Current Eye Research

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Effects of topical FK506 on endotoxin-induced

uveitis (EIU) in the Lewis rat

Naofumi Hikita, Chi-Chao Chan, Scott M. Whitcup, Robert B. Nussenblatt &

Manabu Mochizuki

To cite this article: Naofumi Hikita, Chi-Chao Chan, Scott M. Whitcup, Robert B. Nussenblatt
& Manabu Mochizuki (1995) Effects of topical FK506 on endotoxin-induced uveitis (EIU) in the
Lewis rat, Current Eye Research, 14:3, 209-214, DOI: 10.3109/02713689509033516

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Effects of topical FK506 on endotoxin-induced uveitis (EIU) in the
Lewis rat
Naofumi Hikita'.2, Chi-Chao Chan', Scott M. Whitcup', Robert B. Nussenblatt' and Manabu Mochizuki2
'Laboratory of Immunology, National Eye Institute, NIH, Bethesda, MD, USA and 2Department of Ophthalmology, Kurume
University, Kurume, Japan
Abstract
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FK506 is a macrolide antibiotic and a potent immuno-
suppressant. To investigate the effect of topical FK506 on
acute ocular inflammation, we evaluated its action on the
development of endotoxin-induced uveitis (EIU). At two
different concentrations of 0.05% and 0.3%, topical FK506
was applied to Lewis rats with EIU. In aqueous, the mean
number of inflammatory cells per microliter t SEM was
2,389t 1,277, 1,571 '-1- 1,562, 898+-882, and 69% 152 for rats
treated with vehicle alone, 0.05%, 0.3% FK506, and 1%
prednisolone acetate. The median of histological grades was
2, 1.5, 0.8, and 0.5 for animals treated with these 4 different
regimens respectively. Analysis of aqueous protein showed a
small reduction in FK506-treated animals. Mean blood levels
of FK506 were low in rats treated with topical FK506 (0.05%,
0.84 ng/ml; 0.3%, 2.0 ng/ml) suggesting that its therapeutic
effect was not secondary to the systemic absorption of the
drug. Although FK506 is not as effective as prednisolone,
0.3% FK506 produced significant decreases in the mean
aqueous inflammatory cell number and histological inflam-
matory score as compared to control vehicle alone. We conclude
that topical FK506 can suppress EIU in a dose-dependent
fashion and may be an alternative medication for patients with
anterior uveitis and contra-indication to topical steroid. Curr.
Eye Res. 14: 209-214, 1995.
Key words: FK506; endotoxin-induced uveitis; cytokines; T
lymphocyte; adhesion molecules; rat
Introduction
Systemic injection of lipopoly-saccharide (LPS), that consists
of gram-negative bacterial cellular wall, will produce systemic
Correspondence: Chi-Chao Chan, Bldg 10, Rm 10N103, NIWNEI, 10 Center
DR MSC 1858, Bethesda, MD 20892-1858, USA
Received on August 16, 1994; accepted on December 6, 1994
0 Oxford University Press
Current Eye Research
inflammatory response. Injection of a sublethal dosage of LPS
into one hind footpad can induce an acute, anterior uveitis in
certain animals, including species of rats, mice, and rabbits
(1-3). This experimental model is named endotoxin-induced
uveitis (EIU) and is characterized by a transient but intense
acute inflammatory infiltration of neutrophils and monocytes,
as well as protein accumulation in the anterior chamber and
mild posterior vitritis of the eye (1). Recent experiments have
addressed the role of T cells (2). Various cytokines released
by the infiltrating inflammatory cells, such as TNF-a, IFN-y,
IL-1, IL-6, IL-8, have been shown to be important to the
development of EIU (4-6). EIU has become a model for the
study of anterior uveitis in humans and been used in the
evaluation of various therapeutic agents in the treatment of
ocular inflammation.
FK506 is a neutral macrolide lactone isolated from the
fermentation broth of Streptomyces tsukubaensis (7). Like
Cyclosporin A (CsA), FK506 exerts inhibitory effects on T
lymphocytes. It inhibits the synthesis of interleukin (1L)-2
mRNA and mRNAs of other genes up-regulated during T cell
activation. These mRNAs include those of TNF-a and IFN-y
(7). It also inhibits the proliferation and the generation of
cytotoxic T cells in mixed lymphocyte culture (8). CsA and
FK506 attenuate the tissue injury associated with reperfusion
of ischemic tissue (9). This observation suggests that FK506
may be able to ameliorate the reperfusive injury by modulating
the rate of accumulation of neutrophils and macrophages
through the release of cytokines and/or other proinflammat-
ory agents.
Although topical corticosteroids are capable of treating EIU
in rats and anterior uveitis in humans (lo), their adverse
effects, cataracts and glaucoma, are well-known and present
serious drawbacks to therapy. Therefore, the search for new
topical immunosuppressive agents is an ongoing task. In the
present study we examined the effect of topical FK506 on EIU.
 210 N. Hikita et al.
Materials and methods
Animals
Female Lewis rats (Charles River, Raleigh, NC), 8-10 weeks
old and weighing -200 g, were housed in a 12 h lightldark
cycle environment and fed Purina rat chow ad libitum. The
rats were kept under standard pathogen-free conditions, and
this research adhered to the Declaration of Helsinki and The
Guiding Principles in the Care and Use of Animals (DHEW
Publication, NIH 80-23).
Induction of EIU
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EIU was induced by injecting 100 pg of Salmonella typhymur-
ium endotoxin (Defco Laboratories, Detroit, MI), diluted in
0.1 ml of sterile pyrogen-free saline (Mediatech, Hemdon,
VA), into one hind footpad of each animal.
Treatment schedule
In two seperate experiments, EIU was induced in a total of 72
Lewis rats. In each experiment (40 for the first experiment
and 32 for the second experiment), animals were divided into
four groups: group A, vehicle alone; group B, FK506 0.05%;
group C, FK506 0.3%; group D, prednisolone acetate 1%. At
the time of endotoxin injection and every 4 h after, each rat
received 14 pl of the topical solution. A total of 6 dosages of
medication was instilled. All rats were killed 24 h after the
endotoxin injection. FK506 and the vehicle were kindly
provided by Fujisawa Pharmaceutical Co. Ltd., Japan.
Histologic study
In both experiments, the left eyes were enucleated, fixed in
4% glutaraldehyde for 30 min, and then transferred to 10%
buffered formaldehyde overnight. The eyes were cut through
the vertical plane to obtain the pupillary-optic nerve section.
The tissue was embedded in methacrylate and 3 pm thick
sections were stained with hematoxylin and eosin. The slides
were graded according to a scale based on the intensity of
ocular inflammation published previously (1 1).
Cell count and protein content in aqueous humor
Animals were sacrificed 24 h after endotoxin injection. In the
first experiment, immediate aspiration of aqueous humor of
the right eye was performed under a dissecting microscope.
One pl of the aqueous aspirate was placed on a poly-L-lysine
coated slide, air-dried and stained with Giemsa stain. The total
number of inflammatory cells in each slide was counted under
microscopic examination. The remainder of the aqueous sample
was reserved for analysis of protein content. Protein concentra-
tion was determined using the Coomassie blue-dye binding
method with the Coomassie Plus Protein Assay Reagent kit@
(PIERCE, Rockford, IL).
Immunohistochemical study
In the second experiment, half of the right eyes (16 eyes from
4 rats in each group) were collected for immunohistochemical
study. The enucleated eyes were snap frozen and embedded
in the optimal cutting temperature compound (O.C.T., Miles
Laboratory, Naperville, IL). Six pm serial sections through the
pupillary-optic nerve plane were stained using avidin-biotin-
complex immunoperoxidase technique (1 1). The primary anti-
bodies included monoclonal antibodies against ICAM- 1
(CD54), LFA-1 (CDlla/CD18), MHC class I1 (RTlB and
RTlD), CDllc/CD18, CD4, CD8 and CD20. The two
anti-adhesion molecules were gifts from Dr Miyasaka (Tokyo,
Japan). Other antibodies were purchased from Accurate Chem-
ical & Scientific Corporation, Westbury, NY. Mouse IgG
(Sigma Chemical Company, St Louis, MO) was the control
primary antibody. Biotin conjugated goat-anti-mouse IgG
(American Qualex, La Mirada, CA) was the secondary anti-
body. The avidin-biotin-peroxidase complex was applied, and
3,3'-diaminobenzidine-Ni2S04-H202was the substrate. The
stained slides were then interpreted by two independent,
masked observers.
FK506 measurement
In the second experiment, aqueous aspiration was performed
on the other half of the right eyes (4 in each group) for FK506
assay. In addition to the aqueous samples, 1 ml of whole blood
in 20 pl of heparin (1,000 I.U.) from each animal was also
harvested at 24 h after LPS injection. These samples were
stored at -20°C. Enzyme linked immunoassay was carried
out to measure the concentration of FK506 in these samples
by Fujisawa Pharmaceutical Co., Japan.
Statistical analysis
Because the grading of ocular inflammation was not distributed
in a normal manner, nonparametric methods were used to
analyze the data. The grades in each treated group (groups B,
C, D) were compared to the control group (group A) using
the Mann- Whitney rank sum test. A P value of less than 0.05
was used to reject the null hypothesis.
Results
Histopathology of EIU
Both experiments demonstrated similar histopathological
results, Figure 1 presented the data of the first experiment. An
acute inflammatory exudate consisting mainly of polymorpho-
nuclear neutrophils, mononuclear cells and proteinaceous
material was observed in the anterior and posterior chambers
as well as in the vitreous adjacent to the optic nerve head of
the Group A animals. In contrast, eyes of the rats treated with
0.05% FK506 (Group B), 0.3% FK506 (Group C) or 1%
predonisolone acetate (Group D) showed milder histological
evidence of ocular inflammation. While suggesting a dose-
response relationship of topical FK506, the differences between
Group B and C did not reach statistical significance.
 Effects of topical FK.506 211

n= 10 in each group n=iO in each group

-: Median
16
00

14
00
* ** I
12
E
Q1) 0 3 10

00
0 0 00
0 0 an *** € 8
- 6
-
-
0 Q, 0
QI, OD 0 4
W
2
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0
A B C D A B C D

Figure I . Scattergram of ocular inflammation in different groups of
animals with EIU. Data represent the histologic gradings of EIU on
a scale of 0 (no inflammation) to 4 (severe inflammation). A significant
decrease in ocular inflammation is observed in treated rats: *Group
A vs. B, P < 0.02; **Group A vs. C, P < 0.005; ***Group A vs.
D, P < 0.001. (group A, vehicle alone; group B, FK506 0.05%;
group C, FK506 0.3%;group D, prednisolone acetate 1 %).
3500
n= 10 in each group
Figure 3. Histogram of protein concentration in aqueous humor from
treated and control rats with EIU. Statistical difference is seen between
group A and D (*). (group A, vehicle alone; group B, FK506 0.05%;
group C, FK506 0.3%; group D, prednisolone acetate 1%).
(68.8+152, P < 0.001 and 898.5t882, P = 0.012; respect-
ively). Cell counts in group B (0.05% FK506) showed mild
reduction (137 12 1,562), and no significant difference as
compared to the placebo group A (Figure 2).

-
30001
2.
2500
T
Aqueous humor protein concentration
In normal rats, the aqueous humor contains 2-8 mg/ml
(3.2" 1.26 mg/ml) of protein. Protein concentrations in the
aqueous samples of the two treated groups (B and C) with
-
\
2 2000 topical FK506 showed mild reduction ( I 0.894 1.42 mg/ml and
3 1500
12.162 1.73 mg/ml, respectively) (Figure 3). Protein content
in Group A was 13.8821.63 mg/ml. In contrast, protein
concentration of Group D (corticosteroid-treated rats) was
1000 significantly decreased (3.2620.7 mg/ml, P < 0.001).

500
0 molecules (ICAM-1 and LFA-1) and MHC class I1 antigens
A B C D
Figure 2. Histogram of inflammatory cell numbers in one pl of
aqueous humor from treated and control rats with EIU. Significant
differences are seen between group A and C (*) or D (**). (group
A, vehicle alone; group B, FK506 0.05%; group C, FK506 0.3%;
group D, prednisolone acetate 1%).
Immunohistochemistry
The results of immunohistochemical staining for adhesion
correlated with the histological scores of ocular inflammation
and the intensity of the infiltrating cells, in particular, the
macrophage (Figure 4). In general, infiltrating leukocytes
expressed different inflammatory cellular markers, MHC class
I1 antigens and LFA- 1. ICAM- I expression in ocular resident
cells decreased in rats treated with topical FK506 (Figure 4)
and steroids.

FK506 level in blood and aqueous humor

Inflammatory cell count in aqueous humor
Aqueous humor obtained from group A (treated with vehicle
alone) had the highest cell count numbers per p1(2,3892 1,277,
mean2SEM). In contrast, cell counts from group D (0.1%
prednisolone acetate) and group C (0.3% FK506) were low
FK506 levels in blood obtained from each group were: Groups
A and D, 0 ng/ml; Group B, 1.2420.37 ng/ml; Group C,
2.2420.19 ng/ml (Figure 5). In the aqueous, FK506 levels
were: 0 ng/ml for Groups A and D; 2.8920.35 ng/ml and
19.028.63 ng/ml for Groups B and C , respectively.
 212 N. Hikita et al.
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Figure 4. Microphotograph of immunostainings shows macrophages (A) and ICAM-1 expression (B) in the anterior segments of a control rat
with EIU. In contrast, there is minimal positive staining of infiltrating macrophages (C) and ICAM-I (D) in a rat treated with FKS06. (avidin-
biotin-immunoperoxidase, Bar = 0.5 pm).

Discussion n=4 In each group

The results of this study indicate that topical FK506 can
suppress the development of EIU in a dose-dependent fashion.
The blood level of FK506 in animals receiving topical FK506 2.5
II

in this experiment is much lower than that needed for the 6

systemic FK506 treatment in uveitic patients reported previ- 0
ously (13). Higher concentration of FK506 or liposome-bound 1.5
E
FK506 may improve penetration of topically administrated
drug into the aqueous humor and vitreous and produce better
1 8

m
lmO
inhibition on EIU (Whitcup et al., in preparation). The mechan-
ism by which topical FK506 accomplishes this may involve
several of its properties, in particular, its inhibitory effects on Om5
0.0 A B C
cytokines (9, 14-18), adhesion molecules (13, 19-22) and T
lymphocytes (7, 8, 13, 14, 19, 22, 23).
Several cytokines, such as IL-1, IL-6 and TNF-a have been
implicated as important mediators in EIU (4-6). IL-la , IL- Figure 5. FKS06 level in blood remains relatively low in animals
l p and TNF genes are found to be expressed locally in the received topical FKS06 treatments. (group A, vehicle alone; group
resident cells of the iris and ciliary body of rats with EIU (5). B, FKS06 0.05%; group C, FKS06 0.3%; group D, prednisolone
Biphasic release of TNF and IL-6 are reported in aqueous acetate 1%).
 Efsects of topical FK506
humor of animals developing EIU (6). However, an exacerba-
tion of EIU is found in both TNF-a resistant mice and mice
pre-treated with systemic anti-TNF-a antibody (25). Thus,
local production of cytokines seems to be critical in the
development of EIU. FK506 is capable of suppressing IL-1
release and synthesis from human monocytes and alveolar
macrophages activated with LPS (15). In vitro experiments
have demonstrated that FK506 inhibits TNF-a production by
cultured proximal tubular epithelium and human monocyte in
a dose related manner (16,17). In vivo, FK506 has been
documented to suppress TNF-a production and prevent hepatic
or renal injury induced by ischemidreperfusion (9, 18, 26).
FK506 blocks IL-1 and TNF production and release from the
infiltrating inflammatory cells and ocular resident cells of the
animals with EIU. This results in a significant reduction of
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ocular inflammation.
Expression of adhesion molecules and MHC class I1 antigens
have been shown upregulation on ocular tissues even before
the inflammatory cells reach the eyes with EIU (12, 27).
Treatments with anti-ICAM- 1 or LFA- 1 antibodies are effective
to prevent and inhibit EIU in the Lewis rat (12). FK506
can modulate expression of adhesion molecules and class I1
antigens on ocular cells in vitro and in vivo (19,21,24). This
inhibitory mechanism of FK506 can ameliorate the severity
of inflammation in a way similar to its action on rats with
autoimmune thyroid disease (20) and on patients with psoriasis
(22). The immunohistochemical data of the present study also
demonstrate lower expression of ICAM- 1, LFA- 1, RTl B and
RTlD on ocular cells of the rats treated with FK506.
Recently the role of T lymphocytes in EIU has been shown
to be of potential interest (2). Kogiso and associates reported
that anti-CD3 and CD4 antibodies significantly decreased
ocular inflammmation in mice with EIU. FK506, an immuno-
suppressant with selective effect on T lymphocytes, acts
directly to inhibit lymphokine production from these cells
(8,14). This effect may counteract the participation of T
lymphocytes in the development of EIU in the rat.
The comparable effectiveness of topical FK506 to topical
corticosteroids on EIU reveals an interesting dimension of its
pharmacological properties and suggests its potential usefulness
in the treatment of acute anterior uveitis in humans. Topical
corticosteroids are potent in treating EIU in rats (10) and
anterior uveitis in humans. The present study also demonstrated
that prednisolone is more effective than FK506 to inhibit
EIU. However, the adverse side effects induced by topical
corticosteroids are not uncommon, particularly in prolonged
application. Therefore, agents with anti-inflammatory effects
that lack associated side effects are worthy of investigation.
The relatively low blood level of FK506 associated with its
topical administration is less likely to contribute to systemic
renal and hepatic toxicities. Thus, topical FK506 may become
one of the therapeutic alternatives for anterior uveitis, particu-
larly in patients with steroid-induced glaucoma or other contra-
indications to topical corticosteroid therapy.
Acknowledgement
The authors would like to thank Ms Deborah Luyo for her
expert technical assistance.
213
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