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*
Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, and
Department of Molecular Cell Biology, Free University Medical Center, Amsterdam, The Netherlands
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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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, promote cell-mediated immunity against intracellular
pathogens, whereas Th2 cells, producing IL-4, IL-5, and IL-13, are
instrumental in humoral immune responses against extracellular
pathogens (1). Polarized Th1 and Th2 cells develop from
naive Th cell precursors during activation by APCs in the lymph nodes.
Naive Th cells respond only to specialized APC with high expression of
costimulatory molecules, i.e., dendritic cells
(DC)3
(2). Immature DC reside in peripheral tissues as sentinel
cells and sample the environment for possible danger signals. Exposure
to microbial and/or inflammatory products induces the maturation and
migration of the DC to the draining lymph nodes. During this process,
the DC lose their Ag uptake capacity but strongly up-regulate
costimulatory molecules, making the cells qualified to activate naive
Th cells (reviewed in Ref. 2). Functional Th cell
polarization is strongly directed by various soluble or membrane-bound
molecules present during primary stimulation that regulate the
expression of the Th1- and Th2-specific "master switch"
transcription factors T-bet (3) or GATA-3
(4), respectively (5). Most prominent in this
respect are the cytokines IL-12, which induces T-bet expression and
drives Th1 responses (3, 6), and IL-4, which leads to
enhanced GATA-3 expression and to the development of Th2 cells
(4, 7).
The main source of IL-12 are DC. The IL-12-producing capacity of mature
DC in the lymph nodes is pre-established in their tissue of origin and
is strongly influenced by tissue environmental factors, such as
pathogens, during their initial activation as immature DC. For example,
exposure of immature DC to IFN- (8) or to (compounds
of) intracellular pathogens, like Mycobacterium tuberculosis
(9) or pertussis toxin (53), induces
maturation into DC that produce high levels of IL-12 and have a strong
ability to induce Th1 development. In contrast,
PGE2 (10), cholera toxin
(11), and certain helminth compounds (12, 53)
lead to the generation of mature DC with very low levels of IL-12 and
have the potential to induce Th2 cell development. However, when mature
DC produce only limited amounts of IL-12, alternative Th
cell-polarizing mechanisms, either by soluble or by surface-bound
molecules, become more prominent and decisive instead.
One such membrane-bound candidate molecule is ICAM-1. During Ag presentation, ICAM-1 expressed on DC binds to LFA-1 on Th cells. ICAM-1/LFA-1 interaction has a pleiotropic effect, as it not only plays an important role in T cell recirculation and inflammation (13, 14) but also in T cell activation. In particular, ICAM-1/LFA-1 mediated adhesion is a critical event for establishing and strengthening the physical contact between DC and Th cells, leading to optimal Th cell activation (15, 16). Several mouse studies have revealed that the ICAM-1/LFA-1 interaction can also be important for driving Th1 cell polarization, as blockage of this interaction leads to increased Th2 cell development from naive Th cells (17, 18, 19). In humans the role of ICAM-1/LFA-1 ligation in Th cell polarization has not been evaluated previously.
Therefore, the aim of this study was to investigate the involvement of ICAM-1/LFA-1 ligation in the polarization of human Th1 cells. Experiments were performed in an APC-free and in a DC-dependent system to determine to what extent ICAM-1 expression levels on DC are relevant. Also, the relative contribution of ICAM-1/LFA-1 ligation to the actions of the potent polarizing cytokines IL-4 and IL-12 was studied. Finally, we explored the molecular pathway through which ICAM-1/LFA-1 ligation may drive Th1 responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human rICAM-1, rICAM-2, and rICAM-3, coupled to human IgG1-Fc,
were described before (20). Human IL-4 (20 x
108 U/mg) was obtained from Pharma
Biotechnology (Hanover, Germany). Human rIL-12 (sp. act.
1.7 x 108 U/mg) was a gift from Dr. M.
K. Gately (Hoffmann-LaRoche, Nutley, NJ). Human rGM-CSF (sp. act.
1.11 x 107 U/mg) was a gift of
Schering-Plough (Uden, The Netherlands). Human rIFN- (sp. act.
8 x 107 U/mg) and neutralizing rabbit IgG
to human IL-12 were gifts from Dr. P. H. van der Meide (U-cytech,
Utrecht, The Netherlands). The anti-LFA-1 mAbs used were NKI-L15
and NKI-L16 (both mouse IgG2a, anti-CD11a), described before
(21). Blocking mouse IgG1 to human ICAM-1 was obtained
from R&D Systems (Abingdon, U.K.). Mouse mAbs to human CD28
(CLB-CD28/1) and human CD3 (CLB-T3/4E-1XE) were obtained from Central
Laboratory of The Netherlands Red Cross Blood Transfusion Center
(Amsterdam, The Netherlands). Mouse Abs to phosphorylated extracellular
signal-regulated kinase (ERK), Jun NH2-terminal
kinase (JNK)1, and p38 were obtained from New England Biolabs (Beverly,
MA). The p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580
and the ERK inhibitor PD98059 were obtained from Alexis (San
Diego, CA).
In vitro generation and maturation of DC from peripheral blood monocytes
Venous blood from healthy donors was collected in
sodium-heparin-containing tubes (VT100H; Venoject, Terumo Europe,
Leuven, Belgium). PBMC were isolated by density gradient centrifugation
on Lymphoprep (Nycomed, Torshov, Norway). Subsequently, PBMC were
centrifuged on a Percoll (Pharmacia Biotech, Uppsala, Sweden) gradient
to isolate the monocytes, as previously described (8).
Monocytes were washed and seeded in 24-well culture plates (Costar,
Cambridge, MA) at a density of 5 x 105
cells/well. After 60 min of incubation at 37°C, nonadherent cells
were removed and adherent cells were cultured in IMDM (Life
Technologies, Paisley, U.K.) containing gentamicin (86 µg/ml;
Duchefa, Haarlem, The Netherlands) and 1% FCS (HyClone Laboratories,
Logan, UT), supplemented with rGM-CSF (500 U/ml) and rIL-4 (250 U/ml)
to obtain immature DC as described elsewhere (22). At day
3, the culture medium, including the supplements, was refreshed. On day
6, maturation of immature DC was induced by the addition of either LPS
alone (100 ng/ml; Difco, Detroit, MI), LPS plus rIFN- (1000 U/ml),
or poly(I:C) (20 µg/ml; Sigma-Aldrich, St. Louis, MO). After 48
h, full maturation into
CD1a+CD83+ mature effector
DC (mDC) was confirmed by flow cytometric analysis.
Analysis of cell surface molecule expression by flow cytometry
Mouse anti-human mAbs against the following molecules were used: CD1a (OKT6; Ortho Diagnostic System, Beerse, Belgium), CD83 (Immunotech, Marseille, France), ICAM-1, ICAM-2, and ICAM-3 (all three obtained from R&D Systems). Bound mAb were detected by FITC-conjugated goat anti-mouse mAb (Jackson ImmunoResearch Laboratories, West Grove, PA).
Cytokine production by DC
CD1a+CD83+ mDC
(2 x 104 cells/well) were stimulated with
CD40 ligand (CD40L)-expressing mouse plasmacytoid cells (J558
cells, 2 x 104 cells/well; a kind gift from
Dr. P. Lane, University of Birmingham, Birmingham, U.K.), in the
presence of rIFN- (1000 U/ml), in 96-well flat-bottom culture plates
(Costar) in IMDM containing 10% FCS in a final volume of 200 µl.
Supernatants were harvested after 24 h and stored at -20°C
until the levels of IL-12p70 secretion were measured by ELISA.
Isolation of naive Th cells
PBMC from buffycoat (CLB) were isolated by density gradient centrifugation on Lymphoprep (Nycomed), and thereafter CD4+CD45RA+CD45RO- naive Th cells were isolated to high purity (>98% as assessed by flow cytometry) through one-step high-affinity negative selection columns (R&D Systems), according to the manufacturer’s instructions.
Stimulation and culture of naive Th cells
Flat-bottom 96-well culture plates (Costar) were coated with goat anti-human Fc Abs (Jackson ImmunoResearch Laboratories). After blocking of nonspecific protein binding sites with BSA, the wells were incubated with Fc-ICAM-1, Fc-ICAM-2, or Fc-ICAM-3 fusion proteins. Purified naive Th cells were either seeded in the coated wells (2 x 104 cells/200 µl) and stimulated with soluble mAbs to CD3 (0.3 µg/ml) and CD28 (1 µg/ml) or were cocultured with mDC (5 x 103 cells) in 200 µl of culture medium in the presence of superantigen Staphylococcus aureus enterotoxin B (10 pg/ml; Sigma-Aldrich), in 96-well flat-bottom culture plates (Costar). There, where indicated, naive Th cells were preincubated with increasing dosages of either SB203580 (p38 inhbitor) or PD98059 (ERK inhibitor) for 1 h at 37°C, before stimulation with anti-CD3/CD28. At day 5, rIL-2 (10 U/ml; Cetus, Emeryville, CA) was added and the cultures were expanded for the next 9 days.
Cytokine production by Th cells
On day 14, the quiescent Th cells were restimulated with PMA (10
ng/ml) and ionomycin (1 µg/ml; Sigma-Aldrich) for 6 h, the last
5 h in the presence of brefeldin A (10 µg/ml; Sigma-Aldrich),
and single-cell IL-4 and IFN- production was determined by
intracellular flow cytometric analysis. Cells were fixated in 2%
paraformaldehyde (Merck, Darmstadt, Germany), permeabilized with 0.5%
saponin (ICN Biochemicals; Cleveland, OH) and stained with
anti-human IFN--FITC and anti-human IL-4-PE (both from BD
Biosciences, Franklin Lanes, NJ). In parallel, the cells were
stimulated with CD3 and CD28 mAbs to measure total IL-4 and IFN-
secretion by ELISA in supernatants collected after 24 h.
Real-time quantitative RT-PCR analyses of GATA-3 and T-bet expression
For quantitative analysis of GATA-3 and T-bet mRNA expression,
105 naive Th cells were stimulated with
anti-CD3/CD28 for 2 days and lysed for total RNA extraction, using
a NucleoSpin RNA Isolation kit (Macherey-Nagel, Duren, Germany).
First-strand cDNA was synthesized, using a cDNA synthesis kit (MBI
Fermentas, St. Leon-Rot, Germany). For quantification of GATA-3, T-bet,
and, as a control, 
2-microglobulin
(2m) transcripts, real-time quantitative PCR
was performed in a LightCycler (Roche Diagnostics, Mannheim, Germany)
based on specific primers and general SYBR green fluorescence
detection. The primer sequences were the following:
2m S-primer, 5'-CCAGCAGAGAATGGAAAGTC-3';
2m AS-primer, 3'-GATGCTGCTTACATGTCTCG-5'
(melting temperature (Tm) 58°C); GATA-3 S-primer,
5'-CTGCAATGCCTGTGGGCTC3'; GATA-3 AS-primer,
3'-GACTGCAGGGACTCTCGCT-5' (Tm 62°C); T-bet S-primer,
5'-CCCCAAGGAATTGACAGTTG-3'; T-bet AS-primer,
5'-GGGAAACTAAAGCTCACAAAC-3' (Tm 62°C); resulting in the
amplification of PCR products of 391 bp (2m),
350 bp (GATA-3), and 317 bp (T-bet). All reactions were performed using
the LightCycler DNA master SYBR Green I PCR kit (Roche Diagnostics). A
bulk cDNA sample of stimulated human Th cells was used as a standard,
and normalization to 2m was performed for each
sample.
PACE
Phosphospecific cell-based ELISA (PACE) was performed essentially as described before (23). In short, 5 x 105 naive Th cells were stimulated with anti-CD3/CD28 in the absence or presence of Fc-ICAM-1 (500 ng/ml coated in plates, as described above). After 24, 48, and 72 h, cells were fixated with 2% paraformaldehyde (Merck). After permeabilization with 0.1% Triton X-100 (Sigma-Aldrich), endogenous peroxidase was quenched in 1% H2O2 (Merck) and 0.1% natrium azide (Merck) for 20 min. After blockage of specific binding sites with 5% BSA, phosphospecific Abs were added in 0.1% Triton X-100 plus 5% BSA and incubated for 1 h at 37°C, followed by a 1-h incubation at room temperature with polyclonal rabbit anti-mouse Abs, labeled with HRP (New England Biolabs). After extensive washing the cells were transferred to a 96-well plate (Costar) and the HRP substrate 3,3',5,5'-tetramethylbenzidine (BioSource International, Nivelles, Belgium) was added. The reaction was stopped with 2 M H2SO4 after 15 min and the OD450/650 was used as a measurement of phosphorylation.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Primary anti-CD3/CD28 stimulation of human naive Th cells in
the absence of DC and polarizing factors yields a mixed population of
IL-4 and/or IFN- producers. This was assessed on day 10 upon
subsequent restimulation and was detected both on a per cell basis by
intracellular staining (Fig. 1
A, left panel) and
by measurement of cytokine secretion (Fig. 1B, upper
bar). The addition of immobilized Fc-ICAM-1 construct induced a
dose-dependent shift toward the Th1 cell phenotype, with gradually
increased percentages of IFN- producers, decreased percentages of
IL-4 producers (Fig. 1A), and an increased IFN-:IL-4
secretion ratio (Fig. 1B). Specificity of this Th1-driving
effect was demonstrated by the abolishment of Th1 polarization by
addition of blocking Abs against either ICAM-1 or LFA-1 (NKI-L15) at
the start of the culture (Fig. 1C). Isotype control Ab had
no effect (data not shown). Furthermore, mRNA analysis at
t = 48 h indicated a dose-dependent drop in the
expression of the Th2-specific transcription factor GATA-3 and a
dose-dependent increase in the expression of the Th1-specific
transcription factor T-bet, with increasing Fc-ICAM-1 concentrations
(Fig. 1D), which correlates with the increased IFN- and
decreased IL-4 production after restimulation on day 10 (Fig. 1, A and B).
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As expected from the lower affinity for LFA-1, the Th1-driving effect
of ICAM-3 was less pronounced. These results demonstrate a clear
Th1-driving effect of ligation of LFA-1.
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The cytokines IL-4 and IL-12 have powerful capacities to drive Th
cell polarization. Therefore, we tested the relative importance of
LFA-1-mediated polarization in the presence of these potent cytokines.
As shown in Fig. 3A,
increasing concentrations of IL-4 dose-dependently abolished the
Th1-driving effect of ICAM-1/LFA-1 ligation, indicating that this
Th2-driving cytokine overrules the effect of LFA-1 ligation. In the
presence of moderate to high concentrations of IL-12, ICAM-1/LFA-1
ligation does not add to the Th1-polarizing effect of IL-12 (Fig. 3B). These data indicate that Th1 differentiation by LFA-1
ligation is primarily of importance under low cytokine conditions.
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Recent studies in the mouse have pointed toward a role for MAPK
signaling pathways in the control of Th cell responses under low
cytokine conditions. These pathways can be induced by TCR
triggering, which activates ERK, by CD28 ligation, which activates p38
MAPK, or by the combination of TCR triggering and CD28 ligation, which
activates JNK1 and JNK2. JNK1 down-regulates IL-4 production, whereas
JNK2 and p38 MAPK increase IFN- production, and ERK promotes Th2
responses (reviewed in Ref. 25). However, in human T
cells, some of these pathways can have different, even opposing,
effects. For example, p38 MAPK was shown to be important for IL-4
production in effector Th2 cells (26, 27), while
interruption of the ERK pathway strongly increased, rather than
abrogated, IL-4 responses in human Th cells (28).
We addressed the question of whether Th1 polarization by LFA-1 ligation
is mediated via the activation of one or more of these MAPK pathways.
Naive Th cells stimulated with anti-CD3/CD28 in the presence
Fc-ICAM-1 displayed a shift toward Th1 cell development. Addition of a
chemical inhibitor of ERK phosphorylation (PD98059) to
anti-CD3/CD28-activated naive Th cells resulted in a strong
dose-dependent induction of IL-4 production and inhibition of IFN-
production, while in the ICAM-1-stimulated naive Th cells this effect
was reduced and required higher concentrations of PD98059 to yield
equal percentages of IL-4-producing Th cells upon restimulation (Fig. 4A). These data suggest that
ERK activity is increased in ICAM-1-stimulated naive Th cells. This was
indeed confirmed by the analysis of phosphorylated (active) ERK in
48-h-stimulated naive Th cells by PACE (23) (Fig. 4C). p38 MAPK phosphorylation was detectable only after 3
days but did not differ between cells stimulated with and without
ICAM-1 (Fig. 4C; day 3 data not shown). Accordingly,
addition of the chemical p38 MAPK inhibitor, SB203580, did not
differentially affect the Th cell cytokine profile in cells stimulated
with or without ICAM-1 (Fig. 4B), suggesting that p38 MAPK
is not involved in ICAM-1-induced Th1 polarization. Although elevated
levels of phosphorylated JNK1/2 were demonstrated in the
ICAM-1-stimulated Th cells at day 2 (Fig. 4C), the
functional consequences for ICAM-1-driven Th1 polarization cannot
be studied in this experimental setup, as no specific JNK
inhibitor is available yet.
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Next, we addressed the question of whether LFA-1-mediated
signaling indeed contributes to Th1 polarization under more
physiological conditions, i.e., during the interaction of naive
Th cells with DC. We generated different Th1-driving DC1 subsets with
different ICAM-1 expression levels. To this end, maturation was induced
in immature monocyte-derived DC either with the synthetic dsRNA
poly(I:C) or by the combination of LPS plus IFN-. As a comparison,
maturation was induced by LPS alone, yielding nonpolarizing DC0. In
addition to ICAM-1, expression of the other ligands for LFA-1, ICAM-2,
and ICAM-3 were analyzed on both DC1 subsets and the DC0 subset. As
shown in Fig. 5A, ICAM-2 was
hardly expressed on either DC subset and was expressed on ICAM-3 only
moderately, but was elevated on the poly(I:C) DC1. ICAM-1 was more
abundantly expressed and particularly high on the poly(I:C) DC1. In
combination with this high ICAM-1 expression, poly(I:C) DC1 produced
only limited quantities of IL-12p70, as compared with the
LPS/IFN--matured DC1 (Fig. 5B). In fact, IL-12p70
production by the poly(I:C) DC1 hardly differed from the levels
produced by the nonpolarizing LPS-matured DC0. As the impact of LFA-1
triggering on Th1 polarization is most evident under such low cytokine
conditions (Fig. 3
B), we tested to what extent the
Th1-driving capacity of poly(I:C) DC1 (Fig. 5C) was mediated
by ICAM-1 and IL-12p70. Coculture experiments with naive Th
cells demonstrated that blocking the ICAM-1/LFA-1 interaction, either
with anti-ICAM-1 or with anti-LFA-1, clearly reduced the
Th1-driving potential (Fig. 5C). Likewise, neutralization of
IL-12p70 only partially blocked Th1 polarization. Addition of
anti-ICAM-3 instead did not substantially reduce Th1 polarization
in any DC subset (Fig. 5C). Full abrogation of Th1
polarization (to the basal level induced by LPS-matured DC0; Fig. 5C, upper bar) was obtained only by combining
IL-12p70 neutralization and blocking the ICAM-1/LFA-1 interaction,
demonstrating the contribution of ICAM-1 in the Th1-driving mechanism
of poly(I:C) DC1 (Fig. 5C). In contrast, the Th1-polarizing
effect of LPS/IFN- DC1 is fully IL-12 dependent (Fig. 5C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mouse studies (17, 18) with ICAM-1-deficient APC or
blocking Abs to ICAM-1 already indicated a profound inhibitory effect
of ICAM-1 on IL-4 production by activated naive Th cells, suggesting at
least prevention of Th2 development as a consequence of LFA-1/ICAM-1
interaction. The mouse experiments did not indicate an active induction
of IFN- production or Th1 development. In contrast, previous studies
with human memory CD4+ Th cells showed that
repeated costimulation by ICAM-1 did lead to increased secretion of
IFN-, but not IL-4 or IL-5 (29, 30). These results are
confirmed in our present study and are further extended by showing that
initial polarization of human naive Th cells upon LFA-1 costimulation
by ICAM-1 gives rise to memory Th cells that produce high amounts of
IFN- but hardly any IL-4. The question remains what precise
mechanisms are involved in this shift in Th cell polarization. It is
well known that the ligation of LFA-1 by ICAM-1 helps to stabilize the
physical APC-Th cell interaction, allowing for a higher TCR occupancy,
and thereby to enhance or prolong TCR-dependent signals
(31), such as inositol phospholipid hydrolysis, increased
intracellular Ca2+ levels (32, 33),
and phosphorylation of phospholipase C1 (34). Indeed,
it has been reported that LFA-1/ICAM-1 interaction can lower the
required Ag dose for Th cell activation by 10- to 100-fold (35, 36). Several studies support the "strength of signal"
hypothesis as an additional factor influencing Th cell polarization.
According to this hypothesis, triggering of high numbers of TCR will
lead to IFN--producing Th cells, whereas low number TCR triggering
will favor the development of IL-4-producing Th cells
(37). The contribution of LFA-1/ICAM-1 ligation to this
model was elegantly demonstrated by a study of Ruedl et al.
(38) in which LFA-1 triggering by ICAM-1 lowered the
required Ag dose for CD40L up-regulation on the Th cell, which is
essential for induction of IL-12 production, by the Ag-presenting DC,
which, in turn, can promote Th1 responses. Nevertheless, this model can
only in part explain our present data, because 1) poly(I:C) DC1 produce
only low levels of IL-12p70 upon CD40L stimulation, which does
not fully account for the Th1 responses induced by these high
ICAM-1-expressing DC, and 2) naive Th cells stimulated with
anti-CD3/CD28, in the absence of APC, also showed a dose-dependent
increase in Th1 cell polarization with increasing concentrations of
immobilized ICAM-1-Fc. Other studies also suggest that the "strength
of signal" hypothesis is incomplete. For example, Abraham et al.
(39) demonstrated that high-density TCR ligand expression
could not compensate for the lack of LFA-1/ICAM-1 interaction in Th
cell activation and that only TCR engagement together with
costimulation through either LFA-1 or CD28 was sufficient to induce
detectable levels of IL-2 mRNA (40), albeit by
qualitatively different signaling routes (41).
Furthermore, it has been described that LFA-1 ligation on human
CD8+ T cells can up-regulate the activity of
phosphatidylinositol 3-kinase, sphingomyelinase, and JNK, all similar
to pathways induced by CD28 ligation, but not identical, as LFA-1 and
CD28 ligation display a different profile of sensitivity to inhibitors
of phosphatidylinositol 3-kinase (33). Therefore, a likely
explanation would be that the shift in Th1 polarization induced by
LFA-1 ligation is the combined result of several factors, involving
modifications of TCR-dependent signals and perhaps the induction of
unique signaling pathways. In the present study, increased levels of
activated ERK in ICAM-1-costimulated Th cells were demonstrated, both
by experiments with a specific ERK inhibitor and by PACE. As ERK
phosphorylation is induced upon TCR triggering, part of our results may
be explained by LFA-1-induced modifications of TCR-dependent signals,
which may lead to a reduction of IL-4-producing Th cells. In the mouse,
JNK is involved in the induction of IFN- production in Th cells and,
although there are no reports on this effect in human Th cells, it is
tempting to speculate that the elevation of activated JNK observed in
ICAM-1-costimulated Th cells by PACE at day 2 promotes the development
of IFN--producing Th cells and together with increased ERK activity
leads to enhanced Th1 polarization. Because JNK is not activated by TCR
triggering alone but requires costimulation via CD28 or LFA-1, we
hypothesize that the Th1 shift observed in this study is accomplished
in part by modifications of TCR-dependent signaling, e.g., via ERK
activation, but may additionally be the result of specific
ICAM-1-induced signaling, like the activation of JNK.
In this study, we identified a novel IL-12-independent Th1-driving
mechanism, actively operated by poly(I:C) DC1. These cells, obtained by
maturation of monocyte-derived immature DC in the presence of
poly(I:C), are characterized by elevated expression of ICAM-1 and low
levels of IL-12, both equally contributing to the Th1 polarization.
Poly(I:C) is a synthetic dsRNA molecule and may represent a model for
viral infections. In this respect, the poly(I:C)-induced up-regulation
of ICAM-1 on DC and subsequent in vitro Th1 cell polarization is nicely
corresponding to numerous in vivo studies that showed that viral
infections lead to the induction of Th1 responses and increased levels
of ICAM-1 expression (reviewed in Ref. 42). Furthermore,
studies with IL-12p40-/- and
IL-12p70-/- mice (43) and patients
with a functional mutation in the IL-12R (44) demonstrated
that virus-specific Th1 responses could still be mounted, indicating
also that in vivo alternative, IL-12-independent, Th1-driving
mechanisms are operational, especially during viral infections. Based
on our present findings, it is tempting to speculate that the
LFA-1/ICAM-1 interaction is relevant in this respect. In vivo yet other
mechanisms may be involved, because during viral infections high levels
of type I IFNs are produced that, in humans, can induce Th1 responses
as well (45). In our experiments, only minimal expression
of IFN-
was detected in the poly(I:C) DC, and blockage did not
result in an inhibition of Th1 polarization.
In addition to viral infections, under low cytokine conditions in vivo LFA-1/ICAM-1 interaction may drive Th1 responses. Those conditions may occur in newborns, which are characterized by an immature immune system, e.g., indicated by the deficient IL-12p70 production by monocyte-derived cord blood DC (46). Consequently, during primary immune responses the polyclonal contribution of LFA-1/ICAM-1 ligation may be relatively high.
Animal models for Th1-mediated responses or diseases also provide evidence that ICAM-1/LFA-1 interaction may be involved in Th1 polarization in vivo. For example, blocking Abs to LFA-1 and ICAM-1 were shown to prevent allograft rejection, associated with a shift from a Th1 to a Th2 cell cytokine profile (47, 48, 49). Similarly, ICAM-1 deficiency in nonobese diabetic mice blocked accelerated diabetes, in part explained by affecting the generation and/or expansion of islet-specific Th1 cells (50). Also, in an experimental autoimmune encephalomyelitis disease model, ICAM-1-deficient mice showed reduced T cell proliferation and reduced Th1 cytokine production in response to myelin Ag (51). The latter two studies suggest the involvement of ICAM-1 in autoreactive Th1 cell development, although it was not specified which cell type, e.g., DC, was essential in this respect. Furthermore, studies with ICAM-1 knockout mice indicated that ICAM-1 is required for rapid activation of Th1 cells to control early acute phase genital chlamydial infection, suggesting a role for ICAM-1 in the control of infectious diseases (52). Therefore, evidence from both in vitro and in vivo models points toward a role for ICAM-1-driven Th1 development. Blockage of ICAM-1/LFA-1 interaction may therefore provide potent therapeutic possibilities for the treatment of autoimmune diseases or prevention of graft rejection by transplantation, whereas therapeutic triggering may be beneficial to reduce Th2-mediated disease.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Eddy A. Wierenga, Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands. E-mail address: E.A.Wierenga{at}amc.uva.nl
3 Abbreviations used in this paper: DC, dendritic cell; JNK, Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; mDC, mature effector DC; MAPK, mitogen-activated protein kinase; PACE, phosphospecific cell-based ELISA; CD40L, CD40 ligand; 2m, 2 microglobulin; Tm, melting temperature.
Received for publication October 9, 2001. Accepted for publication December 11, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. J. Exp. Med. 179:1109. mitogen-activated protein kinase is activated by CD28-mediated signaling and is required for IL-4 production by human CD4+CD45RO+ T cells and Th2 effector cells. J. Immunol. 162:7110. cytokine profiles dependent on binding to ICAM-1, ICAM-2 or ICAM-3. Eur. J. Immunol. 29:2248.[Medline]
2-integrin LFA-1 signaling through phospholipase C-1 activation. Proc. Natl. Acad. Sci. USA 90:7099.1 (IL-12R1): evidence for an IL-12R1-independent pathway of IL-12 responsiveness in human T cells. J. Exp. Med. 192:517. and IL-18 synergistically enhance IFN- gene expression in human T cells. J. Immunol. 160:6032.This article has been cited by other articles:
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S. Radhakrishnan, K. R. Wiehagen, V. Pulko, V. Van Keulen, W. A. Faubion, K. L. Knutson, and L. R. Pease Induction of a Th1 Response from Th2-Polarized T Cells by Activated Dendritic Cells: Dependence on TCR:Peptide-MHC Interaction, ICAM-1, IL-12, and IFN-{gamma} J. Immunol., March 15, 2007; 178(6): 3583 - 3592. [Abstract] [Full Text] [PDF]
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J.-i. Suzuki, S. Yamasaki, J. Wu, G. A. Koretzky, and T. Saito The actin cloud induced by LFA-1-mediated outside-in signals lowers the threshold for T-cell activation Blood, January 1, 2007; 109(1): 168 - 175. [Abstract] [Full Text] [PDF]
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R. Grenningloh, A. Gho, P. di Lucia, M. Klaus, W. Bollag, I-C. Ho, F. Sinigaglia, and P. Panina-Bordignon Cutting Edge: Inhibition of the Retinoid X Receptor (RXR) Blocks T Helper 2 Differentiation and Prevents Allergic Lung Inflammation J. Immunol., May 1, 2006; 176(9): 5161 - 5166. [Abstract] [Full Text] [PDF]
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K. P. Pavlick, D. V. Ostanin, K. L. Furr, F. S. Laroux, C. M. Brown, L. Gray, C. G. Kevil, and M. B. Grisham Role of T-cell-associated lymphocyte function-associated antigen-1 in the pathogenesis of experimental colitis Int. Immunol., February 1, 2006; 18(2): 389 - 398. [Abstract] [Full Text] [PDF]
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A. E. Morelli, J. P. Rubin, G. Erdos, O. A. Tkacheva, A. R. Mathers, A. F. Zahorchak, A. W. Thomson, L. D. Falo Jr., and A. T. Larregina CD4+ T Cell Responses Elicited by Different Subsets of Human Skin Migratory Dendritic Cells J. Immunol., December 15, 2005; 175(12): 7905 - 7915. [Abstract] [Full Text] [PDF]
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M. Yasutomi, Y. Ohshima, N. Omata, A. Yamada, H. Iwasaki, Y. Urasaki, and M. Mayumi Erythromycin Differentially Inhibits Lipopolysaccharide- or Poly(I:C)-Induced but Not Peptidoglycan-Induced Activation of Human Monocyte-Derived Dendritic Cells J. Immunol., December 15, 2005; 175(12): 8069 - 8076. [Abstract] [Full Text] [PDF]
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T. Owaki, M. Asakawa, N. Morishima, K. Hata, F. Fukai, M. Matsui, J. Mizuguchi, and T. Yoshimoto A Role for IL-27 in Early Regulation of Th1 Differentiation J. Immunol., August 15, 2005; 175(4): 2191 - 2200. [Abstract] [Full Text] [PDF]
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V. Dardalhon, A. S. Schubart, J. Reddy, J. H. Meyers, L. Monney, C. A. Sabatos, R. Ahuja, K. Nguyen, G. J. Freeman, E. A. Greenfield, et al. CD226 Is Specifically Expressed on the Surface of Th1 Cells and Regulates Their Expansion and Effector Functions J. Immunol., August 1, 2005; 175(3): 1558 - 1565. [Abstract] [Full Text] [PDF]
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OD450/650) ±
SEM as compared with OD values from unstimulated naive Th cells
(background OD450/650 = 0.28).