Abstract
purpose. To investigate effects of rolipram, an inhibitor of type 4 phosphodiesterase, on lipopolysaccharide (LPS)-induced uveitis in Wistar rats.
methods. A total of 100 μg LPS was injected into the rat footpad. Rolipram (Wako Pure Chemical, Osaka, Japan) was injected intraperitoneally 30 minutes before administration of LPS. Levels of intracameral protein, cells, E-selectin, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and nitrite were determined. E-selectin, TNF-α, IL-6, and inducible nitric oxide synthase (iNOS) mRNAs and immunohistochemical reactivity of nuclear factor (NF)-κB and TNF-α were also examined in the iris-ciliary body.
results. After LPS injection, intracameral protein and cells increased from 18 to 30 hours later. Rolipram, however, inhibited elevation of protein and cells. After LPS injection, mRNA levels of E-selectin, TNF-α, and IL-6 in the iris-ciliary body increased 3 hours later, and iNOS mRNA increased 6 hours later. Elevation of mRNA levels for E-selectin, TNF-α, and IL-6 was inhibited by rolipram. After LPS injection, intracameral TNF-α and IL-6 levels increased 4 to 6 hours later, and nitrite levels increased 14 to 20 hours later. Elevation of TNF-α and IL-6 levels was decreased by rolipram. Rolipram did not affect iNOS mRNA and nitrite levels. Immunoreactivity of NF-κB was strong 1 hour after LPS injection, and was decreased by rolipram. Immunoreactivity of TNF-α was strong 4 hours after LPS injection and was decreased by rolipram.
conclusions. NF-κB translocation and expression of E-selectin, TNF-α, and IL-6 are involved in the pathogenesis of LPS-induced uveitis and are inhibited by rolipram. The inhibitory effect of rolipram in uveitis may be independent of iNOS synthesis.
Endotoxin (lipopolysaccharide; LPS)-induced uveitis in rats is a known model of human disease.
1 Interleukin (IL)-6, tumor necrosis factor (TNF)-α, E-selectin, and nitric oxide (NO) production have been reported to be involved in the pathogenesis of LPS-induced inflammation and uveitis.
2 3 4 5 6 7 8 9 IL-6 and TNF-α are known as proinflammatory cytokines,
5 and E-selectin contributes to continuing cellular infiltration into the site during inflammation.
4 Immunologic and inflammatory stimuli induce the expression of the inducible isoform of nitric oxide synthase (iNOS), producing NO.
10 LPS has induced translocation of nuclear factor (NF)-κB, an inducible transcription factor that mediates the overproduction of TNF-α and other cytokines.
11
Phosphodiesterase type 4 is reportedly a major cAMP-hydrolyzing isoenzyme in proinflammatory cells, including T-lymphocytes, monocytes, neutrophils, and eosinophils.
12 13 14 15 Rolipram, a type 4 phosphodiesterase inhibitor, suppresses cutaneous inflammation,
16 zymosan-induced inflammation,
17 LPS-induced TNF-α expression,
18 and collagen-induced arthritis.
19 Xu et al.
20 have demonstrated the protective effect of rolipram in experimental autoimmune uveoretinitis and have reported that the protection is independent of IL-10–induced activity. Blease et al.
21 have reported that rolipram, in combination with salbutamol inhibited TNF-α, induces E-selectin expression. Sanz et al.
22 have demonstrated that rolipram inhibits leukocyte–endothelial cell interactions in vivo through P- and E-selectin downregulation. Rolipram is used as an antidepressant.
23 In the present study, in a model of LPS-induced uveitis in Wistar rats, we determined the number of cells and amount of protein in the aqueous humor and the mRNA and protein levels of IL-6, TNF-α, E-selectin, and nitrite and of NF-κB.
To produce uveitis, LPS (Escherichia coli, serotype 055:B5; Sigma-Aldrich, St. Louis, MO) in 100 μg/100 μL pyrogen-free 0.9% sodium chloride was injected into one hind footpad. Rats treated with 0.9% sodium chloride alone served as the control. Rolipram (4-(3-[cyclopentyloxy]-4-methoxyphenyl)-2-pyrrolidinone) was purchased from Wako Pure Chemical (Osaka, Japan), dissolved in 3% dimethyl sulfoxide (DMSO), and injected intraperitoneally 30 minutes before administration of LPS. The treatments were performed between 9 and 11 AM.
After intraperitoneal injection of pentobarbital (50 mg/kg body weight) the eye globe was enucleated and immediately submerged in RNA stabilization reagent (RNALater; Qiagen, Hilden, Germany), followed by isolation of the iris-ciliary body tissue from the stabilized eye globe. The dissected iris-ciliary body tissue was homogenized with a rotor-stator homogenizer in buffer (RLT; Qiagen). Total RNA was extracted with a kit (RNeasy Protect Mini Kit; Qiagen) and treated with RNase-free DNase (Qiagen) to remove any residual genomic DNA. cDNA from each sample was obtained by reverse transcription with random hexamers using reverse transcriptase (Multiscribe; Applied Biosystems Inc., [ABI], Tokyo, Japan).
Based on the database, real-time PCR primers and probes (Nippon EGT, Tokyo, Japan) were designed for E-selectin (GenBank accession no. L25527; http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD): forward primer 5′-GCCATGTGGT-TGAATGTAAAGC-3′, reverse primer 5′-GGATTTGAGGAACATTTC-CTGACT-3′, and 5′-(FAM)TTGACCCAACCTGCCCACG(TAMRA)-3′ (TaqMan probe; ABI); for TNF-α (accession no. NM012675): forward primer 5′-ACAAGGCTGCCCCGACTAC-3′, reverse primer 5′-TCCTGGTATGAAATGGCA AACC-3′, and probe 5′-(FAM)TGCTCCTCACCCACACCGTCAGC(TAMRA)-3′ (TaqMan; ABI); for IL-6 (accession no. NM012589): forward primer 5′-TCAACTCCATCTGCCCTTCAG-3′, reverse primer 5′-AAGGCAACTGGCTGGAAGTCT-3′, and 5′-(FAM)AACAGCTATGAAGTTTCTCTCCGCA(TAMRA)-3′ (TaqMan probe; ABI); for iNOS (accession no. NM012611): forward primer 5′-TGGTCCAACCTGCAGGTCTT-3′, reverse primer 5′-CAGTAATGGCCGACCTGAT-GT-3′, and 5′-(6-FAM)TGCCCGGAGCTGTAGCACTGCAT(TAMRA)-3′ (TaqMan probe; ABI); and for glyceraldehyde 3-phosphate dehydrogenase (GAPDH, accession no. AF106860): forward primer 5′-CCGAGGGCC-CACTAAAGG-3′, reverse primer 5′-GCTGTTGAAGTCACAGGAGACAA-3′, and 5′-(FAM)CATCCTGGGCTACAC TGAGGACCA(TAMRA)-3′ (TaqMan probe; ABI).
cDNA was used to detect real-time PCR products for E-selectin, TNF-α, IL-6, and iNOS with master mix (TaqMan Universal Master Mix; ABI) and a sequence detection system (Prism 7700; ABI) with specific primers and probe. The thermal profile for each primer consisted of 2 minutes at 50°C and 10 minutes at 95°C, followed by 40 cycles for 15 seconds at 95°C and 1 minute at 60°C. To compare expression patterns, mRNA template concentrations of GAPDH and the target genes were calculated using the standard curve method. The expression levels of E-selectin, TNF-α, IL-6, and iNOS mRNA were normalized by the GAPDH mRNA level in each sample, and the changes were expressed as an n-fold increase, relative to the levels in control rats treated with 0.9% NaCl alone.
Rats were anesthetized with an intraperitoneal injection of pentobarbital and were perfusion fixed with 4% cold paraformaldehyde in phosphate-buffered saline (PBS) at various times (n = 4 for each time point). The eyes were enucleated, cut in half, and fixed with 4% paraformaldehyde in PBS for 30 minutes at 4°C.
For subunit p65 of NF-κB, fixed specimens were embedded in paraffin and sectioned at 5 μm. The sections were immunostained with a rabbit avidin-biotin complex (ABC) staining system (Santa Cruz Biotechnology, Santa Cruz, CA), according to the manufacturer’s instructions. Sections were deparaffinized in xylene and hydrated with graded ethanol. Slides were boiled in target retrieval solution buffer (Dako, Kyoto, Japan) in a microwave for 4 minutes and then placed at room temperature for 20 minutes. Endogenous peroxidase activity in the tissue was quenched with 1% hydrogen peroxide in PBS for 10 minutes. After blocking with 1.5% normal goat serum in PBS, slides were incubated with rabbit anti-rat NF-κB polyclonal antibody (Santa Cruz Biotechnology) for 1 hour. Biotinylated secondary antibody (goat anti-rabbit IgG) and avidin-biotinylated horseradish peroxidase-complex reagent was applied, respectively, for 30 minutes each. Also, 3,3′-diaminobenzidine (Dako) was used as a chromogenic substrate. The sections were counterstained with hematoxylin (Wako Pure Chemicals, Osaka, Japan) and mounted (Aquatex; Merck, Darmstadt, Germany). To verify the binding specificity for NF-κB, some sections were also incubated with normal goat IgG or without the primary antibody (data not shown).
For TNF-α, specimens were OCT embedded, frozen, cryosectioned at 10 μm, and subjected to immunofluorescence staining. After blocking with 10% normal bovine serum in PBS, slides were incubated with goat anti-rat TNF-α polyclonal antibody (Santa Cruz Biotechnology) for 1 hour and immunolabeled with Texas red–conjugated bovine anti-goat IgG secondary antibody for 30 minutes. To verify the binding specificity for TNF-α, some sections were also incubated with normal bovine IgG or the primary antibody was omitted (data not shown).
After injection of LPS into the footpad, mRNAs for E-selectin, TNF-α, and IL-6 in the iris and ciliary body increased at 1.5 hours, reached maximum levels (88 ± 5-, 62 ± 8-, and 1250 ± 150-fold, respectively) at 3 hours, and then gradually decreased. After injection of LPS into the footpad, iNOS mRNA in the iris and ciliary body increased at 3 hours, reached maximum level (75 ± 11-fold) at 6 hours, and then gradually decreased.
Inhibition of Expression of mRNAs for E-selectin, TNF-α, IL-6, and iNOS by Rolipram
Inhibition of Protein Levels of TNF-α and IL-6 in the Anterior Chamber by Rolipram
In the present study, protein and cells in the anterior chamber increased 18 to 30 hours after 100 μg of LPS was injected. Our findings of LPS-induced anterior uveitis in Wistar rats were similar to those in Lewis and Wistar rats described by Rosenbaum et al.,
1 and in young Lewis rats described by Hoekzema et al.
2
In our preliminary study, rolipram was injected intraperitoneally 60 or 30 minutes before, at the same time of, or 30 minutes after administration of LPS. Rolipram injected 30 minutes before LPS was most effective in inhibiting LPS-induced elevation of intracameral protein and cells. In our present study, therefore, rolipram was injected 30 minutes before LPS.
Alterations in behavior occurred in some rats treated with rolipram in the present study. The alterations were mild and were observed only in a small number of rats treated with 30-μmol/kg doses. Our findings were similar to those described by Wachtel.
25
Hoekzema et al.
2 reported that after LPS injection, the IL-6 concentration increased in the aqueous humor of Lewis rats. In a study by de Vos et al.
3 TNF-α and IL-6 levels in the aqueous humor of Lewis rats were elevated 4 hours after administration of LPS. Suzuma et al.
4 have reported observing E-selectin immunoreactivity on the vessel walls of the iris 7 hours after LPS treatment in male Lewis rats. Our present findings of LPS-induced expression of mRNAs for E-selectin, TNF-α, and IL-6 and elevation of TNF-α and IL-6 in the aqueous humor in Wistar rats were similar to those in Lewis rats described by these investigators.
2 3 4 Goureau et al.
7 reported that after LPS injection, iNOS mRNA in the iris-ciliary body increased at 2 to 24 hours, and nitrite in the aqueous humor increased in Lewis rats to 18 μM at 16 hours. Increased expression of iNOS mRNA 6 hours after LPS injection and elevated nitrite production 16 hours after LPS injection in our present study were similar. Baeuerle and Henkel
11 reported that LPS induces translocation of NF-κB and mediates the overproduction of TNF-α. In our present study, NF-κB immunoreactivity was strong 1 hour after LPS injection. Ollivier et al.
26 reported that cAMP inhibited NF-κB–mediated transcription in human monocytic cells and endothelial cells. Our findings of LPS-induced translocation of NF-κB and its inhibition by rolipram are compatible with those findings.
11 26
Rolipram suppressed several types of inflammation.
16 17 18 19 Klemm et al.
17 reported that rolipram inhibited endogenous TNF-α production in a murine model of acute inflammation induced by zymosan. In a study by Buttini et al.
18 the level of TNF-α mRNA induced in rat brain by LPS challenge was reduced by intraperitoneal administration of rolipram. Ross et al.
19 have proposed that a major mechanism of action of rolipram in collagen-induced arthritis is suppression of TNF-α activity. In our present study, rolipram inhibited LPS-induced anterior uveitis and suppressed mRNAs for E-selectin, TNF-α, and IL-6 and protein levels of TNF-α and IL-6 in Wistar rats.
Increased NO production has been thought to be involved in LPS-induced uveitis.
7 8 9 In our present study, increased nitrite production was found in LPS-induced uveitis. Also, rolipram decreased LPS-induced elevation of protein and cells in the aqueous chamber, did not inhibit the expression of iNOS mRNA, and increased nitrite production induced by LPS. Dutta et al.
27 reported that the injection of LPS increased iNOS activity in the lung of Long-Evans rats, but pretreatment with rolipram did not affect NOS activity. Ross et al.
19 reported that rolipram inhibited the expression of TNF-α and IL-12, but did not affect NO production in collagen-induced arthritis in mice. Lieb et al.
28 reported that rolipram showed no inhibitory effect on LPS-induced iNOS or NO synthesis. The effects of rolipram on NO production that we observed were similar to those described previously.
20 27 28 It is unclear at present whether rolipram is clinically useful in treating uveitis or not.
In conclusion, the findings in our present study indicate that NF-κB translocation and expression of E-selectin, TNF-α, and IL-6 were involved in the pathogenesis of LPS-induced uveitis and were inhibited by rolipram. In contrast, the inhibitory effect of rolipram on LPS-induced uveitis was independent of iNOS synthesis and NO production in Wistar rats.
Supported in part by Grant-in-Aid for Scientific Research Grant 14571661 and by 21st Century COE Program from the Ministry of Education, Science, Sports and Culture of Japan.
Submitted for publication December 18, 2003; revised March 1 and March 17, 2004; accepted March 23, 2004.
Disclosure:
Z.-L. Chi, None;
S. Hayasaka, None;
X.-Y. Zhang, None;
Y. Hayasaka, None;
H.-S. Cui, 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: Zai-Long Chi, Department of Ophthalmology, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan;
[email protected].
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