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1
*
INSERM U167, and
U416 Institut Pasteur de Lille, France;
Department of Microbiology, University of Western Australia;
§
Lab. Traitement de Données, Centre O. Lambret, Lille, France; and
¶
Department of Medicine, University of Cincinnati, Cincinnati, OH 45221
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA expression, and frequency of
IFN--producing cells. In contrast, the introduction of Der p 1 in
the alum protocol did not affect IL-4 or Ig isotype responses. The
effect of Der p 1 was specific, since coimmunization with tetanus toxin
fragment C did not affect the profile of the response against Sm28-GST.
Furthermore, inactivation of Der p 1 reduced its ability to modify the
immune response profile, suggesting that its protease activity played
an important role in deviating the immune response. Our results suggest
that the Der p 1 has the ability to modify the profile of an immune
response by modulating the balance between the polarizing cytokines
IL-4 and IFN-. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The observation that many allergens, especially aeroallergens, and
parasite Ags possess proteolytic activities (13, 14, 15), has raised the
possibility that this common property may be essential for their
ability to generate IgE and Th2 cytokine responses. Some direct
evidence showing that the proteolytic enzyme papain stimulates IL-4
gene expression has been provided recently in support of this
hypothesis (16). Also, the possibility that the major allergen of the
house dust mite, Dermatophagoides pteronyssinus (Der p
1),3 may modulate IgE expression through
its ability to cleave membrane CD23 to its soluble form has been
proposed in recent studies (17, 18). However, determination of whether
allergens possess Th2-polarizing capacity in vivo is complicated by the
fact that immunization in adjuvants is often needed to elicit an immune
response against allergens, and adjuvants themselves may influence the
type of the immune response generated (19, 20). In previous studies, it
has been shown that a bystander immune response could influence the
type of the response against an irrelevant Ag (21, 22, 23). We reasoned,
therefore, that it would be possible to test the polarizing capacity of
an allergen in an heterologous immunization system. To do that, we
examined whether coadministration of Der p 1 influenced the immune
response against an irrelevant Ag, for instance, the Schistosoma
mansoni glutathione S-transferase (Sm28-GST). In a
previous study, we showed that immunization with Sm28-GST in CFA
induced a nonpolarized immune response, in contrast to immunization in
aluminum hydroxide (AH), which induced a type 2 immune response against
Sm28-GST (19). In this study, we show that administration of Der p 1
together with Sm28-GST in CFA diminished IFN- production and the
specific anti-Sm28-GST IgG2a Ab response. In addition, this
property of Der p 1 seems to be, in part, dependent on its protease
activity, since inactivated Der p 1 presented reduced capacity to
modulate immune responses against Sm28-GST.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to eight-week-old female BALB/c mice were purchased from IFFA-CREDO (L’Arbresle, France). Mice were maintained under pathogen-free conditions throughout the study.
Ags
Escherichia coli expressing recombinant Sm28-GST was cultured in LB medium, and recombinant protein was purified as previously described (24). Recombinant tetanus toxoid fragment c (TTc) was purchased from Boehringer Mannheim (Meylan, France). Der p 1 was isolated by affinity chromatography from fecally enriched spent mite growth medium as previously described (25). The material eluted from the mAb affinity column was further purified by chromatofocusing and the protein identified by using an insoluble protease substrate (Azocoll; Calbiochem, Sydney, Australia).
Enzymatic activity of Der p 1 was inhibited either by heating (95°C, 1 h) (see 26 , or by adding the cysteine protease inhibitor E-64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane; Sigma, St. Quentin, France), at 7 mol/mol of Der p 1.
Immunizations
Mice were injected s.c. in the base of the tail with 20 µg of recombinant Sm28-GST adsorbed on AH (final concentration 0, 6% w/v) or emulsified (v/v) in CFA (Difco Laboratories, Detroit, MI). In most experiments, 20 µg of Der p 1 were injected/mouse, unless otherwise mentioned. In coimmunization experiments Sm28-GST and Der p 1 were present in the same immunization mixture.
Ab measurement by ELISA
IgG1 and IgG2a Ab response to Sm28-GST were measured by ELISA as previously described (19). Briefly, 96-well plates (Immulon II; Dynatech, Denkendorf, Germany) were coated overnight with Sm28-GST (0.25 µg/well) in 0.1 M carbonate buffer, pH 9.6, at 4°C. After washing, serial dilutions of sera were added, in PBS/0.1% Tween 20/0.5% gelatin for 2 h at room temperature. Plates were then washed and biotin-conjugated goat anti-mouse IgG1 or IgG2a Abs (Southern Biotechnology Associates, Birmingham, AL) diluted at 1/45,000 were added for 1 h at room temperature. After further washes, peroxidase-coupled streptavidin (Jackson ImmunoResearch, West Grove, PA) was added for 30 min. After extensive washing, 50 µl of substrate solution (1 mg/ml orthophenylene diamine, Sigma, St. Louis, MO) was added to the wells. The color reaction was terminated by addition of 50 µl of 3 N HCl, and OD was measured at 492 nm.
Reverse transcription-polymerase chain reaction (RT-PCR)
Spleens and inguinal lymph nodes were removed from mice
after cervical dislocation and stored immediately in RNAzol (Bioprobe,
Montreuil sur Bois, France). RNA was extracted according to the
manufacturer’s instructions. Levels of cytokine mRNA were assessed by
a semiquantitative RT-PCR, as previously described (27). RNA was
reverse transcribed using an RNase H-Reverse Transcriptase (Superscript
II; Life Technologies, Gaithersburg, MD). Briefly, 2.5 µg of RNA were
heated 10 min at 70°C, then mixed with 1 mM of each dNTP (Pharmacia
Biotech., Orsay, France), 0.4 mM of random hexamers (pd(N)6,
Pharmacia), 40 U of RNase inhibitor (Pharmacia), and 200 IU of reverse
transcriptase in reaction buffer supplied by the manufacturer. The
mixture was incubated for 1 h at 37°C and 5 min at 95°C, and
then frozen at -20°C until PCR analysis. PCR was performed on the
resultant cDNA to amplify HPRT, IL-4, IL-10, and IFN-. Reaction
mixtures (25 µl) contained 100 ng cDNA, 0.1 mM of each dNTP, 8 to 80
pmol of each primer, 1.5 to 3.5 mM of MgCl2 (depending on
the cytokine) (see 19 and 1.25 U of Taq DNA polymerase (Promega,
Madison, WI). Amplifications were performed as follows: 94°C, 3 min
and 40 s; followed by 21 to 34 amplification cycles (94°C,
20 s; 54°C, 30 s; 72°C, 45 s) determined so that a
linear relation existed between the amount of input cDNA and the final
PCR product. Resulting cDNA was Southern blotted and hybridized with
32P-labeled probes, and radioactive signals were quantified
with a PhosphorImager (Molecular Dynamics, Evry, France). To compare
different samples, the number of amplification cycles that resulted in
a linear correlation between radioactive signal intensity and input
cDNA was defined for each cytokine, as previously described (19, 27).
To correct for sample variations, cDNA for the housekeeping gene HPRT was initially quantified, and a correction factor was applied to each sample. Signal intensities were then compared with those obtained for samples from control mice, and results were expressed as index of stimulation comparing to the control, arbitrarily considered as 1.
Lymphocyte cultures
Spleens and inguinal lymph nodes were isolated from mice on day
7 after immunization, and cells cultured in a concentration of 4
x 106 cells/ml of RPMI 1640 supplemented with 10% FCS
(Boehringer Mannheim), 2 mM L-glutamine, and 25 mM 2-ME.
Cells were stimulated in triplicate with either 20 µg of Sm28-GST, 20
µg of conalbumin (as a negative control), or 5 µg of Con A (Sigma)
per ml. Supernatants from 72-h cultures were collected and frozen until
IL-4 and IFN- were determined.
Lymphokine measurements by ELISA
Concentrations of IL-2, IL-4 and IFN- were determined by
ELISA. Clones producing mAb for detecting IL-2 (JES-1A12 and JES-5H4),
IL-4 (BVD-4 and BVD-6), and IFN- (R46A2 and AN-18) by ELISA (28)
were kindly provided by Dr. A. O’Garra (DNAX). Ninety-six-well plates
(Nunc, Naperville, IL) were coated 2 h at 37°C with capture Abs
(50 to 100 ng/well) in either carbonate buffer or PBS. After washing
with PBS/0.1% Tween 20, supernatants were diluted 1/2 and added
overnight at 4°C. After washing, 25 to 50 ng of biotin-conjugated Ab
were added per well for 90 min at room temperature. After additional
washing, peroxidase-labeled streptavidin was added for 1 h at room
temperature. This was followed by washing and substrate addition.
Supernatants were compared with standard curves, generated by serial
dilutions of rIL-2, IL-4, and IFN- (PharMingen, San Diego, CA). The
sensitivity of these ELISA ranged from 0.3 to 40 ng/ml for IL-2, 1 to
500 pg/ml for IL-4, and from 60 to 16,000 pg/ml for IFN-.
ELISPOT assays
IL-4- and IFN--secreting cells were evaluated by ELISPOT
assay, as previously described (29). Briefly, 96-well plates (Immulon
II) were coated overnight with capture Abs for IL-4 (BVD-4) or IFN-
(R46A2) (500 ng/well) in PBS at 4°C. Single-cell suspensions from
spleens of immunized mice, or from 3-day cultures with Sm28-GST or
conalbumin, were serially diluted (first concentration of 0.5 x
106 cells/well) and incubated in coated plates overnight at
37°C. After extensive washes in PBS and PBS-Tween 0.1%, 200 ng/well
of biotin-conjugated Abs for IL-4 (BVD-6) or IFN- (XMG-6) were added
for 2 h at 37°C. After additional washing,
alkaline-phosphatase-labeled streptavidin (Jackson ImmunoResearch) was
added for 1 h at 37°C. Then, the substrate solution (1 mg/ml of
5-bromo-4-chloro-3-indolyphosphate (Sigma) in a 0.1 M
2-amino-2-methyl-1 propanol (Sigma)) was mixed with 0.6% Sea-plaque
agarose (FMC Bioproducts, Rockland, ME) and 100 µl of this solution
were added per well. Spots were counted using a dissecting
microscope.
Statistical analysis
Because normality of data distribution was not demonstrated, nonparametric tests (Kruskal-Wallis and Mann-Whitney) were used to evaluate for statistical differences in Ig production and cytokine expression among different groups of mice.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a previous study (19), we demonstrated a strong correlation
between specific IgG1 and IgG2a anti-Sm28-GST Abs and IL-4 and
IFN- expression, respectively, in mice immunized against Sm28-GST in
different adjuvant formulations. Thus, mice immunized with Sm28-GST in
CFA made a nonpolarized response characterized by both IL-4 and IFN-
production and specific IgG1 and IgG2a Abs. When Sm28-GST was
administrated in alum, a type 2 response developed with high IL-4 and
IgG1 anti-Sm28-GST titers. In preliminary experiments, we also
observed that, similarly to Sm28-GST, isotypic profiles against Der p 1
were influenced by the adjuvant used (data not shown). However, when
the CFA was used as adjuvant, the anti-Der p 1 IgG1/IgG2a ratio was
higher than the anti-Sm28-GST IgG1/IgG2a ratio, suggesting that the
immune response against Der p 1 presents a more Th2-like profile. Based
on these results, we examined whether affinity-purified Der p 1 would
influence isotypic profiles against coinjected Sm28-GST in CFA. In
initial experiments, similar isotype profiles were observed in 14-,
21-, and 28-day sera from single-dose immunized mice. Because on day 28
higher anti-Sm28-GST titers were observed, only this time point was
examined in subsequent experiments. In six independent experiments,
average and median IgG2a anti-Sm28-GST titers strongly diminished
in Der p 1-coinjected mice; IgG1 titers also diminished but to a lesser
degree (Fig. 1
). For statistical analysis
we first determined, using the Kruskal-Wallis nonparametric rank test,
that inter-experiment variation was not significant. Based on this,
results from all the experiments were grouped and analyzed together.
Comparison of IgG2a anti-Sm28-GST titers in mice immunized with
both Sm28-GST and Der p 1 or with only Sm28-GST showed that the former
had significantly lower titers (p < 0.01;
Mann-Whitney test). In contrast, IgG1 titers were not statistically
different in the two groups of mice. Also, the IgG1/IgG2a ratio
significantly increased (p < 0.02) in
Sm28-GST/Der p 1-immunized mice (data not shown). To eliminate the
possibility that the decrease in the anti-Sm28-GST titers was due
to antigenic competition, control experiments were performed in which
mice were immunized with Sm28-GST plus TTc. Analysis of sera, 28 days
after immunization, showed that IgG1 and IgG2a anti-Sm28-GST titers
were similar in single- and coimmunized animals (data not shown). To
further confirm that antigenic competition was not the reason of the
diminished anti-Sm28-GST titers in Der p 1-coimmunized animals,
mice were immunized with 10 or 40 µg of Der p 1 plus Sm28-GST, or
with the latter alone, in alum (Th2 protocol). With this protocol, Der
p 1 had a dose-dependent positive effect on IgG1 titers, while IgG2a
anti-Sm28-GST titers remained at baseline levels in all groups of
mice (data not shown). This result was confirmed in four independent
experiments, in which IgG1 titers increased from 1.3- to 3.4-fold in
Der p 1-coimmunized mice. However, no statistically significant
difference was demonstrated. A dose effect of Der p 1 was also observed
in the CFA protocol. As shown in Figure 2, 10 µg of Der p 1 had no effect on
IgG titers; with 20 µg a 2.6-fold decrease of IgG2a was observed, but
no statistically significant difference was demonstrated. In contrast,
with 40 µg of Der p 1 both IgG1 and IgG2a diminished but only the
latter showed a statistically significant decrease
(p < 0.05). In contrast to the effect that the
presence of Der p 1 had on Sm28-GST isotypic profiles, no changes on
Der p 1 Ab profiles were observed in coimmunized mice (data not
shown).
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Der p 1 influences cytokine mRNA expression in vivo
Since IgG2a production is promoted by IFN- and inhibited by
IL-4 (30), these results prompted us to examine whether Der p 1
coimmunization would influence the expression of these cytokines. As
expected from a previous study (19), immunization of mice with Sm28-GST
in CFA induced increased IL-4 and IFN- mRNA expression in draining
lymph nodes 7 days after immunizations (Fig. 3). Similar IL-4 mRNA levels were
expressed in Sm28-GST/Der p 1 and in mice immunized with Sm28-GST
alone. At the same time, IFN- mRNA expression decreased to almost
baseline levels in Sm28-GST/Der p 1 immunized mice. This decrease was
statistically significant (p < 0.02) as
demonstrated with the Mann-Whitney test. Similar results were observed
on day 10 after immunization (data not shown). Because IL-10 has been
shown to inhibit IFN- production (31, 32), the expression of mRNA
coding for this cytokine was also examined in the same mice. In fact,
IL-10 mRNA expression did not increase significantly in either group of
mice on day 7 (Fig. 3) or at any other time point examined (4, 8, 24,
48, and 96 h, and 10 days) (data not shown).
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mRNA
expression, kinetic experiments were performed. As shown in Figure 4, IL-4 mRNA expression increased
similarly, as early as 4 h after immunization, in both groups of
mice. At later time points, however (24 and 48 h), the increase in
IL-4 mRNA in Der p 1 coimmunized mice was larger than in the group
immunized with Sm28-GST alone. Statistical analysis of three
independent experiments showed that the difference observed at 48
h was highly significant (p < 0.01). At the
96- and 168-h time points, similar levels of IL-4 mRNA were observed.
IFN- mRNA expression was similar in both groups of mice until the
96-h time point. At 168 h, however, a highly significant
difference (p < 0.01) in IFN- mRNA
expression was observed between the Sm28-GST and the Sm28-GST/Der p 1
immunized mice. IFN- mRNA increased 10-fold in the former group and
only 2-fold in the latter. These results show that Der p 1 exerts a
positive effect on early IL-4 mRNA and a negative effect on late
IFN- expression. Without excluding other possibilities, this result
suggests that the diminution of Th1-associated response in Der p
1-coimmunized mice could be the result of the early increased
production of IL-4, which inhibits later IFN- expression.
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production in single- and coimmunized mice
Experiments were performed to examine whether the Der p 1 effects
on IFN- and IL-4 mRNA expression resulted in similar changes in the
production of these cytokines. To this end we analyzed the frequency of
IL-4- and IFN--producing spleen cells, in mice immunized with
Sm28-GST or with Sm28-GST plus Der p 1 in CFA or in AH ex vivo, by
ELISPOT assay. Splenocytes of Sm28-GST/Der p 1-immunized mice had a
significantly lower frequency (p < 0.05) of
IFN- production as compared with their counterparts that received
only Sm28-GST in both the alum and the CFA immunization protocol (Fig. 5). In fact, the frequency of
IFN--producing cells dropped even lower than that of the control
mice that received only normal saline. In contrast, the frequency of
IL-4 producing cells was not affected by the presence of Der p 1. To
determine whether the Ag-driven cytokine production was also diminished
in Sm28-GST/Der p 1-immunized mice, spleen cells from 7-day immunized
mice were stimulated in vitro with Sm28-GST for 3 days, and the
frequency of IL-4- and IFN--producing cells was determined by
ELISPOT. We found that Sm28-GST-stimulated cells from both Sm28-GST-
and Sm28-GST/Der p 1-immunized mice showed similar frequencies of IL-4
production (data not shown). In contrast, the frequency of
IFN--producing cells in the latter group decreased approximately
threefold in comparison with that observed in the Sm28-GST-immunized
group. Collectively, these results suggest that Der p 1 diminished the
frequency of IFN- but not that of IL-4-producing cells.
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To determine whether the protease activity of Der p 1 is necessary
for its immunomodulatory effect, experiments with the specific cysteine
protease inhibitor E-64 or heat treatment were performed. In the first
experiment (Fig. 6), we examined whether
inactivated Der p 1 modified isotypic responses against the Sm28-GST in
coimmunization experiments. In this experiment, Sm28-GST-immunized mice
showed a relatively high specific anti-Sm28-GST IgG1/IgG2a ratio of
4.2. In active Der p 1 coimmunized mice, both IgG1 and IgG2a titers
diminished, with the latter showing a slightly larger decrease. When
heat-inactivated Der p 1 was used, both IgG1 and IgG2a titers
increased, but only the IgG2a titers demonstrated a statistically
significant increase when compared with those of mice that received
native Der p 1 (p < 0.05). A similar increase
was observed in the group of mice that received Sm28-GST/Der p 1 in
combination with the specific cysteine protease inhibitor E-64. In
addition, Ab titers in control mice that received Sm28-GST plus E64
were similar to those in mice immunized with Sm28-GST alone, excluding
the possibility that E-64, by itself, affects the Ab response (data not
shown). Next, we examined the effect of heat-inactivated Der p 1 on
IFN- expression. Mice immunized with Sm28-GST alone or with Sm28-GST
plus native Der p 1 or heat-inactivated Der p 1 were killed 7 days
later, and spleen cells were cultured for 3 days with Sm28-GST or
conalbumin in control cultures. Cells from Der p 1-coimmunized mice
produced threefold less IFN- than cells from mice immunized only
with Sm28-GST (Fig. 7). Cells from mice
coimmunized with heat-inactivated Der p 1 showed only a 40% decrease
in their capacity to produce IFN-. Statistical analysis showed that
significant differences existed between all groups of mice. These
results were confirmed when the frequency of cells producing IFN-
was examined by ELISPOT assay. Der p 1 reduced the frequency of
IFN--producing cells by 3.5-fold, in contrast to heat-inactivated
Der p 1 in which reduction was only 1.8-fold (data not shown). Thus,
these results strongly suggest that the immunomodulatory effects of Der
p 1 are, in part, due to its protease activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, this question was studied by comparing the
response induced against an irrelevant Ag, the Sm28-GST, in the
presence or absence of the major domestic allergen, Der p 1, in the
immunization mixture. We have observed that, when both Ags emulsified
in CFA were injected in mice, Der p 1 was able to substantially
decrease the Th1-associated IgG2a response against Sm28-GST. In
addition, analysis of IFN- and IL-4 showed that Der p 1 induced an
imbalance in the expression of these cytokines. In particular, the
expression of mRNA coding for IFN- as well as the frequency of
IFN--producing cells was inhibited. It is of interest that when alum
was used as adjuvant, the frequency of IFN--producing cells in Der p
1/Sm28-GST-immunized mice dropped below the levels observed in control
mice. Since IL-4 and IFN- are antagonistic cytokines that
reciprocally inhibit each other’s expression (33), one would expect
that decreased IFN- expression would result in increased IL-4
expression and IgG1 production. To our surprise, we observed a small
but reproducible decrease in IgG1 production in the CFA protocol. Also,
even though an early (48-h) increase in IL-4 mRNA expression was
observed in Sm28-GST/Der p 1-immunized mice, no significant increase
was observed at later time points at either the mRNA level or in terms
of frequency of IL-4-producing cells. A possibility explaining this
observation is that the Der p 1, in addition to affecting IFN-
expression, also affects the expression of other cytokines that are
involved in stimulating IL-4 expression. A good candidate for that is
IL-2 since: 1) IL-2 has been shown to positively influence development
of the IL-4-producing cells (34, 35), 2) IL-2 is a Th1-associated
cytokine (36), and 3) IL-2 is central in the development of the immune
response. According to this hypothesis, the small diminution of the
type 2 response at either the cytokine or the isotypic level is
secondary to the substantial decrease of the Th1 function and the
diminution of the IL-2 production. Our preliminary experiments support
this hypothesis since Con A-stimulated spleen cells from Sm28-GST/Der p
1-immunized mice showed reduced capacity to produce IL-2, in comparison
with mice immunized with Sm28-GST alone (data not shown). Another
possibility is that the Der p 1 inhibits IL-12 production. IL-12,
through its induction of IFN- production, can substantially increase
synthesis and secretion of IgG2a. In addition, although large and
sustained IL-12 responses inhibit IgG1 production (37), smaller and
less sustained IL-12 responses can promote the secretion of all IgG
isotypes, including IgG1 (38, 39). Thus, Der p 1 inhibition of a
relatively limited IL-12 response would have precisely the effects that
have been observed in the present study: marked inhibition of IgG2a
production and modest inhibition of IgG1, with considerable inhibition
of IFN- and a variable effect on IL-4 production. The proposed
inhibitory effects of Der p 1 on IL-12 might result from Der p 1
induction of a cAMP-activating agent, such as PGE2. In
fact, other allergens (40) have been shown to elicit PGE2,
which through the induction of cAMP inhibit IL-12 production (41).
Several other mechanisms may account for the observed effects of Der p
1 on cytokine expression. A recent observation, showing that Der p 1
induces changes in intracellular calcium flux on cultured human
bronchial epithelial cells (C. King and G. Stewart, manuscript in
preparation), brings up the possibility that Der p 1 affects cytokine
production by inducing similar changes on T or other immunocompetent
cells. Der p 1 may also be acting indirectly by modulating
proinflammatory cytokine production by other cell types. In that
respect, it is of interest that Der p 1 stimulates IL-6 production by
epithelial cells (C. King and G. Stewart, unpublished observations), a
cytokine that has been proposed to promote Th2 differentiation
(42).
In addition of having similar effects in reducing IFN- expression in
both the CFA and the alum protocol, Der p 1 had also distinct effects
in these two protocols, especially on IgG1 production that was slightly
reduced in the first and increased in the latter. A possible
explanation for this is that Der p 1 differentially influence the
cytokine pathways that are activated when mice are immunized using the
CFA or the alum as adjuvants (43).
A possibility explaining the diminished anti-Sm28-GST response in mice immunized with Sm28-GST/Der p 1 in CFA is that the Sm28-GST is digested by the Der p 1, and as a consequence of that, diminished antigenic stimulation is provided. This possibility was examined in control experiments using the reference protease trypsin, which possesses a stronger protease activity than Der p 1. In the first place, we determined that mice coimmunized with Sm28-GST plus trypsin in alum produced sixfold higher anti-Sm28-GST IgG1 titers than mice immunized with Sm28-GST alone (data not shown). In addition, using the azocasein test, we have found that trypsin adsorbed in alum retained its full protease activity. Therefore, the presence of an active protease in the immunization mixture does not prevent the development of the immune response against an associated Ag. Finally, the migration pattern of Sm28-GST as demonstrated by PAGE did not change after incubation with Der p 1 (data not shown), suggesting that Der p 1 is unable to digest the Sm28-GST.
Previous studies have implied that the enzymatic activity of allergens
is important for their antigenic properties. In fact, due to their
enzymatic activity, allergens seem to cause epithelial damage in vitro
(44). This finding implies that allergens may increase bronchial
permeability and thereby enable Ag presentation to occur. It has also
been shown that Der p 1 is able to cleave membrane-associated human
CD23 to its soluble form (17, 18). Since soluble CD23 positively
influences IgE production (45, 46), it was proposed that through this
mechanism, Der p 1 stimulates IgE responses. In a recent study,
evidence was presented showing that the allergenicity of the bee venom
phospholipase A2 is dependent on its lipolytic activity,
through which phospholipase A2 induces mast cell
degranulation and IL-4 secretion (40). In addition to the above
findings, our study is the first to suggest that, due to its protease
activity, an allergen, Der p 1, is able to modulate the immune response
at an additional level, by influencing the balance between the
antagonistic cytokines IL-4 and IFN-. However, since in most
experiments Der p 1 was inactivated by heat treatment, which may affect
the three-dimensional structure and the aggregation level of the Der p
1, we cannot exclude the possibility that the enzymatic activity of Der
p 1 is not the only mechanism by which this protein affects cytokine
expression. The use of recombinant Der p 1 lacking enzymatic activity
may clarify this question.
It is noteworthy that Der p 1 was able to modulate the immune response against an irrelevant Ag in both immunization protocols. In the light of our results, it is likely that an initial allergic reaction may positively influence the development of type 2 responses against additional Ags. This may explain in part why many atopic individuals develop allergies to multiple substances. We are presently examining this issue with experiments that use SCID mice reconstituted with PBL from allergic individuals (47).
In conclusion, our data suggest that the major allergen, Der p 1, has
the capacity 1) to induce type 2 responses by modifying the
IL-4/IFN- balance in favor of the former and thereby to influence
isotypic profiles, and 2) to influence a bystander immune response;
also its protease activity is important in modulating the immune
response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Georges Thyphronitis at the present address; Department of Pathophysiology, Medical School, University of Athens, 11527 Goudi, Greece. E-mail address:
3 Abbreviations used in this paper: Der p 1, Dermatophagoides pteronyssinus; Sm28-GST, Schistosoma mansoni glutathione S-transferase; AH, aluminum hydroxide; TTc, tetanus toxoid fragment c; HPRT, hypoxanthine phosphoribosyltransferase; ELISPOT, enzyme-linked immunospot.
Received for publication May 12, 1997. Accepted for publication October 24, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Proc. Natl. Acad. Sci. USA 86:5580. and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science 236:944.This article has been cited by other articles:
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C. Ebeling, T. Lam, J. R. Gordon, M. D. Hollenberg, and H. Vliagoftis Proteinase-Activated Receptor-2 Promotes Allergic Sensitization to an Inhaled Antigen through a TNF-Mediated Pathway J. Immunol., September 1, 2007; 179(5): 2910 - 2917. [Abstract] [Full Text] [PDF]
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Y. Kikuchi, T. Takai, T. Kuhara, M. Ota, T. Kato, H. Hatanaka, S. Ichikawa, T. Tokura, H. Akiba, K. Mitsuishi, et al. Crucial Commitment of Proteolytic Activity of a Purified Recombinant Major House Dust Mite Allergen Der p1 to Sensitization toward IgE and IgG Responses J. Immunol., August 1, 2006; 177(3): 1609 - 1617. [Abstract] [Full Text] [PDF]
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L. U. Buxbaum, H. Denise, G. H. Coombs, J. Alexander, J. C. Mottram, and P. Scott Cysteine Protease B of Leishmania mexicana Inhibits Host Th1 Responses and Protective Immunity J. Immunol., October 1, 2003; 171(7): 3711 - 3717. [Abstract] [Full Text] [PDF]
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F. Kheradmand, A. Kiss, J. Xu, S.-H. Lee, P. E. Kolattukudy, and D. B. Corry A Protease-Activated Pathway Underlying Th Cell Type 2 Activation and Allergic Lung Disease J. Immunol., November 15, 2002; 169(10): 5904 - 5911. [Abstract] [Full Text] [PDF]
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 S. Saarelainen, T. Zeiler, J. Rautiainen, A. Narvanen, M. Rytkonen-Nissinen, R. Mantyjarvi, P. Vilja, and T. Virtanen Lipocalin allergen Bos d 2 is a weak immunogen Int. Immunol., April 1, 2002; 14(4): 401 - 409. [Abstract] [Full Text] [PDF]
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 H. Hammad, A.-S. Charbonnier, C. Duez, A. Jacquet, G. A. Stewart, A.-B. Tonnel, and J. Pestel Th2 polarization by Der p 1-pulsed monocyte-derived dendritic cells is due to the allergic status of the donors Blood, August 15, 2001; 98(4): 1135 - 1141. [Abstract] [Full Text] [PDF]
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K. G. Tournoy, J. C. Kips, and R. A. Pauwels The Allergen-Induced Airway Hyperresponsiveness in a Human-Mouse Chimera Model of Asthma Is T Cell and IL-4 and IL-5 Dependent J. Immunol., June 1, 2001; 166(11): 6982 - 6991. [Abstract] [Full Text] [PDF]
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 O. Fahy, H. Hammad, S. Sénéchal, J. Pestel, A.-B. Tonnel, B. Wallaert, and A. Tsicopoulos Synergistic Effect of Diesel Organic Extracts and Allergen Der p 1 on the Release of Chemokines by Peripheral Blood Mononuclear Cells from Allergic Subjects . Involvement of the MAP Kinase Pathway Am. J. Respir. Cell Mol. Biol., August 1, 2000; 23(2): 247 - 254. [Abstract] [Full Text]
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 C. DUEZ, J. KIPS, J. PESTEL, K. TOURNOY, A.-B. TONNEL, and R. PAUWELS House Dust Mite-induced Airway Changes in hu-SCID Mice Am. J. Respir. Crit. Care Med., January 1, 2000; 161(1): 200 - 206. [Abstract] [Full Text]
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 L. Gough, O. Schulz, H. F. Sewell, and F. Shakib The Cysteine Protease Activity of the Major Dust Mite Allergen Der P 1 Selectively Enhances the Immunoglobulin E Antibody Response J. Exp. Med., December 20, 1999; 190(12): 1897 - 1902. [Abstract] [Full Text] [PDF]
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