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*
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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 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-ß2 (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-ß2 has been most intensively studied for its capacity to
inhibit T cell-dependent responses. TGF-ß2 is constitutively produced
by intraocular cells [epithelium of iris and ciliary body (I/CB),
retinal pigment epithelium] (13, 14, 15), and the vast
majority of TGF-ß2 present in normal AqH exists in its latent, rather
than its active, form (6, 7). Accordingly, current
evidence suggests that TGF-ß2 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, 18, 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, 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-ß 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-ß.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
For EAU induction, B10. A mice were immunized s.c. in the nape of neck, thigh, and footpad with 50 µg 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.
AqH collection and analysis
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-µl 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 µl 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.
Determination of cytokine levels in AqH
TGF-ß2 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-ß2. 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- 1ß, and IL-6 were estimated
using ELISA kits from R&D Systems (Minneapolis, MN) according to the
manufacturer’s instructions.
TGF-ß bioassay
To measure total TGF-ß, AqH samples were added to Mv1Lu cells (CCL-64; American Type Culture Collection, Manassas, VA) as described previously (4). In brief, 1 x 105 cells in 200 µl 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 µl 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 µl of 10x 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-ß1 (R&D Systems) were prepared in the same plates as the assayed experimental samples. A standard curve of TGF-ß concentration vs cpm was used to estimate TGF-ß in AqH samples. After acid activation, each 5 µl of AqH containing TGF-ß was diluted to 100 µl with assay medium.
Assay of T cell proliferation
Spleens were removed from naive BALB/c mice and pressed through nylon mesh to produce single-cell suspensions. RBC were lysed with Tris-NH4Cl. 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 µg/ml streptomycin (BioWhittaker), and 1 x 10-5 M 2-ME (Sigma) and supplemented with 0.1% BSA (Sigma), ITS+ culture supplement [1 µg/ml iron-free transferrin, 10 ng/ml linoleic acid, 0.3 ng/ml Na2Se, and 0.2 µg/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 x 104 enriched T cells, hamster anti-mouse CD3e IgG (2C11; PharMingen) (final concentration is 1.0 µg/ml), and 5 µl of AqH or PBS as 20% v/v. Total reaction volume was kept constant at 25 µl. The cells were pulsed with 2.5 µl of 20 µCi/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-ß2 (R&D system), IL-6 (PharMingen), or control polyclonal IgG (ICN Pharmaceuticals, Lisle, IL and PharMingen, respectively).
RNA preparation and ribonuclease protection assay (RPA)
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 µg 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-ß1, and TGF-ß2 as well as two housekeeping gene products, L32
and GAPDH. A total of 5 µg 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 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-1ß, 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-1ß, IL-6, and
IL-2 were detected; whereas the content of IL-1ß 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-1ß 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-1ß. Because
the content of IL-6 in 11-day AqH was almost 1000-fold greater than the
content of IL-1ß, we first examined the potential role of
IL-6.
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BALB/c T cells (2 x 104) were
suspended in serum-free medium to which anti-CD3 Abs (1.0 µg/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, anti-IL-6 Abs (20 µg/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-1ß Abs were substituted for
anti-IL-6 Abs. AqH from 11-day EAU eyes failed to suppress T cell
proliferation in the presence of anti-IL-1ß 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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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.
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TGF-ß2 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-ß1, we next examined AqH from EAU-inflamed
eyes for the presence of both isoforms, TGF-ß1 and TGF-ß2, 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-ß1 or anti-TGF-ß2 Abs were added. Whereas
anti-TGF-ß1 Abs only neutralize TGF-ß1, anti-TGF-ß2 Abs
neutralize both TGF-ß2 and the heterodimeric isoform TGF-ß1.2. 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-ß. At 11, 17, and 28 days, AqH from eyes with EAU contained 2- to
3-fold higher concentrations of total TGF-ß. The elevated total
TGF-ß 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-ß. Moreover, the elevated levels of TGF-ß
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-ß Abs were added to the bioassay to AqH
samples collected on days 11 and 28 (Fig. 4B), a large
proportion of the total TGF-ß proved to be the TGF-ß2/1.2 isoforms.
Only on day 17 did AqH contain a significant amount of the TGF-ß1
isoform. Even on this day, AqH contained significantly elevated levels
of TGF-ß2. As an independent assessment of TGF-ß2 content in AqH,
samples were assayed for TGF-ß2 using a sandwich ELISA. As revealed
in Fig. 4C, TGF-ß2 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-ß 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-ß2 was even higher.
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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-ß2, neutralizing anti-TGF-ß2 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-ß2 Abs. We have previously
reported that anti-TGF-ß 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-ß2.
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As a means of determining the source of increased levels of
TGF-ß isoforms in AqH during EAU, I/CB tissues were removed from
inflamed eyes on days 11, 17, and 28. As before, the tissues were
analyzed for mRNA expression by RPA. In Fig. 3
A (again), an
autoradiograph depicts mRNA for TGF-ß1 and TGF-ß2 plus GAPDH.
Densitometric analysis of this gel is presented in Fig. 6. Levels of TGF-ß2 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-ß1 mRNA levels were quite low in normal I/CB
and increased levels (2-fold increase) were only detected in
samples collected at 17 days. These findings indicate that
transcription of TGF-ß2 mRNA changed little during EAU, even though
activity of this growth factor was markedly elevated during the
disease. Thus, the increased TGF-ß 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-ß2 mRNA translation and posttranslational processing, rather than
enhanced transcription of the TGF-ß2 gene in ocular
tissues. It is also likely that intraocular transcription of TGF-ß1
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-ß1 production to ocular parenchymal cells or to leukocytes
that have heavily infiltrated into the posterior compartment of the eye
at this time.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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, 8, 9, 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-ß. The paradox of high TGF-ß 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-ß 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, 34, 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-ß1 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, 39, 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.
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 anti-inflammatory corticosteroids
(44). Administration of IL-6 protected mice from
demyelinating disease induced by a virus (45). More
important, pathologic concentrations of IL-6 have been shown to inhibit
T cell responses via TGF-ß, either by inducing de novo synthesis of
active TGF-ß or by activating macrophages which in turn synthesize or
release enzymes capable of activating latent TGF-ß (46).
Thus, the high levels of IL-6 we observed in AqH from eyes of mice with
EAU on day 11 may contribute to the accumulation of TGF-ß in this
fluid. In an indirect manner, early IL-6 production by ocular cells at
the time of onset of EAU may lead to progressive TGF-ß accumulation
in the ocular microenvironment, and this may influence the subsequent
course of the disease.
Neutralizing anti-IL-6 Abs restored the ability of 11-day AqH to suppress T cell activation, correlating with enhanced levels of TGF-ß. In fact, at all time points assessed, AqH from mice with EAU contained significantly higher levels of TGF-ß than normal AqH. Moreover, unlike that of normal AqH, the immunosuppressive activity of AqH from eyes inflamed with EAU is due predominantly to its content of TGF-ß2. Endogenous production of TGF-ß2 by ocular parenchymal cells is constitutively high, and levels of TGF-ß2 mRNA did not rise in ocular tissues during the course of EAU. Therefore, the increase in activity ascribed to TGF-ß2 found in AqH samples collected during EAU probably reflects increased efficiency of posttranslational processing of the gene product and/or accumulation of TGF-ß2 due to decreased loss via the trabecular meshwork.
Although TGF-ß2 was the dominant isoform in AqH throughout EAU, significantly elevated levels of TGF-ß1 were observed on day 17. Several sources for this TGF-ß1 can be considered. First, the blood-ocular barrier is broken at this point, and plasma contains TGF-ß1 (47). Second, the breached blood-ocular barrier permits blood-borne leukocytes to enter the eye, and these cells can produce TGF-ß1. It is relevant that Kiefer et al. (48) have demonstrated that T cells and macrophages in the spinal cord of animals with EAE produce TGF-ß. In addition, cells of I/CB, which normally produce only TGF-ß2, can activate their own TGF-ß1 production in the presence of inflammation and injury. Our evidence indicates that mRNA for TGF-ß1 is increased mildly in I/CB tissues removed from eyes with EAU at 17 days. Because neutralizing Abs directed at TGF-ß2, rather than TGF-ß1, abolished the immunosuppressive activity of 17-day EAU AqH, the enhanced immunosuppressive activity observed in this AqH arises predominantly from its TGF-ß2 content.
The coexistence of immunosuppressive AqH and progressively intense and
destructive inflammation in eyes of mice with EAU on day 17 is a
paradox. It may be relevant that TNF- levels in AqH were highest on
day 17, commensurate with increased levels of TNF- mRNA in I/CB.
Thus, the inflammation present at 17 days may be driven by an excess of
TNF-. We suspect that the severity of EAU at day 17 might be even
greater if intraocular levels of TGF-ß were not significantly
elevated, and that this may account for why the disease begins to
resolve between 17 and 28 days.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. J. Wayne Streilein, Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02114. E-mail address:
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.
Received for publication August 23, 1999. Accepted for publication November 11, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-melanocyte stimulating hormone as a potential immunosuppressive factor in aqueous humor. Curr. Eye Res. 11:1199.[Medline]
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Y. Ke, G. Jiang, D. Sun, H. J. Kaplan, and H. Shao Ocular Regulatory T Cells Distinguish Monophasic from Recurrent Autoimmune Uveitis Invest. Ophthalmol. Vis. Sci., September 1, 2008; 49(9): 3999 - 4007. [Abstract] [Full Text] [PDF]
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 M. Kitamura, K. Iwabuchi, N. Kitaichi, S. Kon, H. Kitamei, K. Namba, K. Yoshida, D. T. Denhardt, S. R. Rittling, S. Ohno, et al. Osteopontin Aggravates Experimental Autoimmune Uveoretinitis in Mice J. Immunol., May 15, 2007; 178(10): 6567 - 6572. [Abstract] [Full Text] [PDF]
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 K. G.-J. Ooi, G. Galatowicz, V. L. Calder, and S. L. Lightman Cytokines and Chemokines in Uveitis - Is there a Correlation with Clinical Phenotype? Clin. Med. Res., December 1, 2006; 4(4): 294 - 309. [Abstract] [Full Text] [PDF]
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 H. Kitamei, K. Iwabuchi, K. Namba, K. Yoshida, Y. Yanagawa, N. Kitaichi, M. Kitamura, S. Ohno, and K. Onoe Amelioration of experimental autoimmune uveoretinitis (EAU) with an inhibitor of nuclear factor-{kappa}B (NF-{kappa}B), pyrrolidine dithiocarbamate J. Leukoc. Biol., June 1, 2006; 79(6): 1193 - 1201. [Abstract] [Full Text] [PDF]
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M. A. Toscano, A. G. Commodaro, J. M. Ilarregui, G. A. Bianco, A. Liberman, H. M. Serra, J. Hirabayashi, L. V. Rizzo, and G. A. Rabinovich Galectin-1 Suppresses Autoimmune Retinal Disease by Promoting Concomitant Th2- and T Regulatory-Mediated Anti-Inflammatory Responses J. Immunol., May 15, 2006; 176(10): 6323 - 6332. [Abstract] [Full Text] [PDF]
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S. Sugita, T. F. Ng, P. J. Lucas, R. E. Gress, and J. W. Streilein B7+ Iris Pigment Epithelium Induce CD8+ T Regulatory Cells; Both Suppress CTLA-4+ T Cells J. Immunol., January 1, 2006; 176(1): 118 - 127. [Abstract] [Full Text] [PDF]
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S. J. Curnow, F. Falciani, O. M. Durrani, C. M. G. Cheung, E. J. Ross, K. Wloka, S. Rauz, G. R. Wallace, M. Salmon, and P. I. Murray Multiplex Bead Immunoassay Analysis of Aqueous Humor Reveals Distinct Cytokine Profiles In Uveitis Invest. Ophthalmol. Vis. Sci., November 1, 2005; 46(11): 4251 - 4259. [Abstract] [Full Text] [PDF]
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S. J. Curnow, D. Scheel-Toellner, W. Jenkinson, K. Raza, O. M. Durrani, J. M. Faint, S. Rauz, K. Wloka, D. Pilling, S. Rose-John, et al. Inhibition of T Cell Apoptosis in the Aqueous Humor of Patients with Uveitis by IL-6/Soluble IL-6 Receptor trans-Signaling J. Immunol., October 15, 2004; 173(8): 5290 - 5297. [Abstract] [Full Text] [PDF]
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S. J. Curnow, K. Wloka, J. M. Faint, N. Amft, C. M. G. Cheung, V. Savant, J. Lord, A. N. Akbar, C. D. Buckley, P. I. Murray, et al. Topical Glucocorticoid Therapy Directly Induces Up-Regulation of Functional CXCR4 on Primed T Lymphocytes in the Aqueous Humor of Patients with Uveitis J. Immunol., June 1, 2004; 172(11): 7154 - 7161. [Abstract] [Full Text] [PDF]
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S. Yamagami, S. Yokoo, T. Mimura, and S. Amano Effects of TGF-{beta}2 on Immune Response-Related Gene Expression Profiles in the Human Corneal Endothelium Invest. Ophthalmol. Vis. Sci., February 1, 2004; 45(2): 515 - 521. [Abstract] [Full Text] [PDF]
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G. Alexander, H. Carlsen, and R. Blomhoff Strong In Vivo Activation of NF-{kappa}B in Mouse Lenses by Classic Stressors Invest. Ophthalmol. Vis. Sci., June 1, 2003; 44(6): 2683 - 2688. [Abstract] [Full Text] [PDF]
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H. Yamagami, S. Yamagami, T. Inoki, S. Amano, and K. Miyata The Effects of Proinflammatory Cytokines on Cytokine-Chemokine Gene Expression Profiles in the Human Corneal Endothelium Invest. Ophthalmol. Vis. Sci., February 1, 2003; 44(2): 514 - 520. [Abstract] [Full Text] [PDF]
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 T. Reinhard, H. Bonig, S. Mayweg, D. Bohringer, U. Gobel, and R. Sundmacher Soluble Fas Ligand and Transforming Growth Factor {beta}2 in the Aqueous Humor of Patients With Endothelial Immune Reactions After Penetrating Keratoplasty Arch Ophthalmol, December 1, 2002; 120(12): 1630 - 1635. [Abstract] [Full Text] [PDF]
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K. Ohta, K. Nakayama, T. Kurokawa, T. Kikuchi, and N. Yoshimura Inhibitory Effects of Pyrrolidine Dithiocarbamate on Endotoxin-Induced Uveitis in Lewis Rats Invest. Ophthalmol. Vis. Sci., March 1, 2002; 43(3): 744 - 750. [Abstract] [Full Text] [PDF]
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