Taxus cuspidata (Japanese yew) pollen nasal allergy
Shiroh Maguchia*, Satoshi Fukuda ba Department of Otolaryngology, Teine Keijinkai Hospital, 1-12 Maeda, Teine-ku, Sapporo 060-8638, Japanb Department of Otolaryngology, School of Medicine, Hokkaido University, N15W7, Kita-ku, Sapporo 006-8555,
Japan*Corresponding author. E-mail address: smaguchi@fa2.so-net.ne.jp (S. Maguchi)

Abstract
We have investigated the Taxus cuspidata pollinosis that has never been studied before.
We applied immunoblotting method in order to detect specific IgE antibody against T. cuspidata
using its pollen itself. Out of 18 patients with seasonal allergic rhinitis suffering mainly in Apriland May, 5 patients were sensitized with this pollen. Furthermore, we were able to diagnose one
case as T. cuspidata pollinosis from the clinical and laboratory findings. While the birch pollen is
the main causative pollen in the spring, T. cuspidata is one of the minor allergens causing pollinosis
in our district.
Keywords: pollinosis, Japanese yew, Taxus cuspidata, immunoblotting

1. Introduction

spring from late April until early June, andmany pollinosis patients see otolaryngologists
during this time.
However, the number of nasal allergy
patients begins to increase actually from early
April every year. As the pollen of birch is not
airborne early April, we assumed that the causeof the pollinosis at that time was alder pollenthat scatters at the beginning of April and thatit had a cross reactivity with birch pollen [1].

It is well known that the birch (Betula
platyphylla var. japonica) is a representativecause of the tree pollinosis in Hokkaido, theisland of northern part of Japan, whereas
Japanese ceder (Cryptomeria japonica) pollen isa major allergen causative of tree pollinosis inJapan [1]. This is the same as observed innorthern Europe [2]. The birch pollen keeps
scattering heavily every year in Hokkaido in-1-

In 1998, in order to verify thishypothesis, we surveyed 68 nasal allergy
patients whose nasal symptoms began early orin mid April, before the flowering of birch. The
result was that the causative pollen was mainlyalder pollen as expected, which was followed byelm and poplar pollen. However,approximately 20 % of those patients indicatedno sensitization to birch, alder, poplar, elm, or
mite allergens [unpublished data].
The airborne pollen grains for floweringin Sapporo in April are those of alders (Alnus),
yews (Taxus), elms (Ulmus), poplars (Poplus)
and birches (Betula). It is impossible to findthe sensitivity against Taxus pollen in those fivekinds of pollen, because there are neither
commercially available tests to examine its
specific IgE antibody nor diagnostic extracts
from that pollen. Therefore, we assumed thepossibility that the patients who began nasal
allergy symptoms before flowering of birch andhad no sensitivity to alders, elms, poplars orbirches were sensitized to Taxus pollen.
Then, we introduced a technique of
immunoblotting for the detection of Taxus
pollen-specific IgE using the pollen grain itself,
and investigated whether the pollinosis due toTaxus cuspidata is present or not in Sapporo.

Fig. 1. Taxus cuspidata (Japanese yew) in April. It has
many small, solitary flowers.

Taxus cuspidata (Japanese yew) is avery slow-growing and long-lived tree. Itreaches a height of 25 m and a diameter of 2 m.
It is widely distributed in Japan, Sakhalin,
Kuril, Korea and the north-eastern section ofChina. Although it is widely distributed fromthe north to the south in Japan, it is abundantin Hokkaido. The leaves are flat, 1.3 - 4 cm long,and pale on the underside. Narrow and sharply
pointed, they are arranged spirally around thetwig. Small, solitary flowers appear in thespring (Fig. 1). The diameter of its pollen is20-26µm and has a little square form (Fig. 2).

2. Materials and methods
2.1. Taxus cuspidata (Japanese yew)-2-with immunoblotting technique according to
the method of Takahashi et al [3], with somemodification.
The pollen of Taxus cuspidata werecollected directly from flowering plants in April
1998, and kept at -4°C until use. Birch(Betula platyphylla var. japonica) pollen andalder (Alnus maximowiczii) pollen were used for
positive control tests, which had been collected
in 1991 and stored at -4°C.
Respectively small amounts of thethree kinds of pollen (birch, alder and yew) wereplaced 10 mm apart from each other on anitrocellulose membrane (5 x 50 mm, Bio-Rad
Laboratory, CA, USA), which had been treatedwith 0.05M Glycine buffer pH 7.6 containing
20% methanol for 1h to transfer the pollen
allergens. The membrane was then washedand blocked with TRIS-buffered saline pH 7.8(TBS) containing 4% bovine serum albumin for
1h at 37°C. The membrane was rinsed threetimes with TBS containing 0.25% Tween 20(washing buffer) and treated with the patient’s
undiluted serum overnight at room
temperature. The membrane was thenwashed with a large volume of washing buffer
and treated with biotinylated anti-human IgE(Kirkegaard & Perry Lab., Inc., Gaithersburg,MD, USA) for 1h at 37°C. After vigorous
washing with washing buffer, it was treatedwith biotinylated avidine alkaline phosphatasecomplex (Vectastain ABC-AP. AK-5000 of
Vector Laboratories. Inc. Burlingame, CA, USA)for 30 min at 37°C. Again, the membrane waswashed with a large volume of washing buffer,and then treated with BCIP/NBT(5-bromo-4-chloro-3-indolyl
phosphate/nitroblue tetrazolium), aphosphatase substrate kit (Kirkegaard & Perry
Lab., Inc., Gaithersburg, MD, USA) for 30 min
at 37°C. The allergens were revealed as purple
spots as a result of this procedure. The
amount of specific IgE antibody was estimatedfrom the degree of the coloring in three steps, -,+, 2+, respectively.

Fig. 2. The pollen of Taxus cuspidata (Japanese yew).
(400x)

2.2. Analysis of airborne pollen
The Durham type pollen samplers aresituated in Hokkaido University, which islocated in the center of Sapporo city. The
analysis of pollen samples was carried out
microscopically throughout the pollen seasonin 1998 and 1999. The numbers of each pollen
type were counted within 4 cm2 on the slides
after dying with Carberla solution (glycerin 5 ml,ethanol 10 ml, 2 drops of saturated fuchsine
and distilled water 15 ml).
2.3. Measurement of IgE antibody
2.3.1. Patient sera
Serum samples were obtained from 18
patients with seasonal allergic rhinitis living inor around Sapporo City and suffering mainly in
April and May every year. The samples werestored at -80°C until use.
2.3.2. Measurement of IgE antibody byimmunoblotting
Since the diagnostic extracts for Taxus
cuspidata are not commercially available, weapplied a method to measure its IgE antibody-3-Sweden) was performed according to themanufacture’s instructions to measure IgE
antibodies against the two kinds of pollen,
birch and alder.

2.2.3. Measurement of IgE antibody by CAP
radioallergosorbent test (CAP RAST)A CAP RAST (Pharmacia, Uppsala,Fig. 3. Airborne pollen counts of Taxus and Betula in 1998 and 1999.

Fig. 4. The membrane with immunoblotting of specific IgE against T. cuspidata, A. maximowiczii and B. platyphylla.

3. Results

pollen, 3748/ 4 cm2 in 1998 and 1415 /4 cm2in 1999.

3.1. Airborne Taxus pollen counts
3.2. Specific IgE antibody examined withimmunoblotting and CAP RAST

Airborne Taxus pollen counts areshown in Fig. 3. Japanese yew started its
flowering early April in 1998 and middle in
April in 1999, approximately 2 weeks before thebeginning of birch flowering in both years.
The total counts of yews were 584 /4 cm2 in1998 and 405 /4cm2 in 1999, and it is one
third to one sixth of the total counts of birch

We have examined 18 sera of seasonal
nasal allergy patients. The membrane withimmunoblotting of specific IgE against Taxus
cuspidata, Alnus maximowiczii and Betula
platyphylla is shown in Fig. 4. The specificIgE antibody levels expressed by the degrees of-4-

coloring and the titer obtained by CAP-RAST
are listed in Table 1. By immunoblotting
method, 5 out of 18 sera (27%) were foundpositive for IgE antibody against Taxus
cuspidata, 16 out of 18 (89%) sera were positive
against Alnus maximowiczii and 14 out of 18sera (78%) were positive against Betula
platyphylla.
The relationship between IgE antibody
against birch by CAP-RAST and the appearanceof spots by immunoblotting with Betula
platyphylla is shown in Fig. 5, where a goodcorrelation was observed. However, there
were 2 sera which were negative by CAP-RAST
but positive by immunoblotting method (No. 1and No. 16).

On the other hand, there was nocorrelation between IgE antibody against birchby CAP-RAST and the appearance of spots byimmunoblotting with Taxus cuspidata as
shown in Fig. 6.
Out of six patients (No. 1, 3, 4, 6, 14,15) whose nasal allergy symptoms began earlyor middle in April, before the flowering of birch,
only two patients had specific IgE antibody
against Taxus cuspidata. Furthermore, 3patients (No. 1, 3, 14) who were negative forbirch or alder by CAP-RAST were also negativefor Taxus cuspidata by immunoblotting, but
unexpectedly positive for Alnus maximowiczii or
Betula platyphylla by immunoblotting.

Immunoblotting
Serum No.

CAP RAST(KU/l)

Taxus
Alnus
Betulacuspidata maximowiczii platyphylla

Alder

Birch
<0.34
13.30
<0.34

1-++2
3+-+++-

<0.34
N.D.
<0.34

45+-+-+-

7.93
<0.34

8.46
<0.34

62+2+2+

N.D.

33.60

7-2+2+82+++

31.80
N.D.

54.40
4.18

910-++++

N.D.
15.70

8.00
24.70

1112+-++++

N.D.
N.D.

4.77
8.44

1314-++2+-

N.D.
N.D.

36.30
<0.34

1516-++2++48.30
N.D.

78.30
<0.34

1718-++N.D.
6.19

<0.34
9.05

N.D.=not done
Table 1. The specific IgE antibody levels measured from the degrees of coloring by immunoblotting and the titerobtained by commercially available CAP RAST.

-5-4. A case reportcorrelation between IgE antibodies against
birch detected by CAP-RAST determination andthe appearance of spots by immunoblottingwith Betula platyphylla showed, thismeasurement is fully reliable (Fig. 5). In
addition, we decided that there was no crossreactivity between Betula and Taxus, as there
was no correlation between IgE antibodies
against those two kinds of pollen (Fig. 6).

There was a case which we were able todiagnose definitely as Taxus cuspidata
pollinosis from the clinical symptom, IgE
antibody against its pollen, and the result of
provocation test.
The patient was 43-year-old female
who had been complained of nasal allergy
symptoms every year in the spring since 1985.
She visited an outpatient clinic with acomplaint of serous nasal discharge and nasal
congestion on May 28th, 1991. Her nasal
symptoms began early April in 1997 and 1998,
and middle in April in 1999, before theflowering of birch. Laboratory examination
revealed many eosinophils in her nasal
discharge and her CAP-RAST scores were:house dust, 0; mite, 0; birch, 3; alder, 3; elm, 0;
poplar, 0; orchard, 0; and mugwort, 0. IgE
antibody against Taxus cuspidata was + by
immunoblotting method (No. 4), and nasal
provocation test using the grains of the pollen
was also positive. From these results, we
diagnosed her as having Taxus cuspidata
pollinosis.
5. Discussion

Fig. 5. The relationship between IgE antibody against

Although Takahashi et al. reported thepresence of IgE antibody against Taxus pollen
in the sera of nasal allergy patients [2], there
have been no report about the definite Taxus
pollinosis patient.
In our study, we reported a case ofTaxus pollinosis patient, which presented a
specific IgE antibody and the nasal symptoms
were easily provocated by Taxus pollen.
Therefore, we decided that the patient’scomplaints in early April were due to Taxus
pollen.
Since the diagnostic extracts of Taxus
cuspidata pollen are not commercially available,
we applied immunoblotting technique in orderto detect specific IgE antibody. As thebirch by CAP-RAST and the appearance of spots byimmunoblotting with Betula platyphylla.

The arrowindicates two sera which were negative by CAP-RAST
but positive by immunoblotting method.

By this immunoblotting method, 5 outof 18 sera (27%) were positive for IgE antibody
against Taxus cuspidata. This result indicatesthat Taxus cuspidata is one of the allergens
that cause pollinosis in the spring in ourdistrict. However, the ratio of 27% isapparently low, compared to the ratio of Alnus
maximowiczii (89%) or Betula platyphylla (78%).
Furthermore, all 5 sera, positive for Taxus
cuspidata, were simultaneously positive for
Alnus maximowiczii and Betula platyphylla. It-6-indicates to us that we should take Taxus
cuspidata pollen as a minor allergen to cause
pollinosis in our district.
At first, we assumed that the patients
who began nasal allergy symptoms before
flowering of birch and had no sensitivity
detected by commercially available CAP-RAST
had Taxus cuspidata pollinosis. However, seraof such 3 patients were negative for Taxus
cuspidata but positive for Alnus maximowiczii
or Betula platyphylla by immunoblotting (Fig.5).
It demonstrates that immunoblotting is moresensitive than commercially available
CAP-RAST determination in the measurementof IgE antibody. The allergens used wereBetula verrucosa for CAP-RAST of birch andAlnus incana for alder. Both grow in Europe
and they are different from the Japanese birch
and alder in species, even though they areincluded in the same genus. We assume thatthe low sensitivity observed in CAP RAST
determinations might be due to the difference.

Fig. 6. The relationship between IgE antibody against
birch by CAP-RAST and the appearance of spots byimmunoblotting with Taxus cuspidata.

5. Conclusion
References
In Hokkaido, the northern island ofJapan, the most important causative allergenof pollinosis in spring is birch pollen. However,Taxus cuspidata pollen scatters every year
before birch flowering.
We investigated whether the Taxus
cuspidata pollen causes pollinosis in ourdistrict by immunoblotting method, since we
could not detect specific IgE antibody using
commercially available test. In conclusion, we
found a definite case of pollinosis due to thispollen. Although Taxus cuspidata pollen is
not a major pollen causing pollinosis, weshould take it as one of minor allergens thatcauses nasal allergy in early spring in Hokkaido.

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1993; 96:1-9.
[2] D'Amato G, Spieksma FThM, Liccardi G,Jäger S, Russo M, Kontou-Fili K, et al.
Pollen-related allergy in Europe. Allergy 1998;
53:567-578.
[3] Takahashi Y, Nilsson S, Berggren B.
Aeroallergen immunoblotting with human IgE
antibody. Grana 1995; 34:357-360.

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