Aqueous humor suppression of dendritic cell function helps maintain immune regulation in the eye during human uveitis.
ABSTRACT Noninfectious uveitis is characterized by a dysregulated inflammatory or immune response in the eye. It is unclear whether this represents a failure of immune privilege or an overwhelming inflammatory drive that has exceeded the capacity of regulatory mechanisms that are still functioning. The authors investigated immune regulation in the human eye during intraocular inflammation (uveitis) and its impact on dendritic cell (DC) function and subsequent T-cell responses.
Myeloid DCs were isolated from the aqueous humor (AqH) and peripheral blood of patients with active uveitis and characterized by flow cytometry. The effect of uveitis AqH was interrogated in an in vitro model of peripheral blood monocyte-derived DCs from healthy controls.
Myeloid DCs isolated from uveitic AqH were characterized by elevated major histocompatibility complex classes I and II (MHC I/II), but reduced CD86 compared with matched peripheral blood DCs. Exposure of peripheral blood monocyte-derived DCs from healthy controls to the inflammatory AqH supernatant recapitulated this phenotype. Despite interferon gamma (IFNγ)-dependent upregulation of MHC I, inflammatory AqH was overall suppressive to DC function, with reduced CD86 expression and diminished T-cell responses. This suppressive effect was equal to or greater than that induced by noninflammatory AqH, but was glucocorticoid independent (in contrast to noninflammatory AqH).
These data indicate that the ocular microenvironment continues to regulate DC function during uveitis, despite IFNγ-driven upregulation of MHC expression, supporting the hypothesis that immune regulation within the eye is maintained during inflammation.
- SourceAvailable from: jleukbio.org[show abstract] [hide abstract]
ABSTRACT: The delicate visual axis that makes precise vision possible is highly vulnerable to the destructive potential of immunogenic inflammation. Immune privilege of the eye is the experimental expression of the way in which evolution has coped with the countermanding threats to vision of ocular infections and ocular immunity and inflammation. Ocular immune privilege has five primary features that account for its existence: blood:ocular barriers, absent lymphatic drainage pathways, soluble immunomodulatory factors in aqueous humor, immunomodulatory ligands on the surface of ocular parenchymal cells, and indigenous, tolerance-promoting antigen-presenting cells (APCs). Three manifestations of ocular immune privilege that have received the most extensive study are the intraocular microenvironment, which is selectively anti-inflammatory and immunosuppressive; the prolonged acceptance of solid tissue and tumor allografts in the anterior chamber; and the induction of systemic tolerance to eye-derived antigens. Anterior chamber-associated immune deviation is known to arise when indigenous, ocular APCs capture eye-derived antigens and deliver them to the spleen where multicellular clusters of these cells, natural killer T cells, marginal zone B cells, and gammadelta T cells create an antigen-presentation environment that leads to CD4(+) and CD8(+) alpha/beta T cells, which as regulators, suppress induction and expression of T helper cell type 1 (Th1) and Th2 immune expression systems. The ways the eye influences local and systemic immune responses to ocular antigens and pathogens carry risks to and benefits for mammalian organisms. As loss of sight is a powerful, negative-selecting force, the benefits of ocular immune privilege outweigh the risks.Journal of Leukocyte Biology 09/2003; 74(2):179-85. · 4.57 Impact Factor
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ABSTRACT: To begin a process of standardizing the methods for reporting clinical data in the field of uveitis. Consensus workshop. Members of an international working group were surveyed about diagnostic terminology, inflammation grading schema, and outcome measures, and the results used to develop a series of proposals to better standardize the use of these entities. Small groups employed nominal group techniques to achieve consensus on several of these issues. The group affirmed that an anatomic classification of uveitis should be used as a framework for subsequent work on diagnostic criteria for specific uveitic syndromes, and that the classification of uveitis entities should be on the basis of the location of the inflammation and not on the presence of structural complications. Issues regarding the use of the terms "intermediate uveitis," "pars planitis," "panuveitis," and descriptors of the onset and course of the uveitis were addressed. The following were adopted: standardized grading schema for anterior chamber cells, anterior chamber flare, and for vitreous haze; standardized methods of recording structural complications of uveitis; standardized definitions of outcomes, including "inactive" inflammation, "improvement'; and "worsening" of the inflammation, and "corticosteroid sparing," and standardized guidelines for reporting visual acuity outcomes. A process of standardizing the approach to reporting clinical data in uveitis research has begun, and several terms have been standardized.American Journal of Ophthalmology 10/2005; 140(3):509-16. · 3.63 Impact Factor
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ABSTRACT: Immune-mediated inflammation and allograft rejection are greatly reduced in certain organs, a phenomenon called 'immune privilege'. Immune privilege is well developed in three regions of the body: the eye, the brain and the pregnant uterus. Immune-mediated inflammation has devastating consequences in the eye and brain, which have limited capacity for regeneration. Likewise, loss of immune privilege at the maternal-fetal interface culminates in abortion in rodents. However, all three regions share many adaptations that restrict the induction and expression of immune-mediated inflammation. A growing body of evidence from rodent studies suggests that a breakdown in immune privilege contributes to multiple sclerosis, uveitis, corneal allograft rejection and possibly even immune abortion.Nature Immunology 05/2006; 7(4):354-9. · 26.20 Impact Factor
Aqueous Humor Suppression of Dendritic Cell Function
Helps Maintain Immune Regulation in the Eye during
Alastair K. Denniston,1,2Paul Tomlins,1,2Geraint P. Williams,1,2Sherine Kottoor,1,2
Imran Khan,2Kadambari Oswal,2Mike Salmon,1Graham R. Wallace,1,2Saaeha Rauz,1,2
Philip I. Murray,1,2and S. John Curnow1,2
PURPOSE. Noninfectious uveitis is characterized by a dysregu-
lated inflammatory or immune response in the eye. It is unclear
whether this represents a failure of immune privilege or an
overwhelming inflammatory drive that has exceeded the ca-
pacity of regulatory mechanisms that are still functioning. The
authors investigated immune regulation in the human eye dur-
ing intraocular inflammation (uveitis) and its impact on den-
dritic cell (DC) function and subsequent T-cell responses.
METHODS. Myeloid DCs were isolated from the aqueous humor
(AqH) and peripheral blood of patients with active uveitis and
characterized by flow cytometry. The effect of uveitis AqH was
interrogated in an in vitro model of peripheral blood mono-
cyte-derived DCs from healthy controls.
RESULTS. Myeloid DCs isolated from uveitic AqH were charac-
terized by elevated major histocompatibility complex classes I
and II (MHC I/II), but reduced CD86 compared with matched
peripheral blood DCs. Exposure of peripheral blood monocyte-
derived DCs from healthy controls to the inflammatory AqH
supernatant recapitulated this phenotype. Despite interferon
gamma (IFN?)–dependent upregulation of MHC I, inflamma-
tory AqH was overall suppressive to DC function, with reduced
CD86 expression and diminished T-cell responses. This sup-
pressive effect was equal to or greater than that induced by
noninflammatory AqH, but was glucocorticoid independent (in
contrast to noninflammatory AqH).
CONCLUSIONS. These data indicate that the ocular microenviron-
ment continues to regulate DC function during uveitis, despite
IFN?-driven upregulation of MHC expression, supporting the
hypothesis that immune regulation within the eye is main-
tained during inflammation. (Invest Ophthalmol Vis Sci. 2012;
system.1–4This phenomenon, known as immune privilege,
appears to be important in protecting these vital structures
from immune-mediated inflammatory damage that would result
in critical loss of function. The key contributors to immune
privilege in the eye appear to be ocular sequestration (the
blood–ocular barriers, limited lymphatic drainage),3an immu-
nosuppressive ocular microenvironment (due to regulatory
molecules such as transforming growth factor-beta [TGF-?],
?-melanocyte stimulating hormone [?-MSH], and cortisol5–9),
Fas-FasL–induced apoptosis,10,11and active immune deviation
(such as described in anterior chamber–associated immune
deviation [ACAID]12). During uveitis, a condition characterized
by intraocular inflammation involving the uveal tract, these
natural protective mechanisms either fail or are overwhelmed,
with resultant leakage of cells and proteins into the optically
clear aqueous humor (AqH) that circulates within the front of
the eye. Subsequent damage to the delicate intraocular struc-
tures results in sight loss, with uveitis overall representing 15%
of total blindness in the developed world.13The most common
form of uveitis is anterior uveitis that, although carrying a
lower rate of acute visual loss than that of those types affecting
the posterior segment of the eye (intermediate, posterior, and
panuveitis), is of importance on account of its incidence and
the long-term sight-threatening complications experienced
by a significant minority of sufferers.13Fundamentally it
remains unclear whether uveitis represents the “failure” of
immune privilege due to a reduction in the efficacy of one or
more of the protective mechanisms, or the “overpowering”
of immune privilege due to an inflammatory drive that ex-
ceeds the capacity of otherwise normally functioning regu-
Dendritic cells (DCs) are bone-marrow–derived leukocytes
that have a pivotal role in presenting antigen, and link innate
and adaptive immune responses. They respond to the presence
of pathogens and inflammation at peripheral tissue sites, mi-
grating to secondary lymphoid organs where they present
antigen to either naïve or memory T cells, leading to T-cell
proliferation and differentiation toward effector and memory
cells.20–23More recently it has been recognized that, depend-
ing on the signals they have received, DCs may adopt a number
of different phenotypes capable of inducing a range of broadly
he eye is one of a number of sites within the body dem-
onstrating a highly regulated relationship with the immune
From the1Centre for Translational Inflammation Research, Col-
lege of Medical and Dental Sciences, University of Birmingham Re-
search Laboratories, Queen Elizabeth Hospital Birmingham, Birming-
ham, United Kingdom; and the
School of Immunity and Infection, College of Medical and Dental
Sciences, University of Birmingham, Birmingham and Midland Eye
Centre, Birmingham, United Kingdom.
Supported in part by a Medical Research Council United Kingdom
(UK) Clinical Training Fellowship G0600416 (AD), a Wellcome Clinical
Research Training Fellowship (GPW), and a Marie Curie Early Stage
Researcher Fellowship MEST-CT-2005-020996 (SK). In addition, the
Academic Unit of Ophthalmology is supported by the Birmingham Eye
Foundation Registered (UK) Charity 257549.
Submitted for publication October 15, 2011; revised November
29, 2011; accepted December 26, 2011.
Disclosure: A.K. Denniston, None; P. Tomlins, None; G.P.
Williams, None; S. Kottoor, None; I. Khan, None; K. Oswal, None;
M. Salmon, None; G.R. Wallace, None; S. Rauz, None; P.I. Murray,
None; S.J. Curnow, None
Corresponding author: S. John Curnow, Centre for Translational
Inflammation Research, College of Medical and Dental Sciences, Uni-
versity of Birmingham Research Laboratories, Queen Elizabeth Hospital
Birmingham, Mindelsohn Way, Edgbaston, Birmingham, B15 2WB, UK;
2Academic Unit of Ophthalmology,
Immunology and Microbiology
Investigative Ophthalmology & Visual Science, February 2012, Vol. 53, No. 2
Copyright 2012 The Association for Research in Vision and Ophthalmology, Inc.
stimulatory or regulatory responses, according to their expres-
sion of key surface molecules (such as the costimulatory mol-
ecules CD80/86) and altered cytokine production.22,24It is
thought that these DC phenotypes may be plastic, enabling
appropriate responses to a changing environment.22
In the ocular microenvironment, and in particular the AqH,
DCs or any other potential antigen-presenting cells (APCs) are
exposed to a number of molecules that are either stimulatory
or suppressive. Animal studies have suggested that the domi-
nant regulatory molecules are TGF?25,6and ?-MSH,7,8with
effects on macrophage inflammatory activity and on the gen-
eration of Th1 responses. We have recently shown that in
humans, under resting conditions, the endogenous glucocorti-
coid cortisol (together with TGF?2) significantly contributes to
AqH inhibition of DC function.9During uveitis there are sig-
nificant changes in the ocular microenvironment. Analysis of
the cytokine profile of uveitis AqH has identified increased
levels of a number of proinflammatory cytokines (such as IL-6
and IFN?25,26). This, coupled with a fall in TGF? levels,25
pointed to the likelihood that uveitis AqH would be stimula-
tory. Alternatively, it was possible that AqH might continue to
be immunosuppressive due to the persistence, influx, or pro-
duction of immunoregulatory molecules in response to the
inflammation. In this study, we aimed to interrogate whether
the ocular microenvironment retains its regulatory function
during uveitis, specifically with regard to the role and function
of dendritic cells and consequent T-cell responses.
MATERIALS AND METHODS
Patients with active uveitis involving the anterior segment of the eye
were recruited for this study. Local ethical committee approval was
granted and, after informed consent, all samples were collected and
stored according to the Human Tissue Act 2004 (United Kingdom).
These studies conform to the Declaration of Helsinki. The uveitis
cohort comprised 80 patients with noninfectious uveitis: 70 patients
with anterior uveitis and 10 with panuveitis. Of the 70 patients with
anterior uveitis, 52 were idiopathic and 18 were HLA-B27 related. Of
the 10 patients with panuveitis, 7 had Fuchs’ heterochromic uveitis, 2
were idiopathic, and 1 had Vogt–Koyanagi–Harada syndrome. Disease
was classified as acute first episode (n ? 17), recurrent (n ? 62), or
chronic (n ? 1). At the time of sampling 54/80 patients were receiving
no treatment. In the remaining patients treatment comprised topical
corticosteroid alone (n ? 22), oral prednisolone and topical cortico-
steroid (n ? 2), and intravenous methylprednisolone and topical
corticosteroid (n ? 2).
Duration of clinical symptoms at the time of sampling was 5.9 ?
4.9 days (mean ? SD). Disease activity in the anterior chamber of the
eye was measured clinically with a biomicroscope and scored in
accordance with the Standardization of Uveitis Nomenclature criteria,
resulting in a value between 0 and 4.27AqH sampling was performed
according to our published protocol.28,29AqH was centrifuged at 300g
for 5 minutes, after which the supernatant was removed and frozen in
aliquots at ?80°C, whereas the cellular component was stained for
flow cytometry. Peripheral blood was taken by venipuncture into
Peripheral blood was taken from normal healthy volunteer donors with
the exclusion of any individuals with a history of inflammatory disease,
infection at the time of sampling, or systemic immunosuppression.
AqH was obtained from otherwise healthy patients by paracentesis
before routine cataract surgery (noninflammatory AqH), excluding any
individuals with a history of inflammatory disease (ocular or systemic),
as well as any taking ocular medication. AqH was centrifuged at 300g
for 5 minutes, after which the supernatant was removed and frozen in
aliquots at ?80°C.
Generation of Monocyte-Derived DCs and
Isolation of Myeloid DCs
Peripheral blood mononuclear cells (PBMCs) were isolated by density-
gradient centrifugation using a commercial density-gradient media (Fi-
coll-Paque; GE Healthcare [formerly Amersham Biosciences], Bucking-
hamshire, UK) according to the manufacturer’s instructions, and were
washed three times with RPMI 1640 to remove platelets. Monocytes
were isolated from PBMC using a commercial cell-separation reagent
(MACS CD14 MicroBeads; Miltenyi Biotec, Surrey, UK) as described by
the manufacturer (?98% purity), and cultured for 6 days (37°C, 5%
CO2, humidified) in RPMI, 10% pooled human AB? male serum (Bio-
sera, Ringmer, UK), 500 U/mL recombinant human IL-4 (ImmunoTools
GmbH, Friesoythe, Germany), and 1000 U/mL GM-CSF (ImmunoTools)
at 2.5 ? 106cells/T25 flask (Sarstedt Ltd, Leicester, UK). At day 3, 2 mL
medium was removed and 2.5 mL fresh medium was added. At day 6
the nonadherent monocyte-derived DCs were harvested.
Culture of DCs in the Presence of AqH
DCs were washed and resuspended in serum-free medium: RPMI 1640
medium, 1% liquid media supplement (Sigma-ITS?3), 1% nonessential
amino acid solution, and 1 mM sodium pyruvate (all Sigma-Aldrich,
Gillingham, UK). DCs were placed in triplicate in a round-bottom
96-well plate (Greiner, Gloucester, UK) at 20 000 cells per well, and
cultured in the presence or the absence of 50% human AqH. AqH was
used at 50%, as previously described, to ensure greater precision when
resuspending cells that could otherwise be variably contaminated with
The role of cortisol was tested with 10?7M ?98% HPLC cortisol
(hydrocortisone; Sigma-Aldrich) and the role of dexamethasone with
10?7M water-soluble dexamethasone (Sigma-Aldrich); both were in-
hibited with 10?7M of the glucocorticoid inhibitor RU486 (Mifepris-
tone; Sigma-Aldrich). The role of IFN? was tested with recombinant
human IFN? at 0.1–100 ng/mL (ImmunoTools) inhibited with an IFN?
blocking antibody at 10 ?g/mL (R&D Systems, Abingdon, UK). The
role of IL-6 was tested with recombinant human IL-6 at 0.01–1 ?g/mL
(ImmunoTools) inhibited with an IL-6 receptor blocking antibody at
1 ?g/mL (R&D Systems). After a 48-hour culture, DC supernatants
were harvested, and the cells were either stained for flow cytometry or
washed three times in RPMI and 10% heat-inactivated fetal calf serum
(HI-FCS) for use in an allogeneic proliferation assay.
Allogeneic Proliferation Assay
Naïve CD4?T cells, memory CD4?T cells, and CD8?T cells were
isolated from PBMC using a cell-separation reagent T-cell isolation kit
(MACS; Miltenyi Biotec) as described by the manufacturer and labeled
with 1 ?M long-term stain (CFSE [carboxyfluorescein diacetate succin-
imidyl ester]; Invitrogen, Paisley, UK) for 10 minutes at 37°C. Cells
were then washed three times in RPMI, 10% HI-FCS, rested overnight,
and washed once further before being placed in triplicate in the
washed DC-culture plates at 100,000 cells per well in RPMI and 10%
HI-FCS. After a 4-day culture, supernatants were harvested, cells were
stained with the dead cell exclusion dye propidium iodide, and prolif-
eration of live cells was analyzed with a flow cytometer. The number
of cells that had proliferated was calculated by gating on cells with a
lower level of positive CFSE staining than unstimulated cells and
assessing total numbers using counting beads (Invitrogen).
Multiplex Bead Immunoassay
Culture supernatants were analyzed using a multiplex bead immuno-
assay (Human Cytokine Twenty-Five-Plex; Biosource, Nivelles, Bel-
gium) detecting (range, pg/mL) IL-2 (0.8–2,500), IL-10 (4.5–15,000),
IL-13 (0.8–3,500), IL-17 (1.4–921), tumor necrosis factor-alpha (TNF?;
2.4–600), and IFN? (2.5–3,500). The procedure was carried out ac-
IOVS, February 2012, Vol. 53, No. 2
Regulation of Dendritic Cell Function in Human Uveitis889
cording to the manufacturer’s instructions. Samples were analyzed
using a microbead analyzer (Luminex 100; Luminex Corp., Austin, TX).
Identification of myeloid DCs and other populations of interest in AqH
and peripheral blood was achieved on the basis of forward scatter/side
scatter profile, and labeling with a combination of APC-anti-BDCA-1
(CD1c; AD5–8E7; Miltenyi Biotec), APCCy7-anti-CD14 (M?P9; BD
Pharmingen), PE-anti-CD86 (2331; BD Biosciences, Oxford, UK), ECD-
HLA-DR (Immu-357; Beckman Coulter, High Wycombe, UK), PECy7-
CCR5 (2D7/CCR5; BD Pharmingen), anti-HLA-A,B,C (Pacific Blue, W6/
32; BioLegend), FITC-CD19 (LT19; ImmunoTools) and FITC-CD56
(MEM-188; ImmunoTools). Monocyte-derived DCs were labeled with a
combination of FITC-anti-CD80 (2D10; Biolegend, San Diego), PE-anti-
CD83 (HB15e; AbD Serotec, Oxford, UK), PE-Cy5 anti-CD86 (2331; BD
Biosciences), anti-HLA-A,B,C (Pacific Blue, W6/32; BioLegend), PE-
Texas Red–anti-HLA-DR (Immu-357; Beckman Coulter), PECy7-CCR5
(2D7; BD Pharmingen), FITC-CCR7 (150,503; R&D Systems) for 20
minutes at 4°C, in PBS 2% bovine serum albumin (Sigma-Aldrich). The
extent of positive staining was determined using an isotype-matched
negative control antibody (data not shown). All antibodies were used
at predetermined optimal dilutions. Dead cells were stained with
propidium iodide (Sigma-Aldrich) according to the manufacturer’s in-
structions, and excluded from further analysis. Cells were analyzed
using a Cyan 3 laser nine-color flow cytometer with commercial soft-
ware (Summit; Beckman Coulter).
Human AqH was diluted 25-fold before testing in a cortisol acetylcho-
linesterase competitive EIA assay (Cayman Chemical Co., Ann Arbor,
MI) according to the manufacturer’s instruction. Absorbance was mea-
sured with a multiwell plate reader (Anthos HT 111; Anthos Labtec
Instruments, Salzburg, Austria). Standard curves were plotted using a
four-parameter logistic equation fitted to the logarithmic transforma-
tion of the standard concentrations versus the percentage cortisol
All data were analyzed using a commercial software package (Graph-
Pad Prism 4; GraphPad Software Inc., San Diego, CA). The figure
legends provide details of the specific statistical tests used.
Myeloid Dendritic Cells Are Found in Human
AqH during Active Uveitis with a Distinct
Myeloid DCs were identified by flow cytometry in both the
PBMC fraction and the AqH. Myeloid DCs were defined accord-
ing to forward/side light scatter profile and characteristic ex-
pression of BDCA-1(CD1c)?CD19?CD56?CD14?/low(Fig. 1).
Myeloid DCs comprised 0.6 ? 0.7% (mean ? SD) of total AqH
cells. This compares with the peripheral blood, where 0.3 ?
0.6% (mean ? SD) of the PBMC fraction were myeloid DCs.
Myeloid DCs in uveitic AqH expressed significantly higher
levels of major histocompatibility complex classes I and II
(MHC I/II), but lower levels of the costimulatory molecule
CD86 compared with the matched peripheral blood samples
(Fig. 1). CD14?cells (monocytes/macrophages) were also
present (3.3 ? 3.9% [mean ? SD] of AqH cells; 7.9 ? 5.5%
[mean ? SD] of the PBMC fraction).
Treatment of Monocyte-Derived DCs with Uveitis
AqH Induces an MHCIhiCD86loPhenotype
Having identified that DC in the uveitic AqH appeared to have
a distinct phenotype, we sought to identify whether this was due
to differential recruitment of MHChiCD86loexpressing DCs or a
consequence of exposure to the intraocular microenvironment.
To investigate the role of the intraocular microenvironment we
tested the effects of uveitis AqH on the phenotype of monocyte-
derived DC in vitro. Treatment of monocyte-derived DCs with
uveitis AqH supernatant resulted in significant upregulation in
class I MHC and downregulation of class II MHC (specifically
HLA-DR) and the costimulatory molecule CD86 (Fig. 2). CD80
and CD83 were expressed at low levels in all cases, showing no
significant increase or decrease on exposure to uveitis AqH
(data not shown).
Uveitis AqH–Induced Upregulation of MHCI on
DCs Is IFN? Dependent
To identify the mechanism by which uveitis AqH–induced
upregulation of class I MHC, we investigated the role of a
number of molecules that had previously been noted to be
elevated in active uveitis, notably IFN? and IL-625,26; addition-
ally, IFN? is known to be capable of MHC upregulation.30We
therefore tested the effects of the recombinant molecule and
the consequence of blocking its effects within AqH.
Recombinant IFN? caused a dose-dependent increase in
MHC (class I and class II) and CD86 (Fig. 3A). There was
differential sensitivity of molecular expression to IFN? with
upregulation of class I MHC occurring from concentrations of
IFN? of 0.1 ng/mL, HLA-DR from 1 ng/mL, and CD86 upregu-
lation, being present only at the higher concentrations of 10
and 100 ng/mL. AqH induction of class I MHC was shown to be
IFN? dependent, with significant reversal with the addition of
an IFN? blocking antibody. As previously observed, AqH treat-
ment downregulated HLA-DR but HLA-DR expression was fur-
ther reduced in the presence of IFN? blockade. CD86 levels
that were reduced in the presence of AqH (as previously
noted) were unaffected by IFN? blockade (Fig. 3B).
In a similar set of experiments investigating the potential
contribution of IL-6, the effects of uveitis AqH on DC pheno-
type were not recapitulated by the addition of recombinant
IL-6, and were not reversed (nor augmented) by IL-6 blockade
(data not shown).
Cortisol Levels Are Elevated in Uveitis AqH
Despite the proinflammatory IFN?-dependent upregulation of
class I MHC, the predominant effect of uveitis AqH appeared to
be inhibitory, with downregulation of class II MHC and CD86,
which we have previously shown to be due to cortisol and
TGF? for noninflammatory AqH.9To our knowledge the pres-
ence of cortisol in human AqH during uveitis has not previ-
ously been determined. In a series of 13 untreated uveitis AqH
samples we observed the concentration of cortisol in uveitis
AqH to be significantly elevated above noninflammatory AqH
levels. Cortisol levels in uveitis AqH ranged from 1884 to
25,536 pg/mL, with a median (interquartile range [IQR]) of
5468 (3905–12,300) pg/mL, compared with a median (IQR) of
2820 (1650–4297) pg/mL for noninflammatory AqH (Fig. 4A).
Cortisol levels in AqH increased with severity of uveitis as
measured by the standard clinical parameter of anterior cham-
ber activity (an estimate of the number of cells in the AqH,
graded from 0 [no inflammation] to 4 [most severe] as de-
scribed earlier27) (Fig. 4B; linear trend test, P ? 0.01).
Control of DC Expression of CD86 by
Glucocorticoids Present in AqH
The observation that cortisol levels were elevated during active
uveitis (Fig. 4), coupled with our earlier observations regarding
the inhibitory role of cortisol within AqH on DC expression of
CD86 and induction of T-cell responses,9led to the hypothesis
that the ability of AqH to regulate DC function during uveitis
890Denniston et al.
IOVS, February 2012, Vol. 53, No. 2
peripheral blood and aqueous humor of patients with active anterior uveitis. The gating strategy comprised identifying cells of appropriate scatter profile that
were BDCA-1(CD1c)?CD19?CD56?and CD14?/lo. (A) Representative histograms and (B) median fluorescence intensity (MFI) scans of MHC and CD86 from
matched PBMC and AqH of six or more patients with acute anterior uveitis. Wilcoxon matched-pairs analysis; *P ? 0.05; **P ? 0.01; NS, not significant.
Myeloid DCs can be identified in AqH during active uveitis and have a distinct MHChiCD86lophenotype. Myeloid DCs were identified in the
IOVS, February 2012, Vol. 53, No. 2
Regulation of Dendritic Cell Function in Human Uveitis891
was also linked to endogenous intraocular cortisol, augmented
by exogenous (i.e., therapeutic) glucocorticoids in the case of
samples from patients who were being treated with cortico-
steroid drops. We therefore tested the inhibitory effects of
uveitis AqH in the presence or the absence of a glucocorticoid
inhibitor (RU486), recognizing that for these “treated uveitis”
AqH samples, glucocorticoid inhibition would block the com-
bined effect of endogenous cortisol and the exogenous gluco-
corticoid treatment, such as dexamethasone (Figs. 5A–C). Both
untreated and treated uveitis AqH caused significant reduction in
CD86 expression (Fig. 5C). As previously shown,9we confirmed
that the inhibitory effects of noninflammatory AqH were reversed
by RU486, but similar reversal was not seen for the uveitis groups;
there was no effect for untreated uveitis AqH and only a partial
reversal for treated uveitis AqH (Fig. 5C). Uveitis AqH–induced
changes in class I and class II MHC were not affected by the
addition of RU486 (Figs. 5A, 5B).
Uveitis AqH Inhibits DC Capacity to Induce
Proliferation of CD4?and CD8?T Cells
Having identified that the inflamed ocular microenvironment
induced both a “stimulatory” upregulation of MHC (class I and
class II in vivo; class I only in vitro) and an “inhibitory” down-
regulation of CD86, we sought to investigate the functional
consequences of this altered phenotype and the impact of
treatment on DC function.
The addition of uveitis AqH significantly inhibited DC ca-
pacity to induce T-cell proliferation, whether naïve or memory
CD4?or CD8?(Fig. 6). Inhibition with uveitis AqH was of a
magnitude similar to that seen with noninflammatory AqH.
Effect of In Vivo Glucocorticoid Treatment on
DC-Induced T-Cell Responses
Having noted that exposure to uveitis AqH caused similar
inhibition of DC function to noninflammatory AqH, we sought
to observe whether the inhibitory effects of uveitis AqH were
affected by the presence of glucocorticoid treatment. In addi-
tion, we sought to determine whether T-cell cytokine produc-
tion was also affected.
Exposure of DCs to uveitis AqH resulted in lower levels of
proliferation regardless of whether the AqH was a “treated” or
“untreated” sample (Fig. 7A), although this was not statistically
significant (P ? 0.18). Similarly, exposure of DCs to uveitis
AqH, whether “untreated” or “treated,” resulted in lower levels
of IL-2, IL-10, IL-13, IFN?, and TNF? (Fig. 7B); correlation with
proliferation was high for all these cytokines (Fig. 7C). IL-17
was near baseline from all cultures and showed no significant
effect of AqH treatment.
This study addresses an important question regarding immune
regulation in an immune privileged site under inflammatory
conditions. Specifically, we have investigated whether DC reg-
ulation in the human eye is maintained during intraocular
inflammation. During active uveitis, we found that myeloid
cyte-derived DCs with uveitis AqH
MHCIhiCD86loprofile similar to that
seen in UvAqH myeloid DCs. Monocyte-
derived DCs were cultured for 48 hours
in the presence or the absence of 50%
uveitic or noninflammatory AqH. (A)
Representative histograms and (B) MFI
scans of MHC and CD86 for nine uveitis
AqH (acute anterior; open circles) and
six noninflammatory AqH (filled cir-
cles), representative of three separate
experiments; for medium alone the
mean ? SD of MFI scans for triplicate
cultures are given. One-way ANOVA
with Bonferroni post hoc test for
multiple comparisons; data passed a
Kolmogorov–Smirnov test for nor-
mality; *P ? 0.05; **P ? 0.01; ***P ?
0.001; NS, not significant.
Treatment of naïve mono-
892 Denniston et al.
IOVS, February 2012, Vol. 53, No. 2
DCs from the anterior chamber expressed high levels of MHC
but low levels of CD86. This phenotype was recapitulated in
vitro by exposure of monocyte-derived DCs to uveitis AqH
supernatant and this resulted in a suppressed function of these
cells. Furthermore, the regulatory effects of uveitis AqH ap-
peared to be distinct from the cortisol and TGF?2-dependent
mechanism we have previously demonstrated for noninflam-
matory AqH.9Maintenance of these regulatory mechanisms
suggests that, for DC function at least, immune privilege within
the eye is maintained in the presence of inflammation.
Although numerous leukocyte populations may be modified
by the unusual microenvironment of an immune privileged
site, DCs are of particular interest due to their pivotal role in
the adaptive immune response. Previous animal and cadaveric
studies31,32have identified ocular APC with DC-like properties,
but to our knowledge this is the first study to identify BDCA-1?
myeloid DCs in the AqH of human patients with uveitis. Our
study parallels the findings in rodents in which both DCs and
macrophage-like populations are identified in uveal tissue.33–35
We have similarly identified both a CD14?monocyte/macro-
phage population and a separate CD14?/lowBDCA-1?myeloid
DC population. DCs were found to be less abundant than
CD14?monocyte/macrophages in uveitic aqueous humor, par-
alleling the findings in rodent uveal tissue,36but their role
should not be underestimated because DCs are far more potent
than macrophages in their antigen presentation capacity, can
fulfill both regulatory and stimulatory roles, and are unique in
their ability to induce naïve T-cell responses.37One of the key
factors for DCs controlling the activation of naïve T cells is the
expression of coreceptors CD80/CD86. It is clear that not only
is CD86 not upregulated by uveitis AqH, but that the levels are
well below those induced on normal maturation of these den-
Our finding that uveitis AqH continues to be inhibitory to
DC function is an important addition to the predominantly
murine literature regarding the retention or otherwise of im-
mune privilege during uveitis. Rodent models of uveitis such as
experimental autoimmune uveitis (EAU), endotoxin-induced
uveitis (EIU), and Mycobacterium tuberculosis adjuvant–in-
duced uveitis (MTU) vary widely in disease severity, time
course, and eventual outcome, reflecting very different immu-
nologic processes. In both EAU and EIU the ability of the ocular
microenvironment to suppress anti-CD3–driven T-cell prolifer-
ation in vitro was lost at the onset of disease but recovered at
the peak of disease.15,16In contrast, a variant of the EIU model
suggests that severe intraocular inflammation is compatible
with maintenance of immune privilege. When LPS is injected,
not systemically but into the vitreous cavity of BALB/c mice, it
was observed that even at the peak of intraocular inflammation
these eyes permit the proliferation of allogeneic tumor cells
and support ACAID in vivo, and their AqH would still strongly
inhibit T-cell activation in vitro.19Furthermore, in the MTU
model, intravitreal injection of M. tuberculosis adjuvant into
the eyes of BALB/c mice resulted in an intense anterior uveitis
but similar preservation of immune privilege behavior.18Our
study provides novel evidence in humans that retention of an
immune privileged microenvironment (or at least some com-
ponents thereof) is consistent with active inflammation.
It is noteworthy that although uveitis AqH was overall
suppressive on human DCs, the presence of significant levels
of IFN? was associated with a limited proinflammatory activity
resulting in elevated MHC class I expression by DCs. We, and
others, have previously demonstrated elevated IFN? levels in
human uveitic AqH that correlate with the severity of inflam-
mation.25In our study we observed a median (IQR) IFN?
concentration of 0.6 (0.005–16.3) ng/mL IFN? in idiopathic
uveitic AqH compared with 0.065 (0.005–0.375) ng/mL in
noninflammatory AqH.25It is interesting to note that there are
differences between DC expression of MHC class II observed
regulation of MHC, but are insufficient to overcome AqH-induced
regulation of HLADR or CD86. Monocyte-derived DCs were cultured in
the presence or the absence of (A) 0.1–100 ng/mL IFN? or (B) 50%
UvAqH, with or without an IFN?-blocking antibody. (A) and (B) are
each representative of three separate experiments. (B) comprises nine
patients with uveitis (acute anterior; open circles); mean ? SD of MFI
scans for triplicate cultures are given except for individual AqH sam-
ples. Wilcoxon matched-pairs analysis; *P ? 0.05; NS, not significant.
Increasing IFN? levels in AqH during uveitis promote up-
tory AqH. Cortisol levels in noninflammatory (CAqH) and uveitic AqH
(untreated; UvAqH) were measured by ELISA. (A) Cortisol levels in
noninflammatory AqH versus UvAqH and (B) cortisol levels according
to the cellular activity of an AqH sample; cellular activity is measured
clinically, as discussed. (A) and (B) comprise 13 patients with uveitis
(acute anterior; open circles) and 17 control patients (noninflammatory
AqH; filled circles); Mann–Whitney U test (A) and linear trend test for
all four columns (B); **P ? 0.01.
Cortisol levels are elevated in UvAqH versus noninflamma-
IOVS, February 2012, Vol. 53, No. 2
Regulation of Dendritic Cell Function in Human Uveitis893
in vivo versus in vitro. The reasons for this are unclear, but
possible explanations include a differential sensitivity of cul-
tured cells to the balance of IFN? and inhibitory factors present
in AqH or, less likely, loss of IFN? activity from the uveitis AqH
specimen during preparation. In rodent models of uveitis,
more attention has been given to IL-6. Development of disease
is associated with increasing IL-6 levels and is implicated in the
loss of regulation seen in both the EAU and EIU models, where
IL-6 blockade resulted in restoration of the normal regulatory
properties of AqH in vitro.15,16Although we have also noted
elevated IL-6 levels in human uveitis AqH, and that these inhibit
T-cell apoptosis within the uveitic eye, blockade of IL-6 in
uveitis AqH did not significantly affect DC phenotype.38
Our observation of the persistence of the regulatory prop-
erties of human AqH during uveitis is also intriguing since it
appears to be operating by an alternative mechanism to the
dominant cortisol/TGF?2 pathway of noninflammatory
AqH.9In mice the predominant regulatory molecules are
neuropeptides such as ?-MSH and vasoactive intestinal pep-
tide (VIP), with TGF?2 becoming increasingly dominant
once inflammation triggers its activation from the predomi-
nant latent form.15,16We have previously observed that in
humans, at least in our in vitro DC model, ?-MSH and VIP
were not found to be significantly inhibitory at physiological
levels.9In humans active TGF?2 levels decrease in uveitis,
falling from a median of 353 (range, ?40 to 497) pg/mL to
a median of 86 (?40 to 667) pg/mL.25Since this lower
uveitis level is significantly below the concentration at
which we have found TGF? to inhibit DC function,9TGF?
was not considered further in this context. Conversely,
cortisol levels in human AqH increased during uveitis (Fig.
4), although the suppressive effects of uveitis AqH on DC
function were not reversed by glucocorticoid blockade. Nev-
ertheless, given the sensitivity of DCs to these levels of corti-
sol,9it is likely that this continues to be a significant regulator
of DC function, but that the presence of novel alternative
regulatory molecules provides redundancy in the system.
As part of this study we have sought to optimally model DC
function in the uveitic human eye by the use of human DCs
cultured in the presence of human uveitic AqH; however, this
brings with it a number of limitations. We recognize that AqH
present in AqH. Monocyte-derived DCs were cultured in the presence
or the absence of 50% noninflammatory (CAqH) or uveitis AqH
(UvAqH), with or without the glucocorticoid blocker, RU486. (A), (B),
and (C) comprise seven patients with untreated and seven patients
with treated uveitis (acute anterior or panuveitis; open circles) and
seven control patients (noninflammatory AqH; filled circles), cortisol or
dexamethasone, were used as positive controls. Mean ? SD of MFI
scans for triplicate cultures are given except for individual AqH sam-
ples. Wilcoxon matched-pairs analysis; *P ? 0.05; NS, not significant;
Dex, dexamethasone; Med, medium alone.
Control of DC expression of CD86 by glucocorticoids
for CD4?and CD8?T cells. CFSE-labeled allogeneic naïve CD4?T cells
were cultured for 4 days with monocyte-derived DCs, which had been
pretreated with medium or 50% AqH (noninflammatory [CAqH] or
uveitic [UvAqH]). (A) CFSE versus side scatter (SS) for live cells and (B)
number of divided cells; each plot is representative of three separate
experiments with the mean ? SD of triplicate cultures for cultures
without AqH (i.e., T-cell cultures with iDC or medium only). Twenty-
four patients with uveitis (acute anterior or panuveitis; untreated or
treated; open circles) and six control patients (noninflammatory AqH;
filled circles) shown. One-way ANOVA with Bonferroni post hoc test for
multiple comparisons (B). Normality of distribution demonstrated by
Kolmogorov–Smirnov test. *P ? 0.05; **P ? 0.01; ***P ? 0.001; NS, not
UvAqH inhibits DC capacity to induce T-cell proliferation
894Denniston et al.
IOVS, February 2012, Vol. 53, No. 2
primarily reflects the ocular microenvironment of the anterior
segment and, thus, one should be cautious of extrapolating our
findings to posterior segment disease; also that the behavior of
resident or newly recruited DCs to the anterior segment in vivo
may be significantly more complex than modeled by our in
vitro studies of human AqH. It should be noted that our model
seeks to establish the behavior of DCs that have been condi-
tioned within the ocular microenvironment before engaging
with a naïve T cell, for example in a draining lymph node. It is
interesting to speculate whether the presence of AqH at the
time of engagement of DCs with either memory (or possibly
even naïve) T cells within the eye would continue to cause
inhibition or skewing of T-cell function (as discussed earlier),
although the scarcity of human uveitis AqH samples has so far
limited these additional studies.
Our finding that, in humans, uveitis AqH continues to be
inhibitory to DC function is an important addition to the
predominantly murine literature regarding the retention or
otherwise of immune privilege during uveitis. Even in the
presence of clinically severe inflammation, AqH is capable of
significantly suppressing DC maturation, retaining this regula-
tory role despite IFN?-driven upregulation of MHC expression,
and appearing to globally establish DC capacity to induce
adaptive T-cell responses. Importantly, these data from the
uveitic eye support the hypothesis that many of the mecha-
nisms that constitute immune privilege are maintained during
inflammation (at least with regard to DC function) and may
have implications for our understanding of the relationship of
immune privilege and inflammation in other sites.
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