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*
Graduate Program in Immunology and
Department of Ophthalmology, University of Texas Southwesterm Medical Center, Dallas, TX 75235
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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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and are involved in cell-mediated
inflammatory responses (1). In direct contrast, Th2 cells secrete IL-4,
IL-5, IL-6, IL-9, IL-10, and IL-13 and are efficient promoters of Ab
responses (1). There is much evidence that the cytokine products of
each individual Th- cell subset inhibit both the
differentiation and effector functions of the other. For example,
IFN- has been shown to prevent Th2 cell proliferation, whereas IL-10
profoundly inhibits the synthesis of Th1 cytokines (2, 3). Therefore,
the emergence of a Th2-type response typically results in the
inhibition of Th1 differentiation and the down-regulation of
Th1-mediated immune responses.
The preferential development of a particular Th- cell
subset has been correlated directly with either susceptibility or
resistance to certain disease states. In the case of infection with
Leishmania major, it has been shown that C57BL/6
mice develop a Th1-dominated response and recover from disease, whereas
BALB/c mice develop a Th2-type response and die from the infection
(4, 5, 6, 7). Susceptibility to disease in BALB/c mice has been associated
directly with the down-regulation of a protective, Th1-mediated
DTH3 response. Although Th1
responses are crucial for recovery from many infectious diseases, DTH
can inflict significant collateral damage to normal host tissues.
Th1-mediated lesions can be tolerated in many organs; however, other
tissues lack regenerative capacities and cannot retain their
physiologic function as a consequence of unbridled DTH (8). This is
particularly true of the eye, in which most of the crucial tissues
necessary for vision lack the capacity to regenerate. The expression of
DTH-mediated immunity in the eye often culminates in blindness (9, 10, 11).
However, Ags introduced into the anterior chamber of the eye
characteristically evoke a spectrum of immune responses in which the
induction and expression of DTH are profoundly suppressed (12). This
unique immunoregulatory phenomenon has been termed anterior
chamber-associated immune deviation (ACAID) and is one of several
important mechanisms for sustaining immune privilege in the anterior
chamber of the eye (13, 14, 15). Recent studies have suggested that Ags
delivered into the anterior chamber of the eye are processed by
resident APC that preferentially migrate to the spleen, where they
induce the generation of T cells that down-regulate DTH (16). The
down-regulation of Th1 immunity appears to be the consequence of
Th2-mediated cross-regulation of Th1 responses (17, 18).
CD4+ T cells from the spleens of mice manifesting ACAID
display a Th2-like cytokine profile in which IL-10 production is
increased, while IFN- secretion is strongly inhibited
(17, 18).
A small, but significant, category of Ags will not induce ACAID, and
instead provoke strong DTH following anterior chamber presentation (11, 19, 20, 21, 22, 23, 24, 25, 26). The cytokine pattern in these mice is characteristic of a
Th1-dominated response in which splenic CD4+ T cells
produce increased amounts of IL-2 and IFN-, but insignificant
amounts of IL-10 and no detectable IL-4 (18). Thus, Ags delivered into
the anterior chamber of the eye can elicit either Th1- or Th2-like
systemic immune responses.
Wilbanks and co-workers (27, 28) have suggested that TGF-ß, present in the aqueous humor, alters the behavior and Ag-presenting characteristics of APC within the eye. Peritoneal macrophages exposed to TGF-ß in vitro, at the same concentration that is present in the aqueous humor, present Ag to T cells in vivo in a manner that leads to the down-regulation of DTH and the development of an immunologic phenotype that is characteristic of ACAID.
In the present study, we wished to utilize the in vitro model of the eye to test the hypothesis that under the influence of TGF-ß, APC can elaborate a Th2-type cytokine, IL-10, which prevents the induction of Th1 immunity. A corollary of this hypothesis predicts that Ags that do not induce ACAID prevent the secretion of IL-10 by TGF-ß-modified APC and allow for the production of cytokines, such as IL-12, that promote the emergence of Th1 immunity.
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Materials and Methods |
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Six- to ten-week old C57BL/6 and BALB/c mice were obtained from the mouse colony at University of Texas Southwestern Medical Center at Dallas. C57BL/6 IL-10 knockout mice (C57BL/6-IL-10tm1Cgn) and C57BL/6 IL-4 knockout mice (C57BL/6J-IL-4tm1Cgn) were purchased from The Jackson Laboratory (Bar Harbor, ME).
Tumor cells
UV5C25 is a highly immunogenic, UV light-induced fibrosarcoma tumor cell line originally derived in BALB/c mice (kindly provided by Dr. Margaret Kripke, M. D. Anderson Tumor Institute, Houston, TX). The immunologic characteristics of UV5C25 tumor cells have been described (20). P815 is a mastocytoma tumor line derived in DBA/2 mice. UV5C25 tumor cells were maintained in complete MEM medium (JRH Biosciences, Lenexa, KS) containing 10% heat-inactivated FBS (HyClone Laboratories, Logan, UT), 2 mM L-glutamine (JRH Biosciences), 1 mM sodium pyruvate (JRH Biosciences), 2 mM MEM vitamins (JRH Biosciences), and 1% penicillin-streptomycin-fungizone solution (BioWhittaker, Walkersville, MD). P815 tumor cells were maintained in complete DMEM medium (JRH Biosciences) containing 10% heat-inactivated FBS (HyClone Laboratories), 2 mM L-glutamine (JRH Biosciences), 1 mM sodium pyruvate (JRH Biosciences), 2 mM MEM vitamins (JRH Biosciences), and 1% penicillin-streptomycin-fungizone solution (BioWhittaker). UV5C25 tumor cells in complete MEM were sonicated (1 min) with a sonic dismembrator (Artek Systems Corp., Farmingdale, NY).
Intracameral inoculation
Mice were anesthetized with 0.66 mg of ketamine hydrochloride (Vetalar; Park-Davis and Co., Detroit, MI) given i.p. A glass micropipette (approximately 80 µm diameter) was fitted onto a sterile infant-feeding tube (no. 5 French; Professional Medical Products, Greenwood, SC) and mounted onto a 0.1-ml Hamilton syringe (Hamilton Co., Whittier, CA). A Hamilton automatic dispensing apparatus was used to dispense 5 µl of a 20 mg/ml OVA (Sigma Chemical Co., St. Louis, MO) solution in sterile PBS (= 100 µg OVA) or 2 x 107 UV5C25 cells/ml in complete MEM medium (= 1 x 105 cells) or 2 x 107 P815 cells/ml in complete DMEM medium (= 1 x 105 cells) into the anterior chamber via the glass micropipette.
Preparation of peritoneal exudate cells
Peritoneal exudate cells (PEC) were collected by peritoneal lavage of normal mice that were injected i.p. with 1 to 1.5 ml of 3% thioglycolate medium 5 days earlier.
In vitro model of the anterior chamber (eye-in-a-dish)
In a previously described model of the anterior chamber of the eye, TGF-ß-treated PEC were found to mimick the function of ACAID APC (27). PEC from normal mice were collected and suspended in complete RPMI 1640 (JRH Biosciences) containing 10% FBS (HyClone Laboratories), 2 mM L-glutamine (JRH Biosciences), 10 mM HEPES buffer solution (JRH Biosciences), 1 mM sodium pyruvate solution (JRH Biosciences), 1% nonessential amino acids solution (BioWhittaker), and 1% penicillin-streptomycin-fungizone solution (BioWhittaker). PEC were incubated on plastic tissue culture dishes (Falcon 3803; Becton Dickinson Labware, Lincoln Park, NJ) at 37°C in 5% CO2 for 2.5 h. Nonadherent cells were washed off with HBSS (BioWhittaker). Adherent cells, the vast majority of which were macrophages, were collected by incubating the plates at 4°C for 15 min and gently dislodging the cells with a Nitex filter swab (Tetko, Briarcliff Manor, NY). Adherent PEC were resuspended in complete RPMI medium and aliquoted at 1 to 1.5 x 106 cells/well in a 24-well plastic tissue culture plate (Falcon 3047; Becton Dickinson Labware). In some experiments, 5 mg/ml OVA (Sigma Chemical Co.) was added to each well. In other experiments, 1 x 105 sonicated UV5C25 cells in complete MEM medium were added to each well. Human rTGF-ß2 (Genzyme Diagnostics, Cambridge, MA) in complete RPMI was added to some wells (at 2 ng/ml). This treatment caused these cells to subsequently function like ocular ACAID APC. Control wells received an identical aliquot (100 µl) of complete RPMI alone. Cell cultures were incubated at 37°C in 5% CO2 overnight. The next day, all cells were collected, washed with HBSS, resuspended in complete RPMI, and infused i.v. (1–3 x 105 cells in 150 µl) into naive, syngeneic recipients.
Subcutaneous inoculations
In some experiments, mice were immunized by s.c. injection of OVA (125 µg) emulsified 1:1 in CFA (0.5 mg Mycobacterium/ml; Behring Diagnostics, La Jolla, CA) in a total volume of 50 to 100 µl. In other experiments, mice were immunized by s.c. injection of 0.5 to 2 x 106 UV5C25 cells suspended in 0.1 to 0.3 ml complete MEM or 3 x 106 P815 cells suspended in 0.2 ml complete DMEM.
DTH assay
Seven days after s.c. immunization, both ear pinnae of experimental and control animals were measured with a Mitutoyo engineer’s micrometer immediately before challenge. In experiments with OVA, 400 µg OVA in 20 µl sterile PBS (pH 7.4) was injected s.c. into the left ear pinnae. The right ear pinnae received 20 µl sterile PBS alone (negative control). In other experiments, X-irradiated (3000 cGy) UV5C25 cells suspended in 20 µl complete MEM (= 8 x 105 cells) were injected s.c. into the left ear pinnae. The right ear pinnae received 20 µl complete MEM alone (negative control). In other experiments, X-irradiated (3000 cGy) P815 cells suspended in 20 µl complete DMEM (= 8 x 105 cells) were injected s.c. into the left ear pinnae. The right ear pinnae received 20 µl of complete DMEM alone (negative control). Both ear pinnae were measured 24 h later, and the difference in ear pinnae size was used as a measure of DTH. Results were expressed as: specific ear swelling = (24-h measurement - 0-h measurement) for experimental ear - (24-h measurement - 0-h measurement) for control ear.
Assessment of cytokine production
Adherent PEC (3 x 106 cells/well) were cultured for 18 to 24 h. Supernatants (1 ml) were collected and tested for the presence of IL-10 and IL-12 by double mAb sandwich ELISA. Primary and secondary mAb for each mouse cytokine were: IL-10, JES5.2A511 (rat IgG1) and SXC-1 (rat IgM); IL-12, Red-T (hamster IgG)/G297-289 (rat IgG2a) and C17.8 (rat IgG2a). Activity in culture supernatants was compared with purified murine (m) IL-10 and rmIL-12 standards. The hybridoma producing anti-IL-10 mAb (JES5.2A511) was kindly provided by Dr. J. Abrams (DNAX, Palo Alto, CA). Biotinylated anti-IL-10 mAb (SXC-1), anti-IL-12 mAb mixture (Red-T/G297-289), rmIL-12, and biotinylated anti-IL-12 mAb (C17.8) were purchased from PharMingen (San Diego, CA). D10 culture supernatant containing mIL-10 was kindly provided by Dr. N. Street (Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX). Fifty microliters of each primary mouse mAb, anti-IL-10 at 3 µg/ml and anti-IL-12 at 8 µg/ml, were coated on sterile ELISA microplates (Corning Glass Works, Corning, NY). The plates were covered and incubated at 4°C overnight. Each well was blocked with 200 µl of PBS containing 1% BSA (Sigma Chemical Co.). Plates were incubated for 3 h at room temperature. Standards and samples (100–200 µl/well) were incubated at 4°C overnight. All samples were run in triplicate. Biotinylated rat anti-mouse secondary mAb (1–2 µg/ml) was added at 100 µl/well and incubated at room temperature for 60 min. Peroxidase-conjugated streptavidin (0.25–0.5 µg/ml) (Jackson ImmunoResearch Laboratories, West Grove, PA) was added (100 µl/well) and incubated at room temperature for 45 min. Plates were washed with 0.05% Tween-20 (Sigma Chemical Co.) in PBS between each step. Finally, 2,2-Azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid; ABTS; 1 mg/ml; Sigma Chemical Co.) substrate with 0.003% H2O2 was added at 100 µl/well and allowed to develop at room temperature for 10 to 90 min. Plates were read with an ELISA Microplate Reader (Molecular Devices, ThermoMax, Menlo Park, CA) at an OD of 405 nm.
mAb treatment
Adherent PEC (1–3 x 106 cells/well) were incubated overnight, as described. In some experimemts, anti-IL-10 mAb (20 µg/ml; JES5.2A511) was added to the cultures. Control wells received an isotype-matched mAb (20 µg/ml; 11B11, rat IgG1; American Type Culture Collection, Rockville, MD). In other experiments, anti-IL-12 mAb (10 µg/ml; Red-T/G297-289) was added to the well. Control wells received an irrelevant control mAb (10 µg/ml; 11B11). The next day, all cells were collected, washed, resuspended in complete RPMI, and infused i.v. (1.5–3 x 105 cells in 150 µl) into naive, syngeneic recipients.
IL-10 cytokine treatment
Adherent PEC (1–3 x 106 cells/well) were incubated overnight with or without mIL-10 (5 or 10 U mIL-10/ml). Other cultures received 10 ng/ml rmIL-10 (PharMingen, San Diego, CA). The next day, all cells were collected, washed, resuspended in complete RPMI, and infused i.v. (1.5–4 x 105 cells in 150 µl) into naive, syngeneic recipients.
Statistics
Unless otherwise indicated, all experimental and control groups contained five animals (n = 5). Differences between groups were analyzed by Student’s t test. p values < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experiments were performed to confirm that the in vitro model of
ACAID mimicked the in vivo counterpart. Accordingly, PEC (APC) from
BALB/c mice were pulsed in vitro for 24 h with OVA in the presence
or absence of the ocular cytokine, TGF-ß. Ag-pulsed APC were injected
i.v. into normal, syngeneic BALB/c mice. APC recipients, as well as
normal untreated mice, were immunized s.c. with 125 µg OVA 7 days
later. DTH responses were assessed 7 days after immunization. The
results demonstrate that APC pulsed with OVA in the presence of TGF-ß
induced down-regulation of OVA-specific DTH and prevented the hosts
from responding to the normally immunogenic s.c. injection of OVA (Fig. 1
A). The suppression of
OVA-specific DTH was comparable with that found in mice primed in the
anterior chamber with OVA before s.c. immunization (Fig. 1B). Thus, the in vitro model of ACAID produced
down-regulation of OVA-specific DTH that was of the same magnitude as
that produced by anterior chamber presentation of Ag.
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A). APC recipients
mounted DTH responses that were indistinguishable from those in mice
primed s.c. or in the anterior chamber of the eye (Fig. 2B). Thus, the in vitro model of ACAID recapitulates
the in vivo counterpart using both ACAID- and non-ACAID-inducing
Ags.
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Previous studies have demonstrated that cytokines elaborated by
APC can profoundly influence the elicitation of a Th1 vs Th2 response
(29, 30). The next experiments examined the effect of TGF-ß on
the cytokines secreted by Ag-pulsed APC in vitro. We began by testing
the hypothesis that TGF-ß renders the APC tolerogenic by enhancing
their production of the Th2-inducing cytokine, IL-10. BALB/c APC were
pulsed in vitro for 24 h with OVA or sonicated UV5C25 Ag in the
presence or absence of TGF-ß. Culture supernatants were collected and
tested for the presence of IL-10 and IL-12 by sandwich ELISA. In some
experiments, the APC were subsequently injected i.v. into normal BALB/c
mice to verify function. As shown in Figure 3, APC exposed to OVA and TGF-ß
secreted significantly higher levels of IL-10 than APC exposed to OVA
in the absence of TGF-ß. As previously shown in Figure 1
A,
these APC induced suppression of OVA-specific DTH when injected i.v.
into normal BALB/c mice. APC exposed to UV5C25 tumor Ag and TGF-ß,
however, did not produce significantly higher levels of IL-10 than
UV5C25 Ag alone (Fig. 3). In addition, the total quantity of IL-10
produced by APC exposed to UV5C25 Ag was significantly lower than that
produced by APC exposed to OVA in the presence or absence of TGF-ß.
These results indicate that TGF-ß stimulates the production of IL-10
by APC, but this effect is highly dependent on the nature of the Ag
encountered. Ags that do not induce ACAID in vivo (i.e., UV5C25 tumor
Ags) inhibit the production of IL-10 by APC in vitro.
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). Although TGF-ß impaired the
secretion of IL-12 by APC pulsed with UV5C25 tumor Ags, the amount of
IL-12 produced was comparable with that produced by APC pulsed with OVA
in the absence of TGF-ß (Fig. 4). It is noteworthy that in both
cases, these APC promoted the development of Ag-specific DTH following
adoptive transfer (Figs. 1A and 2A). Thus,
it appears that Ag presentation may require a certain threshold of
IL-12 secretion to promote the induction of Th1 immunity. Likewise, the
data suggest that a minimal level of IL-10 secretion is associated with
the down-regulation of Th1 responses and/or preferential induction of
Th2 responses. Although TGF-ß, and presumably the intraocular
environment, influences the behavior of APC in promoting a Th2-type
systemic immune response, certain Ags (e.g., UV5C25 tumor) can redirect
APC cytokine secretion to favor the development and emergence of Th1
immunity. We next wanted to determine whether the secretion of either
cytokine had a direct effect on the acquired phenotype of the
APC.
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To test the possibility that the induction of IL-10 secretion by
TGF-ß is involved in suppressive, Th2-inducing Ag presentation, we
removed IL-10 by incubating APC in the presence of a neutralizing
concentration of anti-IL-10 mAb. BALB/c APC were incubated for
24 h with OVA and with or without TGF-ß. Wells received either
anti-IL-10 mAb (20 µg/ml) or an isotype-matched Ab control.
Ag-pulsed APC were injected i.v. into normal, BALB/c mice. As expected,
APC incubated with OVA, TGF-ß, and control Ab induced suppression of
DTH (Fig. 5). By contrast, anti-IL-10
mAb treatment reversed the effects of TGF-ß on APC function; APC
incubated with OVA, TGF-ß, and anti-IL-10 mAb induced positive
DTH to subsequent challenge (Fig. 5). Therefore, TGF-ß-induced IL-10
secretion by the APC is necessary for that APC to acquire a
suppressive, Th2-inducing phenotype. Removal of IL-10 during the early
stages of Ag processing blocks this transformation in vitro.
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Because the removal of IL-10 from cultures containing TGF-ß
blocked the generation of suppressive, Th2-inducing APC (ACAID APC), it
appeared that the effect of TGF-ß was mediated directly through the
secretion of IL-10. To confirm the role of IL-10, we added mIL-10 to
APC cultures in vitro. We hypothesized that, in the absence of TGF-ß,
the addition of mIL-10 to the cultures would still generate ACAID APC.
We began our studies by adding a biologically relevant 10 U mIL-10/ml
culture supernatant. As before, APC incubated with OVA and TGF-ß
induced suppression of DTH (Fig. 6A). APC incubated with
OVA and mIL-10 in the absence of TGF-ß were able to induce
suppression of OVA-specific DTH in vivo (Fig. 6A).
This was confirmed in additional studies using 5 U mIL-10/ml (data not
shown). Because the crude supernatant containing mIL-10 also contained
nominal levels of IL-3, IL-4, and IL-5, we wanted to eliminate any
possible role for these cytokines. Accordingly, purified rmIL-10 was
used in subsequent assays. APC cultured with OVA and 10 ng/ml rmIL-10
also suppressed OVA-specific DTH (Fig. 6B).
Interestingly, APC pulsed with UV5C25 Ag in the presence of TGF-ß and
mIL-10 or mIL-10 alone were able to induce suppression of
UV5C25-specific DTH (Fig. 6C). This strongly suggests
that the failure of UV5C25 tumor Ags to induce IL-10 secretion by APC
in vitro is a key factor in the elicitation of Th1 immunity. In
summary, these results show that the effects of TGF-ß are mediated
through IL-10 and that a nominal level of exogenous IL-10 is sufficient
to confer a suppressive phenotype on APC in vitro.
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As mentioned, previous studies in normal mice had shown that ACAID
correlates with increased production of IL-10 in the spleen (18).
Curiously, these studies were unable to detect the presence of IL-4
protein or mRNA in these animals. To confirm both our present findings
and this earlier work, we utilized mice that had a homozygous mutation
in the gene for IL-4 or IL-10 (31, 32). OVA was injected into the
anterior chamber of IL-4- and IL-10-deficient mice. Seven days later,
mice were s.c. immunized with OVA in CFA. DTH was assessed 7 days after
s.c. immunization. As shown in Figure 7A, deletion of the IL-4 gene
did not affect the induction of ACAID. In direct contrast, deletion of
the gene for IL-10 prevented the induction of ACAID to OVA (Fig. 7B). Additional experiments were performed to confirm
that these results were not limited to soluble Ags, but would also
apply to particulate Ags known to induce ACAID. P815 mastocytoma
induces the down-regulation of DTH responses to DBA/2 alloantigens when
inoculated into the anterior chamber of allogeneic mice (33). The
results shown in Figure 7C indicate that, like the soluble
Ag OVA, the particulate Ag P815 (DBA/2; H-2d) failed to
induce ACAID in IL-10 knockout mice (C57BL/6; H-2b).
Therefore, the production of IL-10 is necessary for the ACAID state in
vivo. We next wanted to confirm the role of IL-10 in the generation of
suppressive, Th2-inducing ACAID APC in vitro. IL-10-deficient APC were
incubated with OVA, either in the presence or absence of TGF-ß, and
injected i.v. into normal, syngeneic C57BL/6 mice. As shown in Figure 8A, IL-10-deficient APC were
unable to induce ACAID. These results confirm the requirement for IL-10
production by the APC for the induction of the suppressive state. We
next wanted to determine whether the secretion of IL-10 was necessary
solely for the APC to obtain a suppressive phenotype (i.e., autocrine
effects) or whether IL-10 secretion was also necessary for the APC to
induce a suppressive, Th2-type response in the spleen (i.e., autocrine
and paracrine effects). Accordingly, IL-10-deficient APC were incubated
for 24 h with exogenous mIL-10 and injected i.v. into normal,
syngeneic C57BL/6 mice. The results show that IL-10-deficient APC
incubated with OVA, TGF-ß, and mIL-10 were able to induce suppression
in normal, syngeneic C57BL/6 mice (Fig. 8B).
Moreover, exposure to exogenous mIL-10 in the absence of TGF-ß also
conferred a suppressive phenotype on IL-10-deficient APC in vitro.
Thus, the in vitro data suggest that the requirement for IL-10 in the
down-regulation of Th1 immunity may be local and limited to initial
ocular Ag processing and may not be necessary during subsequent
presentation of peptide fragments by APC to T cells in lymphoid
tissues.
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Discussion |
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Various parameters have been shown to influence the differentiation of
a particular Th response, including Ag type, Ag dose, the type of APC,
and the presence of cytokines (34, 35). For example,
Schistosoma mansoni eggs preferentially elicit a Th2
response (36), whereas Listeria monocytogenes stimulates a
Th1 response (37). The type of APC also has a profound effect on the
resultant immune response. Langerhans cells and dendritic cells
normally present Ags such that Th1 immunity is induced (38, 39),
whereas B cells tend to induce a Th2 response (40). A major factor in
the disparity in APC function is the elaboration of particular
cytokines upon Ag encounter. One of the most important factors in Th
differentiation is the cytokine milieu. The most potent cytokine
inducer of Th2 cells is IL-4. Several types of immune cells can produce
IL-4 in the initial immune response, including
NK1.1+CD4+ T cells, 
T cells,
CD8+ T cells, and/or mast cells (35, 41). Because our
previous data had shown that IL-4 was not induced in ACAID animals and
our present data indicate that ACAID can be induced in IL-4 knockout
mice, we predicted that another cytokine capable of inducing Th2
development was involved. As previously mentioned, IL-10 is known to
inhibit the induction of Th1 immunity and allows for the elaboration of
a Th2 response. Since IL-10 can be produced by certain APC (42, 43), we
hypothesized that under the influence of TGF-ß, the preferential
production of IL-10 by the APC leads to the development of Th2 immunity
in ACAID. In addition, we hypothesized that the autoregulatory effects
of IL-10 on APC function might be a critical factor in the ability of
an APC to present Ag in a suppressive manner (3).
In direct contrast to IL-4 or IL-10, IL-12 strongly induces the
development of Th1 cells (44). Activation of monocytes/macrophages
usually results in the elaboration of proinflammatory, IL-12 cytokine
(30). The effect of IL-12 is twofold. One effect is the direct
stimulation of Th1 differentiation (41). Another effect is the
stimulation of IFN- production by T and NK cells. IFN- is a
potent inducer of Th1 immunity both by the additional stimulation of
IL-12 secretion and the inhibition of IL-4 function (29, 30). On the
other hand, IL-10 and IL-4 are powerful inhibitors of IL-12 production
(45, 46). Because previous experiments had measured increased levels of
IFN- in the immunizing or non-ACAID state, we predicted that the
preferential secretion of IL-12 by the APC was responsible for the
resultant Th1 immune response.
Our data show that at least one factor in the anterior chamber of the eye, TGF-ß, has a profound effect on the cytokines produced by Ag-stimulated APC. We have shown that the presence of TGF-ß can significantly enhance the production of IL-10, as measured by sandwich ELISA. In direct contrast, TGF-ß was also found to significantly suppress the production of the proinflammatory cytokine, IL-12 (p70). These cytokine data were confirmed with C57BL/6 APC (data not shown). These results suggest that TGF-ß contributes to the maintenance of immune privilege by predisposing an APC to the preferential production of IL-10 cytokine, which in turn might down-regulate or alter certain Ag-presenting functions leading to the generation of a suppressive, Th2-type response. The simultaneous inhibition of IL-12 secretion contributes to this response. In addition to the effect of TGF-ß alone, it appears that the Ag itself can have a significant impact on the APC cytokine profile. We have found that soluble OVA induces a twofold increase in the production of IL-10 as compared with UV5C25 tumor Ags. In direct contrast, UV5C25 tumor Ags stimulated a twofold increase in the production of IL-12 as compared with OVA. In addition, UV5C25 tumor Ags appear to completely inhibit the TGF-ß-induced production of IL-10. UV5C25 Ags did not completely override the suppressive effects of TGF-ß on IL-12 secretion, however. We conclude then that the immune privileged eye is endowed with at least one factor, TGF-ß, that has a profound effect on local APC cytokine production. This effect is the concomitant enhancement of IL-10 and suppression of IL-12 secretions. However, the effect of TGF-ß can be inhibited or circumvented depending on the specific Ag encountered.
The systemic suppression of DTH to OVA and the systemic immunization to
UV5C25 Ags can be directly correlated with the initial APC cytokine
profile. We have shown that the suppressive, ACAID state correlates
with increased IL-10 and decreased IL-12 secretion in vitro.
Conversely, we have shown that the immunizing, Th1 state correlates
with decreased IL-10 and increased IL-12 secretion in vitro. Our data
also indicate that there may be a minimal threshold of cytokine
secretion necessary for an APC to be Th1 or Th2 inducing in nature. The
results in Figure 3 suggested that IL-10 secretion 
5 U/ml culture
supernatant in vitro may be necessary for eventual suppressive,
Th2-inducing Ag presentation in vivo. Subsequent experiments in which
APC were pulsed with OVA and 3 or 5 U/ml mIL-10 in vitro have confirmed
that 5 U/ml, but not 3 U/ml, mIL-10 is sufficient for the induction of
ACAID in vivo (data not shown). IL-12 secretion 500 pg/ml is
indicative of eventual Th1 immunity in vivo (Fig. 4).
We had hypothesized that the effect of TGF-ß on APC function was mediated through IL-10 and that the autoregulatory effects of IL-10 were a critical component of ACAID Ag presentation. To test this hypothesis, we removed IL-10 from the APC cultures. Even in the presence of TGF-ß, treatment with neutralizing anti-IL-10 Ab in vitro completely reversed subsequent ACAID Ag presentation in vivo. These data indicated that the suppressive effect of TGF-ß on macrophage APC was mediated directly through the enhanced secretion of IL-10. If this were true, then the addition of IL-10 in the absence of TGF-ß would induce a suppressive phenotype. We have shown that the addition of 10 U mIL-10/ml of culture supernatant in vitro completely substituted for the effect of TGF-ß alone. Because the D10 supernatant used in these studies also contained nominal levels of IL-3, IL-4, and IL-5 in addition to IL-10, we wanted to rule out any effect of these ancillary cytokines. The addition of 10 ng rmIL-10/ml culture supernatant also effectively substituted for D10 supernatant or TGF-ß alone. In total, these data show that the presence of IL-10 in the APC microenvironment is critical for eventual ACAID Ag presentation. These data demonstrate that the effect of TGF-ß on macrophage APC can be directly mediated through the induced secretion of IL-10. Interestingly, the addition of 10 U/ml mIL-10 to UV5C25-pulsed APC induced ACAID to UV5C25 tumor Ags in vivo. This indicates that a lack of suppression to certain Ags, such as UV5C25, in the immune privileged eye is the direct result of insufficient secretion of IL-10. It therefore appears that the secretion of IL-10 serves as a primary determinant in the initial decision to respond in a Th1- vs Th2-mediated manner. Teleologically, the eye may be generally protected against harmful immune responses to innocuous agents, such as soluble proteins. However, if a more threatening agent is encountered, such as a growing tumor or a pathogen, circumvention of ACAID would be preferred over death. Unfortunately, weakly immunogenic tumors capable of inducing ACAID can escape immune surveillance and grow progressively, eventually destroying the eye and killing the host.
Although IL-12 is not reported to have a direct effect on macrophages, we wanted to determine whether neutralizing Ab to IL-12 affected the immunizing phenotype of ocular APC pulsed with UV5C25 Ag. As expected, the neutralization of IL-12 in the culture supernatant did not have any direct effect on APC function in vivo (data not shown). This finding contrasts with the suggested role for IL-10, since IL-10 is shown to have a direct effect on APC in the microenvironment of the in vitro eye. It is probable that the enhanced production of IL-12 in the non-ACAID state primarily serves as a stimulant to Th1 differentiation in the spleen.
A previous report had shown that systemic anti-IL-10 Ab treatment in vivo abrogates ACAID (18). We have confirmed those results in mice deficient in the gene for IL-10. We were unable to induce ACAID to OVA or P815 tumor line injected into the anterior chamber of IL-10 knockout mice. Furthermore, our in vitro studies showed that in the presence or absence of TGF-ß, APC from IL-10 knockout mice could not generate ACAID to OVA. In addition, we have confirmed that the production of IL-10, but not IL-12, is impaired in IL-10 knockout mice (data not shown). These results demonstrate that the ACAID APC must have the capability to produce IL-10 for the generation of the suppressive state in vivo. Our in vitro data indicate that the production of IL-10 is necessary in the local, ocular environment. We propose that the effect of IL-10 in the local environment is to feedback on the APC in an autoregulatory loop and thereby alter normal Ag presentation function. We have also tested whether the production of IL-10 by the APC is necessary once that cell has migrated from the eye to lymphoid organs such as the spleen. The addition of exogenous IL-10 to the IL-10-knockout APC cultures restored the ability of these cells to generate suppression in wild-type mice. We conclude that, although IL-10 is requisite in the local environment of the eye, the production of IL-10 by the APC is not necessary once that cell migrates to lymphoid tissues such as the spleen.
Many cell types have been reported to produce IL-10 cytokine, including keratinocytes (47), T cells (3), B cells (42), mast cells (48), and monocytes (43). Since its discovery, countless studies have shown that IL-10 can have a profound effect on an immune response. It has been shown that IL-10 generally inhibits cell-mediated immune responses such as contact hypersensitivity and DTH, while promoting the development of Ab responses (29, 49). Apart from the direct effect on T cells, these studies have concluded that the alteration of macrophage function by IL-10 plays a key role in the preferential development of Th2-type responses. One study with experimental allergic encephalomyelitis in SJL mice has demonstrated that depletion of IL-4 or IL-10 in nonresponder mice leads to the expansion of previously nonexistent, responding Th1 cells (50). This indicates that the presence of certain cytokines before Ag exposure can regulate Ag presentation and eventual Th development. We have shown definitively that a minimal level of IL-10 cytokine present at the time of Ag exposure can induce APC to subsequently present Ag to a population of T cells that suppress DTH.
TGF-ß is expressed in multiple immune privileged sites, including the anterior chamber of the eye (51), the vitreous humor (52), the central nervous system (53), the testes (54), and the adrenal cortex (55). Apart from its other immunomodulatory effects, we predict that the presence of TGF-ß contributes to immune priviege in each of these sites by the described alteration of APC cytokine secretion and function. The mechanism whereby TGF-ß induces IL-10 secretion remains a mystery. However, it is noteworthy that in other models of ACAID, Ag-bearing cells introduced into the anterior chamber undergo apoptosis following exposure to Fas ligand expressed within the eye and elaborate IL-10 before succumbing to apoptotic death (56). We have preliminary evidence that either aqueous humor or TGF-ß also induces apoptosis of APC in vitro. Thus, it is possible that within the anterior chamber, Ag-bearing cells are exposed to at least two conditions that could culminate in apoptosis and the elaboration of IL-10. The presence of duplicate pathways for inducing IL-10 production seems quite plausible considering the redundancy of the immune system. The presence of an IL-10-enriched milieu would favor the development of a Th2 response. The suppression of IL-12 and probably other proinflammatory cytokines also contributes to this effect by discouraging the differentiation and activation of Th1 cells.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jerry Y. Niederkorn, Department of Ophthalmology, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9057. E-mail address:
3 Abbreviations used in this paper: DTH, delayed-type hypersensitivity; ACAID, anterior chamber-associated immune deviation; m, murine; PEC, peritoneal exudate cell(s).
Received for publication July 17, 1997. Accepted for publication November 6, 1997.
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, p < 0.0003 for medium
control and positive control vs negative control; **p
< 0.006 for positive control vs OVA, i.c. and negative control.
, p < 0.0004 for
mIL-10 and TGF-ß + mIL-10 vs positive control.
