Arch. Oto-Rhino-Laryng. 211, 25--33 (1975) 9 by Springer-Verlag 1975
Response of the Human Nasal Mucous Membrane to Anti-Human IgE Serum M. O k u d a Department of Otolaryngology Wakayama )s
College, Wakayama, Japan
Received March 18, 1975
Summary. The response of the nasal mucous membrane to anti-human IgE serum was examined. The application of anti-IgE sera to the nasal mucous membrane induced a nasal Mlergy-like symptom as well as nasal eosinophilia. This symptom was observed in the majority of nasal allergy cases and in some vasomotor rhinitis cases, while not in non-Mlergic normal persons. The nasal eosinophilia, however, increased not only in allergic patients, but in nonallergic persons in whom no nasal symptoms were induced. Such a nasal reaction was not induced with anti-IgA sera or anti-IgG sera. The character of the reaction was presumed to be specific to anti-IgE and was predominantly a reversed type anaphylaxis in the nasal mucous membrane. The sensitivity of the nasal mucous membrane to anti-IgE decreased in the course of the hyposensitization with house dust. The cause of this phenomenon was obscure. Zusammen/assung. Durch Berfihrung des Anti-Menschen-IgE-Serums auf die Oberflgche der Nasenschleimhaut treten allergieartige Symptome und Lokaleosinophilie auf. Solche Symptome finder man meistens bei F/illen yon Nasenallergie und in wenigen Fiillen der sogenann)en vasomotorischen Rhinitis, aber man finder sic nicht bei nichtallergischen Patienten. Die Naseneosinophilie tritt nicht nur bei allergischen Patienten, sondern auch bei nichtallergischen Patienten auf, die bei der obigen Beriihrung keine Symptome gehabt haben. Solche Nasenreaktionen waren aus Anti-Menschen-IgA-Serum oder -Anti-IgG-Serum nicht hervorgegangen. Der Charakter der Reaktion ist spezifisch ffir Anti-IgE-Serum, und hauptsgchlich denkt man an Reaktion "reversed type anaphylaxis" in der Nasenschleimhaut. Die Sensibilitiit der Nasenschleimhaut zur Anti-IgE nimmt wiihrend der Hyposensitisation durch Hausstauballergien ab. Der Grund dieses Phiinomens ist unklar. A u n i q u e i m m u n o g l o b u l i n , IgE, was discovered b y Ishizaka (1966) as a carrier of h u m a n reaginie antibodies. A n a n t i - s e r u m to this i m m u n o g l o b u l i n either i n d u c e d a reversed t y p e atopie skin r e a c t i o n b y its i n t r a d e r m a l i n j e c t i o n or caused the release of chemical mediators of a n a p h y l a x i s from leucocyte or lung to which it was applied in vitro, according to I s h i z a k a et al. (1968, 1969, 1970). I n our previous e x p e r i m e n t a n t i - h u m a n I g E r a b b i t s e r u m (a-IgE) also produced a local eosinophilia b y the skin window m e t h o d of g e b u c k (1972) or characteristic histological changes of atopie allergy i n the skin, mucous m e m b r a n e of the oral c a v i t y a n d the trachea b y its i n j e c t i o n into each tissue (1973). The p r e s e n t exp e r i m e n t was u n d e r t a k e n to s t u d y the response of the n a s a l m u c o u s m e m b r a n e to a-IgE.
Material and Methods The subjects examined consisted of 128 cases with nasal allergic symptoms before hyposensitization, 38 cases with nasal allergies in the course of the hyposensitization, and 20 normal cases without nasal symptoms, as indicated in Table 1, 2 and 3.
26
M. Okuda Table 1. Age distribution
Age
10--20
21 --30
31--40
41 --50
51 --60
Total
Group 1
22
25
5
4
Group 2
9
24
6
9
3
51
Group 3
2
9
5
4
1
21
Normal
4
8
5
2
1
20
56
Table 2. Sex distribution Sex
Male
Female
Total
Group i
35
21
56
Group 2
32
19
51
Group 3
11
10
21
Normal
13
7
20
Table 3. Offending allergens Allergen
House dust
Pollen
Fungus
Group 1 Group 2
Others
46
1
4
5
33
2
9
7
The diagnosis of nasal allergy was established b y such test as history taking, skin test, nasal provocative test with allergen extracts, and nasal smear test. The skin test was performed using over 15 allergen extracts (Torii, J a p a n ) including house dust, pollen a n d fungus. Of t h e offending allergens detected, house dust was t h e most frequent, as indicated in Table 3. The nasal provocative test was carried out as previously reported (1973), using t h e various kinds of allergen extracts suspected as offending allergen b y the skin test. The procedure of t h e test was as follows: a small r o u n d disc, 3 X 3 m m in diameter and No 5 A in quality (Toyo Roshi, J a p a n ) containing a certaine a m o u n t of allergen extract, was kept dry. A control disc which was similar to the above disc b u t without t h e allergen extract, was also prepared. The control disc was first placed on the surface of t h e anterior portion of t h e inferior t u r b i n a t e on one side of t h e nose, a n d then, when no reaction occurred within 5 rain, a disc with allergen extract (allergen disc) was applied to t h e same site of the inferior t u r b i n a t e in the same m a n n e r as the control disc. I n this test symptoms such as 1) sneezing or itching, 2) excessive secretion, a n d 3) swelling of t h e nasal mucous m e m b r a n e were induced. Symptoms 2) a n d 3) were observed b y rhinoseopy. The result obtained was evaluated as positive when a n y two or all three of t h e symptoms were induced, a n d evaluated as negative when one or no symptoms appeared, or when the nasal symptoms appeared immediately, usually within 30 see, after the disc was applied. The smear test was made as follows: three nasal smears for each person were prepared a n d observed b y microscopy according to a Hansel's staining m e t h o d (1953). The degree of eosinophilia was estimated using Hansel's criteria (1953): a few scattered eosinophils were recorded as 1 + ; larger collections or clumps were noted as 2 ~- ; very dense collections of eosinophils were 3 ~ or 4~-. Each subject diagnosed as allergic was positive in two or all three of the above mentioned clinical tests such as the skin, nasal test with allergen extract a n d t h e smear test.
Response of the H u m a n Nasal Mucous Membrane to A n t i - H u m a n IgE Serum
27
The response of t h e nasal mucous m e m b r a n e to a-IgE was examined b y t h e following end point m e t h o d (a-IgE test). Fresh dilutions of a-IgE were made from 1:10 to 1 : I 0 0 0 0 in ten fold series on each day of testing fl'om t h e stock solution (Behringwerke, West Germany). A few drops, ca. 0.01 ml of each dilute were dropped on each disc of filter paper which was used as a control in t h e nasal provocative test (a-IgE disc). Discs with a 1:10 concentration of a n t i - h u m a n IgG r a b b i t serum (a-IgG) or anti-IgA serum (a-IgA) were also made. For 3 days before the test a n y drug against allergy were withheld from the person to be tested. The test commenced with t h e use of a n a-IgG disc or a-IgA disc, Each disc was placed gently on the surface of the anterior portion of the inferior turbinate, carefully to minimize the irritation to the mucous m e m b r a n e ; t h e n t h e test was followed b y a challenge from a n a-IgE disc on t h e opposite side of t h e nose tested with a n a-IgG or a-IgA disc. The increasing concentrations of a-IgE in t h e series from 1:10000 to 1:10 were tested at time intervals of 10 min on t h e alternating sides of t h e nose until a positive reaction oecured. The criteria of a positive reaction were the same as in the nasal provocative test with allergen extract. The end point was considered as a concentration of a-IgE necessary to produce a positive reaction. The above test was carried out not only in the 128 cases of allergy before hyposensitization b u t also in 38 cases among t h e m once every m o n t h repeatedly for 6 m o n t h s in the course of hyposensitization with house dust. An example of t h e most frequent plan of hyposensitization used is indicated in Table 4. The sensitivity of t h e nasal mucous m e m b r a n e to b r a d y k i n i n was tested. A disc w i t h b r a d y k i n i n (Sandoz, Swiss) which contained 0.01 mI of the concentration of 100 ~zg per ml (bradykinin disc) was applied to t h e inferior t u r b i n a t e of 24 cases the day before the a-IgE test. I t s procedure and criteria of positive reaction were just the same as those of the a-IgE test. Changes in t h e degree of nasal eosinophilia following t h e I g E test were examined in 16 of the allergic a n d 12 of the normal persons. The excessive nasal secrete filling the nasal cavity was first removed b y suction, a n d t h e n 3 nasal smears were made with the secrete collected from t h e surface of t h e inferior tm'binate with a cotton swab. Ten mim~tes later, a disc, 10 • 5 m m in size, which contained 0.04 ml of stock non-diluted a-IgE, was introduced on the surface of the inferior turbinate. I m m e d i a t e l y after the onset of the positive reaction the disc was removed. Then 1, 2, 3 a n d 4 hrs following the reaction t h e nasal secrete was collected from the challenged site of the inferior turbinate, a n d nasal smears were made and observed as has already been described. The relations between the degree of a-IgE reaction a n d the serum I g E level were investigated. IgE levels were determined b y the single radial radio-immunodiffusion method according to A r b e s m a n (1).
Table 4. A frequent schedule of hyposensitization with house dust allergen Frequency of injection
Concentration of allergen
Dose of allergen (ml)
2/week
1 : 1000
0.02--0.03--0.05--0.07 --0.1--0.15--0.2--0.3--0.5
2/week
1 : 100
0.05--0.07--0.1 --0.15--0.2 --0.3--0.5
2/week
1 : i0
0.05--0.07--0.1 --0.15--0.2 --0.3--0.5
i/week
1 : 10
0.5--0.5--0.5--0.5
1/2 weeks
1 : 10
0.5--0.5--0.5--0.5
1/month
1 : 10
0.5--0.5 . . . . . .
House dust extract used contained 0.36 ~zg/ml of total nitrogen or 0.18 tzg/ml of protein nitrogen in t h e concentration of 1 : 1000. I n t r a d e r m a l injections were made for the t r e a t m e n t .
28
M. Okuda Results
The results from the a - I g E test were analyzed in the following groups determined on the basis of the skin and provocative tests with allergen extract. Group 1 was considered as allergic rhinitis with positive skin and provocative tests. Group 2 was possibly allergic with positive skin b u t negative provocative test. Group 3 was so-called vasomoter rhinitis with negative skin and provocative tests. The n u m b e r of the cases was 56 in Group 1, 51 in Group 2, 21 in Group 3 and an additional 20 normal cases. The distribution of age and sex a m o n g each group was slightly different as indicated in Table 1 and 2 ; females were the most frequent in Group 3 and the cases of 10--20 years of age were the m o s t frequent in Group 1. The main offending allergen detected was house dust, as indicated in Table 3. A m o n g these groups positive responses were obtained in 85.4~ of Group 1, in 50.60/0 of Group 2, in 28.60/0 of Group 3 and in none of the normal control group. On the other hand, no positive nasal reaction was produced in a n y group in the a - I g A or a-IgG test. The end point of a - i g E for the positive reaction was 1 : 1000 in the m a j o r i t y of the eases in Group 2 and 3, while 1 : 1000 and 1 : 10000 in Group 1 as in Table 5. The s y m p t o m produced was similar to t h a t seen in the nasal provocative test with allergen extract or in the case of nasal allergy. Of 80 eases with positive reactions 17 showed all three s y m p t o m s of nasal allergy such as sneezing, rhinorrhea and swelling of the nasal mucous membrane, 41 showed a n y two of the symptoms, and 22 showed only one as indicated in Table 6.
Table 5. End point of anti-IgE serum for positive nasal reaction End point for positive response
Group 1 Group 2 Group 3 Normal
1:10
1:10 2
l:10 ~
1:10 4
5 4 0 0
6 6 1 0
25 15 5 0
12 1 0 0
Negative response
Total
8 25 15 20
56 51 21 20
The positive reactions were obtained in the majority of Group i, in the half of Group 2, in 28.60/0 of Group 3, and in none of the normal control group.
Table 6. Nasal symptoms produced in anti-IgE test Itching or sneezing
Rhinorrhea
Swelling
*
*
*
*
*
* *
Total 17 31
*
2
*
8
Response of the Human Nasal Mucous Membrane to Anti-Human IgE Serum
29
Table 7. Relationship between nasal bradykinin test and the end point in a-IgE test End point of a-IgE
Bradykinin test
Positive Negative
i : 10
1 : 10 ~
1 : 103
1 : 104
3 5
1 2
4 3
4 2
The bradykinin test induced a positive nasal reaction in half of the 24 cases. The higher the concentration of a-IgE required for positive reaction, the lower the incidence of positive reaction in the bradykinin test.
Table 8. Changes in nasal eosinophilia following the challenge with anti-IgE After challenge Allergic
Before challenge
0 1 -t2+ 3+
Non-allergic
1+
2+
3+
6
4
1 1 1 1
4+
0
1+
2+
3+
4
5
1
2
2
Three hours after the a-IgE test nasal eosinophilia increased in 15 of 16 allergic persons, and appeared in 8 of 12 non-allergic persons who had no eosinophilia before the test and showed no positive nasal symptom in the test.
The b r a d y k i n i n disc i n d u c e d a p o s i t i v e n a s a l r e a c t i o n in h a l f of t h e 24 eases e x a m i n e d . W h e n t h e r e a c t i o n to b r a d y k i n i n was c o m p a r e d to t h a t of a - I g E in t h e same person, t h e following t e n d e n c y was o b s e r v e d : t h e higher t h e conc e n t r a t i o n of a-IRE r e q u i r e d for p o s i t i v e reaction, t h e lower t h e incidence of p o s i t i v e r e a c t i o n in the b r a d y k i n i n t e s t as i n d i c a t e d in T a b l e 7. N a s a l eosinophilia a p p e a r e d a n d increased following t h e a-IRE test. One h o u r a f t e r t h e t e s t the smears showed o n l y scarce cell c o m p o n e n t s w i t h o u t eosinophil, b u t 2 or 3 hrs l a t e r eosinophilia increased n o t o n l y in t h e allergic g r o u p b u t also in t h e non-allergic group. T h e t i m e a t which t h e m a x i m u m n u m b e r of eosinop h i l i a were seen a t 2 or 3 hrs a f t e r t h e t e s t in 23 of 25 cases e x a m i n e d . I n c r e a s e d eosinophilia were o b s e r v e d in 15 o f 16 allergic persons a t 3 hrs, a n d in 8 o f 12 nonallergic persons, who h a d no eosinophilia before the a - I g E t e s t a n d showed no p o s i t i v e n a s a l s y m p t o m s in t h e a - l g E t e s t as in T a b l e 8. The s e r u m I g E level was c o m p a r e d to t h e e n d p o i n t of a-IRE for a p o s i t i v e n a s a l reaction, b u t no close r e l a t i o n b e t w e e n t h e m was o b t a i n e d ; t h e m e a n I g E level was 916.3 u n i t s / m l in 8 cases w i t h a 1:10 c o n c e n t r a t i o n o f t h e e n d p o i n t ; t h e level was 562.6 in 15 cases w i t h 1:100; 766.3 in 16 cases w i t h 1 : 1 0 0 0 ; 975.0 in 4 cases w i t h a 10000; 708.0 in 23 cases w i t h a n e g a t i v e r e a c t i o n b y the challenge w i t h a 1 : 10 c o n c e n t r a t i o n of a - I g E as i n d i c a t e d in Fig. 1.
9g':~!g u.~ po~o!pu.r ss pou.~msxo osso I u! g i u o pomsoaoop p u s ~, u.t p o ~ u ~ q o u n pou.~mo~ 'sos~so I 8 u.r pos~oaou! N~I-qS go ~u.~od p u o oq~ q~snp o s n o q q%tat uo.r0~sz.~.~suosod.~q $o os2noo oqc~ u I
tto!~uzD~.suos-ods q jo osanoo oqc~u! uoDoeoa pssqstt oAt.~.rsod ~ zo~ mnaos ~I~i-!~u~ jo ~u!od puo oq~ ur so~uvqo "g'~.r~[ (q~uo~) ~uom~ee~• ~o ~e~s eqr 5ui~oilozl ecU!.L OI
6
8
L
9
S
~
~
~, ,'x\
.
Z
I
".x :l.e ~ 8 N
9
\ \
Ot
201
gOI
i~oT
301fi~uv ~LO ]~U!Od puq ,~01.
sOl.
~0~
OL
(--) ~OL
eot
,,lie
D.o
eo',.
9
9
e. .oo
9
9
9
66,,, 8
,,
- ~OLXg
i,, oQ~
rn i.
t;
I 9
.o ee
iP
gQ
t
~t
9
IW/f3
oo
-,~0!. x g
~Pn~Io "/~
08
Response of the Human Nasal Mucous Membrane to Anti-Human IgE Serum
31
Discussion D a t a presented in this paper show t h a t a nasal allergy-like s y m p t o m as well as nasal eosinophilia were induced b y the challenge with a-IgE to the nasal mucous membrane. This s y m p t o m was observed mostly in allergic rhinitis cases and in some cases of vasomotor rhinitis, while not in normal non-allergic persons. I t was induced with neither a-IgA nor a-IgG. The end point of a-IgE for the positive reaction was not closely related to the serum I g E level, but was somewhat related to the sensitivity of the nasal mucous membrane to bradykinin. I n the course of the t r e a t m e n t the end point increased in m a n y cases. Now a question arises as to whether or not the above reaction was specific to a-IgE and was of allergic origin. The specifity of the reaction m a y be supported b y the following results from the present experiment: the s y m p t o m produced was very similar to t h a t seen in nasal allergy or in the nasal provocative test with allergen extract. The similar symptom, however, can also be induced by nonspecific mechanical or chemical irritation. I f it is not specific, the reaction usually appears immediately, within 30 see following the challenge, according to our experience in the nasal provocative test with allergen extract. I f not-specific, it must be induced not only with a-IgE but with a-IgG or a-IgA, since the same non-specific factor of irritation is contained commonly in these three anti-sera and the same stimulation is given to the nasal mucous membrane. I f it is not specific, the s y m p t o m is not associated with the rises of nasal eosinophilia, since local eosinophilia are generally approved as a characteristic sign of local allergy. A further discussion, nervertheless, is necessary before the final conclusion. W h y is the reaction produced more frequent in the allergic group than in the non-allergic group? According to Neweomb and Ishizaka (1969), the skin reaction from the injection with a-IgE is produced in the same degree either in allergic or non-allergic persons. I f so, the reaction in the nasal mucous membrane must be the same as in the skin. Actually, the reaction oecured in the normal mucous membrane. Nasal eosinophilia, an important sign of allergy, appeared in nonMlergie persons as well as allergic persons, although nasal symptoms were not observed in the non-allergic group. The production of the nasal s y m p t o m m a y require m a n y factors other than the combination of I g E in tissue with a-IgE given. One of the factors is the degree of the sensitivity of the nasal mucous membrane to the chemical mediators of allergy. The ease with increased sensitivity to bradykinin showed a stronger nasal reaction in the present experiment although histamine was weferable to bradykinin for such a test. Presumably both ~he amount of chemical mediator released and the sensitivity to it is higher in allergic persons than in non-allergic ones in the a-IgE test. The intensity of the nasal reaction to a - I g E was not related to the serum I g E level in the present experiment, in spite of the fact t h a t the increase in serum I g E level is one of the special characteristics of allergy. The same result was reported b y IIalliwell (1973) and J a r r e t t (1973) regarding the relationship between the skin reaction to a-IgE and the serum I g E level. Such a contradictory relation was also observed between the nasal reaction in the provocative test with allergen extract and the serum I g E level in our previous experiment (1974) Is it atopie or not if the present nasal reaction is specific? The skin reaction is revealed b y Ishizaka (1968, 1969, 1970) to be a reversed type of atopie skin
32
M. Okuda
reaction since aggregated I g E induces the same reaction as that by I g E itself, and the absorption of the antiserum with I g E does not. The reaction to a-IgE is not observed in newborn infants without detectable serum I g E according to Stevenson (1971). The nasal mucous membrane contains IgE. Therefore the same mechanism of reaction as t h a t proposed by Ishizaka (1968) in the skin might occur in the nasal mucous membrane in the present experiment. Actually, the nasal reaction was induced only with a-IgE and not with a-IgA or a-IgG in the present experiment. Is the present reaction pure atopie or not, if it is an IgE-mediated reaction? In our previous experiment (1973, 1974) skin was examined histologically following injection with a-IgE. The sections from these skins were characterized by findings similar to those observed in atopic nasal mucous membrane: degranulation of mast cell, eosinophilia, edema and local circulatory disturbance. On the other hand, following injection with a-IgA or a-IgG, only slight edema and infiltration of neutrophil were observed. The above differences in the changes in the a-IgE and in a-IgA or a-IgG suggest that the predominant part of the reaction was atopic in character in the skin, although the possibility of the introduction of non-specific factors can not completely be excluded since the commercial a-IgE used contained a non-specific irritable substance together with a-IgE itself. A curious and attractive phenomenon obtained was the decreases in the sensitivity of the nose to a-IgE in the course of hyposensitization with house dust. W h y does the injection with house dust induce the decreases? The injection can't produce an antibody to a-IgE, since a common antigenesity is not known between them. Is there a possibility for the production of antibody to antisera i.e. rabbit sera by a repeated a-IgE test? The protein amount in the serum used in each test was 0.7 ~g. I t m a y be insufficient to produce the antibody to the rabbit serum. Other possible factors for this phenomenon, based on the results of the treatment, m a y be the decrease in the sensitivity of the nasa] mucous membrane to non-specific irritation, the number of local mast cells and the amount of chemical mediator released from the mast cell. Further study of these factors is necessary for final conclusions. Conclusion The application of anti-IgE sera to the nasal mucous membrane induced a nasal allergy-like s y m p t o m as well as nasal eosinophilia. This s y m p t o m was observed in the majority of nasal allergy cases and in some of vaso-motor rhinitis cases, while not in non-allergic normal persons. The nasal eosinophilia, however, increased not only in allergic patients, but also in non-allergic persons in whom no nasal symptoms were induced. Such a nasal reaction was not induced with a-IgA sera or a-IgG sera. The character of the reaction was presumed to be specific to anti-IgE and was predominantly a reversed type anaphylaxis in the nasal mucous membrane. The sensitivity of the nasal mucous membrane to antiI g E decreased in the course of the hyposensitization with house dust. The cause of this phenomenon was obscure. References Arbesman, C. E., et al. : Measurement of serum IgE by a one step single radial radiodiffusion method. J. Allergy 49, 72 (1972)
Response of the Human Nasal Mucous Membrane to Anti-Human IgE Serum
33
Halliwell, R. E. W. : The localization of IgE in canine skin. J. Immunol. 110, 442 (1973) Hansel, F. K. : Clinical Allergy. St. Luois: Mosby 1953 Ishizaka, K., Ishizaka, T.: Physicochemical properties of reaginic antibody. Association of reaginic activity with immunoglobulin other than rA or rG globulin. J. Allergy 37, 169 (1966) Ishizaka, K., Ishizaka, T.: Induction of erythema wheal reaction by soluble antigen-rE antibody complexes in humans. J. Immunol. 101, 68 (1968) Ishizaka, K., Ishizaka, T.: Reversed type allergic skin reactions by anti-rE globulin antibodies in humans and monkeys. J. Immunol. 100, 554 (1968) Ishizaka, K., Ishizaka, T. : Immnnomeehanism of reversed type reaginic hypersensitivity. J. Immunol. 103, 588 (1969) Ishizaka, K., et al. : Biologic activities of aggregated immunoglobulin E. J. Immunol. 104, 854 (1970) Jarrett, E. E. E., Stewart, D. C.: The significance of circulating IgE. Immunology 24, 37 (1973) Newcomb, R. W., Ishizaka, K.: Skin reaction to anti-rE antibodies in atopic, non-atopic, and immunologically deficient children and adults. J. Allergy 43, 292 (1969) Okuda, M., et al. : Studies on local eosinophilia in humans. Wakayama Med. Report 15, 152 (1972) Okuda, M., et al.: Experimental atopic allergy. Otologia Fukuoka 19, Suppl. 2, 451 (1973) Okuda, M., et al.: Nasal provocation with antigen in nasal allergy. Excerpta Med. Congr. Series 300, 52 (1973) Okuda, M. : Fundamental and clinical studies on nasal allergy. Otologia Fukuoka 20, Suppl. 2, 297 (1974) Stevenson, D. D.. et al.: Development of IgE in newborn human infants. J. Allergy 48, 61 (1971) Minoru Okuda, M. D. Dept. Otolaryngology Wakayama Medical College
Wakayama Japan
3 Arch. Oto-/~hino-:Laryng.,Vol. 211
Response of the Human Nasal Mucous Membrane to Anti-Human IgE Serum M. O k u d a Department of Otolaryngology Wakayama )s
College, Wakayama, Japan
Received March 18, 1975
Summary. The response of the nasal mucous membrane to anti-human IgE serum was examined. The application of anti-IgE sera to the nasal mucous membrane induced a nasal Mlergy-like symptom as well as nasal eosinophilia. This symptom was observed in the majority of nasal allergy cases and in some vasomotor rhinitis cases, while not in non-Mlergic normal persons. The nasal eosinophilia, however, increased not only in allergic patients, but in nonallergic persons in whom no nasal symptoms were induced. Such a nasal reaction was not induced with anti-IgA sera or anti-IgG sera. The character of the reaction was presumed to be specific to anti-IgE and was predominantly a reversed type anaphylaxis in the nasal mucous membrane. The sensitivity of the nasal mucous membrane to anti-IgE decreased in the course of the hyposensitization with house dust. The cause of this phenomenon was obscure. Zusammen/assung. Durch Berfihrung des Anti-Menschen-IgE-Serums auf die Oberflgche der Nasenschleimhaut treten allergieartige Symptome und Lokaleosinophilie auf. Solche Symptome finder man meistens bei F/illen yon Nasenallergie und in wenigen Fiillen der sogenann)en vasomotorischen Rhinitis, aber man finder sic nicht bei nichtallergischen Patienten. Die Naseneosinophilie tritt nicht nur bei allergischen Patienten, sondern auch bei nichtallergischen Patienten auf, die bei der obigen Beriihrung keine Symptome gehabt haben. Solche Nasenreaktionen waren aus Anti-Menschen-IgA-Serum oder -Anti-IgG-Serum nicht hervorgegangen. Der Charakter der Reaktion ist spezifisch ffir Anti-IgE-Serum, und hauptsgchlich denkt man an Reaktion "reversed type anaphylaxis" in der Nasenschleimhaut. Die Sensibilitiit der Nasenschleimhaut zur Anti-IgE nimmt wiihrend der Hyposensitisation durch Hausstauballergien ab. Der Grund dieses Phiinomens ist unklar. A u n i q u e i m m u n o g l o b u l i n , IgE, was discovered b y Ishizaka (1966) as a carrier of h u m a n reaginie antibodies. A n a n t i - s e r u m to this i m m u n o g l o b u l i n either i n d u c e d a reversed t y p e atopie skin r e a c t i o n b y its i n t r a d e r m a l i n j e c t i o n or caused the release of chemical mediators of a n a p h y l a x i s from leucocyte or lung to which it was applied in vitro, according to I s h i z a k a et al. (1968, 1969, 1970). I n our previous e x p e r i m e n t a n t i - h u m a n I g E r a b b i t s e r u m (a-IgE) also produced a local eosinophilia b y the skin window m e t h o d of g e b u c k (1972) or characteristic histological changes of atopie allergy i n the skin, mucous m e m b r a n e of the oral c a v i t y a n d the trachea b y its i n j e c t i o n into each tissue (1973). The p r e s e n t exp e r i m e n t was u n d e r t a k e n to s t u d y the response of the n a s a l m u c o u s m e m b r a n e to a-IgE.
Material and Methods The subjects examined consisted of 128 cases with nasal allergic symptoms before hyposensitization, 38 cases with nasal allergies in the course of the hyposensitization, and 20 normal cases without nasal symptoms, as indicated in Table 1, 2 and 3.
26
M. Okuda Table 1. Age distribution
Age
10--20
21 --30
31--40
41 --50
51 --60
Total
Group 1
22
25
5
4
Group 2
9
24
6
9
3
51
Group 3
2
9
5
4
1
21
Normal
4
8
5
2
1
20
56
Table 2. Sex distribution Sex
Male
Female
Total
Group i
35
21
56
Group 2
32
19
51
Group 3
11
10
21
Normal
13
7
20
Table 3. Offending allergens Allergen
House dust
Pollen
Fungus
Group 1 Group 2
Others
46
1
4
5
33
2
9
7
The diagnosis of nasal allergy was established b y such test as history taking, skin test, nasal provocative test with allergen extracts, and nasal smear test. The skin test was performed using over 15 allergen extracts (Torii, J a p a n ) including house dust, pollen a n d fungus. Of t h e offending allergens detected, house dust was t h e most frequent, as indicated in Table 3. The nasal provocative test was carried out as previously reported (1973), using t h e various kinds of allergen extracts suspected as offending allergen b y the skin test. The procedure of t h e test was as follows: a small r o u n d disc, 3 X 3 m m in diameter and No 5 A in quality (Toyo Roshi, J a p a n ) containing a certaine a m o u n t of allergen extract, was kept dry. A control disc which was similar to the above disc b u t without t h e allergen extract, was also prepared. The control disc was first placed on the surface of t h e anterior portion of t h e inferior t u r b i n a t e on one side of t h e nose, a n d then, when no reaction occurred within 5 rain, a disc with allergen extract (allergen disc) was applied to t h e same site of the inferior t u r b i n a t e in the same m a n n e r as the control disc. I n this test symptoms such as 1) sneezing or itching, 2) excessive secretion, a n d 3) swelling of t h e nasal mucous m e m b r a n e were induced. Symptoms 2) a n d 3) were observed b y rhinoseopy. The result obtained was evaluated as positive when a n y two or all three of t h e symptoms were induced, a n d evaluated as negative when one or no symptoms appeared, or when the nasal symptoms appeared immediately, usually within 30 see, after the disc was applied. The smear test was made as follows: three nasal smears for each person were prepared a n d observed b y microscopy according to a Hansel's staining m e t h o d (1953). The degree of eosinophilia was estimated using Hansel's criteria (1953): a few scattered eosinophils were recorded as 1 + ; larger collections or clumps were noted as 2 ~- ; very dense collections of eosinophils were 3 ~ or 4~-. Each subject diagnosed as allergic was positive in two or all three of the above mentioned clinical tests such as the skin, nasal test with allergen extract a n d t h e smear test.
Response of the H u m a n Nasal Mucous Membrane to A n t i - H u m a n IgE Serum
27
The response of t h e nasal mucous m e m b r a n e to a-IgE was examined b y t h e following end point m e t h o d (a-IgE test). Fresh dilutions of a-IgE were made from 1:10 to 1 : I 0 0 0 0 in ten fold series on each day of testing fl'om t h e stock solution (Behringwerke, West Germany). A few drops, ca. 0.01 ml of each dilute were dropped on each disc of filter paper which was used as a control in t h e nasal provocative test (a-IgE disc). Discs with a 1:10 concentration of a n t i - h u m a n IgG r a b b i t serum (a-IgG) or anti-IgA serum (a-IgA) were also made. For 3 days before the test a n y drug against allergy were withheld from the person to be tested. The test commenced with t h e use of a n a-IgG disc or a-IgA disc, Each disc was placed gently on the surface of the anterior portion of the inferior turbinate, carefully to minimize the irritation to the mucous m e m b r a n e ; t h e n t h e test was followed b y a challenge from a n a-IgE disc on t h e opposite side of t h e nose tested with a n a-IgG or a-IgA disc. The increasing concentrations of a-IgE in t h e series from 1:10000 to 1:10 were tested at time intervals of 10 min on t h e alternating sides of t h e nose until a positive reaction oecured. The criteria of a positive reaction were the same as in the nasal provocative test with allergen extract. The end point was considered as a concentration of a-IgE necessary to produce a positive reaction. The above test was carried out not only in the 128 cases of allergy before hyposensitization b u t also in 38 cases among t h e m once every m o n t h repeatedly for 6 m o n t h s in the course of hyposensitization with house dust. An example of t h e most frequent plan of hyposensitization used is indicated in Table 4. The sensitivity of t h e nasal mucous m e m b r a n e to b r a d y k i n i n was tested. A disc w i t h b r a d y k i n i n (Sandoz, Swiss) which contained 0.01 mI of the concentration of 100 ~zg per ml (bradykinin disc) was applied to t h e inferior t u r b i n a t e of 24 cases the day before the a-IgE test. I t s procedure and criteria of positive reaction were just the same as those of the a-IgE test. Changes in t h e degree of nasal eosinophilia following t h e I g E test were examined in 16 of the allergic a n d 12 of the normal persons. The excessive nasal secrete filling the nasal cavity was first removed b y suction, a n d t h e n 3 nasal smears were made with the secrete collected from t h e surface of t h e inferior tm'binate with a cotton swab. Ten mim~tes later, a disc, 10 • 5 m m in size, which contained 0.04 ml of stock non-diluted a-IgE, was introduced on the surface of the inferior turbinate. I m m e d i a t e l y after the onset of the positive reaction the disc was removed. Then 1, 2, 3 a n d 4 hrs following the reaction t h e nasal secrete was collected from the challenged site of the inferior turbinate, a n d nasal smears were made and observed as has already been described. The relations between the degree of a-IgE reaction a n d the serum I g E level were investigated. IgE levels were determined b y the single radial radio-immunodiffusion method according to A r b e s m a n (1).
Table 4. A frequent schedule of hyposensitization with house dust allergen Frequency of injection
Concentration of allergen
Dose of allergen (ml)
2/week
1 : 1000
0.02--0.03--0.05--0.07 --0.1--0.15--0.2--0.3--0.5
2/week
1 : 100
0.05--0.07--0.1 --0.15--0.2 --0.3--0.5
2/week
1 : i0
0.05--0.07--0.1 --0.15--0.2 --0.3--0.5
i/week
1 : 10
0.5--0.5--0.5--0.5
1/2 weeks
1 : 10
0.5--0.5--0.5--0.5
1/month
1 : 10
0.5--0.5 . . . . . .
House dust extract used contained 0.36 ~zg/ml of total nitrogen or 0.18 tzg/ml of protein nitrogen in t h e concentration of 1 : 1000. I n t r a d e r m a l injections were made for the t r e a t m e n t .
28
M. Okuda Results
The results from the a - I g E test were analyzed in the following groups determined on the basis of the skin and provocative tests with allergen extract. Group 1 was considered as allergic rhinitis with positive skin and provocative tests. Group 2 was possibly allergic with positive skin b u t negative provocative test. Group 3 was so-called vasomoter rhinitis with negative skin and provocative tests. The n u m b e r of the cases was 56 in Group 1, 51 in Group 2, 21 in Group 3 and an additional 20 normal cases. The distribution of age and sex a m o n g each group was slightly different as indicated in Table 1 and 2 ; females were the most frequent in Group 3 and the cases of 10--20 years of age were the m o s t frequent in Group 1. The main offending allergen detected was house dust, as indicated in Table 3. A m o n g these groups positive responses were obtained in 85.4~ of Group 1, in 50.60/0 of Group 2, in 28.60/0 of Group 3 and in none of the normal control group. On the other hand, no positive nasal reaction was produced in a n y group in the a - I g A or a-IgG test. The end point of a - i g E for the positive reaction was 1 : 1000 in the m a j o r i t y of the eases in Group 2 and 3, while 1 : 1000 and 1 : 10000 in Group 1 as in Table 5. The s y m p t o m produced was similar to t h a t seen in the nasal provocative test with allergen extract or in the case of nasal allergy. Of 80 eases with positive reactions 17 showed all three s y m p t o m s of nasal allergy such as sneezing, rhinorrhea and swelling of the nasal mucous membrane, 41 showed a n y two of the symptoms, and 22 showed only one as indicated in Table 6.
Table 5. End point of anti-IgE serum for positive nasal reaction End point for positive response
Group 1 Group 2 Group 3 Normal
1:10
1:10 2
l:10 ~
1:10 4
5 4 0 0
6 6 1 0
25 15 5 0
12 1 0 0
Negative response
Total
8 25 15 20
56 51 21 20
The positive reactions were obtained in the majority of Group i, in the half of Group 2, in 28.60/0 of Group 3, and in none of the normal control group.
Table 6. Nasal symptoms produced in anti-IgE test Itching or sneezing
Rhinorrhea
Swelling
*
*
*
*
*
* *
Total 17 31
*
2
*
8
Response of the Human Nasal Mucous Membrane to Anti-Human IgE Serum
29
Table 7. Relationship between nasal bradykinin test and the end point in a-IgE test End point of a-IgE
Bradykinin test
Positive Negative
i : 10
1 : 10 ~
1 : 103
1 : 104
3 5
1 2
4 3
4 2
The bradykinin test induced a positive nasal reaction in half of the 24 cases. The higher the concentration of a-IgE required for positive reaction, the lower the incidence of positive reaction in the bradykinin test.
Table 8. Changes in nasal eosinophilia following the challenge with anti-IgE After challenge Allergic
Before challenge
0 1 -t2+ 3+
Non-allergic
1+
2+
3+
6
4
1 1 1 1
4+
0
1+
2+
3+
4
5
1
2
2
Three hours after the a-IgE test nasal eosinophilia increased in 15 of 16 allergic persons, and appeared in 8 of 12 non-allergic persons who had no eosinophilia before the test and showed no positive nasal symptom in the test.
The b r a d y k i n i n disc i n d u c e d a p o s i t i v e n a s a l r e a c t i o n in h a l f of t h e 24 eases e x a m i n e d . W h e n t h e r e a c t i o n to b r a d y k i n i n was c o m p a r e d to t h a t of a - I g E in t h e same person, t h e following t e n d e n c y was o b s e r v e d : t h e higher t h e conc e n t r a t i o n of a-IRE r e q u i r e d for p o s i t i v e reaction, t h e lower t h e incidence of p o s i t i v e r e a c t i o n in the b r a d y k i n i n t e s t as i n d i c a t e d in T a b l e 7. N a s a l eosinophilia a p p e a r e d a n d increased following t h e a-IRE test. One h o u r a f t e r t h e t e s t the smears showed o n l y scarce cell c o m p o n e n t s w i t h o u t eosinophil, b u t 2 or 3 hrs l a t e r eosinophilia increased n o t o n l y in t h e allergic g r o u p b u t also in t h e non-allergic group. T h e t i m e a t which t h e m a x i m u m n u m b e r of eosinop h i l i a were seen a t 2 or 3 hrs a f t e r t h e t e s t in 23 of 25 cases e x a m i n e d . I n c r e a s e d eosinophilia were o b s e r v e d in 15 o f 16 allergic persons a t 3 hrs, a n d in 8 o f 12 nonallergic persons, who h a d no eosinophilia before the a - I g E t e s t a n d showed no p o s i t i v e n a s a l s y m p t o m s in t h e a - l g E t e s t as in T a b l e 8. The s e r u m I g E level was c o m p a r e d to t h e e n d p o i n t of a-IRE for a p o s i t i v e n a s a l reaction, b u t no close r e l a t i o n b e t w e e n t h e m was o b t a i n e d ; t h e m e a n I g E level was 916.3 u n i t s / m l in 8 cases w i t h a 1:10 c o n c e n t r a t i o n o f t h e e n d p o i n t ; t h e level was 562.6 in 15 cases w i t h 1:100; 766.3 in 16 cases w i t h 1 : 1 0 0 0 ; 975.0 in 4 cases w i t h a 10000; 708.0 in 23 cases w i t h a n e g a t i v e r e a c t i o n b y the challenge w i t h a 1 : 10 c o n c e n t r a t i o n of a - I g E as i n d i c a t e d in Fig. 1.
9g':~!g u.~ po~o!pu.r ss pou.~msxo osso I u! g i u o pomsoaoop p u s ~, u.t p o ~ u ~ q o u n pou.~mo~ 'sos~so I 8 u.r pos~oaou! N~I-qS go ~u.~od p u o oq~ q~snp o s n o q q%tat uo.r0~sz.~.~suosod.~q $o os2noo oqc~ u I
tto!~uzD~.suos-ods q jo osanoo oqc~u! uoDoeoa pssqstt oAt.~.rsod ~ zo~ mnaos ~I~i-!~u~ jo ~u!od puo oq~ ur so~uvqo "g'~.r~[ (q~uo~) ~uom~ee~• ~o ~e~s eqr 5ui~oilozl ecU!.L OI
6
8
L
9
S
~
~
~, ,'x\
.
Z
I
".x :l.e ~ 8 N
9
\ \
Ot
201
gOI
i~oT
301fi~uv ~LO ]~U!Od puq ,~01.
sOl.
~0~
OL
(--) ~OL
eot
,,lie
D.o
eo',.
9
9
e. .oo
9
9
9
66,,, 8
,,
- ~OLXg
i,, oQ~
rn i.
t;
I 9
.o ee
iP
gQ
t
~t
9
IW/f3
oo
-,~0!. x g
~Pn~Io "/~
08
Response of the Human Nasal Mucous Membrane to Anti-Human IgE Serum
31
Discussion D a t a presented in this paper show t h a t a nasal allergy-like s y m p t o m as well as nasal eosinophilia were induced b y the challenge with a-IgE to the nasal mucous membrane. This s y m p t o m was observed mostly in allergic rhinitis cases and in some cases of vasomotor rhinitis, while not in normal non-allergic persons. I t was induced with neither a-IgA nor a-IgG. The end point of a-IgE for the positive reaction was not closely related to the serum I g E level, but was somewhat related to the sensitivity of the nasal mucous membrane to bradykinin. I n the course of the t r e a t m e n t the end point increased in m a n y cases. Now a question arises as to whether or not the above reaction was specific to a-IgE and was of allergic origin. The specifity of the reaction m a y be supported b y the following results from the present experiment: the s y m p t o m produced was very similar to t h a t seen in nasal allergy or in the nasal provocative test with allergen extract. The similar symptom, however, can also be induced by nonspecific mechanical or chemical irritation. I f it is not specific, the reaction usually appears immediately, within 30 see following the challenge, according to our experience in the nasal provocative test with allergen extract. I f not-specific, it must be induced not only with a-IgE but with a-IgG or a-IgA, since the same non-specific factor of irritation is contained commonly in these three anti-sera and the same stimulation is given to the nasal mucous membrane. I f it is not specific, the s y m p t o m is not associated with the rises of nasal eosinophilia, since local eosinophilia are generally approved as a characteristic sign of local allergy. A further discussion, nervertheless, is necessary before the final conclusion. W h y is the reaction produced more frequent in the allergic group than in the non-allergic group? According to Neweomb and Ishizaka (1969), the skin reaction from the injection with a-IgE is produced in the same degree either in allergic or non-allergic persons. I f so, the reaction in the nasal mucous membrane must be the same as in the skin. Actually, the reaction oecured in the normal mucous membrane. Nasal eosinophilia, an important sign of allergy, appeared in nonMlergie persons as well as allergic persons, although nasal symptoms were not observed in the non-allergic group. The production of the nasal s y m p t o m m a y require m a n y factors other than the combination of I g E in tissue with a-IgE given. One of the factors is the degree of the sensitivity of the nasal mucous membrane to the chemical mediators of allergy. The ease with increased sensitivity to bradykinin showed a stronger nasal reaction in the present experiment although histamine was weferable to bradykinin for such a test. Presumably both ~he amount of chemical mediator released and the sensitivity to it is higher in allergic persons than in non-allergic ones in the a-IgE test. The intensity of the nasal reaction to a - I g E was not related to the serum I g E level in the present experiment, in spite of the fact t h a t the increase in serum I g E level is one of the special characteristics of allergy. The same result was reported b y IIalliwell (1973) and J a r r e t t (1973) regarding the relationship between the skin reaction to a-IgE and the serum I g E level. Such a contradictory relation was also observed between the nasal reaction in the provocative test with allergen extract and the serum I g E level in our previous experiment (1974) Is it atopie or not if the present nasal reaction is specific? The skin reaction is revealed b y Ishizaka (1968, 1969, 1970) to be a reversed type of atopie skin
32
M. Okuda
reaction since aggregated I g E induces the same reaction as that by I g E itself, and the absorption of the antiserum with I g E does not. The reaction to a-IgE is not observed in newborn infants without detectable serum I g E according to Stevenson (1971). The nasal mucous membrane contains IgE. Therefore the same mechanism of reaction as t h a t proposed by Ishizaka (1968) in the skin might occur in the nasal mucous membrane in the present experiment. Actually, the nasal reaction was induced only with a-IgE and not with a-IgA or a-IgG in the present experiment. Is the present reaction pure atopie or not, if it is an IgE-mediated reaction? In our previous experiment (1973, 1974) skin was examined histologically following injection with a-IgE. The sections from these skins were characterized by findings similar to those observed in atopic nasal mucous membrane: degranulation of mast cell, eosinophilia, edema and local circulatory disturbance. On the other hand, following injection with a-IgA or a-IgG, only slight edema and infiltration of neutrophil were observed. The above differences in the changes in the a-IgE and in a-IgA or a-IgG suggest that the predominant part of the reaction was atopic in character in the skin, although the possibility of the introduction of non-specific factors can not completely be excluded since the commercial a-IgE used contained a non-specific irritable substance together with a-IgE itself. A curious and attractive phenomenon obtained was the decreases in the sensitivity of the nose to a-IgE in the course of hyposensitization with house dust. W h y does the injection with house dust induce the decreases? The injection can't produce an antibody to a-IgE, since a common antigenesity is not known between them. Is there a possibility for the production of antibody to antisera i.e. rabbit sera by a repeated a-IgE test? The protein amount in the serum used in each test was 0.7 ~g. I t m a y be insufficient to produce the antibody to the rabbit serum. Other possible factors for this phenomenon, based on the results of the treatment, m a y be the decrease in the sensitivity of the nasa] mucous membrane to non-specific irritation, the number of local mast cells and the amount of chemical mediator released from the mast cell. Further study of these factors is necessary for final conclusions. Conclusion The application of anti-IgE sera to the nasal mucous membrane induced a nasal allergy-like s y m p t o m as well as nasal eosinophilia. This s y m p t o m was observed in the majority of nasal allergy cases and in some of vaso-motor rhinitis cases, while not in non-allergic normal persons. The nasal eosinophilia, however, increased not only in allergic patients, but also in non-allergic persons in whom no nasal symptoms were induced. Such a nasal reaction was not induced with a-IgA sera or a-IgG sera. The character of the reaction was presumed to be specific to anti-IgE and was predominantly a reversed type anaphylaxis in the nasal mucous membrane. The sensitivity of the nasal mucous membrane to antiI g E decreased in the course of the hyposensitization with house dust. The cause of this phenomenon was obscure. References Arbesman, C. E., et al. : Measurement of serum IgE by a one step single radial radiodiffusion method. J. Allergy 49, 72 (1972)
Response of the Human Nasal Mucous Membrane to Anti-Human IgE Serum
33
Halliwell, R. E. W. : The localization of IgE in canine skin. J. Immunol. 110, 442 (1973) Hansel, F. K. : Clinical Allergy. St. Luois: Mosby 1953 Ishizaka, K., Ishizaka, T.: Physicochemical properties of reaginic antibody. Association of reaginic activity with immunoglobulin other than rA or rG globulin. J. Allergy 37, 169 (1966) Ishizaka, K., Ishizaka, T.: Induction of erythema wheal reaction by soluble antigen-rE antibody complexes in humans. J. Immunol. 101, 68 (1968) Ishizaka, K., Ishizaka, T.: Reversed type allergic skin reactions by anti-rE globulin antibodies in humans and monkeys. J. Immunol. 100, 554 (1968) Ishizaka, K., Ishizaka, T. : Immnnomeehanism of reversed type reaginic hypersensitivity. J. Immunol. 103, 588 (1969) Ishizaka, K., et al. : Biologic activities of aggregated immunoglobulin E. J. Immunol. 104, 854 (1970) Jarrett, E. E. E., Stewart, D. C.: The significance of circulating IgE. Immunology 24, 37 (1973) Newcomb, R. W., Ishizaka, K.: Skin reaction to anti-rE antibodies in atopic, non-atopic, and immunologically deficient children and adults. J. Allergy 43, 292 (1969) Okuda, M., et al. : Studies on local eosinophilia in humans. Wakayama Med. Report 15, 152 (1972) Okuda, M., et al.: Experimental atopic allergy. Otologia Fukuoka 19, Suppl. 2, 451 (1973) Okuda, M., et al.: Nasal provocation with antigen in nasal allergy. Excerpta Med. Congr. Series 300, 52 (1973) Okuda, M. : Fundamental and clinical studies on nasal allergy. Otologia Fukuoka 20, Suppl. 2, 297 (1974) Stevenson, D. D.. et al.: Development of IgE in newborn human infants. J. Allergy 48, 61 (1971) Minoru Okuda, M. D. Dept. Otolaryngology Wakayama Medical College
Wakayama Japan
3 Arch. Oto-/~hino-:Laryng.,Vol. 211
