| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Centre for Immunology and Cancer Research, University of Queensland, Princess Alexandra Hospital, Brisbane, Queensland, Australia
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, IL-6, or
TGF-ß. GM-CSF, IL-1ß, and TNF- were all able to restore APC
function. Whereas the effects of IL-10 on PB DC were shown to be
mediated by IL-10R1, neither PB nor RA SF DC constitutively expressed
IL-10R1 mRNA or detectable surface protein. In contrast, IL-10R1
protein was demonstrated in PB and SF DC whole cell lysates, suggestive
of predominant intracellular localization of the receptor. Thus, DC
responsiveness to IL-10 may be regulated through modulation of cell
surface IL-10R1 expression or signaling. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
DC are potent APC whose function is specialized for the stimulation of the primary immune response. The functional capacity of DC is tightly regulated and is closely related to the process of DC differentiation. In man, DC isolated from rheumatoid arthritis (RA) synovial fluid (SF) provide an easily accessible model of functional DC that have differentiated from precursors in vivo (6). We and others have previously demonstrated expression of both IL-10 mRNA and protein in RA synovial cells (7, 8, 9, 10). Nevertheless, DC derived from RA synovium have been demonstrated to be differentiated phenotypically and functionally, in that they express markers of maturation, as well as markers of activation, including CD80 and CD86 (6). It has previously been proposed that such activated synovial DC play a key role in the perpetuation of the rheumatoid immune response (11). In view of the high levels of IL-10 detected within the rheumatoid synovium, it appeared paradoxical that DC in this location expressed markers of activation and differentiation in vivo and were fully functional in vitro. It was therefore of interest to examine the effects of IL-10 upon rheumatoid SF DC in vitro. The data demonstrate that, in contrast to normal peripheral blood (PB) DC precursors, rheumatoid SF DC are resistant to the immunosuppressive effect of IL-10 in vitro.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
SF was obtained from 12 patients with RA. Five were taking methotrexate, one sulfasalazine, one hydroxychloroquine, four penicillamine, and one i.m. gold, and two were on no disease-modifying drug. Four of the patients were also on low dose prednisone. PB was obtained from normal volunteers.
Culture medium
All cell cultures were conducted in medium RPMI 1640 (Sigma, St. Louis, MO) supplemented with penicillin G (200 U/ml), gentamicin (10 µg/ml), L-glutamine (0.3 mg/ml), and 10% FCS (Commonwealth Serum Laboratories, Melbourne, Australia).
Cells and reagents
Human TF1 cells expressing either an irrelevant murine
(ecotropic) receptor or recombinant human IL-10R (hIL-10R) were
provided by Dr. K. Moore, DNAX, Palo Alto, CA (12). TF1
cells express low levels of endogenous IL-10R at the cell surface. TF1
cell lines were maintained in culture medium supplemented with human
GM-CSF (20 U/ml), and, for selection purposes, 2 µg/ml puromycin
(Sigma) was added to TF1-hIL-10R. The following mAb were used:
FITC-conjugated Leu-M1 (Becton Dickinson, San Jose, CA) and
PE/Cy5-conjugated Tuk4 (Caltag, South San Francisco, CA), directed
against CD14 on human monocytes; PE-conjugated Leu-M9, directed against
CD33 on human myeloid cells; Leu-11b, directed against CD16
(Fc
RIII); Leu-12, directed against CD19 (Becton Dickinson); L243,
directed against monomorphic determinants of HLA-DR (American Type
Culture Collection (ATCC), Manassas, VA); OKT3, directed at the CD3
complex on human T cells (ATCC); B70, directed against CD86/B7-2
(PharMingen, San Diego, CA); BB1, directed against CD80/B7-1 (Ancell,
Bayport, MN); 3F9, directed against the IL-10-binding epitope of the
human IL-10R (provided by Dr. K. Moore; Ref. 12); and
19F1, directed against human and viral IL-10 (ATCC). Control Abs
included mouse IgG1 (Dako, Carpinteria, CA), rat Ig (Dako), FITC-IgG1
and PE-IgG1 (Becton Dickinson), and biotinylated anti-mouse Ig and
biotinylated anti-rat Ig (Dako). PE-conjugated streptavidin and
HRP-streptavidin were purchased from Dako. Recombinant human GM-CSF was
a gift from Schering-Plough, Sydney, Australia; recombinant TNF- and
recombinant IL-1ß were obtained from the World Health
Organization International Laboratory for Biological Standards,
(Hertfordshire, U.K.). Recombinant human IL-10 was expressed in
Escherichia coli and purified (13).
DC, monocyte, monocyte-derived DC (MDDC), and T cell preparation
PBMC and SF MNC were prepared as previously described (14). Briefly, heparinized venous blood from healthy adult donors, or SF from patients with RA, was sedimented over ficoll diatrizoate (Pharmacia, Uppsala, Sweden). Cells were washed and then incubated with neuraminidase-treated sheep red blood cells (NSRBC). The rosetting and nonrosetting populations were separated on ficoll diatrizoate gradients. After lysis of the red cells with 1 M ammonium chloride, PB rosetting cells were passed over a nylon wool column. Pure populations of responder T cells were prepared by magnetic immunodepletion with anti-CD14, anti-CD16, and anti-HLA-DR, followed by GAMIg-conjugated magnetic beads (Miltenyi Biotec, San Francisco, CA) as described. Greater than 98% of the recovered T cells expressed CD3 by flow cytometric analysis.
PB non-T cells were myeloid enriched by magnetic immunodepletion using
anti-CD19, anti-CD3, and anti-CD16, followed by
GAMIg-conjugated magnetic beads as described. By flow cytometric
analysis, 98% of these cells lacked expression of CD19, CD3, and CD16,
and 80–95% expressed CD33. SF non-T cell populations contained less
than 10% of T, B, and NK cells and 
30% each of DC, monocytes, and
granulocytes as described previously (14).
SF non-T cells or myeloid-enriched PB non-T cells were incubated with FITC-conjugated anti-CD14 and PE-conjugated anti-CD33 on ice for 30 min and washed. Gates were set, and DC were sorted on an Epics Elite Flow Cytometer (Coulter Electronics, Hialeah, FL) as CD33+CD14dim cells and monocytes as CD33+CD14bright cells as described previously (6). The threshold was set to abort dead cells, debris, and granulocytes as determined by light scatter. Postsort analysis showed that <3% of the cells sorted as negative were positive, and >95% of the cells sorted as positive were positive.
For generation of MDDC, myeloid-enriched PB non-T cells were cultured at 1.3 x 106 cells/ml in the presence of GM-CSF (800 U/ml) and IL-4 (400 U/ml) for 7 days as previously described (54).
Staining of non-T cells for flow cytometric analysis
Freshly isolated SF non-T cells or myeloid-enriched PB non-T cells were incubated with optimal concentrations of mAb or rat Ig on ice for 30 min, washed twice, then incubated with biotinylated anti-rat Ig for 30 min on ice, washed twice, then finally incubated with streptavidin-PE, FITC-conjugated-anti-HLA-DR, and PE/Cy5-conjugated anti-CD14 with or without PE/CY5-conjugated anti-CD19 for 30 min on ice. After staining, all cells were fixed with 1% paraformaldehyde (Sigma) and analyzed on an Epics Elite Flow Cytometer using a single argon laser. DC and monocytes were gated as HLA-DR+ CD14-/dim CD19- cells and HLA-DR+CD14bright cells, respectively. In some experiments, cultured sorted DC were stained with biotinylated anti-CD86 followed by streptavidin PE/Cy5 and HLA-DR-FITC and analyzed for two colors as above.
Induction of T cell responses in MLR and DC differentiation in vitro
DC were incubated in 96-well round-bottom polypropylene dishes (Costar, Cambridge, MA) for 18 h at 37°C in the presence or absence of various cytokines or mAb as described in the text. Cells were washed and counted and either added to the MLR or stained for flow cytometry. For the MLR, cells were resuspended at 5 x 106 cells/ml in medium containing 0.08 mg/ml mitomycin C (Sigma) to inhibit cell proliferation. Cells were incubated at 37°C in the dark for 20 min, then washed three times in Hanks buffered saline solution. Various numbers of APC were incubated in triplicate in round-bottom 96-well tissue culture plates (Costar) with 105 freshly isolated purified normal allogeneic PB T cells at 37°C for 5 days. T cell proliferation was measured by the uptake of [3H]thymidine (1 µCi/well; 6.7 Ci/mM, ICN, Costa Mesa, CA), which was added during the final 18 h of the culture period. Cells were harvested onto glass fiber filter paper with an automated 96-well harvester (Wallac, Turku, Finland), and [3H]thymidine incorporation was determined by liquid scintillation spectroscopy. The responses are reported as the mean cpm ± SEM for triplicate wells.
Acridine orange assay for apoptosis
Cell cultures were stained with acridine orange, a fluorescent nuclear binding dye that facilitates the distinction of apoptotic cells from healthy cells based on changes in nuclear morphology (15). Cell counting was undertaken within 5 min of the addition of 10 µg/ml acridine orange to the culture. Apoptotic cells were identified on the basis of cellular shrinkage and condensed and highly fluorescent chromatin.
RNA isolation and RT-PCR analysis
Total RNA was isolated using RNAzol (Biotecx, Houston, Tx) according to the manufacturer’s instructions. First strand cDNA was synthesized using oligo(dT)20 (Pharmacia Biotech) as a primer and AMV-RT (Promega, Madison, WI) as previously described (16). Complementary DNA was mixed with 25 pmol each of forward and reverse oligonucleotide primers, 200 µM each of dGTP, dATP, dCTP, and dTTP (Perkin-Elmer, Norwalk, CT) in 10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl2, 50 mM KCl, and 0.1% Triton X-100 in a total volume of 25 µl. The reaction mix was heated at 95°C for 7 min before adding two units of Taq polymerase (Perkin-Elmer), followed by 35 cycles of 95°C (1 min), 63°C (1 min), 72°C (1 min 30 s), and a final extension step at 72°C for 7 min. The IL-10R primer sequences were: sense primer, 5'-GTA CCA CAG CAA TGG CTA CC-3'; and antisense primer, 5'-CAC GGT GAA ATA CTG CCT GG-3' (17). The GAPDH primers were: sense primer, 5'-ACC ACA GTC CAT GCC ATC AC-3'; and antisense primer, 5'-CAC GGT GAA ATA CTG CCT GG-3' (Clontech, Palo Alto, CA). PCR products were analyzed by electrophoresis on a 1.8% agarose gel and visualized by staining with ethidium bromide. To confirm product specificity, purified fragments were prepared, using a PRISM Ready Reaction DyeDeoxy Terminator Cycle sequencing kit (Applied Biosytems, Foster City, CA), for analysis on the Applied Biosystem DNA Sequencing System.
Immunoblotting
After sorting or incubation, cells were washed in ice cold PBS, and protein extracts from either whole cell lysates or purified membranes were prepared. For lysates, cells were resuspended at 2 x 107 cells/ml in Nonidet P-40 (NP-40) lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris (pH 8.0), 1 mM PMSF (Sigma)) and incubated for 30 min on ice. The lysate was centrifuged at 600 x g for 10 min, and the supernatant was collected. Lysates were diluted 1:1 with 2x gel-loading buffer (50 mM Tris-HCl, 2% w/v lauryl sulfate, 0.1% bromophenol blue, 10% w/v glycerol, and 5% v/v 2-ME (pH 8.0)) and heat denatured. For membrane purification, cells were resuspended at 2 x 107 cells/ml in ice cold homogenization buffer (10 mM Tris (pH 7.6), 0.5 mM MgCl2, 10 µg/ml leupeptin (ICN), 10 µg/ml aprotinin (ICN), 2 µg/ml pepstatin (ICN), and 1 mM PMSF), transferred to a clean, chilled Dounce homogenizer, and homogenized by delivering 30 strokes. Efficiency of homogenization was monitored by phase contrast microscopy. The homogenate was transferred to a clean microfuge tube, and one-quarter volume of restoration buffer (10 mM Tris-HCl (pH 7.6), 0.5 mM MgCl2, 0.6 M NaCl, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 2 µg/ml pepstatin, and 1 mM PMSF) was added. The nuclear fraction was removed by centrifugation at 500 x g for 5 min at 4°C. The supernatant was centrifuged at 13,500 x g for 1 h at 4°C, and pelleted membrane protein was washed in TBS supplemented with 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM PMSF and resuspended in 2 x gel-loading buffer at 1 x 107 cells/ml. Lysate and membrane preparations were loaded (105 or 106 cell equivalents/lane, respectively, or in some experiments, 25 µg protein/lane) alongside prestained protein m.w. standards (5 µl/lane; Bio-Rad, Hercules, CA) on an 8% polyacrylamide gel and elect-rophoresed for 1 h at 150 V. Protein was electrophoretically transferred to nitrocellulose membranes (Amersham, Sussex, England), which were blocked in 10% skim milk powder in TBS. Membranes were incubated in either 3F9 mAb (0.5 µg/ml in TBS containing 0.5% milk powder) or irrelevant mAb, washed twice in 0.1% Tween 20 in TBS (TBS-T), then incubated in biotin-conjugated anti-rat Ig in TBS-T, washed again, and incubated with streptavidin-conjugated HRP. After washing and a 1-min chemiluminescent enhanced chemiluminescence (ECL) reaction (Amersham), membranes were exposed to Hybond x-ray film (Amersham).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The first experiments compared the effect of IL-10 on the APC
function of freshly isolated DC derived either from normal PB or RA SF.
To specifically examine the effect of IL-10 on DC and not on T cells,
DC APC function was examined after their differentiation in the
presence of varying concentrations of IL-10.
CD33+CD14dim DC were sorted
from normal PB or RA SF and preincubated for 18 h in the presence
or absence of IL-10, and the ability of the washed DC to stimulate
purified allogeneic PB T cells was examined. As seen in Fig. 1
A, preincubation of PB DC in
the presence of IL-10 induced a dose-dependent reduction in their APC
function. To exclude the possibility that death of DC induced by IL-10
was responsible, cells from parallel 24- and 48-h MLR cultures were
stained with trypan blue. Equivalent numbers of viable DC were added to
the MLR after preincubation in either medium or IL-10, and equivalent
numbers of viable DC remained under both conditions for 48 h (data
not shown). Furthermore, 24- and 48-h cultures of DC, which had been
preincubated in either medium or IL-10, were examined for apoptosis
using acridine orange staining. The number of apoptotic cells was
similar in DC cultures irrespective of prior IL-10 exposure (data not
shown). In contrast to PB DC, there was no functional effect of
preincubation of SF DC in the presence of IL-10 (Fig. 1B).
The data indicate that incubation of PB but not RA SF DC in the
presence of IL-10 down-regulates the APC function of the DC.
|
Previously IL-10 has been shown to down-regulate expression of
HLA-DR and CD86 expression by PB DC. To determine whether this was also
true for rheumatoid SF DC, freshly isolated normal PB DC or rheumatoid
SF DC were incubated with varying concentrations of IL-10 for 18
h, and the expression of cell surface molecules was subsequently
examined. As shown in Fig. 2, HLA-DR and
CD86 expression was down-regulated in a dose-dependent fashion by
incubation in IL-10. However, there was no effect on the expression of
these cell surface molecules in the case of RA SF DC (Fig. 3). Consistent with previous reports,
there was no effect of IL-10 on CD80 expression by either cell type
(data not shown) (4). The data demonstrate that rheumatoid
SF DC are resistant to down-modulation of MHC class II molecule and
CD86 expression by IL-10 and further suggest that these DC are not
susceptible to IL-10 in vitro.
|
|
Since differentiation of PB DC precursors in the presence of IL-10
led to suppression of their APC function, and SF DC had been exposed to
IL-10 in vivo, but were still functional APC, the next experiments
examined whether proinflammatory cytokines present within RA synovium
might influence the differentiation of DC so as to render them
resistant to IL-10. Therefore, PB DC precursors were cocultured with or
without IL-10 in the presence of either GM-CSF, IL-1-ß, TNF-,
IL-6, or TGF-ß. Cells were then washed and used to stimulate purified
allogeneic T cells in MLR. As seen in Fig. 4, coculture in the presence of IL-10 and
either GM-CSF, IL-1-ß, or TNF-, but not IL-6 or TGF-ß, reversed
the effect of IL-10 on DC APC function. Furthermore, the effects of
IL-10 on PB DC HLA-DR and CD86 expression were reversible by coculture
in the presence of GM-CSF, IL-1ß, and TNF- (data not shown).
Hence, the data suggest that differentiation of DC in the presence of
GM-CSF, IL-1ß, or TNF- can alter the ability of these DC to
respond to IL-10.
|
Two components of the IL-10 receptor (IL-10R1 and IL-10R2) have
been identified. However, their expression by PB DC has yet to be
characterized (17, 18). The next experiments therefore
examined whether IL-10R1 was involved in IL-10-mediated suppression of
PB DC and monocyte APC function. Freshly purified PB DC or monocytes
were preincubated for 18 h with or without IL-10 (100 U/ml) in the
presence or absence of anti-IL-10R1 mAb 3F9 (5 µg/ml) or control
rat Ig. This mAb recognizes the IL-10-binding epitope of IL-10R1. Cells
were then washed and used to stimulate purified allogeneic T cells in
MLR. 3F9, but not rat Ig effectively blocked the effects of IL-10 on PB
DC and monocyte APC function, indicating that IL-10R1 is essential for
IL-10 signaling of these cell populations (Fig. 5).
|
Since SF DC derived from patients with RA appeared resistant to
the down-modulatory effects of IL-10 and since IL-10R1 was shown to
mediate IL-10 signaling of PB DC, the next experiments examined whether
this resistance was due to modified IL-10R1 expression by RA SF DC.
Cell surface expression of IL-10R1 by normal PB MNC and RA SF DC and
monocytes was examined by flow cytometry using mAb 3F9. Cell surface
IL-10R1 was expressed at low levels by normal PB T cells as previously
reported (12) and by RA SF monocytes (Fig. 6, C and D,
respectively). In contrast, IL-10R1 expression was detected at low
levels on normal PB monocytes in some but not all donors but was not
detectable on the surface of normal PB or SF DC (Fig. 6, A,
B, and F, respectively). Furthermore, cell
surface IL-10R1 was not detected after incubation of DC in either
medium or any of the cytokines used in the functional assays shown in
Fig. 4
(data not shown). Significant levels of IL-10 are found in RA
SF, and previous studies have demonstrated that murine macrophages may
display significant quantities of functional cell surface IL-10
(19). Thus, to determine whether the 3F9-binding epitope
was blocked by IL-10 bound in vivo, freshly isolated normal PB and RA
SF monocytes and DC were stained with anti-IL-10 mAb. Whereas low
levels of surface IL-10 were detected on RA SF monocytes (Fig. 6E), no IL-10 was detected on RA SF DC (Fig. 6G),
PB DC, or monocytes (data not shown). Therefore, IL-10 did not appear
to be bound to RA SF DC via cell surface IL-10R1. The data demonstrate
that RA SF monocytes expressed an increased level of surface IL-10R1 as
compared with normal PB monocytes, some of which is bound by IL-10. In
contrast, the level of expression by normal PB monocytes of some
individuals, and all PB and RA SF DC, was undetectable by flow
cytometry using mAb 3F9. Recently, human PB DC were found to bind IL-10
in vitro (20). Since binding studies demonstrate 300
IL-10R1 per T cell and that this can be detected flow cytometrically
(Refs. 21 and 22 , and Fig. 6C),
the data suggest, surprisingly, that freshly isolated DC express fewer
than 300 IL-10R1 at the cell surface. Finally, since this low level of
expression was not detectable in the current assay, a comparison
between PB and SF DC surface Il-10R1 expression could not be
made.
|
Since cell surface IL-10R1 expression was undetectable on both PB
and SF DC, the next experiments examined whether IL-10R1 was
transcriptionally regulated by the SF DC. Using human IL-10R1-specific
oligonucleotides, RT-PCR demonstrated the expression of IL-10R1 mRNA by
freshly isolated PB T cells as has been previously reported
(17). In contrast, as found for IL-10R1 protein
expression, IL-10R1 mRNA was inconsistently detected in freshly
isolated PB or SF monocytes and not detected in freshly isolated PB or
SF DC (Fig. 7A). Previously,
PB monocytes and macrophage cell lines have been reported to express
IL-10R1 mRNA (17, 21). To determine whether the lack of
expression by myeloid cells was due to a loss of message during cell
purification or a lack of sensitivity of the RT-PCR technique,
CD4+ T cells were sorted and cDNA titrated.
IL-10R1 mRNA was not degraded as a result of the rigorous purification
protocol required for obtaining pure monocyte and DC populations, since
cDNA derived from normal PB CD4+ T cells purified
by sorting generated an IL-10R1 amplicon. Complementary DNA derived
from an equivalent number of monocytes and DC derived from the same PB
sample, however, failed to produce an IL-10R1 PCR fragment (Fig. 7B). Furthermore, the lack of IL-10R1 mRNA expression in DC
and monocytes is unlikely to be a result of insensitivity of the RT-PCR
assay or an alternately spliced mRNA. First, titration of the sorted
CD4+ T cell cDNA revealed that an IL-10R1 PCR
fragment could be produced from as few as 32 T cells (data not shown).
Second, an alternative set of IL-10R1-specific oligonucleotides failed
to improve the sensitivity of the assay (data not shown). To determine
whether IL-10R1 mRNA might be expressed in either myeloid precursors or
upon DC differentiation, either cultured DC or myeloid-enriched non-T
cells (which contain DC, monocytes, and a small percentage of myeloid
precursors) were examined (54). Whereas IL-10R1 mRNA was detectable in
myeloid-enriched non-T cells (Fig. 7A), it was not detected
in cDNA derived from cultured DC (Fig. 7C). The data
therefore suggest that IL-10R1 mRNA is expressed at early stages but is
undetectable at later stages of myeloid differentiation.
|
The previous experiments demonstrated that both cell surface
IL-10R1 and IL-10R1 mRNA were undetectable in PB DC and monocytes, yet
these cells are capable of responding to IL-10 through IL-10R1 in
vitro. Immunoblotting using anti-IL-10R1 mAb 3F9, or control rat
Ig, was therefore used to determine whether IL-10R1 were present in the
cells. An immunoreactive 100-kDa protein was detected in whole cell
lysates of sorted PB DC and monocytes but not of equivalent numbers of
T cells. The 3F9 immunoreactive protein was of an identical size to the
immunoreactive IL-10R1 detected in TF1-hIL-10R lysates (Fig. 8A). No immunoreactive protein
was detected when 3F9 was replaced with control rat Ig (data not
shown). These data demonstrate that immunoblotting is insufficiently
sensitive to detect the small number of T cell surface IL-10R1.
Nevertheless, IL-10R1 protein was readily detected in PB monocytes and
DC lysates, although the intensity of the signal was variable between
donors. Therefore, in these cells, IL10-R1 must be intracellular.
Moreover, IL-10R1 was even more abundant in lysates from equivalent
cell numbers of SF monocytes and DC (Fig. 8A). To determine
whether the difference in IL-10R1 expression between PB and SF DC and
monocytes was due to a difference in cell size and activation state,
cell lysates were normalized for protein, electrophoresed, and blotted.
In this case, myeloid-enriched SF protein preparations contained more
IL-10R1 than an equivalent amount of myeloid-enriched PB protein (data
not shown). Although the flow cytometric analysis demonstrated that at
least some of the SF monocyte IL-10R1 was expressed on the membrane,
the abundant signal obtained by immunoblotting suggests that a larger
proportion was intracellular. The immunoreactivity of whole cell lysate
protein derived from 105 cells was therefore
compared with that of membrane protein derived from
106 cells. Whereas IL-10R1 was detected in cell
lysates prepared from either MDDC, freshly purified PB DC or monocytes,
or RA SF MNC, immunoreactive protein was detectable only in the
TF1-hIL-10R cell line membrane preparations (Fig. 8B). The
control membrane protein CD31 was readily detectable in membrane
preparations from each cell type (data not shown). The data therefore
indicate that, in contrast to T cells, IL-10R1 is predominantly
intracellular in DC and monocytes. Furthermore, these cells are likely
to regulate their response to IL-10 by transporting IL-10R1 between an
intracellular compartment and the cell surface.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production (23, 24, 25, 26, 27). IL-10 is
present at low levels in normal serum and has been demonstrated,
alongside proinflammatory cytokines, in a wide variety of inflammatory
and infective situations (28, 29, 30, 31). Given that IL-10 is
rapidly produced in the immune response, particularly in response to
TNF- induction in monocytes and IFN-
in T cells
(32), the mechanisms whereby DC continue to function as
effective APC are poorly understood. This study examined the regulation
of DC responsiveness to IL-10 using as a model the relatively mature DC
that can be easily obtained from the inflammatory autoimmune source RA
SF. The data demonstrate that, in contrast to normal PB DC precursors,
rheumatoid SF DC are resistant to the immunosuppressive effects of
IL-10. Furthermore, differentiation of PB DC in the presence of the
proinflammatory cytokines TNF-, GM-CSF, and IL-1ß rendered the DC
unresponsive to IL-10. Although the exact mechanism of this resistance
of SF DC is still not clear, the current studies suggest that DC
responsiveness to IL-10 may be regulated through modulation of cell
surface IL-10R1 expression or signaling. The hIL-10R, a member of the class II cytokine receptor family, comprises two subunits, IL-10R1 and IL-10R2 (18, 33, 34). The expression of IL-10R1, the ligand binding subunit and most widely studied of the subunits, is largely restricted to hemopoietic cells. In all cell types examined, only hundreds of IL-10R1 molecules per cell have been detectable (21, 22, 35). In contrast, IL-10R2 expression appears less restricted; however, its expression and function remain to be fully elucidated. The current studies suggest that the mechanism of regulation of surface IL-10R1 expression and IL-10 responsiveness of DC might differ from that of lymphocytes. In this regard, mature circulating T cells express abundant IL-10R1 mRNA, hundreds of IL-10R1 molecules on the cell surface, and very few or no receptors intracellularly. In vitro activation results in diminished IL-10R1 mRNA and surface protein expression (17). In contrast, constitutive expression of either IL-10R1 mRNA or surface protein was below the threshold of detection in PB DC, was inconsistently detected in PB monocytes, and was detectable in PB preparations enriched for myeloid precursors. Intracellular IL-10R1 protein was detectable in freshly isolated PB monocytes and DC. Thus, it appears likely that early myeloid precursors transiently produce IL-10R1 that is stored intracellularly for transport to the cell surface. However, there are likely to be other signals for intermittent IL-10R1 mRNA production in monocytes and DC. In this regard, constitutive IL-10R1 mRNA expression by PB monocytes purified by adherence has previously been shown (17). Preliminary data indicate that freshly purified normal PB monocytes up-regulate IL-10R1 mRNA in response to adherence for 2 h in vitro (data not shown). Furthermore, the increased IL-10R1 immunoreactivity of SF DC, as compared with PB DC, implies that IL-10R1 mRNA was expressed at some stage during the differentiation of DC precursors in the synovial environment.
The functional capacity of DC is tightly regulated and is closely
related to the process of DC differentiation. This process involves the
up-regulation of MHC and costimulatory molecule expression, migration
of mature or maturing DC to the paracortex of secondary lymphoid
organs, and down-regulation of Ag uptake and processing capacity
(36, 37). Coculture of freshly isolated PB DC in the
presence of IL-10 and either GM-CSF, IL-1ß, or TNF- prevented the
immunosuppressive effects of IL-10 on PB DC APC function. It is of
interest that GM-CSF, TNF-, and IL-1 have been shown previously to
represent key cytokines involved in the differentiation and
up-regulation of function of DC precursors derived from many tissue
sources, including the blood (38, 39, 40, 41). Therefore, these
proinflammatory cytokines present within RA synovium, and indeed at
many inflammatory sites, appear to modulate the immunosuppressive
effects of IL-10 on DC. However, neither IL-6 nor TGF-ß could
modulate the effects of IL-10 on PB DC precursors in this in vitro
system. In support of these findings, IL-6 appears to be important in
the early differentiation of the DC lineage within the bone marrow but
has little effect on DC function at the later differentiation stages
(42). Similarly, TGF-ß has been demonstrated to play a
critical role in the early development of LC and PB DC; however, it
also has been shown to exert inhibitory effects on DC function
(43, 44, 45). Although not examined here, it is also possible
that the location and kinetics of exposure of differentiating,
migrating DC to various pro- and antiinflammatory cytokines might be
critical to the outcome of an immune response. Taken together, the data
suggest that, during the differentiation of DC within the inflammatory
synovial environment, the capacity to transport intracellular IL-10R1
to the cell surface or to signal through IL-10R1 is modulated, possibly
through combined or sequential exposure to IL-10 and certain
proinflammatory cytokines.
Previous studies indicate that LC differentiated in the presence of keratinocytes are less susceptible than fresh LC to IL-10-mediated suppression and that addition of IL-10 to the MLR has little effect unless added early (27, 46). Although it might be interpreted from these studies that mature DC are resistant to IL-10, the current data support the notion that IL-10 resistance is not related to the "mature phenotype" alone. Rather, certain factors are required to be present and to signal DC during the process of differentiation to mediate IL-10 resistance. In support of this, in vitro differentiated PB DC that express a fully mature phenotype are also susceptible to suppression by IL-10 (4). Thus, IL-10 resistance does not strictly correlate with functional maturity but is more likely to be related to DC differentiation signals affecting expression of surface IL-10R1.
The current data reflect the complexity of interactions between
cytokines present in RA synovium on the outcome of the immune response.
IL-10 is likely to be important for the differentiation of RA synovial
B cells into plasma cells and the production of Ig and rheumatoid
factor (47, 48). IL-10 has also been shown to induce
cartilage destruction, mediated both directly and through TNF- and
IL-1ß (49). Several studies suggest that synovial
monocytes are less susceptible than PB monocytes to the
immunosuppressive effects of IL-10 (50, 51, 52). Furthermore,
expression of MHC class II by SF monocytes appears to be more resistant
to IL-10-mediated suppression than proinflammatory cytokine secretion
(50). Taken together, these and the current studies
suggest that various cell types within RA synovium are responsive to
IL-10 to varying degrees. Furthermore, the overall balance between
proinflammatory and antiinflammatory cytokines in the synovium is
likely to be less important than the determination of specific key
interactions for specific cell types.
The current data have important implications for the potential efficacy
of IL-10 immunotherapy in RA. Clinical trials of IL-10 immunotherapy in
RA are in progress. On balance, the available data might predict that
IL-10 would ameliorate disease but not induce remission, as is the case
in the collagen-induced arthritis model (53). With regard
to our hypothesis that TNF- is likely to play a significant role in
the orchestration of DC escape from IL-10, it is of interest that IL-10
provides little additional benefit to that induced by anti-TNF-
Abs in the collagen-induced arthritis model (53), and it
is possible that part of the mechanism of action of anti-TNF- in
murine and human disease relates to enhanced cellular responsiveness to
endogenous IL-10. In summary, the data provide evidence for control of
the IL-10R as an important DC escape mechanism from IL-10 and suggest
that manipulations that circumvent this control could enhance
responsiveness to either endogenous or exogenous IL-10.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Ranjeny Thomas, Centre for Immunology and Cancer Research, University of Queensland, Princess Alexandra Hospital, Brisbane, Queensland, 4102, Australia. E-mail address:
3 Abbreviations used in this paper: DC, dendritic cell; LC, Langerhans cell; RA, rheumatoid arthritis; SF, synovial fluid; hIL-10R, human IL-10R; PB, peripheral blood; MDDC, monocyte-derived DC.
Received for publication July 22, 1999. Accepted for publication August 30, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, but strongly enhanced by interleukin-10. Eur. J. Immunol. 25:1943.[Medline]
(IFN-), IL-10, IL-12 and transforming growth factor-ß (TGF-ß) mRNA in synovial fluid cells from patients in the early and late phases of rheumatoid arthritis (RA). Clin. Exp. Immunol. 103:357.[Medline]
induces apoptosis of human adipose cells. Diabetes 46:1939.[Abstract]
production by peripheral blood mononuclear cells in response to viral stimulation. J. Immunol. 160:5861. in human monocyte IL-10 synthesis. J. Immunol. 151:6853.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  W. Barchet, J. D. Price, M. Cella, M. Colonna, S. K. MacMillan, J. P. Cobb, P. A. Thompson, K. M. Murphy, J. P. Atkinson, and C. Kemper Complement-induced regulatory T cells suppress T-cell responses but allow for dendritic-cell maturation Blood, February 15, 2006; 107(4): 1497 - 1504. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. J. O'Sullivan and R. Thomas CD40 Ligation Conditions Dendritic Cell Antigen-Presenting Function Through Sustained Activation of NF-{kappa}B J. Immunol., June 1, 2002; 168(11): 5491 - 5498. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Page, S. Lebecque, and P. Miossec Anatomic Localization of Immature and Mature Dendritic Cells in an Ectopic Lymphoid Organ: Correlation with Selective Chemokine Expression in Rheumatoid Synovium J. Immunol., May 15, 2002; 168(10): 5333 - 5341. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Ding, L. Qin, D. Zamarin, S. V. Kotenko, S. Pestka, K. W. Moore, and J. S. Bromberg Differential IL-10R1 Expression Plays a Critical Role in IL-10-Mediated Immune Regulation J. Immunol., December 15, 2001; 167(12): 6884 - 6892. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Avdiushko, D. Hongo, H. Lake-Bullock, A. Kaplan, and D. Cohen IL-10 receptor dysfunction in macrophages during chronic inflammation J. Leukoc. Biol., October 1, 2001; 70(4): 624 - 632. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. G. Schwacha, C. P. Schneider, K. I. Bland, and I. H. Chaudry Resistance of macrophages to the suppressive effect of interleukin-10 following thermal injury Am J Physiol Cell Physiol, October 1, 2001; 281(4): C1180 - C1187. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Crepaldi, S. Gasperini, J. A. Lapinet, F. Calzetti, C. Pinardi, Y. Liu, S. Zurawski, R. de Waal Malefyt, K. W. Moore, and M. A. Cassatella Up-Regulation of IL-10R1 Expression Is Required to Render Human Neutrophils Fully Responsive to IL-10 J. Immunol., August 15, 2001; 167(4): 2312 - 2322. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Corinti, C. Albanesi, A. la Sala, S. Pastore, and G. Girolomoni Regulatory Activity of Autocrine IL-10 on Dendritic Cell Functions J. Immunol., April 1, 2001; 166(7): 4312 - 4318. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
