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* Department of Immunotoxicology, Institute for Risk Assessment Sciences, and
Immunology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands; and
Tanox Pharma B.V., Amsterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production, influx of CD11c+ and
F4/80+ cells, and prevention of architectural disruption of
the lymph node, but no effect on IgG2a Ab and TNF-
secretion or B
and CD4+ T cell proliferation). The findings indicate that
the CD40-CD154 costimulatory interaction is a prerequisite in
drug-induced type 2 responses and is only marginally involved in type 1
responses. The observed expression patterns of CD80 and CD86 on
different APC (B cells in type 2 and dendritic cells in type 1) may be
responsible for this discrepancy. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In conjunction with IL-4, cross-linking of CD40 is able to stimulate B cell survival, proliferation, and maturation into germinal center (GC)3 cells (13, 14, 15, 16). Moreover, in CD154-deficient mice, IgG1 Ab responses (characteristic for type 2 responses in mice) to viruses were severely decreased and no memory B cells were found, whereas IgG2a, IgG2b, and IgG3 Ab-producing cells (representing type 1 responses) were still present (17). This indicates that without CD154 triggering, type 2 mechanisms leading to Th2-type Ab production are disabled while type 1 responses are still operational. Additionally, Kishimoto et al. (18) concluded that CD154 ligation was decisive in an allogeneic Th2 response but had a minor impact on regulation of the allogeneic Th1 response. Finally, CD4+ T cell priming was not impaired in the absence of CD154 ligation in the Th1-mediated autoimmune oophoritis system (19).
In contrast, only the development into Th1, and not Th2, cells was impaired after in vitro stimulation to OVA peptide when using OVA-specific T cells from CD154-deficient mice (20). Blocking of CD154 in NOD/shi mice also impaired the development of islet-reactive Th1 cells (7). Moreover, the CD40-CD154 interaction was demonstrated to be crucial for in vivo priming of Th1 cells by IL-12-producing APC in SJL/J mice (21), and supplementary expression of CD154 directs the response to respiratory syncytial virus toward a type 1 phenotype (22). At last, anti-CD154 Ab was found to induce tolerance to 2,4-dinitrofluorobenzene-induced contact hypersensitivity, a typical type 1 phenomenon (23), but CD154-deficient mice showed normal delayed-type hypersensitivity responses to OVA (24).
These contradictory data may be the consequence of comparing studies
with different experimental set-ups: transplantation models,
spontaneous or induced autoimmune diseases, or the use of transgenic
mice, in which responses are all under extensive control by diverse
regulatory mechanisms, which complicates the interpretation of the
importance of CD40-CD154 in the sensitization phase in type 1 vs type 2
responses. Therefore, we intended to study the involvement of CD154 in
type 1 vs type 2 immune responses in a relatively simple model using a
predefined Ag in a wild-type strain of mice, the reporter Ag popliteal
lymph node assay (RA-PLNA). The PLNA, originally developed to
investigate immunomodulating properties of autoimmunogenic chemicals,
was extended with the use of a reporter Ag (trinitrophenyl (TNP)-OVA)
to allow detection of dichotomous immune responses elicited by
chemicals. Previously, we have shown that the response to TNP-OVA
parallels the type of response expected to occur in the case of the
xenobiotic itself (25). Notably, the response to TNP-OVA
in the presence of the lupus-inducing chemicals
HgCl2 and diphenylhydantoin (DPH;
anti-epileptic drug) is type 2 (IgG1 Ab-secreting cells (ASC), GC
formation, influx of IL-4-producing CD4+ cells)
(26), whereas the response to the same Ag elicited by the
anti-neoplastic diabetogenic chemical streptozotocin (STZ) is type
1 (IgG2a ASC, influx of IFN--producing CD8+
cells and macrophages) (27).
In the present study the RA-PLNA was used to investigate the role of CD154 in both type 1 and type 2 responses induced by STZ and DPH or D-penicillamine (D-Pen; anti-rheumatic drug), respectively, by simultaneously treating drug-exposed mice with a CD154 antagonist. Our findings clearly show that type 1 and type 2 immune responses are differently regulated by CD40-CD154 interactions and support the hypothesis that the dichotomy in immune responses is at least partially dependent on different responsiveness to costimulatory molecules provided by different APC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specific pathogen-free female BALB/c mice (6–12 wk old) were obtained from the Utrecht University breeding facility (Gemeenschappelÿk Dier Laboratorium, Utrecht, The Netherlands) and randomly assigned to specific treatment. Mice were allowed to settle for 1 wk and were maintained under barrier conditions in filter-topped Macrolon cages with wood chips bedding, at a mean temperature of 23 ± 2°C, 50–55% relative humidity, and a 12-h light/dark cycle. Drinking water and standard laboratory food pellets were provided ad libitum. The experiments were conducted according to the guidelines of the animal experiments committee of the Veterinary Faculty of Utrecht University.
Chemicals and reagents
Chemicals were obtained from Sigma-Aldrich (Zwijndrecht, The
Netherlands) unless stated otherwise. Saline (0.9%; B. Braun
Melsungen, Melsungen, Germany) and citrate buffer (0.1 M citric acid
and 0.1 M disodium hydrogen phosphate, pH 6) were used to dilute test
chemicals. TNP-OVA and blocking agent TNP-BSA were prepared as
previously described by Albers et al. (26). MR1 was used
as anti-CD154 Ab, which was a gift from Tanox Pharma (Amsterdam,
The Netherlands). For ELISPOT analyses, alkaline phosphatase
(AP)-conjugated goat anti-mouse, human adsorbed IgG1, IgG2a, and
IgM and AP-conjugated rat anti-mouse IgE were obtained from
Southern Biotechnology Associates (Birmingham, AL). For the development
of spots, fresh stock solutions of para-nitroblue tetrazolium and
5-bromo-4-chloro-3-indolylphosphate toluidine salt in dimethylformamide
(BDH Laboratory Supplies, Poole, U.K.) were prepared and diluted in
Tris buffer (100 mM Trizma base, 100 mM NaCl, and 5 mM
MgCl2·6H2O, pH 9.5). For
immunochemistry, acetone and
H2O2 were obtained from
Merck (Darmstadt, Germany), and Earl’s balanced salt solution buffer
was purchased from Life Technologies (Breda, The Netherlands). Rat
anti-mouse B220 (RA3-6B2) was used to stain B cells. AEC was
obtained from Janssen (Beerse, Belgium) and rabbit anti-rat HRP was
obtained from DAKO (Glostrup, Denmark). For cytokine measurements, IL-4
and IFN- capture and detecting Abs were obtained from BD
PharMingen (Hamburg, Germany), TNF- was purchased from BioSource
(Camarillo, CA), streptavidin coupled to HRP was obtained from CLB
(Amsterdam, The Netherlands), and casein was purchased from BDH
Laboratory Supplies.
The following Abs used for surface marker staining in tri-color flow
cytometry were purchased from BD PharMingen: CD3
CyChrome (CY;
145-2C11), CD4 FITC (RM4-5), CD8a PE (53-6.7), CD19 PE (1D3), CD80 FITC
(16-10A1), CD86 FITC (GL1), CD40 FITC (3/23), CD11c FITC (HL3), CD54
FITC (3E2), streptavidin CY, rat-anti-mouse MHC-II biotin (NIMR-4),
and anti-macrophage marker PE (F4/80) from Caltag Laboratories
(Burlingame, CA).
RA-PLNA
Naive mice were injected s.c. into the right hind footpad with 50 µl of a freshly prepared mixture of the drug together with a subsensitizing dose (10 µg) of TNP-OVA. Drugs were injected in quantities that were immunostimulatory in the PLNA in previous experiments (25): 1 mg D-Pen or 2 mg DPH dissolved in saline or 1 mg STZ diluted in citrate buffer (0.1 M, pH 6) instead of saline to maintain the NO-generating capacity in vivo. Control groups treated with TNP-OVA in either saline or citrate buffer were not different when comparing cell counts, TNP-specific ASC numbers or expression of costimulatory molecules. Mice were treated i.p. with 300 µg anti-CD154. Seven days after drug injection, mice were killed by cervical dislocation, blood was drawn by orbital puncture, and the popliteal lymph node (PLN) was excised and separated from adherent fatty tissue. PLNs were isolated in ice-cold PBS/1% BSA, and single-cell suspensions were prepared, washed (1000 rpm at 4°C), resuspended in 1 ml PBS/1% BSA, counted using a Coulter counter (Coulter Electronics, Luton, U.K.) and adjusted to 1 x 106 cells/ml. For immunochemistry PLNs were snap-frozen in liquid nitrogen and stored at -70°C until use.
ELISPOT assay
The ELISPOT assay was performed based on the operating procedure described by Schielen et al. (28). Immobilon-P membranes (Immobilon PVDF Transfer; Millipore, Etten-Leur, The Netherlands) were coated overnight with PBS/0.05% Tween/TNP-BSA (10 µg/ml) and blocked for 1 h with PBS/Tween/1% BSA. These membranes were clamped in spot blocks (made in-house), and 5 x 105 cells were centrifuged onto the membranes and incubated for 4 h at 37°C. Membranes were removed from the spot blocks, washed with PBS and PBS/Tween, and incubated overnight at 4°C with AP-conjugated Abs in PBS/Tween. Membranes were washed and incubated with para-nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate toluidine salt reagent to accomplish color development of TNP-specific Ab spots. These spots were counted by two independent observers using a stereomicroscope.
Cell culture and cytokine measurement
Cell suspensions (1 x 105 cells in
100 µl complete RPMI 1640 from Life Technologies supplemented with
10% FCS, 50 mM 
-ME, and 200 mM L-glutamine) were
incubated with 50 µl Con A (15 µg/ml), LPS (6 µg/ml), or medium
in 96-well plates (Highbond 3590; Costar, Cambridge, MA) overnight at
37°C in 5% CO2. After centrifugation for 10
min at 1000 rpm, supernatant was collected and stored at -70°C until
analysis. IFN- and IL-4 were determined by sandwich ELISA. Plates
were coated overnight at 4°C with 1 µg/ml rat-anti-mouse
IFN- or 1 µg/ml rat-anti-mouse IL-4 in 0.05 M carbonate buffer
(pH 9.6), washed with PBS/Tween, and blocked with PBS/Tween/casein for
4 h at room temperature. Samples and IL-4 and IFN- standards
(100 µl) were added in several dilutions and incubated overnight at
4°C. After washing, plates were incubated with 0.25 µg/ml
biotinylated rat anti-mouse IFN- or IL-4 conjugate diluted in
PBS/Tween/casein for 1 h at room temperature. Plates were washed
again and incubated with streptavidin-HRP (0.3 µg/ml) diluted in
PBS/Tween/casein for 45 min at room temperature.
For TNF- ELISA, 96-well plates were coated with 1.25 µg/ml rat
anti-mouse TNF- in PBS (100 µl, overnight) and after washing
with PBS/Tween were blocked with PBS/0.5% BSA for 2 h at room
temperature. Plates were washed, and TNF- standards and samples were
added in several dilutions. Immediately, biotinylated anti-TNF-
diluted in PBS/Tween/0.5% BSA was added and incubated for 2 h at
room temperature. Plates were washed and incubated with streptavidin
(0.3 µg/ml) diluted in PBS/Tween/0.5% BSA for 45 min at room
temperature.
After the final washes, TMB substrate (0.1 mg/ml) was added, and the color reaction was stopped after 10 min with 2 M H2SO4. Absorbance was measured at 450 nm using an ELISA reader ELX800 (Bio-Tek Instruments, Winooski, VT).
Flow cytometry
For flow cytometric analysis, 1 x 105 cells in PBS/BSA were centrifuged, resuspended, and incubated with predetermined dilutions of FITC-, PE-, and CY-conjugated mAbs in 96-well plates (30 min in darkness at 4°C). Samples incubated with biotin-conjugated mAbs were once again centrifuged and incubated with streptavidin-CY in the same way. Cells were washed, resuspended, stored in formalin (0.1%), and analyzed within 18 h. Samples were analyzed on a FACScan with standard FACSflow using CellQuest software (BD Biosciences, Franklin Lakes, NJ).
Immunochemistry
Cryostat sections (6 µm) were fixed in acetone and incubated with predetermined dilutions of rat anti-mouse B220 mAb in PBS/1% BSA at room temperature for 1 h. After three washes with PBS/Tween, sections were incubated (at room temperature for 1 h) with polyclonal peroxidase-conjugated rabbit anti-rat Ig in PBS/10% normal mouse serum. After washes with PBS/Tween and acetate buffer, peroxidase-based staining was performed with 3-amino-9-ethylcarbazole or diaminobenzidine, and counterstaining was performed with hematoxylin.
Statistics
Values deviating >2 SD from group means were considered outliers and were not included in statistical analyses. Preceding analyses absolute cell numbers were log-normally transformed. Multiple comparison of group means were analyzed using one-way ANOVA with Bonferroni as post-hoc test. A value of p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The definitions of type 1 and type 2 responses are based on
differentiation of Th cells in either Th1 or Th2 cells, which are
characterized by their pattern of cytokine secretion (29)
that determines the IgG isotype that is secreted by B cells. In mice,
IFN- and IgG2a are generally used as type 1 prototypes, whereas IL-4
and IgG1 indicate a type 2 response (30). Although the
Th1/Th2 paradigm is probably an oversimplification, as cells and
mediators of one type regulate the characteristic features of the other
type, we use these prototypic parameters to differentiate type 1 from
type 2 drug-induced responses to TNP-OVA.
Our present observations confirm earlier results described by Albers
and colleagues (25, 26) showing that coinjection of
TNP-OVA with DPH or D-Pen elicits type 2 responses, whereas exposure to
STZ stimulates a type 1 response. Responses evoked by DPH and D-Pen are
characterized by elevated levels of TNP-specific IgM, IgG1, and IgE ASC
numbers (Fig. 1
A). In this
study we additionally show that IL-4 levels were increased after in
vitro restimulation of PLN cells with Con A, while IFN- and TNF-
levels were not different from control values (Fig. 1B),
indicating that Th2 cells activated B cells to proliferate and secrete
TNP-specific Abs.
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A) indicative of Th1 cell-dependent B cell priming.
Indeed, extremely high levels of IFN- and TNF-, but not IL-4,
confirmed Th1 involvement (Fig. 1B).
We further identified the type of response by characterizing the cell
types present in the draining lymph node. All chemicals significantly
increased PLN cellularity (Table I), but the compositions of
various cell types present in the PLN as well as the PLN architecture
were completely different between type 1 and type 2 responses.
CD4+ and CD8+ T cells
increase in concert after D-Pen and DPH injection (Table I), but T:B
cell ratios are significantly decreased compared with controls due to
extensive proliferation of B (CD19+) cells.
Immunohistochemical examination of the draining PLN showed GC in lymph
nodes from animals injected with D-Pen, indicating B cell maturation
and differentiation into memory cells (Fig. 2B). STZ exposure increased
absolute numbers of CD4+ T and
CD8+ T and B cells (Table I). The response was
characterized by a strong influx of CD8+ T cells
as the Th/Tc balance changed in favor of Tc and the T:B cell ratio was
significantly increased compared with controls. Interestingly, a rather
disturbed structure of the lymph node was observed, showing B cells
scattered through the entire PLN tissue, and no GC were present (Fig. 2E). Numbers of CD11c+ and
F4/80+ cells were increased in STZ-mediated
responses. These data prove that RA-PLNA using adjuvating drugs
provides a model to study both type 1 and type 2 immune parameters in
response to a well-defined Ag in the same mouse strain. We used this
experimental set-up to study the role of CD154 in both types of
response.
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To study the role of CD40-CD154 ligation in drug-induced type 1
and type 2 responses, mice were treated i.p. with anti-CD154 mAb.
Anti-CD154 treatment decreased total PLN cell numbers in both D-Pen-
and DPH-induced responses (inhibition, 63 and 54%, respectively), but
not in responses to STZ (Table I), compared with those in
anti-CD154-nontreated mice. In type 2 responses primarily B cell
numbers were affected, as CD19+ cell numbers
decreased significantly with 58% in D-Pen-exposed animals, whereas the
decreases in CD4+ and CD8+
T cell numbers were 31 and 34%, respectively (not significant).
Anti-CD154 completely prevented GC formation in these animals (Fig. 2C). For DPH the B and CD4+ and
CD8+ T cell numbers were 52, 65, and 40% lower
(all significant), respectively.
CD4+:CD8+ ratios were
unchanged compared with those in nontreated animals, indicating that
the proliferation of both cell types was equally affected by
anti-CD154 mAb.
In the type 1 response anti-CD154 did not affect the proliferation
of B and CD4+ T cells, but relative numbers of
CD8+ T cells were slightly lower compared with
those in mice without anti-CD154 treatment. Interestingly, fewer
CD11c+ and F4/80+ cells
were present in the PLN of anti-CD154-treated mice compared with
drug-exposed mice that were not injected with anti-CD154.
Additionally, anti-CD154 prevented the architectural disruption of
the PLN in STZ-exposed mice (Fig. 2F).
Furthermore, IL-4 secretion, which was induced by D-Pen, was completely
inhibited by treatment with anti-CD154 mAb (Fig. 3). In the type 1 response, on the other
hand, secretion of TNF- was not inhibited, and IFN- was only
slightly inhibited by anti-CD154. Consistently, TNP-specific Ab
secretion was inhibited by anti-CD154 in type 2, and not in type 1,
drug-induced responses. Anti-CD154 decreased TNP-specific IgM ASC
numbers (per 1 x 106 cells) with >80% in
the case of type 2 responses (Fig. 4).
The numbers of IgG1 and IgE ASC (per 1 x
106 cells) were also significantly reduced in the
case of DPH (87 and 84%, respectively) and D-Pen (84% reduction in
IgG1 and no detectable IgE; Fig. 5). In
responses to STZ no changes were observed in TNP-specific IgM and IgG2a
ASC numbers after CD154 treatment.
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Coinjection of TNP-OVA and D-Pen together with i.p. treatment with
0, 75, 150, 300, or 500 µg anti-CD154 resulted in a
dose-dependent inhibition of the immune response indicated by
cellularity of PLN (data not shown) and the presence of TNP-specific
IgM and IgG1 ASC (Fig. 6). Mice treated
with either 300 or 500 µg anti-CD154 (separate experiments)
showed significantly reduced total PLN cell numbers compared with
drug-exposed mice that did not receive anti-CD154. ELISPOT data
showed that TNP-specific IgG1 and IgM ASC numbers were significantly
reduced after treatment with 300 and 500 µg anti-CD154 compared
with those in groups that received a lower dose. IgG1 ASC numbers were
comparable after 300 or 500 µg anti-CD154 treatment, whereas
numbers of IgM ASC were slightly lower after treatment with 500 µg
anti-CD154 compared with 300 µg. In an additional experiment it
was tested whether a 300-µg dose of anti-CD154 on 2 consecutive
days (days 0 and 1) could inhibit these parameters more effectively,
but the results showed no differences between treatments. A specific
ELISA was performed to assess the amount of anti-CD154 mAb
circulating in the body in time, and anti-CD154 levels were still
detectable in sera 7 days after injection (data not shown).
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Others have already shown that CD40-CD154 ligation is an important
stimulus for dendritic cells (DC) and B cells to up-regulate the
expression of the surface molecules CD80, CD86, and CD54
(31). Therefore, we set out to characterize expression of
these molecules in our model on both B and non-B cells, as shown in
Fig. 7. These graphs show that in type 2
responses relative numbers of CD80+ cells were
unchanged, whereas percentages of CD86+ cells
were significantly increased, but only when analyses were performed for
B cells separately. Relative numbers of CD54-expressing cells were all
significantly increased compared with controls. In the response to
D-Pen, B cells expressed 57, 68, and 94% of the total expression of
CD80, CD86, and CD54, respectively. In DPH-elicited responses these
numbers were even higher: 88, 61, and 94%, respectively, indicating
that B cells are efficient in Ag presentation and providing
costimulation in type 2 conditions.
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Modulation of CD80, CD86, and CD54 expression by anti-CD154 in drug-induced type 1 vs type 2 responses
Fig. 7 indicates that the expressions of CD86 costimulatory
molecules were differentially regulated in type 2 responses. Anti-CD154
inhibited CD86 expression to control levels in response to DPH, whereas
CD86+ cells were still elevated in response to
D-Pen. In the type 1 response anti-CD154 inhibited CD86 expression,
but CD80 expression remained unaffected. Notably, the inhibition of
cells expressing CD86 was only significant for B cells in type 2
responses, but CD86-expressing non-B cells were decreased in the type 1
response. Anti-CD154 treatment had a diverting impact on the expression
of CD54, as numbers decreased significantly in type 2 responses, but
there was no change in expression in the type 1 response.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In contrast, blocking of CD154 in the case of the type 1 immune
response elicited by STZ did not affect the proliferation of B and
CD4+ T cells, TNF- secretion, and TNP-specific
IgM and IgG2a production. Anti-CD154 only slightly affected the
observed relative increase in the number of CD8+
T cells and IFN- production and inhibited the influx of
F4/80+ and CD11c+ cells
into the PLN. Together these results show that CD40-CD154 interaction
is crucial in type 2 responses and is only marginally important in type
1 responses elicited by drugs with opposing adjuvant potential.
CD40 engagement is an important stimulus for DC and B cells to up-regulate the expression of the costimulatory molecules CD80 and CD86 (15, 33, 34, 35), and, vice versa, ligation of the CD80/86 ligand CD28 on T cells increases the expression of CD154 (36). Activation through these costimulatory molecules together with CD40-mediated up-regulation of cytokine receptors (37) and secretion of proinflammatory cytokines (13, 31) are required for DC and B cells to become efficient APC and to activate T cells (18, 38). In the present study CD86 and CD54 expressions were enhanced in both type 1 and type 2 circumstances, but, interestingly, in elicitation of type 2 responses these molecules appeared particularly on B cells, whereas in the STZ-induced type 1 response expression was found predominantly on non-B cells, which appeared to be CD11c+ and F4/80+. CD80 expression, on the other hand, was increased only in the type 1 response and exclusively on non-B cells. We further show that in anti-CD154-treated mice the expression of CD86 on B cells is inhibited and not different from that in controls in the case of DPH and on non-B cells after STZ exposure. However, the numbers of CD86-expressing B cells in D-Pen-exposed animals are still elevated compared with control values after CD154 treatment. Surprisingly, anti-CD154 had no effect on CD80 expression induced by STZ.
From this study important conclusions can be drawn concerning the involvement of CD80 and CD86 and the cells that function as APC in drug-induced type 1 vs type 2 responses. First, DC and/or macrophages serve as the most efficient APC in type 1 responses, whereas this Ag presentation function is taken over by B cells in type 2 responses. Second, CD86 is not as crucial in all type 2 responses. Third, CD80 expression on non-B cells appears only important in the type 1 response. Finally, this CD80 expression is not affected by anti-CD154 and thus is independent of CD40 interaction with its ligand. From the latter we infer that CD80 signaling may circumvent the need for CD40 triggering in STZ-treated animals.
These conclusions support the idea that CD80 is important in activating Th1 cells (39, 40), whereas CD86 preferentially induces Th2 responses (41, 42, 43). Rather, our results suggest that type 2 responses arise upon CD86 ligation in the absence of CD80, whereas the expressions of both molecules prime for type 1 responses. However, evidence accumulates that many more features, such as involvement of regulatory genes, effects of different APC and Ag, location and duration of exposure, and costimulatory expression pattern in several stages of the response, determine the actual balance between type 1 and type 2 immune responses (44). We are currently looking into the kinetics of CD80 and CD86 up-regulation and expression of other costimulatory molecules on specified APC to investigate their importance in drug-induced sensitization.
In line with the multifactorial aspect of immune response polarization,
we show that anti-CD154 treatment affects some Th1 parameters more
than others; i.e., anti-CD154 did not disturb (IgG2a and TNF-
production or B and CD4+ T cell proliferation) or
only slightly disturbed (CD8+ T cells and IFN-
production) effector functions induced by Th1 cells. The independence
of IgG2a production for the CD40-CD154 interaction is also demonstrated
in CD154-deficient mice (17), but in the same as well as
other studies (17, 45) CTL function of
CD8+ T cells was found to be unaffected. Our
results together with the fact that at least a subgroup of
CD8+ T cells expresses CD154 (13)
indicate that CD8+ T cells, at least for some
functions, can be affected by anti-CD154.
Compared with the slight effects on CD8+ T cell
activation and IFN- production, the observed profound reduction of
STZ-induced migration of CD11c+ and
F4/80+ cells into the lymph node and the recovery
of the disturbed PLN morphology by interference with the CD40-CD154
interaction are remarkable. In previous studies (27) we
have demonstrated that this disturbed PLN morphology is accompanied by
an increase in apoptotic cells, possibly initiated by macrophage
excretion products (enzymes, NO). These observations are in agreement
with data from CD154-deficient mice showing that Langerhans cells fail
to migrate to the draining lymph node (46) and with
studies demonstrating that anti-CD154 treatment prevents the
secretion of tissue-disrupting chemokines and reactive oxygen species,
such as NO (47). It can be concluded that the CD40-CD154
couple strongly determines macrophage influx as well as excretion of
tissue-damaging products, and that the macrophage activation by STZ is
largely independent of Th1 cells. However, part of this
macrophage-stimulating effect of the costimulatory receptor-ligand
couple might be mediated via CD8+ T cells, as we
observed that STZ stimulated these cells to produce large quantities of
IFN- (this study combined with Ref. 27), being a
chemoattractant for macrophages. In agreement with this, anti-CD154
slightly inhibited STZ-induced IFN- excretion by 27% and may thus
contribute to the inhibition of macrophage function by
anti-CD154.
Thus, our results give support to the following concept for dependence
of drug-induced type 1 vs type 2 responses on costimulation. Type 2
responses are initiated by professional APC (48), probably
DC and macrophages (marginal influx, Table I), but, conceivably, Ag
presentation is soon taken over by B cells. This Ag presentation by B
cells strongly depends on CD40-CD154 interaction (49).
Once activated to become effective APC, these B cells may stimulate Th2
cells that, in turn, trigger (the same or other) B cells to produce
IgG1 and IgE. Type 1 responses also start with professional APC
activation, which expresses both CD80 and CD86, but in this situation
DC remain the APC throughout the response. We show that CD86, but not
CD80, expression on macrophage-like DC and the destructive machinery of
these cells are also sensitive to anti-CD154 treatment. On the
other hand, Th1 cell-dependent effector functions
(CD8+ T cell proliferation and IgG2a) seem less
dependent on CD40-CD154 and may somehow be mediated by CD80 ligation.
We cannot exclude that CD86 has an influence on the destructive
macrophage activity or IFN- production by CD8+
T cells.
In summary, our data show that the CD40-CD154 costimulatory interaction is highly important in drug-induced type 2 responses and only marginally involved in the drug-induced type 1 response. We confirm that the PLNA, using a reporter Ag together with adjuvating drugs, provides a suitable model to study specific immune parameters and mechanisms in prototype primary type 1 and type 2 responses. This model has the advantage that these selectively acting drugs condition the immunostimulatory microenvironment in such a way that opposing immune responses arise against the same Ag and on the same genetic background. This allows detection of immune modulation using simple read-out parameters shortly after exposure, thereby minimizing the involvement of complex regulatory mechanisms. However, as different drugs are used to facilitate a specific type of response, we cannot exclude that the specific characteristics of the chemical may be responsible for our observations. In our current experiments we are studying the CD40-CD154 interaction in type 1 and type 2 responses elicited by the same chemical using mice with different H2 haplotypes.
Besides basic immunological mechanistic information, the present findings provide important new insights into the adjuvant activity of allergenic or autoimmunogenic drugs and may help to define early activation molecules as predictive parameters for adverse immune effects of newly developed drugs exerting their effect via either a type 1 or type 2 mechanism.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stefan Nierkens, Department of Immunotoxicology, Institute for Risk Assessment Sciences, Utrecht University, P.O. Box 80176, NL 3508 TD Utrecht, The Netherlands.
3 Abbreviations used in this paper: GC, germinal center; AP, alkaline phosphatase; ASC, Ab-secreting cell; CY, CyChrome; DC, dendritic cell; D-Pen, D-penicillamine; DPH, diphenylhydantoin; PLN, popliteal lymph node; RA-PLNA, reporter Ag PLN assay; STZ, streptozotocin; TNP, trinitrophenyl.
Received for publication November 29, 2001. Accepted for publication February 19, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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J. P. Sanderson, D. J. Naisbitt, J. Farrell, C. A. Ashby, M. J. Tucker, M. J. Rieder, M. Pirmohamed, S. E. Clarke, and B. K. Park Sulfamethoxazole and Its Metabolite Nitroso Sulfamethoxazole Stimulate Dendritic Cell Costimulatory Signaling J. Immunol., May 1, 2007; 178(9): 5533 - 5542. [Abstract] [Full Text] [PDF]
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 J. B. Carey, A. Allshire, and F. N. van Pelt Immune Modulation by Cadmium and Lead in the Acute Reporter Antigen-Popliteal Lymph Node Assay Toxicol. Sci., May 1, 2006; 91(1): 113 - 122. [Abstract] [Full Text] [PDF]
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S. Nierkens, M. Aalbers, M. Bol, R. Bleumink, P. van Kooten, L. Boon, and R. Pieters Differential Requirement for CD28/CTLA-4-CD80/CD86 Interactions in Drug-Induced Type 1 and Type 2 Immune Responses to Trinitrophenyl-Ovalbumin J. Immunol., September 15, 2005; 175(6): 3707 - 3714. [Abstract] [Full Text] [PDF]
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F. van Wijk, S. Nierkens, I. Hassing, M. Feijen, S. J. Koppelman, G. A. H. de Jong, R Pieters, and L. M. J. Knippels The Effect of the Food Matrix on In Vivo Immune Responses to Purified Peanut Allergens Toxicol. Sci., August 1, 2005; 86(2): 333 - 341. [Abstract] [Full Text] [PDF]
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S. Nierkens, M. Aalbers, M. Bol, F. van Wijk, I. Hassing, and R. Pieters Development of an Oral Exposure Mouse Model to Predict Drug-Induced Hypersensitivity Reactions by Using Reporter Antigens Toxicol. Sci., February 1, 2005; 83(2): 273 - 281. [Abstract] [Full Text] [PDF]
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S. Nierkens, L. Nieuwenhuijsen, M. Thomas, and R. Pieters Evaluation of the Use of Reporter Antigens in an Auricular Lymph Node Assay to Assess the Immunosensitizing Potential of Drugs Toxicol. Sci., May 1, 2004; 79(1): 90 - 97. [Abstract] [Full Text] [PDF]
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T. Yasumi, K. Katamura, T. Yoshioka, T.-a. Meguro, R. Nishikomori, T. Heike, M. Inobe, S. Kon, T. Uede, and T. Nakahata Differential Requirement for the CD40-CD154 Costimulatory Pathway during Th Cell Priming by CD8{alpha}+ and CD8{alpha}− Murine Dendritic Cell Subsets J. Immunol., April 15, 2004; 172(8): 4826 - 4833. [Abstract] [Full Text] [PDF]
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C. Sharp, C. Thompson, E. T. Samy, R. Noelle, and K. S. K. Tung CD40 Ligand in Pathogenesis of Autoimmune Ovarian Disease of Day 3-Thymectomized Mice: Implication for CD40 Ligand Antibody Therapy J. Immunol., February 15, 2003; 170(4): 1667 - 1674. [Abstract] [Full Text] [PDF]
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) and with (
) additional i.p.
anti-CD154 treatment. Levels from all drug-exposed mice are
significantly higher than control values. *, Significant differences
(p < 0.05) between group means with and without
anti-CD154 treatment after injection of a specific chemical. The
data presented for each condition are derived from two separate
experiments with four mice per group.
,
significant differences in costimulatory molecule-expressing non-B
cells in drug-exposed groups without anti-CD154 and drug-exposed
groups with anti-CD154. The data presented for each condition are
derived from two separate experiments with four mice per group.