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and CD40 ligand expression by T cells, which results in IL-12 production from peripheral blood mononuclear cells
The Pulmonary Center, Boston University School of Medicine, Boston, Massachusetts
Correspondence: Jeffrey S. Berman, M.D., Professor of Medicine, The Pulmonary Center, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118. E-mail: jberman{at}lung.bumc.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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expression is required to induce a protective
response to many intracellular pathogens. Herein, we demonstrate that
osteopontin stimulation augments the ability of anti-CD3 monoclonal
antibody to induce CD40 ligand (CD40L) and IFN- expression on human
T cells, resulting in CD40L- and IFN--dependent IL-12 production
in vitro. These findings suggest a functional role for
osteopontin in early Th1 responses, namely regulation of T
cell-dependent IL-12 production. Further, osteopontin up-regulation of
CD40L provides mechanistic support for the association of osteopontin
with polyclonal B cell proliferation and humoral autoimmune
disease.
Key Words: human • T lymphocytes • Th1 cytokines • costimulatory molecules • inflammation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The nature of the immune response to intracellular pathogens reflects
the pattern of cytokines released from T cells and monocytes in the
local microenvironment [11
, 12
]. The
inflammatory or Th1 response is characterized by the early production
of interleukin (IL)-12 and interferon (IFN)- and is generally
associated with resistance to intracellular infection
[13
, 14
]. Human and murine hosts that are
deficient in these cytokines or lack functional cytokine receptors are
highly susceptible to mycobacterial diseases and fail to mount an
adequate granulomatous response [15
16
17
]. The early
regulation of IFN- and IL-12 expression in cell-mediated responses
is thought to involve the interaction of antigen-presenting cells
(APCs) with T cells through costimulatory molecules, such as CD40
ligand (CD40L) and the release of early regulatory cytokines
[11
, 12
]. In view of the abundant early
expression of Opn by macrophages and T cells, its ability to
costimulate T cell proliferation, and its association with
cell-mediated immune responses, we hypothesized that Opn may regulate
early IFN- and IL-12 expression.
In this paper, we show that Opn coimmobilized with anti-CD3 monoclonal
antibody (mAb) upregulated IFN- but not IL-4 expression from human
CD3-positive T cells. In the presence of anti-CD3 mAb, Opn augmented
IL-12 expression from peripheral blood mononuclear cells (PBMCs), but
IFN- production was independent of IL-12. In fact, in this system,
IL-12 production from blood monocytes was dependent on the presence of
T cells, and on the expression of CD40L and IFN by T cells. Based on
these results, we propose a role for Opn in the early regulation of T
cell-dependent IL-12 production from APCs at the site of developing
cell-mediated immune responses. In addition, by inducing CD40L on T
cells, we propose a unifying mechanism linking the previously described
association of Opn with disorders of humoral and cellular immunity
[18
, 19
].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antibodies and reagents
For all experiments, we used endotoxin-free Opn, purified from
human milk over an antibody column (kindly donated by Dr. Donald
Senger, Beth Israel Hospital, Boston MA) [21
]. Phorbol
myristate acetate (PMA) and ionomycin were purchased from Sigma (St.
Louis, MO).
The following antibodies were used. 1) Monoclonal anti-human CD3
antibody (Clone HIT3a, Pharmingen, San Diego, CA) was used to ligate
the T cell receptor and in CD3-depletion experiments. 2) Antibodies
used in FACS analysis were mouse anti-human CD3 (clone UCHT-1, Sigma),
mouse anti-human IFN- (clone 4S.B3, Pharmingen), and mouse
anti-human CD40L (CD154, clone TRAP1, Pharmingen). 3) Antibodies used
in cytokine-blocking experiments were mouse anti-human IFN- (clone
B27, Pharmingen), mouse anti-human IL-12 (clone 24910.1, R&D Systems,
Minneapolis, MN), mouse anti-human CD40L (clone TRAP1), and mouse
immunoglobulin (Ig)G1 isotype control (anti-TNP, clone 107.3,
Pharmingen). All antibodies used in culture experiments were azide- and
endotoxin-free.
Costimulation assay and cytokine enzyme-linked immunosorbent assay
(ELISA)
Costimulation assays were performed as previously described
[4
]. Briefly, anti-CD3 mAb (0.3 ug/ml) diluted in
phosphate-buffered saline (PBS) was incubated overnight at 4°C in
48-well, nontissue, culture-treated plates (Falcon #1178, Becton
Dickinson, San Jose, CA). Unbound antibody was removed, and Opn (1
ug/ml PBS) was incubated overnight at 4°C. The doses of anti-CD3 mAb
and Opn were chosen based on optimal responses in previous
costimulatory experiments. Adhesion of Opn was confirmed by ELISA as
previously described [4
]. Resultant wells contained Opn
alone, anti-CD3 alone, or anti-CD3 mAb coimmobilized with Opn. PBS
alone served as a negative control and the combination of PMA (1 ng/ml)
and ionomycin (0.5 uM) as a positive control. Human NWNTs
(0.5x106/ml) were diluted in RPMI 1640 supplemented with
10% heat-inactivated FCS, and penicillin and streptomycin were added
to each well and cultured for 24 h at 37°C. Supernatants were
harvested and analyzed for cytokine concentrations by standard
sandwich-antibody ELISA. IFN-, IL-4, and IL-12 (p70/p40) ELISA
assays were purchased from Biosource (Camarillo, CA) and IL-18 ELISA,
from R&D Systems. ELISA assay was conducted as specified by the
manufacturer. Optical density was measured on a standard ELISA plate
reader at 450 nm. For each experiment, cytokine concentrations were
determined using a standard curve constructed from serial dilutions of
recombinant cytokine (provided by manufacturer). The ELISA sensitivity
ranged from 5–20 pg/ml up to 500–2000 pg/ml, depending on the
cytokine being measured. Results are expressed as the arithmetic
mean ± SD of duplicate wells.
Intracellular FACS analysis for cytokine expression
Intracellular FACS analysis for cytokine expression was
performed on a cell-by-cell basis. Briefly, NWNTs were treated with
Brefeldin A (Golgiplug, Pharmingen) at a dose of 1 ug/ml cells for 30
min at 37°C. Following this, human NWNTs (1x106/ml) were
incubated in the presence of immobilized anti-CD3 mAb (0.3 ug/ml) with
and without coimmobilized Opn (1 ug/ml) in 48-well, nontissue,
culture-treated plates, as described above. At 24 h, cells were
harvested and surface-labeled with quantum red-conjugated mouse
anti-human CD3 mAb in staining buffer (1% FCS/PBS) for 30 min at
4°C. Then, the cells were fixed in formaldehyde, 4% for 20 min at
4°C. Intracellular cytokine expression was assessed by incubation
with fluorescein isothiocyanate (FITC)-labeled anti-human IFN- mAb
or isotype control in 50 ul of permeabilization buffer (saponin, 0.1%
in 1% FCS/PBS) for 30 min at 4°C. The cells were washed, fixed, and
analyzed by flow cytometry (Becton Dickinson).
FACS analysis
To determine concentrations of monocytes and T cells in cell
populations and the expression of CD40L on stimulated CD3-positive T
cells, staining and flow cytometric analysis of cells was performed
using FACScan for two-color analysis. Briefly, 0.5 x
106 cells/tube in PBS containing 0.1% sodium azide were
incubated with different combinations of FITC or Texas red-labeled mAbs
for 30 min at 4°C. CD40L staining was performed by sequential
incubation with saturating concentrations of purified, unlabeled,
anti-CD40L mAb or isotype-control mAb followed by FITC-conjugated goat
anti-mouse (GAM) Ig as a second Ab. Results are expressed as the
percentage of gated cells positive for each of the mAbs using 95%
confidence intervals set for isotype controls. Staining for isotype
controls was included in all experiments and for all conditions.
Statistical analysis
Data are expressed as mean ± SD or mean ± SE of the mean. For cytokine ELISA, results are also
expressed as fold increase over control (i.e., ratio of cytokine
produced in the presence of Opn coimmobilized with anti-CD3, divided by
cytokine produced with anti-CD3 alone). Ratios above one indicate an
augmentation of cytokine production by Opn. Results were compared for
significance using 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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but not IL-4 production from peripheral blood
lymphocytes
expression [2
3
4
, 7
]. As shown in
Figure 1
, NWNTs (0.5x106) stimulated by Opn (1 ug/ml),
coimmobilized with anti-CD3 mAb (0.3 ug/ml), produced significantly
more IFN- than with anti-CD3 activation alone [Opn plus anti-CD3
mAb 1205±42 pg/ml vs. anti-CD3 mAb 600±60 pg/ml
(mean±SD), p=0.05]. There was no detectable
IFN- production following stimulation with Opn alone or with PBS
alone (negative control). The amount of IFN- produced was similar to
that seen on stimulation with PMA (1 ng/ml) plus ionomycin (0.5 uM;
Fig. 1
). Under identical conditions, Opn coimmobilized with anti-CD3
mAb did not augment significantly IL-4 production from T cells when
compared with anti-CD3 mAb alone (unpublished results). In five
experiments using NWNTs from different subjects, when compared with
anti-CD3 mAb alone, Opn coimmobilized with anti-CD3 mAb-augmented
IFN- expression (2.03±0.3-fold increase over stimulation with
anti-CD3 mAb alone, mean±SD, range 1.7–2.4,
n=5) and not IL-4 expression (0.83±0.3-fold increase over
stimulation with anti-CD3 mAb alone, mean±SD, range
0.2–1.1, n=5).
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by T cells.
NWNTs isolated in our laboratory are >90% CD3-positive, and IFN-
expression required ligation of the antigen receptor. Other potential
sources of IFN- in these assays include natural killer (NK) cells
and B cells [22
, 23
]. To demonstrate
IFN- production by CD3-positive T cells in response to Opn
costimulation, we performed intracellular staining and FACS analysis
for IFN- using NWNTs cultured in identical conditions to those
described for ELISA studies. Results (Fig. 2
) show that at 24 h, there was a significant increase in the
fraction of CD3-positive cells expressing IFN- when stimulated by
anti-CD3 mAb plus Opn compared with anti-CD3 stimulation alone (23±5%
vs. 6±1%, mean±SE, respectively; n=2;
p=0.03). These results demonstrated that Opn enhanced
anti-CD3 mAb-dependent IFN- production from CD3-positive T cells.
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from human CD3-positive T cells may
occur by a variety of mechanisms. Opn may directly stimulate T cells to
produce IFN-. Alternatively, Opn might initially stimulate IL-12 or
IL-18 expression, which, in turn, indirectly induces IFN- production
by T cells or NK cells [22
, 24
]. To
determine whether Opn augmented IL-12 or IL-18 production, we measured
IL-12 and IL-18 levels by ELISA in NWNT supernatants cultured in
identical conditions to those described in the IFN- experiments
above. In each case, Opn, coimmobilized with anti-CD3 mAb, induced
significantly greater IL-12 production compared with anti-CD3 mAb alone
(2.9±1.6-fold increase, mean±SD, range 1.4–6.5,
n=11; Fig. 3a
and b
). Opn alone did not induce IL-12 in these
cultures or from freshly isolated adherent monocytes (unpublished
results). Neither Opn coimmobilized with anti-CD3 mAb nor Opn alone
induced detectable IL-18 expression (unpublished results). Thus, Opn,
coimmobilized with anti-CD3 mAb, augmented the production of IFN-
and IL-12 but not IL-18 from PBMCs.
|
production by Opn was not IL-12-dependent, we wished to determine if in our system
Opn-associated IFN- expression by T cells was IL-12-dependent. NWNTs
were preincubated with anti-IL-12 mAb (1–10 ug/ml) or isotype-control
mAb (1–10 ug/ml), and IFN- expression in response to coimmobilized
Opn and anti-CD3 mAb was measured by ELISA at 24 h. IFN-
production with no blocking mAb (806±25 pg/ml, mean±SD)
was not altered significantly by the presence of anti-IL-12 mAb
(827±60 pg/ml) or isotype-control mAb (973±31 pg/ml; unpublished
results). The experiment was repeated twice with similar results.
Anti-IL-12 mAb used at a similar concentration inhibited
IL-12-dependent IFN- production induced by lipopolysaccharide (LPS;
100 ng/ml; unpublished results). In keeping with this observation, we
found that IFN- production preceded that of IL-12 in these assays.
Specifically, IFN- expression, but not IL-12 expression, was
detected in the supernatants of NWNTs costimulated with Opn and
anti-CD3 mAb for 6 h (Fig. 4
). By 24 h, IFN- and IL-12 were produced in these assays
(Fig. 4)
. This experiment was repeated three times with similar
results. These data suggested that IFN- production preceded IL-12
production and was not dependent on IL-12 expression.
|
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-dependent primes APCs to respond to
IL-12-inducing stimuli and that absence of IFN- can result in
diminished IL-12 expression from PBMCs [26
,
27
]. Furthermore, IFN- upregulates CD40 epression on
human monocytes [28
]. We hypothesized that
Opn-associated IL-12 production depended not only on CD40L but also
required the initial production of functional IFN-. Using an
anti-human IFN- mAb, we showed that Opn-dependent IL-12 production
was IFN--dependent. IL-12 production was measured in Opn plus
anti-CD3 mAb costimulation assays, as previously described. Cells were
preincubated with neutralizing concentrations of anti-human IFN- mAb
(1–10 ug/ml) or identical concentrations of isotype-control mAb. The
presence of anti-IFN- but not isotype-control mAb inhibited
Opn-dependent production of IL-12 in a dose-dependent manner
(Fig. 8
). Over three experiments, Opn-associated IL-12 production was
70±15.8 pg/ml (mean±SE). Anti-IFN- mAb (10 ug/ml)
reduced significantly IL-12 expression to 13±7 pg/ml
(mean±SE, p=0.04), and isotype-control mAb (10
ug/ml) did not alter significantly IL-12 production (60±23.5 pg/ml,
mean±SE, P=0.23). Thus, Opn-dependent IL-12
production was IFN--dependent.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and CD40L by T cells. This results in
T cell-, CD40L-, and IFN--dependent IL-12 expression from PBMCs.
These results complement recent studies documenting the role of Opn in
regulating IL-12 and IL-10 expression form murine peritoneal
macrophages and suggest that Opn may function in the early regulation
of Th1 cytokine responses in vivo [29
]. Opn is expressed in host cell-mediated immune responses. It is present in Th1 granulomas, is produced by macrophages infected with mycobacteria, and, in mice, its expression confers resistance to intracellular infections including rickettsia tsutsugamushi. High levels of Opn production occur in early stages of inflammatory reactions and correlate with early T cell and macrophage infiltration [2 3 4 ]. It is therefore likely that Opn functions in the initial stages of cell-mediated immunity. The abundant expression of Opn by mitogen-activated T cells and by macrophages infected with mycobacteria suggests that the effects of Opn on the developing cell-mediated response would be amplified in the local microenvironment of the invading pathogen.
We showed that Opn upregulated IL-12 and IFN- expression from PBMCs
in vitro. The ability of Opn to augment IL-12 expression in
this system was dependent on CD3-mediated costimulation of T cells to
express CD40L and IFN-. This is an interesting finding, considering
the active form of IL-12 is produced only by a limited subset of cells
(primarily monocytes), which does not include T lymphocytes
[25
]. Recent studies have elucidated two distinct
pathways for monocyte IL-12 production in vitro
[26
]. The first is T cell-independent, representing
direct induction by infectious agents [30
]. The other is
T cell-dependent and involves the interaction of CD40L on activated T
cells with its receptor, CD40, on APCs [31
32
33
]. A role
for CD40L in cell-mediated responses in vivo is supported by
the finding that hosts deficient in CD40L exhibit diminished
proliferative and cytokine responses to T cell-specific antigens and
develop progressive Leishmaniasis as a result of defective
IL-12 and IFN- production and impaired macrophage activation
[34
35
36
]. In addition, mice genetically deficient in
CD40L develop a defective granulomatous response to MTb
[36
]. Thus, CD40L:CD40 interaction is considered an
important pathway of early IL-12 production at the time of antigen
presentation and T cell activation [32
,
33
].
IFN- is another important determinant of monocyte IL-12 production
in some systems. For example, resident murine peritoneal macrophages
are unresponsive to LPS at rest but produce IL-12 in response to LPS
following priming with IFN- [37
]. In addition, the
presence of IFN- at the time of stimulation via CD40L has been shown
to amplify IL-12 production from monocytes, and the combination of
IFN- and CD40 with other IL-12-inducing stimuli enhances IL-12
expression in vitro [26
]. There is also
evidence that effective IL-12 expression is dependent on early
functional IFN- production in vivo. Libraty et
al. [38
] demonstrated that the relative presence or
absence of IFN- in the microenvironment of the developing response
to leprosy was a determinant of early monocyte-cytokine responses.
Moreover, using a murine model of M. bovis BCG infection,
Flesch et al. [39
] found that IL-12 was
produced in immunocompetent mice early during BCG infection but not in
mutant mice lacking the IFN- receptor. The mechanism by which
IFN- regulates monocyte/macrophage IL-12 production appears to
involve priming of the IL-12 promoter and upregulating CD40 expression
on APCs [28
, 40
, 41
]. These
data suggest that early IFN- is important for effective
cell-mediated immunity and may regulate early IL-12 expression in
vivo.
Taken together, these data suggest that factors that simultaneously
upregulate CD40L and IFN- would provide an efficient mechanism for
inducing IL-12 expression at sites of cell-mediated responses to
invading organisms such as mycobacteria. We present in vitro evidence
that Opn is such a factor. Our results complement recent work by Ashkar
et al. [29
], which shows that Opn critically
regulates early Th1 cytokine expression by murine peritoneal
macrophages. These studies demonstrated that phosphorylated Opn
directly stimulated macrophages to produce IL-12, although
dephosphorylated Opn inhibited IL-10 expression by LPS-stimulated
macrophages. In Opn-deficient mice, infections characterized by
Th1-cytokine expression (Herpes simplex virus type 1; Listeria
monocytogenes) were associated with defective IL-12 expression,
increased IL-10 expression, and abnormal cell-mediated immunity
[29
]. In view of these findings, we propose a multistage
Opn-dependent pathway for early IL-12 expression as follows. First, Opn
is produced early, following exposure to invading organisms, ensuring
high levels of Opn in the local microenvironment of responding
macrophages and T cells. Second, Opn directly stimulates macrophages to
increase IL-12 and downregulate IL-10 production. Third, Opn
costimulates antigen-activated T cells to express IFN- and CD40L,
thus creating a positive feedback loop, further augmenting IL-12
expression from macrophages in a T cell-dependent mechanism.
The induction of CD40L by Opn costimulation of T cells provides a mechanism linking the association of Opn with humoral and cellular immune responses [18 , 19 ]. The expression of Opn is increased in patients with systemic lupus erythematosus, and Opn levels correlate with the development of the systemic autoimmune syndrome in MRL/lpr mice [42 ]. In these mice, disease and Opn production are T cell-dependent, and Opn drives proliferation of isolated B cells in vitro [18 , 43 ]. Recently, Opn-transgenic mice have been shown to express increased levels of IgG and IgM, and anti-double-stranded DNA autoantibodies [19 ]. The mechanism by which Opn modulates T cell-dependent B cell responses is unknown. As well as inducing IL-12 production from APCs, signals transduced by CD40 are essential for T cell-dependent B cell proliferation and differentiation [44 ]. Mutation of the CD40L gene in humans causes X-linked hyper-IgM syndrome, characterized by absent or low levels of IgG, IgA, and IgE in serum but normal or elevated levels of IgM. Concomitant defects are seen in B cell proliferation, germinal center formation, and development of B cell memory [34 , 44 ]. Our findings that Opn upregulates CD40L expression on T cells provides a possible explanation for the ability of Opn to modulate these T cell-dependent humoral responses as well as cell-mediated immune events.
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ACKNOWLEDGEMENTS |
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Received February 3, 2000; revised March 27, 2000; accepted April 10, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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