Analysis of IgE binding proteins of Celtis tala pollen

Abstract

Celtis tala is a tree, widely distributed in Argentina. Its significance as a source of allergic sensitisation is still unclear. Our aim was to partially characterise the IgE binding proteins of C. tala pollen. Skin prick tests using C. tala pollen extract were carried out in 25 patients with respiratory allergy. Celtis tala extract elicited positive skin reactions in five patients. Sensitised patients showed increased specific IgE levels to C. tala by immunodot. Immunoblotting assay demonstrated IgE binding to proteins of 50 kDa and 55 kDa. Celtis tala pollen extract is allergenically potent requiring approximately 50 ng of self-protein for 50% inhibition of IgE binding in enzyme_linked immunosorbent assay (ELISA) inhibition, whereas no cross-inhibition was detected with other types of pollen widely distributed in Argentina (Helianthus annuus and Prosopis spp.). Thus, these results taken together indicate that proteins of 50 kDa and 55 kDa may be considered as specific allergens of C. tala pollen.

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Analysis of IgE binding proteins of Celtis tala pollen
María Verónica Baroni a; Jorge S. Alvarez b; Daniel Alberto Wunderlin a; Gustavo Alberto Chiabrando a
a Departamento de Bioquímica Clínica, Centro de Investigaciones en Bioquímica Clínica (CIBICI)-CONICET,
Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina b Servicio de Alergia
e Inmunología, Instituto Oulton de Diagnóstico y Tratamiento, Córdoba, Argentina
Online Publication Date: 01 September 2008
To cite this Article Baroni, María Verónica, Alvarez, Jorge S., Wunderlin, Daniel Alberto and Chiabrando, Gustavo
Alberto(2008)'Analysis of IgE binding proteins of Celtis tala pollen',Food and Agricultural Immunology,19:3,187 — 194
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Analysis of IgE binding proteins of Celtis tala pollen
Marı´a Vero´nica Baroni
a
, Jorge S. Alvarez
b
, Daniel Alberto Wunderlin
a
and
Gustavo Alberto Chiabrando
a
*
a
Departamento de Bioquı´mica Clı´nica, Centro de Investigaciones en Bioquı´mica Clı´nica
(CIBICI)-CONICET, Facultad de Ciencias Quı´micas, Universidad Nacional de Co´rdoba, Co´ rdoba,
Argentina;
b
Servicio de Alergia e Inmunologı´a, Instituto Oulton de Diagno´stico y Tratamiento,
Co´ rdoba, Argentina
(Received 27 February 2008; final version received 9 May 2008)
Celtis tala is a tree, widely distributed in Argentina. Its significance as a source of allergic
sensitisation is still unclear. Our aim was to partially characterise the IgE binding
proteins of C. tala pollen. Skin prick tests using C. tala pollen extract were carried out in
25 patients with respiratory allergy. Celtis tala extract elicited positive skin reactions in
five patients. Sensitised patients showed increased specific IgE levels to C. tala by
immunodot. Immunoblotting assay demonstrated IgE binding to proteins of 50 kDa
and 55 kDa. Celtis tala pollen extract is allergenically potent requiring approximately 50
ng of self-protein for 50% inhibition of IgE binding in enzymelinked immunosorbent
assay (ELISA) inhibition, whereas no cross-inhibition was detected with other types of
pollen widely distributed in Argentina (Helianthus annuus and Prosopis spp.). Thus,
these results taken together indicate that proteins of 50 kDa and 55 kDa may be
considered as specific allergens of C. tala pollen.
Keywords: allergen; Celtis tala; pollen; seasonal rhinitis; serum IgE
Introduction
Allergic diseases of the respiratory system caused by pollinosis are quite frequent and have
a great clinical impact in industrialised countries (Ibarrola et al., 2004; ISAAC, 1998),
being in part responsible for rises in the morbidity rate, lack of productivity and increased
costs for the health care system (Weiss & Sullivan, 2001). However, among the 250,000
well-described pollen-producing plant species, less than 100 are significant in terms of
inducing pollen allergy (D’Amato et al., 1998). Pollens from wind-pollinated plants are the
most potent allergen sources, whereas pollens from insect-pollinated plants less frequently
elicit allergic symptoms (Durham, Gould, & Hamid, 1997). The characterisation of
allergens from plant pollens is a necessary task to improve the diagnosis and treatment of
this increased clinical disorder and can help to explain relationships among biologic
function, protein structure, and allergenic activity (Aalberse, 2000; Puc, 2003).
Celtis tala (Gill. ex Planchon), a member of the Ulmaceae family, is an autochthonous
tree in South America and widely distributed in Argentina (Nitiu, 2003). As with all the
species belonging to the Celtis family, C. tala is a typically anemophilous tree (Green &
Dettmann, 2004). The pollen of C. tala has been found to be one of the most important
causes of pollinosis and allergic sensitisation in the north and central areas of Argentina
*Corresponding author. Email: gustavo@bioclin.fcq.unc.edu.ar
Food and Agricultural Immunology
Vol. 19, No. 3, September 2008, 187194
ISSN 0954-0105 print/ISSN 1465-3443 online
#2008 Taylor & Francis
DOI: 10.1080/09540100802190765
http://www.informaworld.com
Downloaded By: [Chiabrando, Gustavo Alberto] At: 17:51 21 August 2008

during the pollination period between August and December (Nitiu, 2003; Nitiu, Mallo, &
Romero, 2003; Teijeiro et al., 2002; Weber, 2005).
Co´rdoba city (31819?S and 64813?W) is located in the Co´rdoba province in the central
region of Argentina. Pollen season in Co´rdoba can be divided in two periods. The first one
goes from August to November with a predominance of arboreal pollen and the second
one goes from October to April with a predominance of grass pollen grains (Teijeiro et al.,
2002). Airborne pollen in the central part of Argentina is mainly represented by Platanus,
Fraxinus,Cupressaceae,Poaceae,Urticaceae,Cyperaceae,Myrtaceae,Celtis, and Casuarina
Morus (Nitiu, 2006). It has been demonstrated that the Celtis airborne pollen content
represented between 1998 and 2000, an average of 2.62% compared to arboreal pollen,
reaching a maximum of 10% in October (Nitiu, 2003). The average pollen count was 1530
grains/m
3
, with a maximum airborne pollen concentration of 275 grains/m
3
(Nitiu, 2003).
In other reports, Celtis pollen is considered to be one of the important pollen types which
exceed 1% of the total relative concentration of pollen present in the atmosphere, together
with other species such us Fraxinus,Poaceae,Cupressus,Ambrosia,Urticaceae,Artemisia,
Myrtaceae,Casuarina,Asteraceae, and Cyperaceae (Nitiu et al., 2003). Considering the
most allergenic species according to skin prick test sensitivity, AugustNovember is a very
risky period in Co´rdoba for patients affected with seasonal symptoms caused by pollen
trees. In the case of grasses and weeds the period extends from October to April (Teijeiro
et al., 2002).
Despite C. tala being described as a potential allergenic (Nitiu et al., 2003; Teijeiro
et al., 2002; Weber, 2005), its allergic components have still not been properly investigated,
which affect the accurate diagnosis and safe immunotherapy of C. tala allergy. The
present study was therefore aimed at identifying IgE binding of C. tala pollen by
immunoblot using hypersensitive sera from allergic patients to C. tala pollen. In addition,
the specific allergens of C. tala with predominant pollen species were delineated by
inhibition assays.
Materials and methods
Patients
Twenty-five patients were recruited from the Service of Allergy and Immunology of Oulton
Institute for Diagnosis and Treatment, Co´ rdoba, Argentina. From August to December of
2003 (spring and summer season), these patients presented allergic symptoms of asthma
and/or rhinitis. The criteria for asthma were symptoms suggestive of it, including self-
reported asthma, dyspnea with wheezing or cough with wheezing, and at least one positive
bronchodilation test (Viinanen et al., 2005). The definition of rhinitis includes a
heterogeneous group of one or more of the following nasal symptoms: sneezing, itching
rhinorrhea, or nasal congestion (Phipatanakul, 2005). The detailed history including age,
sex, duration of illness, family history, onset of symptoms, seasonality, etc. of each patient
was recorded and clinical investigations were carried out with informed consent. The
Human Ethics Committee of the Institute approved the study protocol.
Skin testing
Skin prick tests were carried out on the forearm surface of patients with C. tala pollen
extract reconstituted in phosphate buffer saline (1:500 w/v in PBS: Na
2
HPO
4
10 mM,
KH
2
PO
4
3 mM, NaCl 120 mM, pH 7.5). The patients were also skin prick tested with a
188 M.V. Baroni et al.
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routine battery of aeroallergens of our area (Ceti Laboratories, Co´rdoba, Argentina). The
battery included grass pollen, tree pollen, weed pollen, house dust mite and animal
dander. For skin prick tests, each drop was pricked using a separate sterile lancet, and the
reaction was read after 15 min. Histamine chloride 10 mg/ml was used as a positive
control and the diluent as a negative control (Greer Laboratories, Lenoir, NC). Positive
reaction was defined if the wheal and erythema size were greater than 3 mm and 5 mm
after subtraction of the diameter of the negative control. The same examiner carried out
each skin prick test.
Sera were collected for immunobiochemical assays from patients with positive skin
prick tests to C. tala pollen extract. Sera were also collected from healthy volunteers and
pooled for use as a negative control. The total concentration of serum IgE was determined
using a commercial enzyme immunoassay.
Pollen protein extracts
Polleniferous materials were collected from C. tala (Tala) and four other predominant
pollen species namely, Eucalyptus sp. (Eucalyptus), Prosopis spp. (Algarrobo), Prosopis
caldenia (Calde´n), and Helianthus annuus (Sunflower), which were obtained in fields from
the Province of Co´rdoba (Argentina) during peak flowering season. Collection and
processing of pollen material was done carefully by trained pollen collectors.
The protein extraction was carried out by the method of Park, Ko, Kim, Jeoung, and
Hong (1999). Briefly, 200 mg of pollen was defatted with diethyl ether and then extracted
in 100 mL of carbonate buffer (125 mM NH
4
HCO
3
, 15 mM NaN
3,
pH 7.5) for 24 h at
48C with constant stirring. The supernatant was separated by centrifugation at 27,000 g
for 30 min at 48C, filtered through 0.22 mm membrane under sterile conditions and
lyophilised. Protein concentration was determined according to the method of Bradford
(1976).
Specific IgE detection
Immunodot assay
To detect the specific IgE reactivity to different types of pollens we performed an
immunodot assay. After standardisation analysis, the optimal condition for immunodot
assay was as follows: 4 mg of total protein, extracted from each type of pollen,
was loaded on nitrocellulose membranes. After washing the unbound antigen with tris-
buffered saline (TBS) (250 mM Tris, 750 mM NaCl; pH 7.3), free sites were blocked with
5% defatted dried milk in TBS for 1 h at 378C. This was followed by three washing with
TBS-T (TBS with 0.05% Tween 20) and overnight incubation with individual patients’ sera
(1:5 v/v TBS-T) at 48C. The membrane was washed five times with TBS-T, incubated with
peroxidase-conjugated goat anti-human IgE (Sigma Co., St. Louis) (1:500 v/v in TBS-T)
for 2 h at room temperature and revealed by chemoluminiscence reaction (ECL Reagent,
NEN Life Science Products). To express the relative amount of serum specific IgE against
the different types of pollen extract the intensity of immunodots was quantified by
densitometric analysis using the image software (UVP Life Science Software, Vision
Works†LS Image Acquisition and Analysis) and relative signals were expressed in density
units (DU) as follows: (): 0300 DU; 1: 3011350 DU; 2: 13512400 DU; 3: 2401
3450 DU; and 4: 34514500 DU.
Food and Agricultural Immunology 189
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Immunoblot assay
To recognise allergenic proteins, SDS-PAGE was carried out by the method of Laemmli
(1970) under reducing conditions using 12% polyacrylamide gels. One hundred microgram
protein of pollen extracts was boiled for 5 min at 1008C in two-fold concentration of
sample buffer containing 50 mM dithiotritol. The proteins were eletrophoresed at a
constant voltage of 120 V and bands were detected by staining with 0.1% Coomassie
brilliant blue R-250 or transferred to nitrocellulose membrane in 25 mM Tris pH 8.3
containing 192 mM glycine and 20% methanol (Towbin, Staehelin, & Gordon, 1979).
Standard proteins of known molecular weight (Sigma Co., St. Louis) were also
electrophoresed on the same gel along with sample proteins. Non-specific sites were
blocked with 5% defatted dried milk in TBS for 1 h at 378C. The membrane was washed
with TBS and incubated overnight at 48C with pooled or individual patients’ sera (1:5 v/v
in TBS-T) positive to C. tala extract. After washing five times with TBS-T and once with
TBS, peroxidase-conjugated goat anti-human IgE (1:500 v/v in TBS-T) was added to the
blotted membrane and incubated for 2 h at room temperature. Finally, the blots were
revealed with chemoluminiscence reaction as is indicated above.
Pollen extracts of four predominant pollen species were also transferred onto
nitrocellulose membrane and incubated with pooled patient sera positive to C. tala pollen.
Specific IgE ELISA inhibition
To evaluate the specificity of IgE binding to C. tala pollen extract, ELISA inhibition tests
were carried out. IgE binding was inhibited by pre-incubating the hypersensitive pooled
patients’ sera (1:10 v/v in TBS-T) with various amounts (22000 ng) of C. tala pollen and
other different pollen extracts (H. annuus and Prosopis spp.) for 16 h at 48C. The pooled
patients’ serum was prepared from five individual patients’ sera which were skin prick test
positive to C. tala extract. The pre-incubated mixture was then added to microtitre plate
coated with C. tala pollen protein (1 mg/well) and ELISA was performed. The pooled
serum without an inhibitor was used as a positive control. Percentage inhibition was
calculated as under:
1absorbance with competitor
absorbance of control 100
Statistical treatment of data
The differences between study results were estimated using the t-test for unpaired data sets
and regression analysis (Pearson correlations). Differences were considered significant
when the p-value was 50.05.
Results
Specific IgE estimation
Skin prick test and immunodot assays
From 25 patients admitted to this study, only five patients showed a strong positive
reactivity to C. tala pollen extract by skin prick test. The mean (9standard deviation)
values of the wheal and erythema size in these five patients were 6.24 (92.99) mm and
190 M.V. Baroni et al.
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15.28 (96.86) mm, respectively, which were significantly different to the mean values of
non-sensitised patients (pB0.01). The main clinical characteristics of these five sensitised
patients are presented in Table 1, showing that these patients had increased serum level of
total IgE in respect to normal control. In order to evaluate the presence of specific IgE
against C. tala in individual patients’ sera an immunodot assay was used. Table 2 shows the
results of immunodot assays demonstrating that the five patients studied presented specific
IgE against C. tala pollen. The totality of these C. tala sensitised patients showed distinct
levels of relative signal of specific IgE reactivity against C. tala pollen. Then, from these C.
tala-sensitised patients, we analysed specific IgE reactivity of the individual patients’ sera
against protein extracts from other types of predominant pollen species using immunodot
assays. The totality of these C. tala sensitised patients showed distinct levels of relative
signal of specific IgE reactivity against Prosopis spp., H. annuus,Eucalyptus spp., and
P. caldenia pollen extracts (Table 2).
Immunoblot assays
By immunoblot analysis, we determined the IgE binding components of C. tala pollen
from sensitised patients. Individual patients’ sera presented IgE reactivity against two
protein bands of approximately 5055 kDa (Figure 1A). Interestingly, these proteins were
identified in all of the sensitised patients studied, indicating that 5055 kDa proteins can be
considered the major allergenic components of C. tala pollen. In addition, we evaluated the
IgE binding components of these five patients with immunodot IgE positive reactivity
Table 1. Main clinical characteristics of sensitised patients’ group.
Patient N8Age Sex
a
Diagnose
b
Season
Total IgE
(UI/ml)
c
1 57 M AR Spring 239
2 56 M AR Spring 358
329FARA Spring 410
4 53 F AR Spring/Summer 597
5 45 M AR Spring/Summer 164
a
M, male; F, female;
b
AR, rhinitis; A, asthma;
c
upper limit of normal reference value: 90 UI/ml.
Table 2. Specific IgE levels against C. tala and other predominant plant pollens of individual
patient sera with positive skin prick test to C. tala pollen.
Immunodot
a
Patient N8C. tala Prosopisc spp. H. annuus Eucalyptus spp. P. caldenia
11241
21  1
321  
4311 
541  
a
DU, density units. Negative relative signal (): between 0300 DU, Positive relative signal: 1: between 301
1350 DU, 2: between 13512400 DU, 3: between 24013450 DU and 4: between 34514500 DU.
Food and Agricultural Immunology 191
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against other types of predominant pollens (Table 2). When the immunoblot assay was
performed with electroblotted protein extracts of H. annuus pollen, the pooled serum from
sensitised C. tala pollen patients with positive relative signal for this pollen (patients 1 and
4 in Table 2) only detected an IgE binding component of 55 kDa (Figure 1B). On the other
hand, when the immunoblot assay was performed with electroblotted proteins extracted
from Prosopis spp. pollen, the pooled patients’ serum with positive relative signal for this
pollen (patients 1, 3, 4, and 5 in Table 2) detected an IgE binding component of 38 kDa
(Figure 1C). Immunoblotting of P. caldenia and Eucalyptus spp. pollen extracts with the
pooled patients’ serum with positive relative signal for each type of pollen (patients 1 and 2
in Table 2, respectively), none IgE binding component was detected (data not shown). As
negative control, pooled normal human serum did not show IgE binding components to
any electroblotted protein extracts of C. tala or other type of predominant pollen species
studied (data not shown). Thus, these results indicate that sensitised patients to C. tala
pollen showed serum IgE reactivity against two proteins of 50 kDa and 55 kDa.
ELISA inhibition
Cross reactivity of C. tala allergens was further investigated by means of ELISA inhibiton
assay with pooled patients’ sera (Table 2) using H. annuus and Prosopis spp. pollen extracts
as inhibitors. C. tala extract required approximately 50 ng of self-protein for 50%
inhibition, whereas H. annuus and Prosopis spp. extracts did not show significant IgE
binding inhibition even at 2000 ng of extract (Figure 2). Thus, these results taken together
suggest that proteins of 50 kDa and 55 kDa can be considered as specific allergens of
C. tala pollen.
Figure 1. (A) IgE immunoblot of C. tala pollen extract using sensitised individual patients’ sera.
The numbers indicate the C. tala sensitised patients showed in Tables 1 and 2. (B) IgE immunoblot of
H. annuus pollen extract using pooled sera of patients 1 and 4 of Table 2. (C) IgE immunoblot of
Prosopis spp. pollen extract using pooled sera of patients 1, 3, 4, and 5 of Table 1. In each panel at the
left line are showed the SDS-PAGE profile of C. tala,H. annuus, and Prosopis spp. pollen extract,
respectively. MW: Molecular weight markers.
192 M.V. Baroni et al.
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Discussion
The pollination period of C. tala in our geographic area is from September to December.
Although a few previous reports have reported that C. tala pollen is an important allergen
in people living in north and central areas of Argentina (Nitiu, 2003; Nitiu et al., 2003;
Teijeiro et al., 2002; Weber, 2005), neither its sensitisation rate nor its allergenic component
has been properly studied. This work was designated to detect allergenic components of
C. tala pollen. In this sense, using skin prick tests and immunodot assays we identified
patients sensitised to C. tala pollen. Out of 25 patients with allergic sensitisation to
different types of pollen, five patients showed positive skin prick tests and specific IgE
reactivity to C. tala pollen extracts. These patients also showed some IgE reactivity against
other types of pollens (as determined by skin prick tests and immunodot assays), i.e. H.
annuus,Prosopis spp., Eucalyptus spp. and P.caldenia, which are also widely distributed in
Argentina (Asturias et al., 1998; Nitiu et al., 2003; Teijeiro et al., 2002). Then, by
immunoblot assays we determined that these five sensitised patients presented IgE
reactivity against two protein bands of approximately 50 and 55 kDa, respectively (Figure
1A). On the other hand, in other types of pollen, pooled patients’ sera only showed IgE
reactivity against a single protein of 55 kDa for H. annuus and 38 kDa for Prosopis spp.
pollen extracts (Figures 1B and C). By ELISA inhibition tests we demonstrated that 50 ng
of self-protein was required for 50% inhibition of IgE binding, whereas the IgE binding to
C. tala pollen extracts was not inhibited by H. annuus and Prosopis spp. pollen extracts
(Figure 2). Therefore, we have identified for the first time allergenic components of C. tala
pollen as two proteins of 50 kDa and 55 kDa, which did not present IgE reactivity against
other plant pollen extracts that are widely distributed in the same area of C. tala. Although
further studies are required, including the increase of the number of sensitised patients, this
partial characterisation could be promising for the development of diagnostic tests and the
formulation of immunotherapy vaccines for patients allergic to C. tala pollen.
Acknowledgements
This work was supported in part by grants from SECyT (Secretarı´a de Ciencia y Tecnologı´a de la
Universidad Nacional de Co´ rdoba), FONCyT-ANPCyT (Fondo para la Investigacio´n Cientı´fica y
Tecnolo´gica-Agencia Nacional de Promocio´ n Cientı´fica y Tecnolo´ gica), and CONICET (Consejo de
Investigaciones Cientı´ficas y Tecnolo´gicas de la Repu
´blica Argentina). MVB is a postdoctoral fellow
Figure 2. IgE ELISA inhibition of C. tala pollen extract. C. tala extract (1 mg/well) was coated and
incubated with a mixture containing pooled patients’ sera (1:10 v/v in TBS-T) and 2, 20, 200, and
2000 ng of extract as inhibitors (j,C. tala;m,Prosopis spp.; *, H. annuus). The bound IgE was
determined using anti-human IgE-HRP. The values represent the mean of three independent
experiments.
Food and Agricultural Immunology 193
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of CONICET. DAW and GACh are members of Researcher Career of CONICET. We are grateful to
Dr Paul David Hobson, native speaker, for checking the language of this manuscript.
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