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Analysis of Immunomodulatory Activities of
Aqueous Humor from Eyes of Mice with
Experimental Autoimmune Uveitis
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of April 29, 2017.
Kouichi Ohta, Barbara Wiggert, Satoru Yamagami, Andrew
W. Taylor and J. Wayne Streilein
J Immunol 2000; 164:1185-1192; ;
doi: 10.4049/jimmunol.164.3.1185
http://www.jimmunol.org/content/164/3/1185
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References
Analysis of Immunomodulatory Activities of Aqueous Humor
from Eyes of Mice with Experimental Autoimmune Uveitis1
Kouichi Ohta,* Barbara Wiggert,† Satoru Yamagami,* Andrew W. Taylor,* and
J. Wayne Streilein2*
I
mmune privilege is a constitutive feature of the anterior
chamber of the normal eye (1). In its strictest definition, immune privilege refers to the experimental finding that foreign
tissue grafts placed in the anterior chamber survive for prolonged,
often indefinite, intervals, whereas placement of such grafts at conventional body sites leads to acute, irreversible immune rejection.
The capacity of the ocular microenvironment [especially aqueous
humor (AqH)]3 to suppress immune effector responses and inflammation is believed to make an important contribution to ocular
immune privilege (2, 3).
When studied in vitro, normal AqH displays many immunosuppressive and anti-inflammatory features. With regard to adaptive
immunity, AqH suppresses T cell activation following ligation of
the TCR for Ag, inhibiting proliferation and secretion of IFN-␥
(4). Moreover, primed T cells activated in the presence of AqH are
converted into regulatory cells that suppress activation of bystander T cells in coculture and that inhibit experimental autoimmune uveitis (EAU) when injected into mice immunized with a
*Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, MA 02114; and †Laboratory of Retinal and Molecular Biology,
National Institutes of Health, Bethesda, MD 20892
Received for publication August 23, 1999. Accepted for publication November
11, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grant EY05678.
2
Address correspondence and reprint requests to Dr. J. Wayne Streilein, Schepens
Eye Research Institute, 20 Staniford Street, Boston, MA 02114. E-mail address:
[email protected]
3
Abbreviations used in this paper: AqH, aqueous humor; EAE, experimental allergic
encephalomyelitis; EAU, experimental autoimmune uveitis; I/CB, iris and ciliary
body; IRBP, interphotoreceptor retinoid-binding protein; RPA, ribonuclease protection assay.
Copyright © 2000 by The American Association of Immunologists
uveitogenic regimen of interphotoreceptor retinoid-binding protein
(IRBP) (5). To account for the immunosuppressive properties of
normal AqH, investigators have discovered that this ocular fluid
contains numerous immunomodulatory factors, such as TGF-␤2
(6, 7), ␣-melanocyte-stimulating hormone (8), vasoactive intestinal peptide (9), calcitonin gene-related peptide (10), macrophage
migration inhibitory factor (11), and free cortisol (12). Of these
factors, TGF-␤2 has been most intensively studied for its capacity
to inhibit T cell-dependent responses. TGF-␤2 is constitutively
produced by intraocular cells [epithelium of iris and ciliary body
(I/CB), retinal pigment epithelium] (13–15), and the vast majority
of TGF-␤2 present in normal AqH exists in its latent, rather than
its active, form (6, 7). Accordingly, current evidence suggests that
TGF-␤2 accounts for only a minor portion of the endogenous capacity of normal AqH to suppress T cell activation in vitro.
Existence of ocular immune privilege is believed to serve the
purpose of limiting the extent to which innate and adaptive immunity can cause intraocular inflammation. By limiting intraocular
inflammation, immune privilege preserves the integrity of the visual axis and thereby prevents blindness. Ocular inflammation,
whether expressed within the cornea, or within the uveal tract (iris,
ciliary body, choriocapillaris), is a frequent cause of visual impairment, accounting for ⬃10% of blindness in the western world (16). A
variety of experimental models have been developed in laboratory
animals as a means of studying the pathogenesis of ocular inflammation (17–19). Yet, virtually nothing is known about the extent to
which ocular inflammation interferes with ocular immune privilege.
For this reason, and because we wish to understand the critical factors
that contribute to the existence of ocular immune privilege, we have
examined the immunomodulatory status of eyes of mice in which
intraocular inflammation has been induced experimentally.
In the recent past, we induced EAU by immunization of genetically susceptible B10.A mice with the retinal protein IRBP (20,
0022-1767/00/$02.00
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Aqueous humor (AqH) contains immunosuppressive factors, especially TGF-␤2, that contribute to the immune privileged status
of the anterior chamber. However, this may not be true when the blood-ocular barrier is compromised by ocular inflammation.
To determine the immunosuppressive status of AqH from murine eyes afflicted with experimental autoimmune uveitis, B10.A mice
were immunized with interphotoreceptor retinoid-binding protein. AqH was collected from eyes of affected mice periodically after
immunization and then evaluated for content of TGF-␤, proinflammatory cytokines, and the capacity to suppress anti-CD3-driven
T cell proliferation. mRNA expression of selected cytokines in iris and ciliary body from inflamed eyes was analyzed by ribonuclease protection assay. We found that TGF-␤ levels were significantly increased in AqH from EAU eyes on days 11, 17, and 28.
AqH collected on day 11 (onset of disease) failed to suppress T cell proliferation and contained large amounts of locally produced
IL-6 that antagonized TGF-␤. In contrast, AqH collected at 17 days (when ocular inflammation was progressively severe) reexpressed the ability to suppress T cell proliferation, in this case due to high levels of blood-derived TGF-␤1 and eye-derived
TGF-␤2 in the absence of IL-6. Thus, during the onset of experimental autoimmune uveitis, the ocular microenvironment loses
its immunosuppressive properties due to local production of IL-6. But as inflammation mounts, AqH IL-6 content falls, and the
fluid reacquires sufficient TGF-␤ eventually to suppress immunogenic inflammation. The paradoxical roles of IL-6 in antagonizing
TGF-␤, while promoting TGF-␤ accumulation during ocular inflammation, is discussed. The Journal of Immunology, 2000, 164:
1185–1192.
1186
Determination of cytokine levels in AqH
TGF-␤2 levels in stored AqH and in sera from mice with EAU were assessed with a commercially available ELISA kit (Promega, Madison, WI).
This immunoassay will only detect biologically active TGF-␤2. A total of
1 N HCl was added to samples to acid activation. After incubation for 1 h
at 4°C, the acid was neutralized with a 1:1 mixture of 1N NaOH:1 M
HEPES. IFN-␥ and IL-2 levels in AqH were measured using anti-mouse
mAb pairs: rat IgG1, 18181D, and IgG1, 18112D; and rat IgG2a, 18161D,
and IgG2b, 18172D, respectively (PharMingen, San Diego, CA). TNF-␣,
IL- 1␤, and IL-6 were estimated using ELISA kits from R&D Systems
(Minneapolis, MN) according to the manufacturer’s instructions.
TGF-␤ bioassay
To measure total TGF-␤, AqH samples were added to Mv1Lu cells (CCL64; American Type Culture Collection, Manassas, VA) as described previously (4). In brief, 1 ⫻ 105 cells in 200 ␮l with AqH diluted with Eagle’s
MEM (BioWhittaker, Walkersville, MD) were incubated for 20 h at 37°C
in 5% CO2. To each well, 20 ␮l of 50 Ci/ml [3H]thymidine (NEN-DuPont)
was added, and the plate was incubated for an additional 4 h. After incubation, the media were discarded and 50 ␮l of 10⫻ trypsin-EDTA (BioWittaker) solution was added to each well; then the plate was incubated for
15 min at 37°C. The cells were recovered using Harvester 96 (Tomtec,
Orange, CT), and [3H]thymidine incorporation was measured in cpm using
a 1205 betaplate liquid scintillation counter (Wallac, Gaithersburg, MD).
Cultures of known amounts of pure TGF-␤1 (R&D Systems) were prepared in the same plates as the assayed experimental samples. A standard
curve of TGF-␤ concentration vs cpm was used to estimate TGF-␤ in AqH
samples. After acid activation, each 5 ␮l of AqH containing TGF-␤ was
diluted to 100 ␮l with assay medium.
Assay of T cell proliferation
For EAU induction, B10. A mice were immunized s.c. in the nape of neck,
thigh, and footpad with 50 ␮g of IRBP in 0.2 ml of emulsion mixed 1:1
with CFA (Difco, Detroit, MI) that had been supplemented with Mycobacterium tuberculosis (Difco) to the final concentration of 2.5 mg/ml. Simultaneously, the mice were given 500 ng of pertussis toxin (Sigma, St. Louis,
MO) in 0.1 ml i.p. as an additional adjuvant.
Spleens were removed from naive BALB/c mice and pressed through nylon mesh to produce single-cell suspensions. RBC were lysed with TrisNH4Cl. T cells were subsequently purified by passage through a T cell
enrichment column (R&D System) according to the manufacturer’s directions. The enriched naive T cells (⬎95% Thy-1⫹ cells as measured by flow
cytometry) were suspended in serum-free medium composed of RPMI
1640 medium, 10 mM HEPES, 0.1 mM nonessential amino acids, 1 mM
sodium pyruvate, 100 U/ml penicillin, 100 ␮g/ml streptomycin (BioWhittaker), and 1 ⫻ 10⫺5 M 2-ME (Sigma) and supplemented with 0.1% BSA
(Sigma), ITS⫹ culture supplement [1 ␮g/ml iron-free transferrin, 10 ng/ml
linoleic acid, 0.3 ng/ml Na2Se, and 0.2 ␮g/ml Fe(NO3)3] (Collaborative
Biomedical Products, Bedford, MA). The proliferation assay used was a
modification of one described previously (21). To individual wells of a
96-well V-shaped bottom plate (Corning Glass, Corning, NY), we added
2.0 ⫻ 104 enriched T cells, hamster anti-mouse CD3e IgG (2C11; PharMingen) (final concentration is 1.0 ␮g/ml), and 5 ␮l of AqH or PBS as
20% v/v. Total reaction volume was kept constant at 25 ␮l. The cells were
pulsed with 2.5 ␮l of 20 ␮Ci/ml [3H]thymidine for the final 8 h of the 48-h
incubation (37°C, 5% CO2/95% humidified air mixture). On day 2, the
cells were recovered using a Harvester 96 (Tomtec), and [3H]thymidine
incorporation was measured in cpm using a 1205 betaplate liquid scintillation counter (Wallac). Each sample were cultured in triplicate. In some
assays, AqH samples were neutralized with Ab against TGF-␤2 (R&D
system), IL-6 (PharMingen), or control polyclonal IgG (ICN Pharmaceuticals, Lisle, IL and PharMingen, respectively).
AqH collection and analysis
RNA preparation and ribonuclease protection assay (RPA)
EAU generates intraocular inflammation that develops over a protracted
course; the clinical expression is not uniformly evident until 10 or 11 days
after immunization, and the inflammation usually persists beyond 28 days
(23, 24). AqH was obtained from eyes of B10.A mice for in vitro analysis
on days 0 (control), 11, 17, and 28 after IRBP immunization. AqH was
obtained immediately after sacrifice from eyes using a 30-gauge needle and
10-␮l micropipettes (Fisher Scientific, Pittsburgh, PA) by capillary attraction and pooled into a siliconized microcentrifuge tube (Fisher Scientific).
AqH samples from panels of at least five mice (10 eyes) at each time point
were pooled, centrifuged at 3000 rpm for 3 min, and the cell-free supernatant was frozen immediately at ⫺70°C. On average, a total of 6 ␮l of
AqH was obtained from the two eyes of each mouse. Leukocytes that were
present in the pellet of centrifuged AqH were resuspended in medium,
stained with 0.4% trypan blue solution, and counted by phase-contrast
microscopy. The total protein content in AqH samples was measured using
the bicinchoninic acid protein assay reagent kit (Pierce, Rockford, IL) in
reference to a bovine albumin standard.
Total RNA was extracted by the single-step method using RNA-STAT-60
(Tel-Test, Friendswood, TX). I/CBs were dissected from eyes, homogenized, and centrifuged to remove cellular debris. The RNA pellet obtained
from 10 to 20 eyes was resuspended in nuclease-free water and processed
together as a group. Twenty to 30 ␮g of total RNA was extracted from
I/CB of 40 eyes. Detection and quantification of murine cytokine mRNAs
were accomplished with a multiprobe RPA system (PharMingen) as recommended by the supplier. Briefly, a mixture of [␣-32P]UTP-labeled antisense riboprobes was generated from the mCK-1 and mCK-3b multiprobe
template set (PharMingen). These sets contain anti-sense RNA probes that
can hybridize with target mouse mRNAs encoding TNF-␣, IL-6, IFN-␥,
TGF-␤1, and TGF-␤2 as well as two housekeeping gene products, L32 and
GAPDH. A total of 5 ␮g of total RNA was used in each sample. Total
RNA was hybridized overnight at 56°C with 300 pg of the 32P-labeled
anti-sense riboprobe mixture. Nuclease-protected RNA fragments were purified by ethanol precipitation. After purification, the samples were resolved on 5% polyacrylamide sequencing gels. The gels were dried and
Materials and Methods
Mice
Female B10.A mice (The Jackson Laboratory, Bar Harbor, ME) were purchased at 6 – 8 wk of age. Normal BALB/c mice were obtained from our
domestic, inbred mouse breeding colony. All animals were treated according to the Association for Research in Vision and Ophthalmology resolution on the use of animals in research.
Ag
IRBP was isolated from bovine retinas by Con A-Sepharose affinity chromatography and fast performance liquid chromatography as described previously (22).
Immunization
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21). Whereas AqH from eyes of normal B10.A mice has an extremely low concentration of protein and scarce leukocytes and
suppresses the activation of T cells exposed in vitro to anti-CD3
Abs, AqH collected from eyes of IRBP-immunized B10.A mice at
10 –12 days (just as intraocular inflammation is appearing) contained elevated levels of protein and leukocytes, indicating a
breach in the blood-ocular barrier. Moreover, this AqH failed to
suppress anti-CD3-driven T cell activation in vitro. Posterior ocular inflammation continued to escalate thereafter in eyes of mice
with EAU; by 17 days many of the animals displayed clinical
evidence of retinal vasculitis and moderate to severe retinal detachments. The inflammatory response in the posterior pole of the
eye begins to regress when eyes are evaluated at 28 –30 days. Not
unexpectedly, AqH obtained from eyes with EAU at 17 and 28
days contained high levels of protein and leukocytes, but, unlike
11-day AqH, displayed the capacity to suppress in vitro T cell
activation. In an effort to explain the changes in immunomodulatory properties of AqH from inflamed eyes during the course of
EAU, we have studied AqH samples for cytokine content, and we
have examined I/CB tissues from inflamed eyes for expression of
cytokine mRNA levels. Our results indicate that loss of immunosuppressive capacity of AqH in EAU correlates with the presence
of large amounts of intraocularly produced IL-6, which acts as a
TGF-␤ antagonist, and that reacquisition of the capacity of AqH
from inflamed eyes to suppress T cell activation correlates with
reduction in IL-6 and accumulation of large amounts of active
TGF-␤.
IMMUNOMODULATORY ACTIVITY OF AQUEOUS HUMOR
The Journal of Immunology
1187
subjected to autoradiography. Protected bands were observed after exposure of gels to x-ray film. Specific bands were identified on the basis of
their individual migration patterns in comparison to the undigested probes.
The bands were quantitated by densitometric analysis (NIH Image) and
were normalized to L32 or GAPDH.
Results
Evidence for proinflammatory cytokines in AqH from
EAU-inflamed eyes
B10.A mice received a uveitogenic regimen of IRBP, CFA, and
pertussis toxin. Panels of animals (five mice per panel) were sacrificed on days 0, 11, 17, and 28, and AqH was collected from both
eyes immediately thereafter. These samples were pooled, frozen at
⫺70°C, and then thawed and assayed by sandwich ELISA for
content of IL-1␤, TNF-␣, IL-6, IL-2, and IFN-␥. The results of a
representative experiment (of three) are presented in Fig. 1. As
anticipated, none of these cytokines was detected in normal AqH.
On day 11, IL-1␤, IL-6, and IL-2 were detected; whereas the content of IL-1␤ and IL-2 was in the pg range, IL-6 content was in the
ng range. Six days later (day 17), elevated levels of TNF-␣, IL-2,
and IFN-␥ were found, with only small amounts of IL-6 still
present. Similarly small amounts of TNF-␣, IL-6, and IL-2 were
still present in AqH samples obtained at 28 days. As described
above, only AqH obtained from mice with EAU at day 11 lacked
the capacity to suppress T cell activation in vitro. Because IL-6 and
IL-1␤ were both present on this day, but markedly reduced or
absent from later AqH samples, our next experiments were designed to determine whether the loss of immunosuppression by
11-day EAU AqH was caused by the presence of either IL-6 or
IL-1␤. Because the content of IL-6 in 11-day AqH was almost
1000-fold greater than the content of IL-1␤, we first examined the
potential role of IL-6.
Effect of anti-IL-6 Abs on capacity of AqH from mice with EAU
at day 11 to suppress anti-CD3-driven T cell activation
BALB/c T cells (2 ⫻ 104) were suspended in serum-free medium
to which anti-CD3 Abs (1.0 ␮g/ml) were added. AqH samples
obtained from B10.A mice 11 days after a uveitogenic regimen of
IRBP were added as 20% v/v (PBS was substituted in positive
control cultures) to these lymphocyte cultures. Subsequently, antiIL-6 Abs (20 ␮g/ml, which neutralizes 100 ng/ml of murine IL-6)
or Ig isotype control Abs were added. After a 40-h culture,
[3H]thymidine was added to assess the degree of proliferation. The
results of a representative experiment (of three) are presented in
Fig. 2. As reported previously (21), normal AqH, as well as AqH
removed from eyes of mice with EAU after 17 and 28 days suppressed T cell proliferation, whereas AqH removed from eyes of
EAU mice at 11 days failed to suppress T cell proliferation in the
presence of isotype control Ab. However, when anti-IL-6 Abs
were added to T cell cultures stimulated with anti-CD3 in the presence of 11-day EAU AqH, T cell proliferation was reduced to
baseline. Similar experiments were performed in which anti-IL-1␤
Abs were substituted for anti-IL-6 Abs. AqH from 11-day EAU
eyes failed to suppress T cell proliferation in the presence of antiIL-1␤ Abs (data not shown). These findings suggest that the inability of AqH collected 11 days after the induction of EAU in
B10.A mice to prevent T cell proliferation was due to its content
of IL-6.
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FIGURE 1. Levels of TNF-␣, IL-1␤,
IFN-␥, IL-2, and IL-6 in AqH from eyes
with EAU. AqH was collected and pooled
from both eyes of panels of B10.A mice at
periodic intervals after IRBP immunization.
Each cytokine was assayed with sandwich
ELISA. Amount of only IL-6 is expressed
as ng/ml. Others are expressed as pg/ml.
1188
IMMUNOMODULATORY ACTIVITY OF AQUEOUS HUMOR
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FIGURE 2. Effects of anti-IL-6 Ab on T cell proliferation cultures in the
presence of EAU-AqH. Spleen T cells from naive BALB/c were cultured
with anti-CD3 Ab in serum-free medium containing 20% AqH collected at
selected intervals from eyes of mice with EAU. Anti-IL-6 Ab (or Ig isotype
control) were added to these cultures. [3H]thymidine was added during the
final 8 h of the 48-h incubation. Mean values of triplicate samples of radioisotope incorporation are presented ⫾ SE. Medium, control cultures
containing medium without any AqH. ⴱ, Values significantly lower than
cultures containing control isotype Ig (p ⬍ 0.01).
Evidence that IL-6 mRNA is expressed in I/CB from eyes of
mice with EAU
IL-6, a proinflammatory cytokine and an acute phase reaction protein with pleiotropic effects, is produced by many cells following
appropriate stimulation. After LPS injection and in other systemic
inflammatory states, the liver produces large amounts of IL-6
which appear in the blood plasma. As a way of deciding whether
the high levels of IL-6 in AqH at 11 days in mice with EAU arose
from ocular cells or was of blood origin (the blood-ocular barrier
is breached at this time), I/CB tissues were removed from eyes of
EAU-afflicted mice at 11, 17, and 28 days. These tissues, plus
tissues from eyes of normal mice, were subjected to analysis of
IL-6 mRNA levels using the RPA (25). The results of this experiment are presented in Fig. 3. An autoradiograph of a gel that
covers the molecular size range of IL-6 mRNA transcripts, as well
as of the housekeeping genes, L32 (data not shown) or GAPDH,
are displayed in Fig. 3A. Whereas tissues removed from normal
eyes contained no IL-6 transcripts, IL-6 mRNA was readily detected in I/CB removed at 11, 17, and 28 days. A quantitative
densitometric analysis of this gel is displayed in Fig. 3B. Compared with levels of L32 mRNA, IL-6 mRNA levels rose 30-fold
at 11 days and then gradually declined at 17 and 28 days. Similar
RPAs were conducted for mRNA of TNF-␣, since the TNF-␣ and
IL-6 genes are coordinately regulated in bone marrow-derived
cells such as monocytes and macrophages. The amount of TNF-␣
mRNA in I/CB tissues at 11 days was not elevated, whereas
TNF-␣ mRNA was moderately elevated at 17 days. The markedly
enhanced expression of IL-6 mRNA in 11-day I/CB compared
with TNF-␣ suggests that expression of IL-6 in I/CB is not under
the same regulation as TNF-␣ in these tissues. Because we did not
find high levels of circulating IL-6 in the sera of mice with EAU
at 11 days (data not shown), we interpret these findings to mean
that the remarkably high levels of IL-6 in AqH of eyes of mice
with 11-day EAU resulted from local intraocular production, rather
than by accumulation from circulating IL-6. Our results do not
permit us to distinguish IL-6 production from ocular parenchymal
cells and that from infiltrating leukocytes. Experiments to examine
these possibilities are currently underway.
FIGURE 3. Time course of mRNA production of TNF-␣, IL-6, IFN-␥,
TGF-␤1, and TGF-␤2 in I/CB tissues of eyes afflicted with EAU. Total
RNA was prepared from murine eyes at the indicated times after IRBP
immunization and subjected to RPA. L32 (data not shown) and GAPDH
indicate housekeeping gene. A, Photograph of an agarose gel that is representative of three experiments. B, The bands were quantitated by densitometric analysis and normalized to L32. The data are expressed as arbitrary units compared with normal (day 0) value, which was set at 1.0.
The Journal of Immunology
1189
Evidence for the presence of TGF-␤ in AqH from eyes inflamed
from EAU
Effect of anti-TGF-␤ Abs on capacity of EAU AqH to suppress
T cell proliferation
To determine whether the immunosuppressive activity detected in
AqH obtained from inflamed eyes of mice with EAU on days 17
and 28 was due to its content of TGF-␤2, neutralizing anti-TGF-␤2
Abs were added to cultures of BALB/c T cells suspended in medium containing anti-CD3 Abs plus AqH. Control cultures received Ig isotype Abs. As the findings presented in Fig. 5 reveal,
AqH from eyes of normal mice and from eyes of mice with EAU
at 17 and 28 days inhibited T cell proliferation. Moreover, this
inhibition was relieved in the presence of anti-TGF-␤2 Abs. We
have previously reported that anti-TGF-␤ Abs have no influence
on T cell proliferation in cultures containing AqH collected at 11
days (21). Our current evidence strongly supports the view that the
immunosuppressive properties of AqH collected from inflamed
eyes during EAU arise primarily from its content of TGF-␤2.
Evidence that TGF-␤ mRNA is expressed in I/CB from eyes of
mice with EAU
As a means of determining the source of increased levels of TGF-␤
isoforms in AqH during EAU, I/CB tissues were removed from
FIGURE 4. The levels of total TGF-␤ in AqH from eyes of mice with
EAU. AqH was acid activated as described in Materials and Methods and
then subjected to the mink lung epithelial cell bioassay for total TGF-␤.
The results are expressed as ng/ml of total TGF-␤ (A). The levels of
TGF-␤1 and TGF-␤2 in AqH from eyes of mice with EAU. After acid
activation, AqH was incubated with neutralizing anti-TGF-␤1 and antiTGF-␤2 Ab for 1 h and then subjected to the mink lung epithelial cell
bioassay for total TGF-␤. The results are expressed as ng/ml of TGF-␤ (B).
The levels of TGF-␤2 in AqH from eyes with EAU. Stored AqH was
assayed for content of TGF-␤2 using sandwich ELISA after acid activation. Amount of TGF-␤2 is expressed as ng/ml (C).
inflamed eyes on days 11, 17, and 28. As before, the tissues were
analyzed for mRNA expression by RPA. In Fig. 3A (again), an
autoradiograph depicts mRNA for TGF-␤1 and TGF-␤2 plus
GAPDH. Densitometric analysis of this gel is presented in Fig. 6.
Levels of TGF-␤2 mRNA were found to be constitutively high in
normal I/CB, and these levels remained relatively constant
throughout the three observed time points during EAU. In contrast,
TGF-␤1 mRNA levels were quite low in normal I/CB and
increased levels (2-fold increase) were only detected in samples
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TGF-␤2 is the isoform normally produced intraocularly and
present, in its latent form, in normal AqH. When the blood-ocular
barrier breaks down during the initial clinical stages of EAU in
mice, plasma proteins leak into the anterior chamber. Because
plasma contains significant amounts of TGF-␤1, we next examined
AqH from EAU-inflamed eyes for the presence of both isoforms,
TGF-␤1 and TGF-␤2, using the mink lung cell bioassay. AqH
samples were collected as before from normal eyes and from eyes
of mice with EAU at 11, 17, and 28 days. These samples were then
added to cultures of Mv1Lu mink lung cells, and incorporation of
[3H]thymidine was assessed as evidence of cell viability. In some
cultures, neutralizing anti-TGF-␤1 or anti-TGF-␤2 Abs were
added. Whereas anti-TGF-␤1 Abs only neutralize TGF-␤1, antiTGF-␤2 Abs neutralize both TGF-␤2 and the heterodimeric isoform TGF-␤1.2. The results of representative experiments are presented in Fig. 4. Using this assay (Fig. 4A), normal AqH was found
to contain slightly more than 10 ng/ml total TGF-␤. At 11, 17, and
28 days, AqH from eyes with EAU contained 2- to 3-fold higher
concentrations of total TGF-␤. The elevated total TGF-␤ levels
observed at day 11 indicate that the inability of AqH collected on
that day to suppress T cell activation does not result from a depletion of TGF-␤. Moreover, the elevated levels of TGF-␤ found
in 17- and 28-day AqH samples correlate positively with the ability of these samples to suppress T cell activation. When neutralizing anti-TGF-␤ Abs were added to the bioassay to AqH samples
collected on days 11 and 28 (Fig. 4B), a large proportion of the
total TGF-␤ proved to be the TGF-␤2/1.2 isoforms. Only on day
17 did AqH contain a significant amount of the TGF-␤1 isoform.
Even on this day, AqH contained significantly elevated levels of
TGF-␤2. As an independent assessment of TGF-␤2 content in
AqH, samples were assayed for TGF-␤2 using a sandwich ELISA.
As revealed in Fig. 4C, TGF-␤2 levels reached their highest concentration in AqH samples collected at day 17 of EAU. Together,
these results indicate that although AqH from eyes suffering the
onset of EAU no longer displayed immunosuppressive properties,
the concentration of TGF-␤ was significantly higher than in normal AqH. At 17 days of EAU, when AqH displayed (again) the
ability to suppress T cell activation in vitro, the level of TGF-␤2
was even higher.
1190
IMMUNOMODULATORY ACTIVITY OF AQUEOUS HUMOR
leukocytes that have heavily infiltrated into the posterior compartment of the eye at this time.
Discussion
FIGURE 5. The effect of anti-TGF-␤ Ab on the capacity of AqH from
EAU mice to suppress T cell proliferation. Anti-CD3 proliferation was
done in the presence of 20% AqH of EAU with neutralizing anti-TGF-␤2
Ab as described in Fig. 2. AqH was pretreated with 20 ␮g/ml of antiTGF-␤2 Ab. M, control cultures containing medium without any AqH. ⴱ,
p ⬍ 0.05 compared with level of cpm of without AqH group. ⴱⴱ, p ⬍ 0.05
compared with level of isotype-treated group.
FIGURE 6. Densitometric analysis of mRNA production of TGF-␤1
and TGF-␤2 in I/CB tissues of eyes afflicted with EAU. The bands shown
in Fig. 3A were quantitated by densitometric analysis and were normalized
to GAPDH. The data are expressed as arbitrary units compared with normal (day 0) value, which was set at 1.0.
Downloaded from http://www.jimmunol.org/ by guest on April 29, 2017
collected at 17 days. These findings indicate that transcription of
TGF-␤2 mRNA changed little during EAU, even though activity
of this growth factor was markedly elevated during the disease.
Thus, the increased TGF-␤ that is present in EAU AqH and that
accounts for the ability of that fluid on days 17 and 28 to suppress
T cell activation must result from enhanced efficiency of TGF-␤2
mRNA translation and posttranslational processing, rather than enhanced transcription of the TGF-␤2 gene in ocular tissues. It is also
likely that intraocular transcription of TGF-␤1 contributes in a
minor way to the enhanced immunosuppression observed with
AqH from 17-day EAU eyes. However, our data do not enable us
to assign TGF-␤1 production to ocular parenchymal cells or to
The eye creates a microenvironment (2, 3, 26, 27) that promotes
anterior chamber-associated immune deviation (28 –30) and that is
inhospitable to the expression of immunogenic inflammation (2,
3). Thus, the eye is relatively protected from the vision-damaging
effects of intraocular inflammation. The term “relative” is important in the preceding sentence because the eye can, and does, experience inflammation. In clinical ophthalmology, acute and
chronic uveitis are common afflictions that all too often lead to
visual impairment and blindness. In the laboratory, intraocular inflammation can be evoked in informative model systems in mice,
rats, and rabbits (31).
By harvesting AqH at periodic intervals after induction of EAU
in mice, we have shown that leakage of plasma proteins into AqH
occurred early in the course of EAU and that the fluid promptly
lost its capacity to suppress T cell proliferation. Three factors
could contribute to the loss of immunosuppressive capacity. First,
plasma proteins display protease activity that degrades many of the
immunosuppressive neuropeptides normally present in AqH (e.g.,
␣-melanocyte-stimulating hormone, vasoactive intestinal peptide,
calcitonin gene-related peptide, and migration inhibitory factor)
(7–10). Second, shortly after plasma proteins leaked into AqH,
leukocytes also penetrated into the intraocular microenvironment,
and these cells may destroy immunosuppressive factors in AqH.
Third, the autoimmune attack directed at IRBP in EAU may halt
intraocular production of immunosuppressive factors, generating
an AqH that is no longer inhibitory of T cells.
Despite the fact that AqH removed from mice with EAU at 11
days lacked the capacity to suppress T cell activation in vitro, this
ocular fluid contained increased, rather than decreased, levels of
TGF-␤. The paradox of high TGF-␤ levels in AqH that failed to
suppress T cell activation is apparently resolved by our finding that
AqH at 11 days contains enormous amounts of IL-6. We have
recently demonstrated that IL-6 acts as a TGF-␤ antagonist when
both cytokines are added to in vitro T cell proliferation assays (K.
Ohta, S. Yamagami, A. W. Taylor, and J. W. Streilein, submitted
for publication). Moreover, the capacity of 11-day AqH to suppress T cell activation in the presence of neutralizing anti-IL-6 Abs
confirms that IL-6 is the factor in 11-day AqH that robs the fluid
of its immunosuppressive properties.
IL-6 is a pleiotropic cytokine whose functions include stimulation of Ig secretion, acute-phase protein synthesis, and platelet production (32). IL-6 synthesis can be induced by other cytokines,
especially IL-1 and TNF-␣. In fact, IL-6 has been implicated as a
major mediator of uveitis. It is detectable in ocular fluids of patients and rodents with anterior uveitis (33–35). Although IL-6
does not appear to act as a direct T cell mitogen, it is relevant that
IL-6 is involved in the induction of IL-2 receptor expression as
well as proliferation and differentiation of T cells activated via
ligation of the TCR for Ag. Indeed, IL-6 was found to be more
active in this regard than either IL-1 or TNF-␣ (36). Together, IL-2
and IL-6 have been reported to abolish the inhibitory effect of
TGF-␤1 on DNA synthesis by T cells (37). IL-6-deficient mice
have been reported to be resistant to the induction of experimental
allergic encephalomyelitis (EAE) (38 – 40). Administration of neutralizing anti-IL-6 Ab also reduced the incidence and intensity of
EAE (41). Our evidence indicates that the IL-6 found in 11-day
AqH derives from local production, and our results from RNase
protection assays points to ocular parenchymal cells and/or infiltrating leukocytes as the local source.
The Journal of Immunology
Acknowledgments
We thank Jacqueline M. Doherty for her invaluable help during these experiments and the preparation of this manuscript. We also thank Dr. Munenori Yoshida and Dr. Takeshi Kezuka for assistance with experimental
methods and design.
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Although IL-6 has many proinflammatory effects, it also has the
potential to display anti-inflammatory effects (42). For example,
IL-6 may provide negative feedback that inhibits the production of
IL-1 and TNF-␣ (43), and IL-6 may stimulate the secretion of
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IMMUNOMODULATORY ACTIVITY OF AQUEOUS HUMOR