行政院國家科學委員會專題研究計畫期中進度報告:細胞間素活化轉錄因子 NF-KB 引起眼球內炎性反應的研究（2/3）

行政院國家科學委員會專題研究計畫 期中進度報告

細胞間素活化轉錄因子 NF-KB 引起眼球內炎性反應的研究

（2/3）

中 華 民 國 93 年 5 月 31 日

Expression of Fractalkine and CX3CR1 in Experimental

Autoimmune Anterior Uveitis

I-Mo Fang 1, 2, Chang-Hao Yang1, Chang-Pin Lin1, Chung-May Yang1,

Muh-Shy Chen1

From the 1Department of Ophthalmology, National Taiwan University Hospital, and

the 2Department of Ophthalmology, Taipei Municipal ZhongXiao Hospital, Taipei,

Taiwan

Reprint requests to:

Chang-Hao Yang, MD, PhD

Department of Ophthalmology, National Taiwan University Hospital

Chung-Shan S. Rd. No.7, Taipei, Taiwan

TEL: 886-2-23123456 ext. 2131 Fax: 886-2-23412875

Abstract.

Purpose. To demonstrate the expression and location of fractalkine and receptor,

CX3CR1, in iris/ciliary body and thus establish their roles in experimental

autoimmune anterior uveitis, an animal model of human acute anterior uveitis.

Methods. Uveitis was induced with the injection of melanin-associated antigen

intraperitoneally and into the left footpad simultaneously. Some rats were treated with

a putative NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC; 40 mg/kg/day) every day after immunization. At defined time points, fractalkine and CX3CR1 mRNA

expression were semiquantified by using RT-PCR. Fractalkine in aqueous humor was

determined by ELISA. The cellular sources of fractalkine were determined by

immunhistochemical staining.

Results. Fractalkine mRNA was found to be upregulated in iris/ciliary body 9 days

after immunization, preceding clinical disease onset. CX3CR1 mRNA exhibited peak

levels at day 14, coincident with disease onset. Fractalkine in aqueous humor showed

a similar expression profile to mRNA expression. PDTC markedly inhibited the

expression of fractalkine mRNA in iris/ciliary body and fractalkine protein in aqueous

humor. Immuhistochemical staining revealed fractalkine was prominently expressed

on vascular endothelial cells.

interactions in the genesis of experimental autoimmune anterior uveitis. The

activation of fractalkine gene is, at least in part, via a NF-κB-dependent pathway. Therefore, selective anti-fractalkine or anti- NF-κB therapy may become a therapeutic potential for acute anterior uveitis treatment.

Introduction:

Experimental autoimmune anterior uveitis (EAAU) is a T-cell-mediated autoimmune

disease that serves as an animal model for human acute anterior uveitis. 1,2.

Immunization of Lewis rat with bovine melanin-associated antigen (MAA) and

appropriate adjuvants resulted in a disease with many of the clinical and pathologic

features of the human disease3. EAAU differed particularly from other models

because the inflammation remained exclusively anterior without retina and choroid

involvement4,5 .

Chemokines are small chemoattractant cytokines that induce leukocyte accumulation

at inflammatory sites and modulate inflammatory activities via the recruited cells6,7.

Chemokine are most likely implicated in the pathogenesis of autoimmune diseases.

According to NH2-terminal cysteine motifs, chemokines can be grouped into four

famuly: C, CC, CXC, and CX3C8. Fractalkine, a member of CX3C chemokine, differs

greatly from other chemokines for it can exit either as a soluble form or a

membrane-bound form9. The soluble form acts as a potent chemoattractant for

monocytes/macrophages, T-cells and natural killer cells whereas the membrane-bound

form functions as an adhesion molecule10, 11. Fractalkine exerts its biological activity

CX3CR1 expressed mainly on specific types of leukocyte, which enable these cells to

migrate in response to the concentration gradients of fractalkine14,15

Because of its important biological activities, fractalkine has been previously reported

to play a role in the pathogenesis of diverse inflammatory diseases16. These include

cardicac allograft refection, arthritis, and psoriasis, and toxic neuronal injury 17-20. In

the eye, previous investigators had demonstrated the presence of fractalkine in normal

cultured ocular endothelial cells in vitro21. However, to our knowledge, no

investigations to date have been conducted to evaluate fractalkine expression in ocular

inflammatory disease.

The nuclear factor kappa B (NF-κB) is a transcriptional factor that can regulate the expression of pro-inflammatory genes, including cytokines, adhesion molecules and

chemokines22,23. Previous studies had demonstrated the central role of NF-κB

activation in the regulation of fractalkine gene induction in aortic smooth muscle cells

in vitro24. However, there were no data available about the role of NF-κB in vivo on fractalkine activation.

fractalkine and its receptors, CX3CR1, in the iris/ciliary body in EAAU. In addition,

we examined the effect of NF-κB inhibitor, Pyrrolidine Dithiocarbamate (PDTC), on fractalkine gene and protein expression in vivo to determine whether fractalkine

Materials and Methods

Animals

Lewis rats, 6–8 weeks old and weighing 125-160 g, were used for the experiments.

All animals were treated in accordance with the ARVO statement for the Use of

Animals in Ophthalmic and Vision Research.

Antigen and induction of EAAU

MAA was prepared as previously described by Borekhuyse and Kuhlmann with a

modification18. The iris and ciliary body were carefully obtained from fresh

pigmented bovine eyes. The tissue was gently homogenized and filtered through a

wire mesh to remove cellular debris and connective tissue. The homogenate was

centrifuged at 1.2 × 105g at 4°C for 15 min and washed once with phosphate-buffered saline (PBS) at pH 7.4. The resulting pellet was resuspended in 2% sodium dodecyl

sulfate (SDS) (Bio-Rad, Richmond, CA)and incubated at 70°C for 10 min. After centrifugation, the pellet was washed three times with water. The insoluble antigen

was dried and stored at –20°C.

To induce EAAU, Lewis rats received two separated injections on the same time: 1).

St.Louis, MO) and injected into left hind footpad in a volume of 0.05 ml. 2). MAA

was emulsified with 1µg purified Bordetella pertussis toxin (List, Campbell, CA) and injected intraperitoneally in a volume of 0.05 ml.

Clinical examination

The rats were clinically observed on a daily basis with slitlamp biomicroscopy for

clinical signs of ocular inflammation. Disease severity was clinically assessed with a

scale ranging from 0 to 4: 0 = normal; 1 = slight iris-vessel dilatation and some

anterior chamber cells; 2 = iris hyperemia, with some limitation in pupil dilation,

anterior chamber cells, and a slight flare; 3 = a miotic, irregular, hyperaemic, and

(sometimes) slightly damaged iris, with a considerable flare and cells (especially with

accumulation near the iris); and 4 = a seriously damaged and hyperaemic iris, a miotic

pupil often filled with protein, and cloudy gel-like aqueous humor (AqH).

Tissue preparation

Rats were sacrificed on days 3 (n = 5), 9 (n = 5), 11 (n = 5), 14 (n = 5), 18 (n = 5), and

25 (n = 5) after immunization. The eyes were harvested. Both eyes of the

experimental animals were used, e.g. 10 eyes from 5 rats were examined at each time

point. The eyes were quickly dissected, and the iris and ciliary body were isolated

Preparation of RNA and cDNA

Total RNA was extracted from the iris/ciliary body with Trizol reagent (Life,

Gaithersburg, MD). One microgram of total RNA from each sample was annealed for

5 min at 65°C with 300-ng oligo(dT)(Promega, Madison, WI) and reverse transcribed to cDNA by using 80 U Moloney murine leukemia virus reverse transcriptase

(MMLV-RT)(Gibco, Grand Island, NY) per 50 µg reaction for 1 h at 37°C. The reaction was stopped by heating for 5 min at 90°C.

Polymerase chain reaction

The amplification was performed with a thermocycler. (MJ Research, Waltham, MA)

The 50-µl reaction mixture consisted of 5 µl cDNA, 1 µl of sense and antisense primer, 200 µM of each deoxynucleotide, 5 µl 10× Taq polymerase buffer, and 1.25 U Taq polymerase (Promega, Madison, WI). Fractalkine primers were:

5’-GAATTCCTGGCGGGTCAGCACCTCGGCATA-3’,

5’-AAGCTTTTACAGGGCAGCGGTCTGGTGGT-3’ (DNAFax, Taipei, Taiwan);

CX3CR1 primers were: 5’-AGCTGCTCAGGACCTCACCAT-3’,

5’-GTTGTGGAGGCCCTCATGGCTGAT-3’ (DNAFax, Taipei, Taiwan); β-actin primers were: 5’-CTGGAGAAGAGCTATGAGCTG-3’,

5’-AATCTCCTTCTGCATCCTGTC-3’ (DNAFax, Taipei, Taiwan). Conditions for

amplifying fractalkine and CX3CR1 were as follows: denaturation, 1 min at 94°C, and elongation, 3 min at 72°C. For the annealing temperature, 62°C was designed for fractalkine and CX3CR1. At the end of amplification, the reaction mixture was heated

for 10 min at 72°C and then cooled to 4°C. A 10-µl sample of each polymerase chain reaction (PCR) product was separated by performing gel electrophoresis on 2%

agarose containing ethidium bromide (Sigma, St. Louis, MO) and then analyzed under

ultraviolet light against the DNA molecular length markers. The intensity of the

products was analyzed by using an image analyzer (Digital 1D Science; Eastman

Kodak, Rochester, NY), and the amount of PCR-amplifiable material in each

reverse-transcribed sample was standardized against the amount of a housekeeping

gene rat β-actin.

Quantification of leukocytes in AqH

Aqueous humor (AqH) was collected from the eyes by using a 30-gauge needle

immediately after the animal was sacrificed. The AqH was pooled in silicon-treated

microcentrifuge tubes (Fisher Scientific, Pittsburgh, PA). A volume of 2 µL AqH from one rat was stained with 0.4% trypan-blue solution, and the number of leukocytes was

counted under phase-contrast microscopy.

Quantification of fractalkine in AqH

The levels of fractalkine in the AqH obtained from rats with EAAU were quantitated

on days 3 (n = 5), 9 (n = 5), 11 (n = 5), 14 (n = 5), 18 (n = 5), and 25 (n = 5) after

immunization by using a sandwich enzyme-linked immunosorbent assay (ELISA) kit

(R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. The

ELISA assay was repeated twice. The sample was diluted up to 50l and used for the

tests. Optical density was determined at A450 with a microplate reader (Bio-Rad,

Richmond, CA). The chemokine concentration was determined from standard curves

by using recombinant standards supplied by the manufacturer.

Immunohistochemistry

Formalin-fixed, paraffin-embedded tissue sections were placed on slides,

deparaffinized in xylenes, and rehydrated through graded ethanol into PBS.

Endogenous peroxidase was blocked with 0.3% hydrogen peroxide in methanol, then

the sections were treated with 5% normal rat serum and incubated overnight with

anti–fractalkine immunoglobulin G (IgG) (150 nm/ml)(Santa Cruz Biotechnology,

and an avidin-biotinylated peroxidase complex (Santa Cruz Biotechnology, Santa

Cruz, CA) were used with 3-3’diaminobenzidine as a peroxidase substrate. Sections

were counterstained with hematoxylin, dehydrated, and mounted.

NF-κB inhibitor, PDTC treatment

To examine whether NF-κB is involved in regulating fractalkine gene, ten rats were randomly selective and received intrapertoneal injection of PDTC (40mg/kg) every

day after MAA induction. On14th day of immunization, the day with high levels of

fractalkne mRNA and protein expression, the rats were sacrificed. Fractalkine mRNA

in iris/ciliary body and fractalkine concentration in aqueous humor after PDTC

treatment was determined.

Statistical analysis

Values in the figures and the text are expressed as mean ± SEM. Difference among

the amounts of the chemokine at the different time points were evaluated by one-way

analysis of variance followed by Bonferroni multiple comparison test, as appropriate.

Values of p <0.05 were considered as significant.

Clinical scores of EAAU

Clinical signs for EAAU appeared on day 14 post-immunization. The disease reached

its peak on day 19, with a clinical score of 2.60 ± 0.35. Then, recovery started. The

disease totally recovered by around day 30.

Iris and ciliary-body fractalkine and CX3CR1 mRNA expression

Fractalkine mRNA was upregulation on day 9 and reached its peak on day 11,

preceding onset of clinical disease. Thereafter, fractalkine mRNA declined gradually

but stayed at higher than control level during the course of disease. (Fig. 1A)

CX3CR mRNA, the receptor for fractalkine, was upregulated on day 11. The maximal

expression of CX3CR1 mRNA was on day 14, concurrent with disease onset, then,

returned abruptly to control level on day 18. (Fig. 1B)

Fractalkine levels in aqueous humor

In aqueous humor, fractalkine protein rise significantly on day 9, prior to clinical

disease onset. It reached peak on day 14, concurrent with disease onset. Thereafter it

Quantification of leukocytes in Aqueous humor

Lymphocytes and monocytes were the dominant leukocyte subsets in aqueous humor,

in parallel with the upregulation of fractalkine protein. (Fig.2)

Effects of NF-κB inhibitor, PDTC, on fractalkine mRNA in iris/ciliary body and

fractalkine protein in aqueous humor

On day 14, PDTC treatment resulted in a reduction in fractalkine mRNA in iris/ciliary

(P=0.001). Furthermore, treatment with PDTC significantly reduced the levels of

fractalkine protein in aqueous humor (720 ± 110 pg/ml vs 280 pg/ml ±70, P= 0.001).

Immunohistochemical staining of fractalkine

Immunohistochemical analysis on day 14 revealed that fractalkine was prominently

expressed on endothelial cells of vessel wall in iris/ciliary body (Fig. 4A). Control

sections after omission of primary antibodies showed only background staining (Fig.

4B).

Discussion

the trafficking of monocytes and T cells into sites of inflammation25. In this study, we

demonstrated a strong expression of fractalkine before and during the evolution of

EAAU. Furthermore, upregulation of fractalkine was associated with expression of

cognate receptor; CX3CR1, suggesting that fractalkine and CX3CR1 may participate

in the pathogenesis of rat model of acute anterior uveitis. To our knowledge, this is the

first study to identify the expression of fractalkine in ocular inflammatory disease.

Large leukocytes infiltration, especially monocytes and T lymphocytes, in iris/ciliary

body is the pathological hallmarks of EAAU26. To recruit certain type of leukocytes to

the site of inflammation, multi-step processes that include, establishment of

chemattractant gradient, firm attachment of leukocytes to and then migration across

the vascular endothelial cells should be properly regulated27-29.Previous studies had

demonstrated that fractalkine could act as a chemoattractive cytokine to attract

monocytes and lymphocytes, and an adhesion molecules to promote transendothelial

migration of leukocytes in a variety of inflammatory diseases30,31. In this study, we

found that fractalkine was upregulated prior to the infiltration of monocytes and

lyphocytes, and onset of clinical disease. Given the mechanistic parallels between

various inflammations elsewhere in the body, we could reasonably postulate that the

influx of monocytes and T cells.

Our observations that CX3CR1 mRNA was upregulated during the evolution of

EAAU provided further evidences of fractalkine –CX3CR1 interactions in selective

recruitment of lymphocytes and monocytes into iris/ciliary body. Furthermore,

regulation of CX3CR1 expression represented another level of control in the clinical

course and severity of diseases. To be an adhesion molecule, fractalkine is capable of

binding to leukocytes expressing CX3CR132. CX3CR1 is reported to express on the

surface of monocytes, T lymphocytes, and NK cells, the main infiltrating cells in

EAAU33,34. Therefore, the increases in CXCR3 expression facilitates these leukocytes

to be firmly entrapped by fractalkine-secreting cells and then subsequent to promote

leukocyte accumulation and inflammatory reactions. At the peak of clinical disease,

however, the abrupt decreases in CX3CR1 expression make these CX3CR1-bearing

cells unresponsive to fractalkine and then less attract to the inflammatory sites, which

marked the initiation of disease resolution.

By immunohistochemistry, we observed that vascular endothelial cells of iris/ ciliary

body are the cellular sources of fractalkine in rat model of acute anterior uveitis.

endothelial cells are capable of producing fractalkine35. We postulate that the

localization of fractalkine in vascular endothelium bears strategic meanings in the

leukocyte recuritment from circulation into iris/ ciliary body. It is only induced in

vascular endothelial cells that fractalkine could accessibly contact with leukocytes in

systemic circulation to act as an adhesion molecule in the membrane-bound from, and

could build concentration gradient between local tissue and systemic circulation to act

as a chemoattractant cytokine in soluble form.

The present study revealed that NF-κB inhibitor effectively suppressed fractalkine gene expression, indicating activation of fractalkine gene is, at lease in part, by means

of a NF-κB- dependent mechanism in experimental autoimmune anterior uveitis. Previous in vitro study demonstrated that fractalkine can be induced by

proinflammatory cytokines TNF-α and IL-1 in aortic endothelial cells36. However, TNF-α and IL-1themselves are activated through NF-κB. Therefore, in vivo during EAAU, it is possible that NF-κB influences fractalkine expression by direct activation of fractalkine or indirect alterations of other mediators.

In summary, the study was the first to demonstrate expression of fractalkine during

expression may direct CX3CR1 receptor-expressing mononuclear cell subsets to

inflammatory sites. Furthermore, this study has also identified that the activation of

fractalkine was by a NF-κB-dependent pathway. These findings may not only provide a new sight to clarify the molcecular mechanisms of acute anterior uveitis, but also

indicate that fractalkine or NF-κΒ might be a drug target for therapeutic applications.

Acknowledgements

This study was supported by research grant NSC from the National Science Council,

Republic of China and the Department of Health, Taipei City Government. We are

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Legends:

Fig. 1

Expression of fractalkine (A) and CX3CR1(B) mRNA in the iris/ciliary body from Lewis rats at different time points during the course of EAAU. The control represents the normal rats not been immunized. Bar charts represent the relative intensity of mRNA of fractalkine. Line charts represent the clinical scores of the disease. The intensity of mRNA was analyzed using an image analyzer and the relative intensity was determined by comparison of its intensity with that of β-actin. Data are presented as the mean ±SEM in five rats. * p < 0.05, when compared to normal control.

Fig. 2

The concentration of fractalkine in aqueous humor from Lewis rats at different time

points during the course of EAAU were determined by ELISA. The control represents

the normal rats not been immunized. Bar charts represent the concentrations of

fractalkine in aqueous humor. Line charts represent the clinical scores of the disease.

Data are presented as the mean± SEM in five rats. * p < 0.05, when compared to

normal control.

Fig. 3

Correlations of fractalkine concentration with numbers of different subsets of

represent number of different types of leukocyte in aqueous humor. Line charts

represent the concentration of fractalkine. Data are presented as the mean± SEM in

five rats. * p < 0.05, when compared to normal control.

Fig. 4

Effect of NF-κB inhibitor, Pyrrolidine Dithiocarbamate (PDTC), on fractalkine mRNA expression in iris/ciliary body (A) and fractalkine protein concentration in

aqueous humor (B) of Lewis rats immunized with melanin-associated antigen. The

rats were treated with PDTC after immunization. They were sacrificed on day 14.

Total RNA and aqueous humor was extracted. Columns marked as “Normal”

represent rats without immunization with MAA, while “MAA” represent rats

immunization with MAA. “PDTC-treated” represent rats immunization with MAA

and treated with PDTC (40mg/kg) everyday. The intensity of mRNA was analyzed

using an image analyzer and the relative intensity was determined by comparison of

its intensity with that of b-actin. Data are presented as the mean ± SD in five rats.

Significant differences: *P<0.05

Fig. 5

with experimental autoimmune anterior uveitis on days 14 post-immunization.

Positive staining was found in vascular endothelial cells (arrow) in ciliary body

Days Post-immunization F rac tal k ine/ β -a ctin 0 1 2 3 4 5 Cli n ical S c ore 0 1 2 3 4 3 9 11 14 18 25 Control * * * _* * Fractalkine β-actin Fig. !A Days Post-immunization CX 3 C R1/ β -acti n 0 1 2 3 4 5 C linic a l Sc or e 0 1 2 3 4 3 9 11 14 18 25 Control * * CX3CR1 β-actin Fig. 1B

D ays Post-im m unization Fr actlkine in Aqueous Humor (p g/ ml) 0 200 400 600 800 1000 1200 C linica l S c ore 0 1 2 3 4 3 9 11 14 18 25 Control * * * * * Fig. 2

Days Post-immunization N u m ber of C e lls in Aqueous Humor 0 200 400 600 800 1000 1200 1400 1600 Fr act a lk in e i n Aqu e ou s Hu m o r (pg/ m l) 0 200 400 600 800 1000 1200 lymphocyte monocyte neutrophil fractaline protein 3 9 11 14 18 25 Control Fig 3

Fractalki n e / β− actin 0 1 2 3 4

PDTC-treated MAA Normal

*

*

PDTC-treated MAA Normal

*

*