS treatment. Western blot analysis of β-actin was used as an internal control for the amount of total protein (C). Numbers on the right side indicate the protein size in kDa.
Figure 5.
 
Western blot analysis for STAT3 (A) and phosphorylated STAT3 (B). Each lane was loaded with 50 μg of total protein extracted from a normal retina or samples taken at various time points after LPS treatment. Western blot analysis of β-actin was used as an internal control for the amount of total protein (C). Numbers on the right side indicate the protein size in kDa.
Figure 6.
 
IHC for phosphorylated STAT3 in the retina. (A) Control; (B) 12 hours after LPS treatment. In the control retina, no immunopositive staining is observed in astrocytes (arrows; A), whereas intense nuclear immunostaining is apparent (brown nuclear staining) in the astrocytes (arrows; B). Original magnification,× 400.
Figure 6.
 
IHC for phosphorylated STAT3 in the retina. (A) Control; (B) 12 hours after LPS treatment. In the control retina, no immunopositive staining is observed in astrocytes (arrows; A), whereas intense nuclear immunostaining is apparent (brown nuclear staining) in the astrocytes (arrows; B). Original magnification,× 400.
Figure 7.
 
Colocalization of SPI-3 mRNA and pSTAT3 in the retinal astrocytes. SPI-3 mRNA positive staining (blue cytosolic staining) and pSTAT3 immunoreactive staining (brown nuclear staining) are seen simultaneously in the retinal astrocytes. NFL, nerve fiber layer; GCL, ganglion cell layer. Original magnification,× 400.
Figure 7.
 
Colocalization of SPI-3 mRNA and pSTAT3 in the retinal astrocytes. SPI-3 mRNA positive staining (blue cytosolic staining) and pSTAT3 immunoreactive staining (brown nuclear staining) are seen simultaneously in the retinal astrocytes. NFL, nerve fiber layer; GCL, ganglion cell layer. Original magnification,× 400.
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