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Department of Neuroscience and Immunology, Division of Immunogenetics, Kumamoto University Graduate School of Medical Sciences, Honjo, Kumamoto, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. Anti-DR and DR-restricted
T cells stimulated monocytes to produce relatively higher levels of
proinflammatory monokines, such as IL-1
, whereas anti-DQ/DP and
DQ-/DP-restricted T cells stimulated higher levels of
anti-inflammatory monokine IL-10. IL-10 production was abrogated by
the p38 inhibitor SB203580, but rather enhanced by the
MAP/extracellular signal-related kinase kinase-I-specific inhibitor
PD98059, whereas IL-1 was only partially abrogated by SB203580 and
PD98059. Furthermore, DR-restricted T cells established from PBMC,
which are reactive with mite Ags, purified protein derivative, and
random 19-mer peptides, exhibited a higher IFN-
:IL-4 ratio than did
DQ- or DP-restricted T cells. These results indicate that HLA-DR, -DQ,
and -DP molecules transmit distinct signals to monocytes via MAP
kinases and lead to distinct monokine activation patterns, which may
affect T cell responses in vivo. Thus, the need for generation of a
multigene family of class II MHC seems apparent. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production from T cells
(1). Thus, signals transmitted to monocytes via class II
HLA molecules are involved in determining immune response patterns. It
is highly conceivable that signals transmitted by class II MHC
molecules in B cells, in regulating APC function during cognate T-B
cell interactions, are important for the following reasons: 1)
cross-linking class II molecules induces an increase in intracellular
calcium and cAMP in mouse or human B cell lines (2, 3, 4, 5); 2)
class II MHC-mediated signals lead to homotypic aggregation of B cells
(6); 3) cross-linking HLA-DR molecules on B cells induces
apoptosis (7); 4) class II MHC molecules, without the
intracellular domain expressed on B lymphoma cells, will not lead to an
increase in cAMP and subsequent CD80 up-regulation when stimulated with
a CD28-expressing autoreactive T hybridoma cells (8); and
5) cytoplasmic domain mutants of class II MHC abrogate generation of
intracellular cAMP (9) and translocation of protein kinase
C (PKC)3
(10). Furthermore, by using human peripheral blood
monocytes, cross-linking HLA-DR molecules with staphylococcal
enterotoxins induces phosphorylation of Src family kinases (Lyn, Fgr)
(11). Another study by Kanner et al. (12)
demonstrated that ligation of class II activates Syk and ZAP-70 in B
cells and activated human CD4+ T cells, respectively .
Moreover, engagement of class II molecules on the THP-1 monocyte cell
line with staphylococcal enterotoxin A induced IL-1 and TNF-
(13). Although functional consequences of such DR-mediated
signaling events induced by T cells are largely unknown, these
observations do raise the possibility that signaling through class II
MHC molecules may affect monocyte responses as well, including monokine
secretion, upon TCR-TCR ligand interaction. Our previous investigations on HLA-DR vs -DQ (14) or on I-A vs I-E by others (15) suggested their distinct roles in activating Th/Ts. Thus, HLA-DR functions as an Ir gene for schistosomal Ag-specific immune responses, whereas HLA-DQ functions as an Is gene, being epistatic to DR. However, their roles in activating Th1/Th2/APC have remained elusive. To investigate the consequence of signaling events through distinct subregion products of class II HLA, we tested monokine secretion patterns induced by 1) solid-phase mAbs to HLA-DR, -DQ, and -DP molecules expressed on peripheral blood-adherent monocytes and 2) coculture of peptide-pulsed monocytes with emetine-treated T cell clones of various HLA-restriction patterns.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anti-HLA class II mAb HU4 (anti-HLA-DRB1 + DRB5 IgG2a, monomorphic), L243 (anti-HLA-DRB1 + DRB4 IgG2a, monomorphic), HU11 (anti-HLA-DQ4 + 5 + 6 IgG2a), HU18 (anti-HLA-DQ7 + 8 + 9 IgG2a), or B7/21 (anti-HLA-DP IgG1, monomorphic) (1) were as described. Anti-HLA class II mAbs 1a3 (anti-HLA-DQ IgG2a, monomorphic) (Leinco Technologies, Manchester, U.K.) were purchased. Mouse IgG, IgG1, and IgG2a were purchased for control from BioPur AG (Bubendorf, Switzerland) and Biogenesis (Poole, U.K.). Igs were purified from the ascites form of mAbs using a protein A column (Pierce, Rockford, IL). F(ab')2 of L243 and mouse IgG were prepared using ImmunoPure F(ab')2 Preparation kits (Pierce) with extensive dialysis to remove residual Fc fragments. Genistein (Sigma, St. Louis, MO), GF109203X (Sigma), piceatannol (Sigma), PD98059 (New England Biolabs, Beverly, MA), and SB203580 (Calbiochem, La Jolla, CA) were purchased. Dermatophagoides farinae (Der f) Ags were kindly provided by Torii Pharmaceuticals (Tokyo, Japan). Purified protein derivative (PPD) was purchased from the Japan BCG Laboratory (Tokyo, Japan). Peptides with defined sequences were synthesized using a solid-phase simultaneous multiple peptide synthesizer PSSM-8 (Shimadzu, Kyoto, Japan) based on the F-moc strategy and using a 10-fold molar excess of single F-moc amino acids, then were purified using C18 reversed-phase HPLC. In the case of degenerate peptides, the introduction of randomized sequence positions was done in a double coupling step with equimolar mixtures of F-moc-L-amino acids used in an equimolar ratio with respect to coupling sites of the resins (all positions have 19 amino acid residues, except for Cys).
Human T cell clones
Human CD4+ T cell clone BC20.7 that recognizes DR14 (DRA + DRB1*1405) + residues 84–100 of bacillus Calmette-Guérin (BCG) a protein (BCGap84–100; EEYLILSARDVLAVVSK) has been described previously (16). OT1.1 (17) and DT13.2 (1) are specific for DP5 (DPA1*0201 + DPB1*0501) + p53p153–165 (STPPPGTRVRAMAIYKQS) and DQ6 (DQA1*0102 + DQB1*0602) + Der f Ip18–31 (RSLRTVTPIRMQGG), respectively. T cell clones were fed weekly with 50 U/ml human rIL-2 and 10 U/ml human rIL-4 in the presence of irradiated autologous PBMC prepulsed with each peptide in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% pooled, heat-inactivated normal human male plasma in 24-well flat-bottom culture plates (Falcon; Becton Dickinson, Lincoln Park, NJ). Culture medium and Ab preparations tested for contamination with endotoxin exhibited negative results. Human bleeding and animal experiments (ascites preparation) were in accordance with institutional guidelines.
Preparation of adherent APC
PBMC were freshly prepared from heparinized blood of healthy adult donors using Ficoll-Paque (Pharmacia, Piscataway, NJ). The PBMC were incubated at 3 x 107 cells in 10 ml of 10% human serum/RPMI 1640 for 1.5 h in 90-mm culture-grade plastic petri dishes precoated with heat-inactivated autologous plasma at 37°C in a CO2 incubator. After removing nonadherent cells, the adherent cells were recovered from plates by incubating with ice-cold 0.05% EDTA/PBS for 10 min and repeated pipetting. Monocytes were cultured for 48 h to allow adherence-induced transcription of monokine mRNA to subside (18). This population was composed principally of monocytes and were >90% CD14 positive, as analyzed by FACS (data not shown). HLA class II (DR, DQ, and DP) alleles were determined as described elsewhere (1). HLA types of the two monocyte donors were DRB1*0101/1201 and DRB1*1405/1502, both of which are negative for DRB4.
Stimulation of monocytes
Ten micrograms per milliliter anti-DR Ab (L243), anti-DQ Ab (1a3), anti-DP Ab (B7/21), and mouse IgG (alternatively, IgG1 and IgG2a) were precoated onto 96-well flat-bottom culture plates. Adherent cells were incubated at 6 x 104 cells/well where mAbs are immobilized at 37°C in a CO2 incubator. Culture supernatants were collected at 6, 16, 24, 48, and 72 h and stored in aliquots at -80°C until determinations of lymphokine concentrations.
Alternatively, T cells treated with 0, 10, 30, and 90 µg/ml of de
novo protein synthesis inhibitor emetine (Sigma) (19) for
1 h at 37°C were washed three times with RPMI 1640 medium. Cells
were resuspended in culture medium, incubated for 3 h at 37°C,
and then washed three times with RPMI 1640 medium and cocultured with
peptide-pulsed or mock-pulsed monocytes. Culture supernatants after a
16-h (for IL-12), 24-h (for IL-1, IL-10, IL-18, GM-CSF, and
TNF-), and 48-h (for IL-6) incubation were collected and subjected
to ELISA. Treatment of T cells by emetine abrogated IL-4 production
from BC20.7 (BCGa specific, DR14 restricted) in a dose-dependent
manner; 90 µg/ml emetine treatment resulted in a complete abrogation
of IL-4 production, but not IL-12 produced by peptide-pulsed monocytes
(data not shown). Moreover, culture supernatants of the peptide-pulsed
monocytes stimulated with emetine-treated T cells were positive for
IL-12 production, but not so for mock-pulsed monocytes stimulated with
emetine-treated T cells (data not shown). The interaction between HLA
and peptide alone did not induce monokine production. Results were
similar in case of HLA-DQ-restricted DT13.2 and HLA-DP-restricted OT1.1
(data not shown).
ELISAs
The human IL-4, IFN-, IL-1, 10, 12 (p40 + p70), GM-CSF,
and TNF- ELISA kits (BioSource International, Camarillo, CA) and
human IL-6 ELISA kit (Genzyme, Cambridge, MA) were used for
quantitation of lymphokines in the supernatants according to
manufacturer’s instructions. ELISA kit for IL-18 was kindly provided
by M. Kurimoto (Hayashibara Biochemical Laboratories, Okayama, Japan).
Statistical significance was analyzed using Student’s
t test.
Western immunoblot analysis
Monocytes prepared from PBMC were added to 96-well culture plates in which class II HLA mAbs had been immobilized, followed by centrifugation. After a 10-to 60-min incubation at 37°C, ice-cold 100 µM sodium vanadate/PBS was added for washing, followed by lysing in 50 µl of lysing buffer (150 mM NaCl, 20 mM Tris, pH7.6, 0.5% Nonidet P-40, 2 mM sodium orthovanadate, 1 mM NaF, and 5 mM EDTA plus a protease inhibitor mixture purchased from Sigma). After centrifugation, supernatant fluids of the lysates were electrophoresed on SDS-PAGE gels and transferred to nitrocellulose membrane. After blocking with 10% skim milk and 0.2% Tween 20 in TBS, the membrane was incubated with Abs specific for extracellular signal-related kinase (Erk), c-Jun N-terminal kinase (JNK), and p38 (Santa Cruz Biotechnology, Santa Cruz, CA) or with Abs specific for the activated form of Erk, JNK, and p38 (Upstate Biotechnology, Lake Placid, NY), washed extensively, and subjected to chemiluminescence detection with peroxidase-conjugated anti-mouse IgG Ab using an ECL kit (Amersham, U.K.). Signals were analyzed using the public domain NIH Image program (developed at the National Institutes of Health, Bethesda, MD, and available on the Internet by anonymous FTP from zippy.nimh.nih.gov).
Establishment and analysis of Der f-, PPD-, and X19-reactive T cell lines
Der f (crude mite Ag)-specific short-term T cell
lines were established from PBMC from two donors carrying different HLA
types (donor MA, HLA-DRB1*1405/DRB1*1502; donor NI,
HLA-DRB1*0901/DRB1*1302). HLA-DR-restricted and HLA-DP-restricted T
cell lines were established by coculture either with anti-HLA-DQ
(HU11 and/or HU18) + anti-HLA-DP (B7/21) mAbs or with
anti-HLA-DR (HU4 and L243) + anti-HLA-DQ (HU11 and/or HU18)
mAbs, respectively, in the presence of the crude extract of Der
f. Restriction molecules of these cell lines were confirmed by
inhibition assays with mAbs (data not shown), and all of the cell lines
of expected restriction patterns were used for the analysis. These cell
lines were restimulated with excess concentrations of Ags (10 µg/ml)
and then after a 48-h incubation, culture supernatants were collected
for measurements of IFN- and IL-4 production by ELISA. PPD-specific
short-term T cell lines were established from PBMC of donor MA.
HLA-DR-restricted and HLA-DQ-restricted T cell lines were established
by coculture with anti-HLA-DQ (HU11) + anti-HLA-DP (B7/21) mAbs
or anti-HLA-DR (HU4 and L243) + anti-HLA-DP (B7/21) mAbs,
respectively, in the presence of PPD. X19 (19-mer peptides with random
sequences)-reactive T cell clones were established from PBMC of donor
MA, using X19, IL-4, IL-7, IL-9, IL-15, and agonistic Ab to CD29, under
cloned conditions.4
Restriction molecules were determined by inhibition assays with
mAbs.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We examined the monokine secretion induced by cross-linking class
II HLA molecules using solid-phase mAbs to class II HLA, by which
involvement of cell surface molecules other than HLA is unlikely to
occur. As shown in Fig. 1
A,
the effect of the anti-DQ mAb should be specific, because
liquid-phase 1a3 (simple coculture) did abrogate HLA-DQ-restricted T
cell clonal responses (DT13.2), but not HLA-DR-restricted responses
(BC20.7). However, solid-phase 1a3 markedly stimulated monocytes to
produce IL-1, IL-6, IL-10, IL-12 (p40 + p70), TNF-, and GM-CSF,
whereas Ig subclass-matched control (mouse IgG2a) did not, as shown in
Fig. 1B. IL-18 production was only marginal. The peaks of
IL-1, IL-10, IL-12 (p40 + p70), TNF-, and GM-CSF secretion were
at 16 or 24 h. On the other hand, the IL-6 concentration was
sustained from 16 to 48 h. IL-12 (p40 + p70) showed its peak at
16 h and was no longer detectable at 48 h. We obtained
similar results regarding specific inhibitory activity on Ag
presentation and kinetics when mAbs to DR (L243) and DP (B7/21) were
used (data not shown).
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We next examined the effects of various inhibitors for signal
transduction molecules. As shown in Fig. 2A, PD98059 (MAP/Erk kinase
(MEK)-1 inhibitor) and SB203580 (p38 inhibitor) inhibited
anti-DR-induced IL-1 production from monocytes. Genistein
exhibited a biphasic effect and inhibited IL-1 production at high
concentrations (500 µM). We then studied the phosphorylation of
various kinases by cross-linking class II HLA, among which only MAP
kinases exhibited differential activation by anti-DR, -DQ, and -DP.
We stimulated monocytes directly with solid-phase anti-HLA mAbs,
and cell lysates were subjected to Western blot analysis using Abs to
phosphorylated forms of Erk, JNK, and p38 (anti-pErk,
anti-pJNK, and anti-pp38, respectively). As shown in Fig. 2, B and C, Erk, especially Erk2, was phosphorylated
only by anti-DR mAb (very weak phosphorylation was detected by
anti-DQ or anti-DP in the original film), whereas p38 was
phosphorylated by anti-DR, anti-DQ, and anti-DP mAbs.
Control mouse IgG did not induce phosphorylation of these kinases, and
this was also the case when mouse IgG1 or IgG2a was used (data not
shown). Phosphorylation of JNK molecules was barely detectable and was
not enhanced by stimulation (data not shown). All of these observations
indicate that signaling through DR molecules phosphorylates (i.e.,
activates) Erk and less efficiently p38, whereas signaling through DQ
and DP activates p38 and much less efficiently Erk.
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. As shown in Fig. 3A,
F(ab')2 of L243 markedly induced phosphorylation
of Erk, whereas F(ab')2 of control mouse IgG did
not. Phosphorylation of Erk induced by F(ab')2 of
anti-DR mAb was not inhibited by 10 µg/ml neutralizing Ab to
TNF- (Fig. 3B). Moreover, as shown in Fig. 3C,
the anti-TNF- at 10 µg/ml inhibited the IL-1 production
induced by 10 pg/ml TNF-. Indeed, monocytes stimulated by
F(ab')2 of anti-DR mAb for 60 min never
produced more than 5 pg/ml TNF- (data not shown). Taken together,
neither Fc of Ig nor TNF- is likely to be involved in MAP kinase
activation induced by class II ligation.
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We wanted to determine whether natural TCR-peptide-HLA
interactions would induce monokine secretion by signaling through class
II HLA molecules. T cell clones of various HLA-restriction patterns
were treated with the de novo protein synthesis inhibitor emetine. This
is because it is highly likely that T cell membrane proteins or T cell
soluble factors newly synthesized after activation work on monocytes.
These T cell clones were not reactive to autoantigens or to
alloantigens (1, 16, 17). As shown in Table I, three human Th0 clones of distinct
HLA-restriction patterns, BC20.7 (BCGa specific, DR14 restricted),
DT13.2 (Der f I specific, DQ6 restricted), and OT1.1 (p53
specific, DP5 restricted), were used for emetine treatment 7 days after
the last antigenic stimulation. First, we determined the
ED50 of each clone to be 0.008, 0.18, and 0.10
µM for BC20.7, DT13.2, and OT1.1, respectively (data not shown).
Monocytes were pulsed with peptides, the concentrations of which were
625-fold as much as the ED50 (5, 112.5, and 62.5
µM for BC20.7, DT13.2, and OT1.1, respectively), followed by
coculture with emetine-treated T cells. These peptide concentrations
induced plateau responses of monokine production and the kinetics of
monokine production were practically the same as that observed with
solid-phase mAbs shown in Fig. 1
B (data not shown).
Peptide-pulsed monocytes cocultured with emetine-treated T cells, as
shown in Table I, produced IL-1, IL-6, IL-10, IL-12 (p40 + p70),
GM-CSF, and TNF-. It is noteworthy that the DR14-restricted clone
BC20.7 tends to induce proinflammatory monokines such as IL-1 (105
pg/ml) and TNF- (887 pg/ml) with the IL-10:IL-1 ratio being 1.6,
whereas the DQ6-restricted clone DT13.2 and DP5-restricted clone OT1.1
tend to induce anti-inflammatory monokine IL-10 (787 and 725 pg/ml,
respectively) with the IL-10:IL-1 ratio being 32.8 and 34.5 for
DT13.2 and OT1.1, respectively. The magnitude of response was markedly
different between Ab-induced (Fig. 1B) and T cell-induced
(Table I) secretion of IL-6 and GM-CSF, the mechanisms for which are
yet to be determined. Allogeneic monocytes that do not share
restriction HLA molecules exhibited marginal monokine production in the
presence of emetine-treated T cells, and neutralizing Abs to TNF-
(20), CD154 (21), and CD23 (22)
only partially abrogated IL-12 or IL-10 production (data not
shown).
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ratio under DR, DQ, and DP stimulation was 1.8, 12.8, and
14.4, respectively, indicating that differential monokine production
induced by stimulation through DR, DQ, and DP molecules is observed
even when monocytes are directly stimulated by mAbs. Effects of protein kinase inhibitors on monokine productions
We cocultured peptide-pulsed monocytes and emetine-treated BC20.7
T cells in the presence of several kinase inhibitors. These inhibitors
were dissolved in DMSO and added to the culture medium at a final
content of 0.5%, a content which did not inhibit DR-mediated monokine
production, as shown in Fig. 4A. The MEK-1 inhibitor
PD98059 inhibited IL-1 secretion in a dose-dependent manner, whereas
the PKC inhibitor GF109203X (IC50, 20 nM;
23) or protein tyrosine kinase (PTK) inhibitors did
not clearly do so. Genistein exhibited a biphasic effect and inhibited
IL-1 production at high concentrations (500 µM), which
corroborates the results shown in Fig. 2A. We next examined
the effect of inhibitors specific for individual MAP kinase(s) on the
production of IL-1 and IL-10 using emetine-treated BC20.7 T cells
and peptide-pulsed monocytes. Interestingly, the 50 µM p38 inhibitor
SB203580 (24) markedly inhibited IL-10 production, whereas
PD98059 rather enhanced IL-10 production (Fig. 4B). On the
other hand, both PD98059 and SB203580 only partially inhibited IL-1
(Fig. 4C). Moreover, a combination of PD98059 and SB203580
completely abrogated IL-1. Because these inhibitors did not decrease
cell viability after 24-h culture (Fig. 4D), it is likely
that the kinase inhibitors at the concentration we used exerted
specific inhibitory effects on monocytes. When DQ- and DP-restricted T
cells were used for stimulation, similar effects of PD98059 and
SB203580 on monokine secretion were observed, except that the enhancing
effect of PD98059 on IL-10 was only marginal (data not shown). These
observations suggest that 1) p38 is involved in both IL-1 and IL-10
production induced by ligating DR molecules expressed on monocytes; 2)
the MEK-1-Erk pathway is only partially involved in IL-1 production,
being independent from p38-associated IL-1 production; and 3)
activation of Erk may inhibit p38-mediated IL-10 production (Fig. 5).
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If the phenomenon observed earlier in this study occurs in a
local milieu of T cell differentiation, lymphokine production patterns
of T cells would be affected by restriction HLA molecules. We then
examined the production of IFN- and IL-4 using Der
f-specific T cell lines. As shown in Fig. 6A, DR-restricted T cell lines
produced more IFN- than IL-4, but DP-restricted T cell lines
produced more IL-4 than IFN- (p = 0.02 and
0.04 in donors MA and NI, respectively). mAbs used in this study did
not induce monokine secretion when used as a soluble form (data not
shown). HLA types of MA
(HLA-DRB1*1502-DRB5*0102-DQA1*0103-DQB1*0601/HLA-DRB1*1405-
DRB3*0202-DQA1*0101DQB1*0503) and NI (HLA-DRB1*
0901-DRB4*010-DQA1*0301-DQB1*0303/HLA-DRB1*
1302-DRB3*0301-DQA1*0102-DQB1*0605) were distinct. Then we
examined the production of IFN- and IL-4 from the PPD-specific T
cell lines (Fig. 6B). DR-restricted T cell lines produced
more IFN- than IL-4, but DQ-restricted lines produced more IL-4 than
IFN- (p = 0.04 in donor MA). We then used
X19 (random 19-mer peptide) to confirm the phenomenon observed in
earlier studies because 1) DQ-restricted/Der f-reactive and
DP-restricted/PPD-reactive T cells were not readily established and 2)
X19 can stimulate most CD4+ memory T cells to
proliferate in the presence of cytokines under cloned
conditions.4 Indeed, DR-, DQ-, and DP-restricted
T cell lines were obtained, the cytokine profiles of which again
exhibited similar results (Fig. 6C). When we titrated down
the peptide concentration for DR-restricted responses, we found that
lower concentrations of X19 peptide did not lead to DQ-/DP-restricted
patterns of cytokines (data not shown), which was indeed the case when
emetine-treated BC20.7 T cells were incubated with monocytes in the
presence of lower concentrations of the antigenic peptide (data not
shown). These data indicate that DR-restricted and already activated
peripheral CD4+ T cells carry the Th1-prone
phenotype compared with DQ-/DP-restricted T cells, albeit the
segregation pattern being incomplete.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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:IL-10 ratio than did 1a3 or B7/21 (data not shown). Soluble
anti-class II induced only marginal signaling (data not shown),
unlike many other studies using B cells. This is probably due to lower
class II expression levels on monocytes compared with B cells. When T
cells were preactivated for 16 h with peptide-pulsed monocytes,
treated with emetine, and subjected to coculture with freshly isolated
monocytes, the IL-1:IL-10 ratio remained unchanged and levels of
IL-1 and IL-10 production became even lower (data not shown),
indicating that 1) expression of cell surface molecules induced as a
result of TCR-peptide-MHC interaction contribute little to
IL-1/IL-10 and 2) decreased monokine production induced by activated
T cells may reflect the TCR down-modulation induced by activation.
Indeed, human T cell clones used in this study were stimulated with
peptides every 6–7 days and CD154 did not return to null expression
even on day 7 (1), which should have contributed to IL-12
production.
The observation that IFN-/IL-4 produced by T cells is
associated with HLA-restriction molecules even in freshly isolated
short-term T cell clonal responses to crude protein Ags or randomized
peptide Ags is evidence that the phenomenon is not limited to three T
cell clones used in this study. In other words, while not being
complete as evidenced by Fig. 6, DR-restricted Th2 or DQ-/DP-restricted
Th1 can exist, HLA class II subregions may determine T cell
differentiation patterns or IFN-/IL-4, probably through monocyte
responses. However, one can speculate that the DR-peptide complex
delivers the strongest avidity between TCR, leading to Th1-prone
responses (31, 32). In this regard, absence of
DR-restricted T cell clones with low IFN-/IL-4 as shown in Fig. 6
rules out this possibility, because low-affinity DR-binding peptides
should exist and would activate Th2-prone responses, if the phenomenon
is attributed to avidity alone. Indeed, it is likely that such a
phenomenon is attributed to high IL-12 production through DR signaling
(Table I). In activating naive T cells to generate primed Th1/Th2,
dendritic cells should play pivotal roles (33) and class
II-mediated signaling in dendritic cells awaits to be determined.
We also examined the effect of peptide concentrations on the
IL-10:IL-1 ratio using T cell clones and peptide-pulsed monocytes.
Indeed, emetine-treated BC20.7 T cells (DR-restricted) stimulated
peptide-pulsed monocytes to produce IL-10 and IL-1 at practically a
constant ratio when monocytes were pulsed with lower concentrations of
the peptides (data not shown), thus indicating that weaker
HLA-peptide-TCR interaction does not alter the pattern of monocyte
responses to secrete monokines. It is also known that 1) different
peptide sequences are not required to differentially drive Th1/Th2
directions by class II (34) and 2) differential signaling
through TCR (but not through monokine receptors) can be induced
depending on the peptide concentrations used, which may lead to
differential activation of Th1/Th2 (35, 36). Therefore,
restriction molecule (DR, DQ, or DP) is not the only one but at least
one of the important molecules to determine monocyte responses. Another
possibility to be ruled out is the effect of class II expression
levels. This question can be addressed using class II transfection into
human class II-deficient monocytic cells because class
IIhigh and class IIlow
populations of peripheral monocytes/macrophages may belong to distinct
cell lineages (28), and stimulation of monocytes by
IFN- leads to PTK and PKC activation and hampers natural signaling
processes through class II (37).
All three MAP kinases phosphorylate substrates on
serine/threonine residues. Erks are characteristically activated by
growth factors, whereas JNK/stress-activated protein kinase and p38
kinase are strongly activated by UV irradiation, osmotic stress, and
inflammatory cytokines (38). Thus, they are differentially
activated by stimuli other than class II HLA isoforms. In other words,
ligation of DR, DQ, and DP is another set of stimuli that
differentially activates MAP kinases. In other experimental systems
(24), where monocytes were stimulated with LPS, p38 and
Erk but not JNK was activated, as observed in the present study.
Interestingly, inhibition of p38 suppressed both IL-10 and IL-1,
whereas that of Erk partially suppressed IL-1 but not IL-10
(24), which corroborates results we obtained. Although Erk
was markedly phosphorylated by anti-DR Ab, the involvement in
monokine secretion was only partial. Other consequences, such as
proliferation, might be a major outcome of Erk activation through DR;
however, proliferation was not readily detected using monocytes.
Another set of experiments using fibroblasts is underway to delineate
the biological significance of DR-mediated Erk activation. As shown in
Figs. 2 and 4, the effect of genistein on IL-1 production was
biphasic. The enhancing effect at 100 µM may indicate that certain
PTKs have inhibitory effects on the IL-1 production induced by DR
ligation. On the contrary, it is likely that the suppressive effect at
500 µM reflects the effect on PTKs whereby the ras-raf pathway is
activated.
Syk is associated with Erk (39) and involved in
signaling through MHC molecules (12, 40). We observed a
slight enhancement of monokine production by PKC inhibitor GF109203X
and Syk inhibitor piceatannol (Figs. 2 and 4). The precise mechanisms
are yet to be determined, but one may speculate that they negatively
regulate monokine production induced by ligating class II HLA.
Because transmembrane and intracellular domains are markedly different
among - and -chains of HLA-DR, -DQ, and -DP and MHC molecules
have no immunoreceptor tyrosine-based activation motif, it seems
reasonable to speculate that HLA-DR, -DQ, or -DP molecules are
associated with distinct signal transduction molecules. Differential
endosomal trafficking/recycling (41), differential
signaling in monocyte subsets (28), including contaminated
dendritic cells, and even differential localization in membrane
microdomains (42, 43) would also need to be considered.
Studies currently underway will address these questions using various
monocytic cell lines and mass mapping techniques.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Sho Matsushita, Department of Neuroscience and Immunology, Division of Immunogenetics, Kumamoto University Graduate School of Medical Sciences, 2-2-1 Honjo, Kumamoto 860-0811, Japan.
3 Abbreviations used in this paper: PKC, protein kinase C; PPD, purified protein derivative; Der f, Dermatophagoides farinae; Erk, extracellular signal-related kinase; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; MEK, MAP/Erk kinase; BCG, bacillus Calmette-Guérin; PTK, protein tyrosine kinase.
4 S. Matsushita, Y. Tanaka, T. Matsuoka, and T. Nakashima. Identification of peptide ligands recognized by single CD4T cells, using limiting dilution primary culture with randomized peptides followed by epitope scanning with combinatorial peptide libraries. Submitted for publication.
Received for publication July 3, 2000. Accepted for publication November 15, 2000.
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and its colocalization with HLA and CD48 on T lymphoma cells suggest their functional association with lipid rafts. Proc. Natl. Acad. Sci. USA 97:6013.This article has been cited by other articles:
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) and mouse
IgG2a (
) are immobilized at 37°C in a CO2 incubator.
Culture supernatants were collected at the indicated time points.
