Autoimmune mechanisms in chronic idiopathic urticaria.
- The journal of allergy and clinical immunology. In practice. 09/2013; 1(5):522-3.
Fig 1, A shows the daily percentage of patients with nasal, oc-
ular, and respiratory symptoms as well as the evolution of daily
pollen levels, weighted according to the number of participants
per town and per day. Although the start of the pollen season is
staggered during the month of April in different cities, significant
peaks are detected mostly between mid-May and mid-July. Fig 1,
B-D displays the shape (with a 95% CIs) of the pollen-symptoms
relationship from the generalized additive mixed models, ad-
symptoms increase on the y-axis, expressed as ‘‘ln(OR)’’ (natural
logarithm of the odds ratio). The plot suggests that the effect of
grass pollen on nasal and ocular symptoms is more or less linear,
up to a saturation point of 80 and 90 grains/m3, respectively.
Above these cutoffs, the effects of pollens on these symptoms re-
main relatively constant. The plot for respiratory symptoms
shows lower slopes and wide CIs, and this relationship has been
considered as linear.
With the generalized estimating equation model, the respira-
tory symptoms’ OR for a 10 grain/m3increase in Poaceae is
significant (1.03 [1.01-1.05]). For nasal and ocular symptoms,
piecewise generalized estimating equation models quantified
significant nasal and ocular pollen-symptom relationships up
to the saturation point of 80 and 90 grains/m3(OR 5 1.06
offs, ORs are close to 1 and not significant.
At the start of the season (ie, before peaks occurrence, days on
which pollen levels were below 10 grains/m3), the relationships
between pollens and nasal and ocular symptoms are globally
linear and ORs are high (OR 5 2.68 [1.23-5.82] and 1.86
[0.55-6.30], respectively), consistent with no or very low thresh-
old. For nasal symptoms, the relationship is higher in peoplewith
perennial sensitization (OR 5 4.22 [1.39-12.81]) than in people
without perennial sensitization (OR 5 2.37 [0.85-6.55]).
In the literature, most authors assume linear relationships
between the number of subjects with symptoms in grass-
sensitized people and Poaceae levels. However, our results
show a sharp positive linear trend followed by a plateau. This is
consistent with the findings of a recent study (although the
outcomevariable is different) from Erbas et al.2This study shows
in a time-series analysis that grass pollen has an increasing effect
on asthma hospital admissions up to a saturation point of 30
grains/m3, with this effect remaining stable thereafter. Tobias
et al3also reported nonlinear short-term effects of airborne grass
levels on asthma emergency room admissions in Madrid. In a
panel study performed in 1973, but that did not use adequate sta-
tistical analyses, Davies and Smith4suggested that the number of
grasspollensnecessarytoinduce upperairwaysymptoms inmost
patients was about 50 grains/m3(ie, plateau effect). In Spain,
Brito et al5suggested that threshold levels for the induction of
symptoms in all monosensitized patients to olive pollens was
The higher level of nasal and ocular symptoms, without any
trigger threshold, observed in the whole population at the
beginning of pollination may be explained by a priming effect,
as described by Connell.6This means that during natural
exposure, decreasing amounts of pollen are required to elicit
sponse is therefore increased.
During this same pollination period, the stronger relationship
observed for nasal symptoms in people with animal or dust
nonspecific prepriming phenomenon, as described by Ellis et al,7
using an Environmental Exposure Unit. However, with provoca-
tion nasal tests, the response to repeated allergen challenge can
show large variations from a strongly enhanced response8to a
complete diminution of the response.9
In conclusion, the clinical response in sensitized patients to
Poaceae varies during the grass season. Our data suggest
prepriming and priming effects at the beginning of the season
without any trigger threshold, a linear response up to a saturation
point, then a plateau.
Denis M. Caillaud, MD, PhDa
Sylvie Martin, PharmD, MPHb
Claire Segala, MD, MPHb
Jean-Pierre Besancenot, PhDc
Bernard Clot, PhDd
Michel Thibaudon, PhDc
on behalf of the French Aerobiology Network
Fromathe Pulmonary and Allergology Department, Gabriel Montpied Hospital, Cler-
mont-Ferrand, France;bSepia-Sant? e, 56150, Baud, France;cthe French Network of
Aerobiological Monitoring, RNSA, Brussieu, France; anddthe Federal Office of Me-
teorology and Climatology MeteoSwiss, Payerne, Switzerland. E-mail: dcaillaud@
Supported by Agence Nationale de Securit? e Sanitaire: research convention no. EST
Disclosure of potential conflict ofinterest: The authors declare that theyhave no relevant
conflicts of interest.
1. Bell ML, Samet JM, Dominici F. Time-series studies of particulate matter. Annu
Rev Public Health 2004;25:247-80.
2. Erbas B, Chang JH, Dharmage S, Ong EK, Hyndman R, Newbigin E, et al. Do levels
of airborne grass pollen influence asthma hospital admissions? Clin Exp Allergy
3. Tobias A, Galan I, Banegas JR. Non-linear short-term effects of airborne pollen
levels with allergenic capacity on asthma emergency room admissions in Madrid,
Spain. Clin Exp Allergy 2004;34:871-8.
4. Davies RR, Smith LP. Forecasting the start and severity of the hay fever season. Clin
5. Brito FF, Gimeno PM, Carnes J, Martin R, Fernandez-Caldas E, Lara P, et al. Olea
europaea pollen counts and aeroallergen levels predict clinical symptoms in patients
allergic to olive pollen. Ann Allergy Asthma Immunol 2011;106:146-52.
6. Connell JT. Quantitative intranasal pollen challenge, II: effect of daily pollen chal-
lenge, environmental pollen exposure, and placebo challenge on the nasal mem-
brane. J Allergy 1968;41:123-39.
7. Ellis AK, Ratz JD, Day AG, Day JH. Factors that affect the allergic rhinitis re-
sponse to ragweed allergen exposure. Ann Allergy Asthma Immunol 2010;104:
8. de Bruin-Weller MS, Weller FR, De Monchy JG. Repeated allergen challenge as a
new research model for studying allergic reactions. Clin Exp Allergy 1999;29:
9. Reinartz SM, van Ree R, Versteeg SA, Zuidmeer L, van Drunen CM, Fokkens WJ.
Diminished response to grass pollen allergen challenge in subjects with concurrent
house dust mite allergy. Rhinology 2009;47:192-8.
Available online June 15, 2012.
To the Editor:
Chronic idiopathic urticaria (CIU) is defined as repeated
episodes of hives occurring over a period of 6 or more weeks
without an identifiable cause.1CIU is a common disorder with a
prevalence of 0.5% in the general population.2In the 1980s, the
following intradermal injection of autologous serum was
J ALLERGY CLIN IMMUNOL
814 LETTERS TO THE EDITOR
reported3and has since been regarded as a helpful surrogate for
autoimmunity in CIU.1IgG autoantibodies directed against the
a subunit of the high-affinity IgE receptor (FcεRI) are thought
to be the primary autoimmune mechanism of CIU, and these
are reportedly present in 30% to 60% of the patients with CIU.4-7
We have developed an immortalized human mast cell line
(LUVA) displaying FcεRI that both degranulates and releases
arachidonic acid-metabolites in an FcεRI-dependent fashion.8
These cells express high levels of chymase and tryptase
(CT)-positive and therefore display characteristics of connective
tissue mast cells. We investigated mast cell prostaglandin D2
(PgD2) production and Ca21influxes by LUVA cells in
response to incubation with sera derived from patients with CIU.
Patients with CIU and matched control subjects were recruited
under a protocol approved by our human investigations commit-
tee.9Autologous serum skin tests were performed by using stan-
dard methodology.1Serum (diluted 1:1) was incubated with
LUVA cells (5 3 104) at 378C for 30 minutes, and activation
was determined via enzyme immunoassay as PgD2secretion
(sensitivity >55 pg/mL; Cayman Chemical Co, Ann Arbor,
Mich). To properly assess LUVA activation, we also measured
PgD2concentrations in comparably diluted serum samples and
subtracted this from the LUVA supernatant values. Additional
PgD2assays were performed after incubating LUVA cells over-
night with monoclonal IgE (100 ng/mL; Millipore, Temecula,
Calif) toupregulate FcεRIexpression.8As an alternativemeasure
of mast cell activation by CIU serum, we investigated the induc-
tion of Ca21fluxes by using the Fluo-4NW calcium assay kit
(Molecular Devices, Sunnyvale, Calif). Briefly, loading buffer
with 5 mM probenecid (Sigma, St Louis, Mo) was added to
each well and incubated at 378C for 30 minutes. Cells were stim-
ulated with CIU serum and cytoplasmic [Ca21]i was determined
at an extinction wavelength of 494 nm and an emission
wavelength of 516 nm by using a Flexstation spectrophotometer
(Molecular Devices). ATP (300 mM) and monoclonal anti-FcεRI
antibodies (Upstate, Lake Placid, NY) were used as positive con-
trols, and data are expressed as arbitrary relative fluorescence
units. To evaluate the role of FcεR1-dependent and independent
pathways in cellular activation,10-14LUVA cells were incubated
with the syk inhibitor ER27319 (10-60 mM, Tocris, Ellisville,
otherwise specified, data are expressed as the mean 6 SD and the
differences analyzed by paired t test.
Fourteen patients with CIU were enrolled in the study.
Characteristics of patients enrolled are presented in this article’s
CIU population was predominantly composed of women with an
average age of 40 6 14 years, similar to what other investigators
iously noted to havewell-recognized markers of autoimmunity in
CIU, including anti-FcεRI antibodies (9 of 14), positive autolo-
gous serum skin test results (9 of 14), and/or antithyroid
antibodies (6 of 14).
As the most prominent activation pathway triggered by LUVA
mast cells,8we initially focused on PgD2secretion. We observed
increased de novo PgD2secretion after the incubation of CIU-
derived serum with LUVA cells in comparison to control serum
(363.3 6 125.9 pg/mL vs 243.5 6 94.18 pg/mL; P <.01; Fig 1).
PgD2secretion was modestly (18%) but consistently (14 of 14
subjects) increased after LUVA cells were incubated overnight
with IgE (768.0 6 177.3 pg/mL to 909.0 6 203.1 pg/mL;
P < .001) (see Fig E1 in this article’s Online Repository at
www.jacionline.org). To dissect the role of FcεRI-dependent
and -independent pathways in CIU, PgD2secretion using serum
itor or pertussis toxin, as well as after the overnight incubation of
serum with recombinant human FcεRI as described.9Preincuba-
Similarly, partial inhibition of PgD2secretion was observed with
observed when the syk inhibitor and pertussis toxin were com-
bined (30% 6 8.17% decrease). Better inhibition of activation
was observed after overnight ‘‘capture’’ of anti-FcεRI antibodies
FIG 1. PgD2Production after incubation with control- and CIU-derived se-
rum. Serum was incubated with LUVA cells at 378C for 30 minutes and se-
creted PgD2 assayed by enzyme immunoassay. Data represent net
production of PgD2after subtracting out PgD2present in the applied serum
sample (P < .01).
FIG 2. Inhibition of PgD2secretion by Syk inhibitor and pertussis toxin.
LUVA mast cells were incubated with the syk inhibitor ER27319 (60
mM) or pertussis toxin (100 ng) for 2 hours at 378C. LUVA cells were sub-
J ALLERGY CLIN IMMUNOL
VOLUME 130, NUMBER 3
LETTERS TO THE EDITOR 815
Ca21fluxes (see Fig E2 in this article’s Online Repository www.
jacionline.org). Induction of Ca21influxes was consistently ob-
served across the entire cohort of subjects (P < .0001). As with
the PgD2data, pretreatment of LUVA cells with the syk inhibitor
produced consistent but incomplete inhibition of Ca21influx,
comparable to the extent of inhibition observed by using anti-
FcεRI antibodies (Fig E2).
Previously, basophil activation tests have been considered the
benchmark diagnostic test to identify autoimmune causes of
CIU.17-19However, this approach is limited by the inherent vari-
ability of basophils, which requires rigorous screening of baso-
phils from numerous donors to identify ‘‘releaser’’ basophils
capable of FcεRI-dependent activation, and even among donors
displaying this phenotype, there is day-to-day variability in this
capacity.20Stringent screening is also required to ensure against
useful diagnostic information, CIU is a disease targeting cutane-
ous, largely connective tissue, mast cells. As basophils are not
considered the primary cellular target of CIU, a mast cell model
would be the more ideal framework to study CIU pathophysiol-
ogy. The concept that CIU involves autoimmune mechanisms
that include anti-FcεRI antibodies is strongly supported by our
current studies. A putative contributing role for anti-FcεRI
antibodies is supported by the increase in PgD2secretion after
overnight incubation with IgE, an intervention that increases
cell-surface expression of these receptors and also as the inhibi-
tion of secretion after overnight ‘‘capture’’ of putative anti-FcεRI
antibodies with excess recombinant FcεRI protein.8,21It should
be noted, however, that the upregulation of PgD2secretion in-
duced by IgE incubation cannot categorically distinguish
FcεRI-dependent effects from nonspecific priming of the LUVA
cells. In further support of a role for FcεRI-mediated cell activa-
duction and Ca21fluxes. The extent of this inhibition of Ca21
fluxes was comparable to that produced when cells were stimu-
lated by anti-FcεRI antibodies. Of note, however, pertussis toxin
also reduced PgD2production, presumably reflecting additional
complementary or collateral activation pathways.11This mast
cell line may also prove useful in dissecting additional mast
cell–targeting autoantibodies or other complementary pathways
such as those mediated by complement, bradykinin, or the clot-
ting cascade that might contribute to the development of CIU.
In summary,weutilized a unique connective tissue mast cellline
tostudy the effectsof CIU patient sera on activation, demonstrating
ter preincubation with a syk inhibitor or recombinant human FcεRI
points to a role for FcεRI-dependent autoimmune mechanisms, al-
though additional complementary pathways appear to be involved.
Future studies will be required to determine the relative validity of
diagnostic testing for autoimmune mechanisms in CIU using baso-
phil activation as opposed to assays based on what is arguably the
more relevant cellular target, the connective tissue mast cell.
Jonathon Posthumus, MD
Adrienne Ti~ nana, MD, MPH
Jonathan D. Mozena, MD
John W. Steinke, PhD
Larry Borish, MD
From the Asthma and Allergic Disease Center, Carter Center for Immunology Research,
University of Virginia, Charlottesville, Va. E-mail: firstname.lastname@example.org.
Disclosure of potential conflict of interest: J. W. Steinke has received research support
from the National Institutes of Health (NIH) and Medtronic. L. Borish has received
honoraria and research support from Merck, has consulted for Pfizer, and has received
research support from the NIH. The rest of the authors declare that they have no rel-
evant conflicts of interest.
1. Sabroe RA, Grattan CE, Francis DM, Barr RM, Black AK, Greaves MW. The au-
tologous serum skin test: a screening test for auto-antibodies in chronic idiopathic
urticaria. Br J Dermatol 1999;140:446-52.
2. Charlesworth EN. Urticaria and angioedema. Allergy Asthma Proc 2002;23:
3. Grattan CE, Wallington TB, Warin RP, Kennedy CTC, Bradfield JW. A serological
mediator in chronic idiopathic urticaria: a clinical immunological and histological
evaluation. Br J Dermatol 1986;114:583-90.
4. Hide M, Francis DM, Grattan CE, Hakimi J, Kochan JP, Greaves MW. Auto-anti-
bodies against the high affinity IgE receptor as a cause of histamine release in
chronic urticaria. N Engl J Med 1993;328:1599-604.
5. Fiebiger E, Maurer D, Holub H, Reininger B, Hartmann G, Weisetschlager M,
et al. Serum IgG auto-antibodies directed against the a chain of FcεRI: a selective
marker and pathogenetic factor for a distinct subset of chronic urticaria patients?
J Clin Investig 1995;96:2606-12.
6. Ferrer M, Kinet JP, Kaplan AP. Comparative studies of functional and binding as-
says for IgG anti-FcεRIa (a-subunit) in chronic urticaria. J Allergy Clin Immunol
7. Kaplan AP, Joseph K. Basophil secretion in chronic urticaria: autoantibody-
dependent or not? J Allergy Clin Immunol 2007;120:729-30.
8. Laidlaw TM, Steinke JW, Tinana AM, Feng C, Xing W, Lam BK, et al. Char-
acterization of a novel human mast cell line that responds to stem cell factor
and expresses functional FcepsilonRI. J Allergy Clin Immunol 2011;127:
9. Mozena JD, Tinana A, Negri J, Steinke JW, Borish L. Lack of a role for cross-
reacting anti-thyroid antibodies in chronic idiopathic urticaria. J Investig Dermatol
10. Saito H, Okajima F, Molski TF, Sha’afi RI, Ui M, Ishizaka T. Effects of ADP-
ribosylation of GTP-binding protein by pertussis toxin on immunoglobulin E-de-
pendent and -independent histamine release from mast cells and basophils.
J Immunol 1987;138:3927-34.
11. Oliver JM, Kepley CL, Ortega E, Wilson BS. Immunologically mediated signaling
in basophils and mast cells: finding therapeutic targets for allergic diseases in the
human FcvarepsilonR1 signaling pathway. Immunopharmacology 2000;48:269-81.
12. Kepley CL, Youssef L, Andrews RP, Wilson BS, Oliver JM. Multiple defects in
Fc epsilon RI signaling in Syk-deficient nonreleaser basophils and IL-3-induced
recovery of Syk expression and secretion. J Immunol 2000;165:5913-20.
13. MacGlashan D Jr. Two regions of down-regulation in the IgE-mediated signaling
pathway in human basophils. J Immunol 2003;170:4914-25.
14. Macglashan D, Miura K. Loss of syk kinase during IgE-mediated stimulation of
human basophils. J Allergy Clin Immunol 2004;114:1317-24.
15. O’Donnell BF, Francis DM, Swana GT, Seed PT, Kobza Black A, Greaves MW.
Thyroid autoimmunity in chronic urticaria. Br J Dermatol 2005;153:331-5.
16. Kulthanan K, Jiamton S, Gorvanich T, Pinkaew S. Autologous serum skin test in
chronic idiopathic urticaria: prevalence, correlation and clinical implications.
Asian Pac J Allergy Immunol 2006;24:201-6.
17. Greaves MW, Tan KT. Chronic urticaria: recent advances. Clin Rev Allergy Immu-
18. Yasnowsky KM, Dreskin SC, Efaw B, Schoen D, Vedanthan PK, Alam R, et al.
Chronic urticaria sera increase basophil CD203c expression. J Allergy Clin Immu-
19. Altrich ML, Halsey JF, Altman LC. Comparison of the in vivo autologous skin test
with in vitro diagnostic tests for diagnosis of chronic autoimmune urticaria. Allergy
Asthma Proc 2009;30:28-34.
20. Lichtenstein LM, MacGlashan DW Jr. The concept of basophil releasability.
J Allergy Clin Immunol 1986;77:291-4.
21. Yamaguchi M, Lantz CS, Oettgen HC, Katona IM, Fleming T, Miyajima I, et al.
IgE enhances mouse mast cell Fc(epsilon)RI expression in vitro and in vivo: evi-
dence for a novel amplification mechanism in IgE-dependent reactions. J Exp
Available online June 12, 2012.
J ALLERGY CLIN IMMUNOL
816 LETTERS TO THE EDITOR
FIG E1. Upregulation of PgD2secretion after incubation with IgE. PgD2as-
says were repeated after incubating LUVA cells with monoclonal IgE (100
ng/mL) overnight to upregulate FcεRI expression (*P < .0001).
J ALLERGY CLIN IMMUNOL
816.e1 LETTERS TO THE EDITOR
FIG E2. Induction of Ca21fluxes after incubation with CIU serum. Mast cell
activation by CIU-derived serum was determined by using the Fluo-4NW
calcium assay as described in the text. ATP (300 mM) and monoclonal
anti-FcεRI antibodies were used as positive controls. Varying concentra-
tions of syk inhibitor were utilized to determine optimal concentration.
Each condition was performed in triplicate, and the average of the 3 mea-
surements is presented. Data are expressed as arbitrary relative fluores-
cence units (RFUs). inh, Inhibitor.
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LETTERS TO THE EDITOR 816.e2
TABLE E1. Characteristics of CIU population
PatientAge (y) SexASST* Anti-FcεRI antibodiesy
ASST, Autologous serum skin test; F, female; M, male.
*Positive ASST result defined as wheal more than 5 mm in diameter more than saline controls.
?Positive anti-FcεRI titer defined as more than 3.9 mg/mL.9
?Positive thyroid antibodies defined as a positive thyroglobulin and/or thyroid peroxidase antibody titer.
J ALLERGY CLIN IMMUNOL
816.e3 LETTERS TO THE EDITOR
TABLE E2. Summary of characteristics of the CIU population
Characteristicn 5 14
Age (y), mean 6 SD
Sex (number of females)
ASST (number positive)*
Anti-FcεRI antibodies (number positive)?
Thyroid antibodies (number positive)?
41 6 14
ASST, Autologous serum skin test; EIA, enzyme immunoassay.
*Positive ASST result defined as wheal more than 5 mm in diameter more than saline
?Positive anti-FcεRI titer defined as more than 3.9 mg/mL via EIA as described.9
?Positive thyroid antibodies defined as a positive thyroglobulin and/or thyroid
peroxidase antibody titer.
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LETTERS TO THE EDITOR 816.e4