From the Forschungszentrum Borstel, D-23845 Borstel,
Germany, the § School of Biological Sciences, University of
Wales, Bangor, Gwynedd, LL57 2UW, United Kingdom, ¶ Centro de
Pesquisas René Rachou-FIOCRUZ, Belo Horizonte, Minas Gerais
30190-002, Brazil, and the
Institute of Immunology and
Allergology, Inselspital, University Hospital Bern,
CH-3010 Bern, Switzerland
Received for publication, January 16, 2003, and in revised form, March 4, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The eggs of the parasitic trematode
Schistosoma mansoni are powerful inducers of a T helper
type 2 (Th2) immune response and immunoglobulin E (IgE) production.
S. mansoni egg extract (SmEA) stimulates human basophils to
rapidly release large amounts of interleukin (IL)-4, the key promoter
of a Th2 response. Here we show purification and sequence of the
IL-4-inducing principle of S. mansoni eggs (IPSE).
Stimulation studies with human basophils using SmEA fractions and
natural and recombinant IPSE as well as neutralization and
immunodepletion studies using antibodies to recombinant IPSE
demonstrate that IPSE is the bioactive principle in SmEA leading to
activation of basophils and to expression of IL-4 and IL-13. Regarding
the mechanism of action, blot analysis showed that IPSE is an
IgE-binding factor, suggesting that it becomes effective via
cross-linking receptor-bound IgE on basophils. Immunohistology revealed
that IPSE is enriched in and secreted from the subshell area of the
schistosome egg. We conclude from these data that IPSE may be an
important parasite-derived component for skewing the immune response
toward Th2.
Infection with the parasitic trematode Schistosoma
mansoni leads to a pronounced
Th21 response and to elevated
IgE production both in humans and in experimental animals. The
definition of parasite-derived products capable of skewing the immune
response toward Th2 would not only enhance our understanding of the
defense mechanisms involved in helminth infections but may also lead to
new insights into the pathogenesis of immediate-type hypersensitivity
diseases such as asthma. However, in contrast to our increasing
understanding of how pathogen-derived products can initiate Th1-type
immune responses, there is so far little detailed knowledge about the nature of the parasite-derived molecule(s) and the underlying mechanisms that trigger and/or amplify a Th2-type reaction. In S. mansoni infection, a critical role in inducing a polarized Th2
response is played by the egg stage of the parasite (1), since a Th2
response and IgE production are only observed after egg deposition or
following injection of schistosome eggs (2) or extracts thereof (3)
into naive animals. By contrast, the initial larval (schistosomula) and
adult worm stages rather induce a response skewed to Th1.
It is now firmly established, both in vivo and in
vitro, that the cytokine profile present during an immune reaction
is an important element in directing the response to Th1 or Th2 and that IL-4 is the key cytokine responsible for biasing the immune reaction toward a Th2 phenotype (4-7). In the human system, basophils are a prominent source of IL-4 and IL-13; these cells secrete large
amounts of IL-4 and IL-13 in response to IgE-receptor cross-linking or
activation by a combination of IL-3 and C5a (8, 9). Indeed, human
basophils can be viewed as "innate Th2-type" effector cells, since
IL-4 and IL-13 are expressed in a very restricted manner without
production of any of the cytokines involved in Th1-type immune
responses. We therefore wondered whether saline-soluble S. mansoni egg antigen extract (SmEA) can directly trigger human blood basophils to release IL-4, thus providing a potential mechanism for biasing the immune response to Th2 during the egg stage of S. mansoni infestation. Indeed, our previous studies showed that basophils from healthy nonsensitized donors from Northern Europe rapidly degranulate and release histamine, sulfidoleukotrienes, and
considerable amounts of IL-4 as well as IL-13 in response to exposure
to SmEA (10) but not to extracts of
schistosomula.2 SmEA thus
obviously contains a basophil-degranulating IL-4-inducing principle,
prompting us to further characterize this activity. Subsequent studies
revealed that the active principle is an excretory/secretory glycoprotein interacting with IgE on basophils in an
antigen-nonspecific way (11).
Here we present the isolation, sequencing, and cloning of the bioactive
product, which was named IL-4-inducing principle from S. mansoni eggs (IPSE). Finally, as unambiguous proof of the identity of the cloned S. mansoni product, we demonstrate that the
recombinant protein has full biological activity.
Antigens
S. mansoni eggs were purified as previously described
(12). SmEA was prepared by the methods of Boros and Warren (13) and
Carter and Colley (14) and was obtained from Dr. F. Yousif (Schistosome
Biological Supply Program, Theodor Bilharz Research Institute,
Imbaba, Egypt).
Purification, Culture, and Stimulation of Basophils
Human basophils were purified by a previously described
three-step procedure (15) and used at a mean purity of 99%. Purified basophils were incubated in round-bottom 96-well microtiter plates (Nunc, Roskilde, Denmark) at 37 °C in humidified air containing 6% CO2 at 106 basophils/ml in a final volume
of 100 µl of culture medium (Iscove's modified Dulbecco's medium
supplemented with 100 units/ml penicillin G, 50 µg/ml transferrin, 5 µg/ml insulin, 100 µg/ml streptomycin, 10% fetal calf serum).
Since IL-3 enhances Fc Cytokine and Histamine Assays
IL-4 and IL-13 levels in culture supernatants were determined
using two-site sandwich enzyme-linked immunosorbent assays (Eli-Pair; Diaclone, Besancon, France) essentially according to the
manufacturer's protocol. The sensitivities for IL-4 and IL-13
detection were 0.55 and 1.56 pg/ml, respectively. Histamine was
determined using the methyl-histamine radioimmune assay (Amersham
Biosciences) according to the manufacturer's recommendations.
SDS-PAGE and Blotting
Proteins were separated by SDS-PAGE (12% T, 4% C) under
reducing and nonreducing conditions according to Laemmli (17). For protein detection on the gels, silver staining was performed (18). For
blotting analysis, separated proteins were transferred onto nitrocellulose membrane (Schleicher & Schuell) via semidry blotting (30 min, 0.8 mA/cm2) (19). Free binding sites were blocked by
incubating the membrane with 0.05% (v/v) Tween 20 in 0.1 M
Tris-buffered saline, pH 7.4. The membranes were then incubated with
alkaline phosphatase-labeled Aleuria aurantia agglutinin
(AAA; 1:5000; Vector Laboratories, Burlingame, CA) or with antibodies
(at the concentrations indicated) in 0.1 M Tris-buffered
saline, pH 7.4, containing 0.05% (v/v) Tween 20. Lectin and antibody
binding was visualized by a substrate/chromogen mixture of 0.033%
(w/v) nitro blue tetrazolium and 0.017% (w/v) 5-bromo-4-chloro-3-indolyl phosphate (Serva, Heidelberg, Germany) in
0.1 M Tris-buffered saline, pH 9.5 (20).
Purification of IPSE
IPSE was purified by cation exchange and affinity
chromatography. First, 2 mg of SmEA in 20 mM potassium
phosphate buffer, pH 5.0, were bound to a HiTrap SP-Sepharose column (1 ml; Amersham Biosciences). Elution of bound proteins was carried out
using a linear salt gradient from 0 to 1 M KCl. Effluent
and eluate fractions were analyzed for their IL-4-inducing effect
(applied volume: 5 µl/106 basophils) and (after speedvac
concentration) for their protein banding pattern. The latter was
determined by SDS-PAGE followed by silver staining. Fractions
containing IPSE were pooled and concentrated, and for affinity
chromatography, the buffer was exchanged to 50 mM sodium
phosphate buffer, pH 7.0, by using Centricon Plus-20 centrifugal
filters (Millipore Corp., Bedford, MA).
For affinity chromatography, AAA (2 mg; Vector Laboratories) was
coupled to N-hydroxysuccinimide-activated Sepharose
HiTrap columns (Amersham Biosciences) according to the manufacturer's instructions. After application of pooled IPSE-containing fractions and
washing, bound material was eluted with 0.1 M
L-fucose in 50 mM sodium phosphate buffer, pH
7.0. Fractions containing IPSE were pooled, concentrated by Centricon
Plus-20 centrifugal filters, and stored at N-terminal Sequencing of Purified IPSE
N-terminal sequencing of the first 20 aa of the purified IPSE
was performed on an Procise 492 Sequencer (Applied Biosystems) at
Chromatec Ltd. (Greifswald, Germany).
S. mansoni Egg cDNA Libraries (SmE-libraries)
SmE-library A--
mRNA was isolated from 1 × 106 S. mansoni eggs using the µMACS mRNA
isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. 3 µg of mRNA were obtained and used for construction of a cDNA library in SmE-library B--
The other SmE-library was constructed as
previously described (21). This library has been extensively used for
the generation of egg stage expressed sequence tags (ESTs) (22).
Screening of SmE-libraries Using a Dig-labeled
Oligonucleotide
For screening of the cDNA libraries, a sequence-specific
30-mer oligonucleotide (for sequence, see Fig. 3) was labeled with Dig-ddUTP using the Dig oligonucleotide 3'-end labeling kit (Roche Diagnostics). The labeled oligonucleotide was detected via an AP-conjugated anti-Dig antibody (1:5000; Roche Diagnostics). Plaque lifts and hybridization were performed as described by Sambrook and
Russell (23). Hybridization was carried out overnight at 55 °C in 5× SSC, 1% (w/v) blocking reagent (Roche Diagnostics), 0.1% (w/v) N-lauroylsarcosine, 0.02% (w/v) SDS. Prior to
detection of bound oligonucleotide, filters were washed twice for 5 min at room temperature with 2× SSC, 0.1% (w/v) SDS and twice for 15 min
at 55 °C with 0.2× SSC, 0.1% (w/v) SDS. Positive phage clones were
isolated, and the pBluescript SK plasmids containing the inserts were
obtained by in vivo excision using the helper phage Exassist
(Stratagene). Automated sequencing of double-stranded plasmid DNA was
performed by cycle sequencing using the Big Dye Terminator Cycle
Sequencing ready Reaction Kit on an ABI 377 DNA sequencer (PerkinElmer
Life Sciences).
Expression and Purification of Recombinant IPSE in
Escherichia coli
Recombinant IPSE was expressed as a His tag fusion protein
(His-IPSE) in E. coli. cDNA coding for IPSE was
subcloned into the pProEX HTb expression vector (Invitrogen)
coding for 6 N-terminally located histidine residues and a cleavage
site for TEV protease. For this purpose, PCR was performed with
IPSE-specific primers containing an AT clamp and the following
restriction sites (underlined): EheI for the sense primer
(5'-ATATATGGCGCCGATTCATGCAAATATTGTC-3') and
HindIII for the antisense primer
(5'-ATATATAAGCTTTCATCAGTTCATATGC-3'), respectively.
Conditions for PCR were as follows: denaturation for 30 s at
95 °C, annealing for 30 s at 44 °C, and extension for 1 min
at 72 °C (30 cycles). Subcloning and confirmation of DNA sequences
followed standard procedures (23). E. coli cultures containing the recombinant plasmid were grown at 37 °C to an
A600 of 0.8, and then expression of His-IPSE was
induced by 0.6 mM isopropyl thiogalactoside. After
incubation for an additional 4 h, cells were harvested by
centrifugation and resuspended in 30 ml 50 mM Tris/HCl, pH
8.0, 0.8 mM EDTA, 200 mM NaCl, 25% sucrose. His-IPSE was recovered as insoluble inclusion bodies after breaking the
cells by passaging them twice through a French press (at a gauge
reading of 1000 p.s.i.) and subsequent centrifugation at 7000 × g. The insoluble fraction was washed once with 20 mM Tris/HCl, pH 8.0, 0.5% deoxycholate and once with 20 mM Tris/HCl, pH 8.0, and subsequently solubilized in 8 M urea, 500 mM NaCl, 50 mM sodium phosphate, pH 7.5. His-IPSE was purified by nickel-nitrilotriacetic acid metal affinity chromatography (Qiagen, Hilden, Germany) under denaturing conditions according to the manufacturer's protocol. Using
the Hampton Research FoldIT Screen (Hampton Research, Laguna Niguel,
CA), optimal buffer conditions for refolding of the denatured His-IPSE
were obtained. Briefly, purified His-IPSE was refolded by dialysis at a
concentration of 0.1 mg/ml against FoldIT buffer 3 (modified to pH
10.5) and subsequently against a physiological buffer such as
phosphate-buffered saline (PBS).
Preparation of Monoclonal Antibodies and Murine Antiserum to
IPSE
Monoclonal antibodies and murine antiserum to His-IPSE were
raised by Labsoft Diagnostics AG (Halle, Germany). To obtain monoclonal antibodies, aliquots containing 28 µg of His-IPSE emulsified in 100 µl of saline (154 mM) and 300 µl of complete Freund's
adjuvant were intraperitoneally administered to 10-week-old female
BALB/c mice. The intraperitoneal immunization was repeated after 9 weeks (28 µg of antigen with incomplete adjuvant). The third (after further 2 weeks) and the fourth injection (3 days thereafter) of IPSE
(each 28 µg) were administered in saline (murine antiserum to IPSE
was raised by a similar immunization protocol). One day after the last
booster injection, the animals were killed and blood was taken for
antiserum preparation. The fusion was performed according to a standard
procedure using the mouse nonsecretory cell line P3X63Ag8.653 as fusion partner.
Immunohistological Detection of IPSE
Liver sections (formalin-fixed, paraffin-embedded material) of
mice infected with 150 S. mansoni cercariae 7 weeks
previously were incubated overnight with the monoclonal anti-IPSE
antibody 74 1G2 (undiluted culture supernatant) or (as negative
controls) with murine IgG (100 µg/ml in PBS; Dianova, Hamburg,
Germany) or PBS only. Subsequently, the sections were incubated for
1 h with AP-conjugated F(ab')2 goat anti-mouse IgG
antibodies (1:100; Dianova), followed by staining with Neufuchsin
(Dako, Hamburg, Germany) and hematoxylin (Merck). All steps were
carried out according to routine protocols.
Computer Analysis and Nucleotide Sequence Accession Number
Sequence data were analyzed with DNASIS software (Amersham
Biosciences). The GenBankTM data base (NCBI) was searched
for homologous sequences using the nucleotide and protein BLAST
programs. Secondary structure predictions were performed online using
the "Protein Predict server" available on the World Wide Web at
maple.bioc.columbia.edu/predictprotein. The nucleotide sequence of
IPSE was submitted to the GenBankTM data base and has been
assigned GenBankTM accession number AY028436.
Fractionation of SmEA by Cation Exchange Chromatography--
The
optimal pH for binding the IL-4-inducing activity to the cation
exchange matrix (SP-Sepharose) was determined in pilot experiments (not
shown). At pH 5.0, nearly all activity was retained by the matrix,
whereas the majority of irrelevant proteins were found in the effluent.
A linear salt gradient (0-1 M KCl) was employed for
eluting the bound material. Under these conditions, the peak of the
IL-4-inducing activity was found to elute within the eluate fractions
representing only a minor proportion of the total protein (Fig.
1, a and b).
To assign the IL-4-inducing activity to a particular component,
SDS-PAGE analysis and extended silver staining of speedvac-concentrated chromatographic fractions of SmEA were performed under nonreducing conditions, revealing a faint diffuse double band at 40 kDa that shifted to about 20 kDa under reducing conditions. The staining intensity of the 40-kDa double band correlated with the IL-4-inducing activity of the active fractions (not shown). Previous molecular mass determinations of the IL-4-inducing activity using
electroelution of gel sections of SmEA separated by SDS-PAGE indicated
a range between 31 and 66 kDa (11), further suggesting that the 40-kDa double band or one of its components might represent the bioactive principle.
The IL-4-inducing Principle Binds to A. aurantia
Agglutinin--
Earlier investigations had indicated that the active
principle is a protein because of its sensitivity to protease digestion (11). Since schistosome extracts contain numerous glycosylated components (24), we asked whether the active principle is a glycoprotein and, thus, could be purified by lectin affinity
chromatography. To address this point, pooled active fractions from
cation exchange chromatography were separated by SDS-PAGE, blotted onto
nitrocellulose membrane, and incubated with a panel of nine labeled
lectins with various glycan specificities. Indeed, two lectins bound to
the 40-kDa double band (the putative active principle). One of them, AAA, seemed most suitable for affinity chromatography, since it reacted
only with one additional irrelevant band of ~30 kDa that appeared in
the late active fractions and was also present in the adjacent inactive
fractions (data not shown). To exclude this irrelevant band, only the
early active fractions were pooled for further purification. On AAA
affinity chromatography, the effluent fractions were inactive, whereas
the fractions eluted with 0.1 mM L-fucose
contained IL-4-inducing activity (Fig. 2,
a and b). SDS-PAGE followed by silver staining
(Fig. 2c) or by blotting with labeled AAA (Fig.
2d) revealed that the eluate fractions contained only the
double band that correlated in its intensity with the IL-4-inducing
activity of the respective fractions. The eluate fractions did not
contain other components detectable by staining, indicating a high
purity of the isolated material. Thus, the active principle is most
likely a glycoprotein of ~40 kDa containing N-terminal Sequencing of IPSE--
Attempts to separate the two
bands at 40 kDa were not successful. Nevertheless, N-terminal
sequencing of the pooled eluate fractions from AAA-Sepharose revealed
an unequivocal sequence with minimal background for the first 20 N-terminal aa, suggesting that the double band represents different
posttranslational forms of the same protein. Two aa in
positions 3 and 6 could not be identified due to a low signal
consistent with the presence of cysteines, as later confirmed by
sequencing the IPSE cDNA (Fig. 4).
A similarity search did not reveal any sequences with significant
homology in the public databases, with the exception of one EST (NCBI
nucleotide data base, NCBI accession number AI820476) from an S. mansoni egg cDNA library, sharing an overall similarity of
81% with the N-terminal sequence and 100% identity for aa 7-16.
Isolation of IPSE cDNA from S. mansoni Egg cDNA
Libraries--
To obtain the complete nucleotide sequence, two
different S. mansoni egg cDNA libraries (SmE-library A
and SmE-library B) were screened via plaque lift assay by
hybridization. A nondegenerate 30-mer oligonucleotide spanning 10 homologous nucleotide triplets coding for aa 7-16 was chosen as a
probe (Fig. 3). A total of 12 identical
clones was isolated (six from each library). The complete sequence
(Fig. 3) consisted of 489 nucleotides. Sequence analysis revealed an
open reading frame of 405 nucleotides with a predicted coding capacity
for 134 aa including a signal sequence of 20 aa. The mature protein of
114 aa had two putative N-glycosylation sites at aa 44 and
60, respectively, and seven cysteine residues. Importantly, all 20 aa
of the predicted N terminus of the mature protein completely matched
the aa sequence determined by N-terminal sequencing of the purified
natural product. Upon screening the NCBI protein and nucleotide data
banks, there was a sequence identity of 92% with the EST sequence
mentioned above but no other significant similarities, indicating that
related proteins have not yet been identified. The molecular mass of
the active principle calculated from the sequence was 13.2 kDa, which
is far below the apparent molecular mass of the unreduced purified
double band at 40 kDa.
Expression of Recombinant IPSE--
To confirm that IPSE
cDNA encoded the active principle, recombinant IPSE was expressed
in E. coli as a His tag fusion protein. His-IPSE was
recovered from inclusion bodies under denaturing conditions in 8 M urea and purified via nickel-nitrilotriacetic acid metal
affinity chromatography. The protein was refolded by dialysis against
the FoldIT buffer 3 (Hampton Research) adjusted to pH 10.5 and then
against PBS. Refolding was performed at a low protein concentration
(0.1 mg/ml), because higher concentrations led to severe protein losses
due to precipitation. As assessed by SDS-PAGE under nonreducing
conditions, refolded His-IPSE formed dimers that could be reversed into
monomers under reducing conditions (Fig.
4a). When tested on human
basophils, His-IPSE had a pronounced IL-4-inducing activity (Fig.
4b) and was in this respect equivalent to its natural
counterpart. The dose-response curve had its maximum at 24 ng/ml or
0.75 nM, respectively, and displayed a bell-shaped form
that is similar to that of IgE-mediated basophil activation.
The identity of the recombinant protein with natural IPSE was further
proven by the following experiments. A mouse antiserum raised against
His-IPSE was strongly reactive with both recombinant and natural IPSE
but showed no cross-reactivity to other components of SmEA (Fig.
5a). Moreover, when basophils
were stimulated with SmEA or (data not shown) with His-IPSE, the
addition of a murine antiserum against His-IPSE
dose-dependently inhibited IL-4 production (Fig.
5b). Finally, immunodepletion using the IgG fraction of murine anti-His-IPSE antiserum (10 µl) bound to protein G-Sepharose (4 Fast Flow; Amersham Biosciences; 100 µl of gel slurry as obtained from the manufacturer) removed the IL-4-inducing capacity from SmEA
(100 µg). In contrast, the IgG fraction of normal mouse serum as a
control had no effect (data not shown). Taken together, our data
clearly demonstrate that the recombinant material corresponds to
IPSE.
IPSE Is the Principal Bioactive Component of SmEA and a General
Activator of Human Basophils--
SmEA leads to a general activation
of basophils including degranulation and mediator release (10). To
study whether all of these bioactivities are due to IPSE, basophils
were incubated with whole SmEA, pooled SmEA fractions, and recombinant
His-IPSE in parallel (Fig. 6). Pools 1 and 2, corresponding to effluent and early eluate fractions,
respectively, from cation exchange chromatography, were devoid of
IPSE. Pool 3, corresponding to the late eluate fractions, contained
IPSE at a purity of at least 70%. Degranulation was observed when
basophils were treated with SmEA anti-IgE, pool 3, or recombinant IPSE
but not following incubation with pool 1 or 2. Degranulated basophils
often formed considerable cell clusters, obviously due to
agglutination. With regard to mediator release, pool 3 and recombinant
IPSE, like SmEA and anti-IgE, induced discharge of histamine, IL-4, and
IL-13, whereas pools 1 and 2 did not have any effect. Note that the
production of IL-13 was studied in the absence of IL-3 because the
latter cytokine alone induces considerable production of IL-13.
Together with the complete abrogation of SmEA-induced IL-4 induction
through murine anti-His-IPSE antiserum (see above), these findings
strongly indicate that IPSE is the only bioactive component in SmEA
that activates basophils for degranulation, mediator release, and
expression of immunoregulatory cytokines.
IPSE Is an IgE-binding Factor--
Earlier studies had
demonstrated that IgE has to be present on basophils for IPSE to become
effective. To elucidate whether IPSE might act as an IgE-binding
factor, blotting experiments were performed, where His-IPSE was
submitted to SDS-PAGE, transferred to nitrocellulose membrane, and
incubated with various concentrations of human IgE. Subsequent
incubation with AP-conjugated anti-human-IgE antibody revealed that IgE
was dose-dependently bound by IPSE. Analogous results were
obtained when solid-phase IgE was incubated with increasing amounts of
His-IPSE (Fig. 7). Biacore experiments confirmed these findings.3
Thus, these results clearly indicate that IPSE is an IgE-binding factor.
IPSE Is Enriched in and Secreted from the Subshell Area of the
Schistosome Egg--
We had earlier observed that IL-4-inducing
activity is released from live schistosome eggs kept overnight at
37 °C in culture medium (11), suggesting that IPSE is a secreted
molecule. To further address this point, immunohistological
localization of IPSE was performed in formalin-fixed liver sections of
schistosome-infected mice using the monoclonal anti-His-IPSE antibody
74 1G2. This antibody stains the subshell area of schistosome eggs and
binds to a variety of circumoval inflammatory cells both
intracellularly and/or extracellularly. Interestingly, the subshell
area stained strongest in eggs that were surrounded by relatively few
inflammatory cells (developing granulomas?), with the cells themselves
being not or minimally IPSE-positive. In contrast, the eggs stained weaker in large advanced granulomas, with many circumoval cells being
strongly positive. This suggests that during granuloma development IPSE
is continuously taken up and accumulated by circumoval cells. Analogous
results (not shown) were obtained with a polyclonal rabbit antiserum to
natural IPSE. On the other hand, the antibodies did not nonspecifically
bind to liver cells or structures outside the egg granulomas. Moreover,
there was no nonspecific binding of normal murine IgG (not shown)
and/or the second antibody, respectively, to schistosome eggs or
inflammatory cells (Fig. 8). In line with these findings, Ashton et al. (25) recently demonstrated
that an antiserum raised against excretory/secretory components from schistosome eggs primarily stained the subshell area. Taken together, we conclude that IPSE is enriched in the subshell area of the schistosome egg from which it is secreted into the surrounding tissue, thereby interacting with a variety of circumoval
inflammatory cells possibly being internalized by some of them.
Here we describe the purification, N-terminal sequence analysis,
cloning, and recombinant expression of IPSE, a glycoprotein secreted
from S. mansoni eggs. IPSE triggers basophils from naive human donors to rapidly degranulate, release mediators, and express IL-4 and IL-13, the key cytokines controlling a Th2-type immune response and IgE synthesis.
The protocol for purifying IPSE was a combination of cation exchange
chromatography and affinity chromatography with the lectin AAA. The
purified material had a high specific activity and consisted of two
bands of about 40 kDa as assessed by nonreducing SDS-PAGE. The purity
of the natural bioactive material was further demonstrated by
microsequencing a preparation containing the two bands, which revealed
one unequivocal N-terminal aa sequence, suggesting that both represent
posttranslational variants of the same protein. This conclusion was
supported by the finding that all clones (n = 12)
obtained from the two libraries were 100% identical.
For probing the SmE-libraries, a nondegenerate 30-mer oligonucleotide
was used, corresponding to a region of 10 aa that was identical between
the N-terminal sequence of purified IPSE and the homologous S. mansoni EST retrieved from the data base. Thus, if present, the
cDNA corresponding to the EST should also have been recovered.
However, the fact that from two different SmE-libraries 12 identical
clones corresponding to IPSE were isolated suggests that the respective
sequence represents a dominant form of this molecule, whereas another
highly homologous S. mansoni gene either does not exist or
is expressed in the egg stage only at a very low level.
The mature protein consists of 114 aa, without relevant sequence
homologies with known proteins. Secondary structure predictions (PHD/PROF on the World Wide Web at
maple.bioc.columbia.edu/predictprotein) indicate that the IPSE monomer
forms a compact globular structure composed mainly of Clear evidence that the recombinant protein corresponds to IPSE came
from functional studies. Like natural IPSE, the recombinant analogue
triggered degranulation of basophils and caused release of histamine,
IL-4, and IL-13. The high specific activity of His-IPSE (maximum at
0.75 nM) was in the same range as that of the natural protein. Moreover, the fusion protein formed homodimers just like natural IPSE. Cell activation experiments with whole SmEA, pooled SmEA
fractions, and recombinant IPSE, respectively, strongly suggested that
basophil activation by SmEA was due to IPSE and not to other components
of SmEA. In line with these findings, agglutination of basophils
correlated with the presence of IPSE and not the other components.
Moreover, antibodies raised against His-IPSE were reactive with natural
IPSE (Western blot) and functionally neutralized as well as
immunodepleted natural IPSE from SmEA.
Although IPSE seemed to be a completely novel protein, the similarity
of its appearance in cation exchange fractions and its apparent
molecular weights under reducing and nonreducing conditions as well as
its glycosylation suggested that IPSE might be identical with antigen
It has been proposed that carbohydrate components of SmEA down-regulate
Th1 responses (27) and are also required for SmEA-stimulated Th2
induction. The latter was demonstrated in a murine model of intranasal
sensitization with deglycosylated (periodate-treated) SmEA that,
compared with the native material, failed to induce IL-4, IL-5, IL-10,
and SmEA-specific IgE production (28). Moreover, lacto-N-fucopentaose III, a predominant glycan component of
SmEA that contains Lewisx (Lex), has been found
to function as a Th2-inducing adjuvant in mice when covalently linked
to human serum albumin (29). Since natural IPSE binds to AAA, a lectin
specific for Regarding the mechanism by which IPSE activates basophils, earlier IgE
stripping and resensitization experiments had revealed that IgE has to
be present on the cells for IPSE to exert its effect (10, 11),
suggesting that this schistosome-derived molecule acts via
cross-linking of Fc The type of interaction between IPSE and IgE is not yet known.
Basically, it could be an antigen-specific, lectin-like, or B cell
superantigen-like interaction. Antigen-specific binding is highly
unlikely, since the basophils were from nonsensitized donors, and, in
the case of the recombinant molecule, potentially cross-reactive
carbohydrates were not involved. The possibility that IPSE acts as a
lectin cannot be excluded, but its sequence does not show any
similarity with known animal or plant lectins. Furthermore, although
several lectins can activate basophils (32), the concentrations
required are in the µg/ml range. In contrast, the possibility that
IPSE is a B cell superantigen requires consideration; B cell
superantigens, such as HIV-1 gp120, protein Fv, and protein L (33,
34),2 can bind to IgE and, as multivalent molecules, can
activate basophils, resulting in the release of IL-4 and IL-13.
Furthermore, release of IPSE-induced IL-4 can be inhibited in the
presence of increasing concentrations of human polyclonal IgG.
Polyclonal IgG contains various VH segment family members, and some of
them may compete with corresponding VH segments of IgE for binding to
IPSE. Experiments addressing these points are currently under way.
The in vivo effects of IPSE are not known yet. The
pronounced IL-4 and IL-13 production triggered by this factor in
vitro strongly suggests that IPSE might be involved in initiating
and/or amplifying a Th2 response. Basophils, while accounting for only 1% of human peripheral blood leukocytes (35), have been found to
produce more IL-4 during the first 6 h after antigen activation than lymphocytes (36) and are considered to be an initial source of
IL-4 (4, 16, 37-39). Basophils are mobile blood cells and can readily
reach sites of interaction with pathogens; they may thus be attracted
to sites of schistosome egg deposition. Morphologically intact
granulated basophils are rarely observed in schistosome egg granulomas,
but in view of the powerful activating effect of IPSE, it is quite
likely that basophils in this environment are degranulated ("ghost
cells") and thus are not easily recognized by conventional
histochemical staining. Moreover, basophils share several chemokine
receptors with eosinophils such as CCR2 and CCR3 (40-42), which are
bound by the chemokines MCP-1, MCP-2, MCP-3, and MCP-4 and the
chemokines MCP-3, MCP-4, RANTES, eotaxin I, and eotaxin II,
respectively, suggesting that they could be important participants in
the circumoval immune response, which is dominated by eosinophils for
several weeks. Since IPSE is secreted by schistosome eggs, as concluded
both from its presence in egg culture supernatants and from the
immunohistological finding of IPSE on and in circumoval inflammatory
cells, this factor will obviously interact also with basophils
attracted to the schistosome egg granuloma.
In conclusion, IPSE is a parasitic glycoprotein with a novel and potent
activating effect on human basophils. Because it can rapidly trigger
basophils from nonsensitized human donors to release considerable
amounts of IL-4, IPSE is a good molecular candidate for biasing the
immune response toward the Th2 phenotype in the course of schistosome infection.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RI-mediated (16) and SmEA-mediated (10) IL-4
production of basophils, all wells were supplemented with IL-3 except
the wells to which ionomycin was added. The cells were incubated with
various stimuli at the concentrations indicated. If not stated
otherwise, the supernatants were collected after 18 h and stored
at
20 °C until further investigation. Degranulation of basophils
was assessed via cytospin preparation and May- Gruenwald staining.
80 °C.
ZAP II
by means of the Lambda ZAP II XR library construction kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol. The resulting cDNA library of S. mansoni eggs had 70% inserts and a
titer of 6 × 109 plaque-forming units/ml.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (38K):
[in a new window]
Fig. 1.
Purification of IPSE by cation exchange
chromatography. a, A280 light
absorption by fractions of SmEA after cation exchange chromatography on
SP-Sepharose. Adherent molecules were eluted with a continuous gradient
of 0-1 M KCl. Solid line,
A280; dashed line, salt
gradient. b, IL-4 production by basophils stimulated with
the SmEA fractions obtained by chromatography on SP-Sepharose in
a. Histogram bars indicate the amount
of IL-4 produced by purified basophils cultured with IL-3 alone (10 ng/ml), IL-3 and SmEA (10 µg/ml) or anti-IgE (100 ng/ml), or IL-3 and
a 5 µl/ml concentration of each SP-Sepharose fraction.
-linked fucose.
View larger version (32K):
[in a new window]
Fig. 2.
Purification of IPSE using lectin
chromatography. a, A280 protein
trace of fractionation of pooled active SmEA fractions from
SP-Sepharose on AAA-Sepharose. Adherent factors were eluted with 0.1 M L-fucose. b, IL-4 production by
cultured basophils stimulated with effluent and eluate fractions (1 µl/ml) from AAA-Sepharose in a. c, SDS-PAGE of
eluate fractions from AAA-Sepharose followed by silver staining.
d, SDS-PAGE of eluate fractions from AAA-Sepharose followed
by blotting onto nitrocellulose membrane and incubation with labeled
AAA.
View larger version (36K):
[in a new window]
Fig. 3.
Nucleotide and predicted protein sequence of
IPSE (GenBankTM accession number AY028436). The 489 nucleotides encode a 134-aa protein with a 20-aa signal peptide
(boldface type). The N-terminal end of the mature
protein, whose sequence was determined by protein sequencing, is
boxed in black. From this sequence and the
corresponding EST (AI820476) identified by a BLASTn search against
dbEST, a 30-nt nondegenerated primer (indicated by an arrow)
was designed and used as a probe for screening the SmE libraries. Also
shown are the seven cysteine residues (circled) and the two
potential N-glycosylation sites (white
boxes); aa 73-89 (boxed in gray)
correspond to a "Greek key" motif signature (PS00225) identified by
ScanProsite. The polyadenylation signal is underlined.
View larger version (21K):
[in a new window]
Fig. 4.
Formation of homodimers and
dose-dependent induction of IL-4 production in basophils by
recombinant IPSE. a, SDS-PAGE and silver staining of
His-IPSE under reduced and nonreduced conditions. b, IL-4
production by basophils stimulated with various amounts of His-IPSE or
with a control His tag fusion protein (His-Phl p1),
respectively.
View larger version (23K):
[in a new window]
Fig. 5.
Murine antiserum to recombinant IPSE reacts
with and neutralizes natural IPSE. a, Western blots of
His-IPSE, purified natural IPSE, and SmEA, respectively, were incubated
with murine anti-His-IPSE antiserum (diluted 1:2000). Bound antibodies
were detected with AP-labeled anti-murine IgG. Controls,
binding of second antibodies to IPSE. b, anti-His-IPSE
dose-dependently inhibits SmEA-induced IL-4 production.
Human basophils were stimulated with SmEA (2 µg/ml) in the presence
of various dilutions of murine anti-His-IPSE antiserum. The culture
supernatants were tested for IL-4 content (one representative out of
two experiments).
View larger version (52K):
[in a new window]
Fig. 6.
Basophil degranulation and mediator release
induced by IPSE. a, cytospin preparations of human basophils
incubated for 30 min with medium (Medium), IL-3
(IL-3), or IL-3 + pools 1 (Pool 1), 2 (Pool 2), and 3 (Pool 3)
(each 0.4 µl/ml) from cation exchange chromatography of SmEA
(fractions 6-10, 48-55, and 56-63, respectively) or with 24 ng/ml
recombinant IPSE (His-IPSE). Pool 3 and His-IPSE (like SmEA and
anti-IgE) cause degranulation and, as shown here, often agglutination,
whereas IL-3 alone leads only to shape change. b, release of
IL-4 from basophils following 4-h stimulation with medium, IL-3 (10 ng/ml), SmEA (10 µg/ml) + IL-3, anti-IgE (100 ng/ml) + IL-3, pool 1, 2, or 3 (each 0.4µl/ml) + IL-3, or His-IPSE (24 ng/ml) + IL-3.
c, IL-13 secretion by basophils after culture (40 h) with
medium, SmEA, anti-IgE, pool 1, 2, or 3, or recombinant His-IPSE (all
without IL-3; concentrations as for b). d,
release of histamine (%) from basophils (conditions as for
b). Representative experiments are shown.
View larger version (31K):
[in a new window]
Fig. 7.
Blot analysis showing the binding of IPSE to
IgE. a, highly purified polyclonal human IgE (Biermann,
Bad Nauheim, Germany) was submitted to SDS-PAGE, transferred onto
nitrocellulose membrane, and incubated with various concentrations of
His-IPSE. Bound His-IPSE was detected with monoclonal anti-IPSE
antibodies (clone 74 2G4; supernatant, diluted 1:50), and the latter
was visualized by AP-conjugated goat anti-mouse IgG (1:10,000; Dianova)
followed by nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl
phosphate. b, His-IPSE was submitted to SDS-PAGE,
transferred onto nitrocellulose membrane, and incubated with various
concentrations of IgE. Bound IgE was detected with AP-conjugated mouse
monoclonal anti-human IgE (diluted 1:10,000; BD Pharmingen, Heidelberg,
Germany) followed by nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl
phosphate.
View larger version (150K):
[in a new window]
Fig. 8.
Immunohistological detection of IPSE in
S. mansoni-infected mouse liver. Liver
sections of mice infected with S. mansoni were
incubated with the murine monoclonal anti-His-IPSE antibody 74 1G2
(a-c) or with PBS (d). Bound antibody was
detected with AP-conjugated F(ab)2 goat anti-murine IgG
followed by Neufuchsin and hematoxylin staining. a, general
view showing liver tissue containing several schistosome egg
granulomas. Note staining of eggs and/or circumoval inflammatory cells.
b, longitudinal section of a schistosome egg in a small
(developing?) granuloma with nearly exclusive staining of the subshell
area; the arrows depict egg shell. c,
longitudinal section of a schistosome egg in an advanced granuloma with
extensive staining of circumoval cells and moderate staining of the
egg. d, longitudinal and traverse section of two schistosome
eggs; negative control.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheets,
including a crystallin
/
"Greek key" motif. Thus, IPSE
may have some structural similarity with the
-crystallin-like
superfamily, and indeed an assignment to this superfamily was also
obtained (E value: 4.4e
05) by using the
Superfamily HMM Library and Genome Assignments server (available on the
World Wide Web at supfam.org/SUPERFAMILY). As assessed by SDS-PAGE
analysis performed under reducing and nonreducing conditions,
recombinant IPSE spontaneously formed disulfide-linked homodimers,
consistent with the presence of an uneven number of seven cysteines.
Correspondingly, when natural IPSE was subjected to SDS-PAGE under
reducing conditions, its apparent molecular mass decreased from
40 to 20 kDa. Given a calculated molecular mass of 26.4 kDa for
recombinant homodimeric IPSE, natural IPSE appears to be a homodimeric
glycoprotein with a glycan content of about 30%.
1 found in S. mansoni eggs (26). This
glycoprotein is a candidate antigen for serodiagnosis of S. mansoni infection but had thus far not been further characterized.
Indeed, Western blotting of antigen
1 and IPSE,
respectively, with anti-
1 and anti-IPSE antibodies as
well as N-terminal sequencing of antigen
1 clearly
indicates both molecules to be
identical.4
1-6-linked fucose (30) and for Fuc
1-3-GlcNAc > Lex > sialyl Lex (31), it is possible that
natural IPSE contains lacto-N-fucopentaose III. However, for
the functional activity of IPSE being investigated here, carbohydrates
are not necessary, since unglycosylated recombinant IPSE has a
powerful IL-4-inducing effect. Moreover,
lacto-N-fucopentaose III coupled to human serum albumin
(Biocarb Chemicals, Lund, Sweden) did not activate human basophils when
added over a wide concentration range (0.03-30 µg/ml using 0.3 log
increments; data not shown). Of course, our findings do not exclude the
possibility that carbohydrates can contribute to a Th2 bias by
mechanisms involving cell types other than basophils.
RI-bound IgE. This assumption is supported by
our blotting and Biacore experiments demonstrating dose-dependent binding of His-IPSE to solid-phase IgE and
vice versa. Taken together, IPSE is an IgE-binding factor
that activates basophils presumably via cross-linking receptor-bound IgE.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to A. Ruppel, M. Kastner, W. Kunz, M.-Q. Klinkert, G. Dörfler, U. Zähringer, T. Goldmann, and L. Rink for advice and/or practical help and to W. Martens and S. Adrian for excellent technical assistance.
![]() |
FOOTNOTES |
---|
* This investigation received financial assistance from the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases Grant 970551.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** Supported by Swiss National Science Foundation Grant 32-63550.00.
To whom correspondence should be addressed: Division of
Cellular Allergology, Forschungszentrum Borstel, Parkallee 22, D-23845 Borstel, Germany. Tel.: 49-4537-188440; Fax: 49-4537-188608; E-mail: hhaas@fz-borstel.de.
Published, JBC Papers in Press, March 6, 2003, DOI 10.1074/jbc.M300497200
2 T. Brunner and C. A. Dahinden, unpublished data.
3 S. Blindow, T. Weimar, and G. Schramm, unpublished data.
4 G. Schramm and M. J. Doenhoff, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
Th2, T helper type
2;
IL, interleukin;
aa, amino acid(s);
AAA, A. aurantia
agglutinin;
EST, expressed sequence tag;
Fc RI, high affinity IgE
receptor;
IPSE, interleukin-4-inducing principle from S. mansoni eggs;
PBS, phosphate-buffered saline;
SmEA, saline-soluble
S. mansoni egg antigen extract;
SmE-library, S.
mansoni egg cDNA library;
Dig, digoxigenin;
AP, alkaline
phosphatase;
RANTES, regulated on activation normal T cell expressed
and secreted.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Pearce, E. J., Caspar, P., Grzych, J.-M., Lewis, F. A., and Sher, A. (1991) J. Exp. Med. 173, 159-166[Abstract] |
2. |
Vella, A. T.,
and Pearce, E. J.
(1992)
J. Immunol.
148,
2283-2290 |
3. | Okano, M., Nishizaki, K., Abe, M., Wang, M. M., Yoshino, T., Satoskar, A. R., Masuda, Y., and Harn, D. A., Jr. (1999) Allergy 54, 593-601[CrossRef][Medline] [Order article via Infotrieve] |
4. | Paul, W. E., and Seder, R. A. (1994) Cell 76, 241-251[Medline] [Order article via Infotrieve] |
5. | Seder, R. A., Paul, W. E., Davis, M. M., and Fazekas de St. Groth, B. (1992) J. Exp. Med. 176, 1091-1098[Abstract] |
6. | Hsieh, C. S., Heimberger, A. B., Gold, J. S., O'Garra, A., and Murphy, K. M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6065-6069[Abstract] |
7. | Shimoda, K., Van Deursen, J., Sangster, M. Y., Sarawar, S. R., Carson, R. T., Tripp, R. A., Chu, C., Quelle, F. W., Nosaka, T., Vignali, D. A., Doherty, P. C., Grosveld, G., Paul, W. E., and Ihle, J. N. (1996) Nature 380, 630-633[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Ochensberger, B.,
Rihs, S.,
Brunner, T.,
and Dahinden, C. A.
(1995)
Blood
86,
4039-4049 |
9. |
Eglite, S.,
Pluss, K.,
and Dahinden, C. A.
(2000)
J. Immunol.
165,
2183-2189 |
10. | Falcone, F. H., Dahinden, C. A., Gibbs, B. F., Noll, T., Amon, U., Hebestreit, H., Abrahamsen, O., Klaucke, J., Schlaak, M., and Haas, H. (1996) Eur. J. Immunol. 26, 1147-1155[Medline] [Order article via Infotrieve] |
11. | Haisch, K., Schramm, G., Falcone, F. H., Alexander, C., Schlaak, M., and Haas, H. (2001) Parasite Immunol. 23, 427-434[CrossRef][Medline] [Order article via Infotrieve] |
12. | Doenhoff, M. J., Pearson, S., Dunne, D. W., Bickle, Q., Lucas, S., Bain, J., Musallam, R., and Hassounah, O. (1981) Trans. R. Soc. Trop. Med. Hyg. 75, 41-53[Medline] [Order article via Infotrieve] |
13. | Boros, D. L., and Warren, K. S. (1970) J. Exp. Med. 132, 488-507[Medline] [Order article via Infotrieve] |
14. | Carter, C. E., and Colley, D. G. (1978) J. Parasitol. 64, 285-290[Medline] [Order article via Infotrieve] |
15. | Haisch, K., Gibbs, B. F., Korber, H., Ernst, M., Grage-Griebenow, E., Schlaak, M., and Haas, H. (1999) J. Immunol. Methods 226, 129-137[CrossRef][Medline] [Order article via Infotrieve] |
16. | Brunner, T., Heusser, C. H., and Dahinden, C. A. (1993) J. Exp. Med. 177, 605-611[Abstract] |
17. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
18. | Heukeshoven, J., and Dernick, R. (1988) Electrophoresis 9, 28-32[Medline] [Order article via Infotrieve] |
19. | Kyhse-Andersen, J. (1984) J. Biochem. Biophys. Methods 10, 203-209[CrossRef][Medline] [Order article via Infotrieve] |
20. | Leary, J. J., Brigati, D. J., and Ward, D. C. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 4045-4049[Abstract] |
21. | Oliveira, G. C., and Kemp, W. M. (1995) Mol. Biochem. Parasitol. 75, 119-122[CrossRef][Medline] [Order article via Infotrieve] |
22. | Oliveira, G. C. (2001) Trends Parasitol. 17, 108-109[CrossRef] |
23. | Sambrook, J., and Russell, D. W. (2001) Molecular Cloning , 3rd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
24. | Van Dam, G. J. (1995) Circulating Gut-associated Antigens of Schistosoma mansoni: Biological, Immunological, and Molecular Aspects , pp. 21-46, CIP-Gegevens Koninklijke Bibliotheek, Den Haag, The Netherlands |
25. | Ashton, P. D., Harrop, R., Shah, B., and Wilson, R. A. (2001) Parasitology 122, 329-338[CrossRef][Medline] [Order article via Infotrieve] |
26. | Dunne, D. W., Jones, F. M., and Doenhoff, M. J. (1991) Parasitology 103, 225-236[CrossRef][Medline] [Order article via Infotrieve] |
27. | Velupillai, P., and Harn, D. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 18-22[Abstract] |
28. |
Okano, M.,
Satoskar, A. R.,
Nishizaki, K.,
Abe, M.,
and Harn, D. A., Jr.
(1999)
J. Immunol.
163,
6712-6717 |
29. |
Okano, M.,
Satoskar, A. R.,
Nishizaki, K.,
and Harn, D. A., Jr.
(2001)
J. Immunol.
167,
442-450 |
30. | Debray, H., and Montreuil, J. (1989) Carbohydr. Res. 185, 15-26[CrossRef][Medline] [Order article via Infotrieve] |
31. | Haselhorst, T., Weimar, T., and Peters, T. (2001) J Am. Chem. Soc. 123, 10705-10714[CrossRef][Medline] [Order article via Infotrieve] |
32. | Haas, H., Falcone, F. H., Schramm, G., Haisch, K., Gibbs, B. F., Klaucke, J., Pöppelmann, M., Becker, W.-M., Gabius, H.-J., and Schlaak, M. (1999) Eur. J. Immunol. 29, 918-927[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Patella, V.,
Giuliano, A.,
Bouvet, J. P.,
and Marone, G.
(1998)
J. Immunol.
161,
5647-5655 |
34. |
Patella, V.,
Florio, G.,
Petraroli, A.,
and Marone, G.
(2000)
J. Immunol.
164,
589-595 |
35. | Marone, G., Lichtenstein, L. M., and Galli, S. J. (2000) Mast Cells and Basophils , Academic Press, Inc., San Diego |
36. | Devouassoux, G., Foster, B., Scott, L. M., Metcalfe, D. D., and Prussin, C. (1999) J. Allergy Clin. Immunol. 104, 811-819[Medline] [Order article via Infotrieve] |
37. | Romagnani, S. (1992) Immunol. Today 13, 379-381[CrossRef][Medline] [Order article via Infotrieve] |
38. | Haas, H., Falcone, F. H., Schramm, G., Haisch, K., Gibbs, B. F., Holland, M., Bufe, A., and Schlaak, M. (1999) Int. Arch. Allergy Immunol. 119, 86-94[CrossRef][Medline] [Order article via Infotrieve] |
39. | Marone, G., Florio, G., Triggiani, M., Petraroli, A., and De Paulis, A. (2000) Crit. Rev. Immunol. 20, 477-496[Medline] [Order article via Infotrieve] |
40. | Kaplan, A. P. (2001) Int. Arch. Allergy Immunol. 124, 423-431[CrossRef][Medline] [Order article via Infotrieve] |
41. | Dunzendorfer, S., Kaneider, N. C., Kaser, A., Woell, E., Frade, J. M., Mellado, M., Martinez-Alonso, C., and Wiedermann, C. J. (2001) J. Allergy Clin. Immunol. 108, 581-587[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Uguccioni, M.,
Mackay, C. R.,
Ochensberger, B.,
Loetscher, P.,
Rhis, S.,
LaRosa, G. J.,
Rao, P.,
Ponath, P. D.,
Baggiolini, M.,
and Dahinden, C. A.
(1997)
J. Clin. Invest.
100,
1137-1143 |