The clinical and immunologic of immunotherapy*
specificity
Philip S. Norman, M.D., and Lawrence M. Lichtenstein, M.D., Ph.D. With the technical assistance of Joanne Tignall Baltimore, Md.
In order to study the spec.$city of immunotherapy for respiratory allergy, a group of patients sensitive to both ragweed and grass pollens were selected. From 87 volunteers with a history of both spring and fall hay fever, 42 patients with evidence of strong sensitivity by basophil histamine release to both ragweed pollen and mixed grass pollen extracts were selected for study. On the basis of the histamine release data, the patients were divided into two groups matched for sensitivity to both grass and ragweed pollens. In 1970, May and June symptom diaries showed the two groups to suffer quite similar severity of symptoms during the grass pollination season. One group of patients was started on a preseasonal course of immunotherapy with alum-precipitated aqueous extract of ragweed pollen while the other group received placebos containing histamine. By fall there had been a considerable rise in IgG-blocking antibodies to ragweed in the treated group. Symptom diaries in August and September showed that the treated group showed significantly less severe symptoms than the placebo group. Both groups received booster injections at 2-wk intervals from the fall of 1970 to the fall of 1971. Doses in the treated group were raised to attempt to administer the largest possible dose. Again there was no difference in the symptoms reported by the two groups during the grass pollination season, but an even greater difference emerged between the two groups during the ragweed season. The following year (1972) the same results were obtained. These data demonstrate that treatment with ragweed pollen extracts has little or no e$ect on grass pollen symptoms and confirm that immunotherapy is clinically as well as immunologically specihc. Antibody responses to the second year of high-dosage booster injections was not greater than responses to a comparatively short preseasonal course given the first year.
It is a logical assumptionthat treatment of respiratory allergies by injections of allergenic extracts (immunotherapy,desensitization)producessymptom relief as a result of immunologically specific responses.This view is buttressedby the often repeated observationthat immunotherapyleadsto development of specific blocking antibodies now known to be of the IgG class.le3Furthermore,specific IgG and secretory IgA antibodies appear in nasal secretions,4and specific IgE antibodiesin serum may be reduced.5*6 From the Division of Clinical Immunology, Department of Medicine, The Johns Hopkins University School of Medicine at the Good Samaritan Hospital. Supported by Grants AI 04866 and AI 08270 from the National Institute of Allergy and Infectious Diseases,National Institutes of Health. Received for publication Sept. 12, 1977. Accepted for publication Feb. 8, 1978. Reprint requeststo: Philip S. Norman, M.D., The Good Samaritan Hospital, 5601 Loch Raven Blvd., Baltimore, Md. 21239. *Publication No. 303 from the O’Neill ResearchLaboratories, The Good Samaritan Hospital, 5601 Loch Raven Blvd., Baltimore, Md. 21239. Vol. 61, No. 6, pp. 370-377
Only Lowell and Franklin,’however, have madeclinical observationsbearingon specificity. In their study of patients with multiple sensitivities and receiving injections of multiple allergens, immunization with ragweed extract was withdrawn or withheld in some of the patientswhile being given in otherswith similar clinical severity. The fall symptoms were significantly less severe in the patients who received the ragweedextract than in those who did not, confirming that treatment with other allergensdid little for ragweed-inducedsymptoms. After techniques were developed for quantitative study of IgE-mediatedhistamine releasefrom basophils in vitro, several laboratories have studied the effect of immunotherapyon this IgE-mediatedreaction. Pruzanskyand Patterson*reportedthat high dosage of immunotherapywith ragweed could induce a state of no or low basophil reactivity to specific antigen. Studies from our laboratory indicated that such changesoccurred in a significant portion of treated patients and only rarely in untreated patients. Although patientswho developedreducedor absentcell
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@ 1978 The C. V. Mosby Co.
VOLUME NUMBER
61 6
Specificity of immunotherapy
TABLE I. Patients who completed of treatment Plrcebo
No. of patients M a le F e m a le M e a nage Range M e a nageonset Range
21 14 7 35 26-48 15 7-35
first season
RAGWEED EXTRACT ; MIXED &ASS
371
1
EXTRACT . i3c
Alumgredpiteted ragweed extract
20 14 6 35.5 24-55 14 3-43
: t -4l
reactivity often fared very well during natural ragweedexposure,they did not invariably do ~0.~9lo A further study by Lichtensteinand Levy” showedthat children who had receivedimmunotherapywith ragweed and developedreducedcellular reactivity to ragweedalso invariably had low cellular reactivity to an immunologicallyunrelatedantigen,timothy grass. O n the other hand, patientswhosecells reactedwith greater than 5 0 % releaseof histamine to ragweed extract showed a similarly high releaseto timothy extract. Theseobservationssuggestedthat diminished cellular reactivity after treatmentwas to allergensnot used in treatment and therefore immunologically nonspecific.The reductionof cellular reactivity occurredafter treatmentdespitethe continuedpresence of IgE antibodiesin the serum. Furthermore,May, Schumacher,and W illiams’* demonstratednearly parallel fluctuations in leukocyte sensitivity to two unrelated allergens in patients receiving injection therapywith oneof the allergens.In one case,during therapywith one allergen,completeloss of reactivity occurredto two unrelatedallergens.Such data suggest that there is a nonspecific element to immunotherapyin addition to the well-recognized specific elementsand raise the possibility that treatment with one allergenmay benefit diseasedue to anotherallergen. Lichtensteinand Levy” recognizedthat their study did not represent a definitive demonstrationof nonspecificdesensitizationof cells andsuggestedthat a prospectivestudy shouldbe donein which patients sensitiveto more than one allergenwould be treated with one allergen while both cellular and clinical reactionsto both the treatmentallergenand another allergenwould be observed. The presentstudywas organizedwith this objective in m ind. Patients doubly sensitive to ragweedand grassallergenswere selectedand treatmentwas instituted with ragweedallergenalone. Unfortunatelyfor our goal, none of the patientstreatedover threesea-
01 ’
i t :
.
I I TREATED
J
PLACEBO TREATED PLACEBO
FIG. 1. Cell sen&iv@ for ragaf%d graes pollen extracts in r mated and pl p&mts. The bar indicstes the geometric meen for e W h group. sons deveioped a significant reduction in cellular reactivity to ragweed;hencethe specificity of loss of reactivity could not be studied. The clSn~crm1 results, however,showeda high degreeof sped&iv in rhat fall (ragweed) symptomswere si~~~~ iwrprloved for three consecutiveseasonswhereass@ng (grass) symptomswere unimproved.
MAT Patientswereselected fromindividuaLwhoresponded to a newspaper storyaskingfor volunteers with “hay fever.” Respondents weresenta questionnaire elicitingthetim e of yearof hayfeversymptoms. Thosewhoindicti that they had sneezing andstuffy, runnynose,with or withouteye symptoms in thelatespringandthefall but v&t&~ symptomsat othertimesof the year,werecalled-tfifor further study.Eighty-seven in&ii&&s underwent ~j~#~~~ testing. The resultsof peripheralblood leukocytehistamine releaseto m ixedgrassextract,qe GroupI, ragwe&d extract,andantigenE havebeenreported rateof reactivityto the purifiedantigens in patientsreactiveto the crudeextracts werealso subjects for a previousstudyo which showedgoodagreement (w&h a small n+un&rof clearexceptions) betweenskin testsandhistaminerelease with bothragweed andgrassextractsandg& agreement betweenRASTandskin tes&with ragwec?a extracts.”O f the 87 patients,4’2hadrelease of 5 0 %or moreof cellular
372
J. ALLERGY CLIN. IMMUNOL. JUNE 1978
Norman and Lichtenstein 1970
1971
J&.L&
& Aq to Sept
SPRING hv to June
Aug to Sept I
ept. -
17%
l
I 20
21
21
0 20
&ii
13 NUMBER
OF
16
12
16
MTIENTS
Placebo 0 Treated o
FIG. 2. Individual mean seasonal symptom scores for each group of patients in the spring and fall of 1970,1971, and 1972. The cross-bar indicates the mean for each group.
histamine to I Fg protein/ml or less of both ragweed and grass extracts and agreed to participate in a therapeutic study.
Alum precipitate Alum-precipitated mixed ragweed extract was furnished by Center Laboratories, Port Washington, N. Y., as a 20,000 PNU/ml concentrate. Subsequently, the ragweed extract was found by Center Laboratories to contain 0.019 mg antigen E/ 10,000 PNU by double diffusion in gel of the alum precipitate redissolved in a potassium citrate-citric acid buffer.
Placebos Diluent was used for placebo injections. To duplicate local wheal and erythema reactions, histamine was added. A solution of 0.5 mg histamine/ml was prepared and labeled 10,000 PNU. Tenfold dilutions were made so that a graded increase in local reactions occurred as the dose was increased. As a placebo identical in appearance to the alum precipitate (a cloudy yellow suspension) could not be prepared, it was necessary to maintain the blind by the method of Lowe11 and Franklin.‘S A physician (P. S. N.) or a technician (J. T.) administered the injections, filling the syringe in another room out of sight of the patient. The two solutions could not be differentiated in a syringe made of translucent plastic without close inspection. Clinical evaluations were performed during the season by other physicians not aware of the treatment received by the patients, and symptom diaries were scored by computer.
Pollen counts Daily pollen counts were taken from an Ogden rotating slide sampler placed in a suburban location in Baltimore. No attempt was made to distinguish between various species of trees and grass. Tree pollen counts are shown in 197 I and 1972 (Figs. 6 to 8) at the time the predominant pollen was Oak.
Symptom
diaries
Patients filled out the daily symptom diary forms described previously for 9 or IO wk during the grass pollination season and 8 wk during the ragweed pollination season. The forms report the duration of sneezing, rhinitis, conjunctivitis, and cough for each 12-hr period, along with antihistamine and other medication use.r6 The forms were collected and checked for completeness weekly. Any inconsistencies were rectified by discussion with the patient. The symptoms were scored numerically and entered into a computer which calculated an average daily symptom score for each patient and an average score for each group of patients for each day of the season.
Blocking
antibodies
IgG antibodies were measured by the inhibition of histamine release as reported previously.6 Briefly, cells from a ragweed-allergic donor were challenged with a concentration of antigen E sufficient to cause 60% to 80% histamine release either in normal (AB) serum or in dilutions of the sera to be tested. The resulting data were plotted on percent inhibition vs serum concentration, and the reciprocal of the
VOLUME NUMBER
61 6
Specificity
of immunotherapy
373
2400
2000
ICOO 5 1200
HO0
8 5 -: g
400
I
II 12
16
20
24
28
I
4
4
8
MAY
I
I1
12 16 JUNE
20
1
I
24
28
0
1970
FIG. 3. Daily average symptom
scores and grass pollen count in the spring of 1970.
serum concentration that caused 5 0 % inhibition of histamine release was defined as the antibody titer.
The 42 patientsselectedfor study were paired on the basisof histaminereleaseto both grassand ragweed extracts in or&r to developtwo groups with similar sensitivity to both allergens.TableI showsthe age, sex, and age of onset of hay fever in the two groups. One memberof each pair was assignedat random to receive treatmentwith alum-precipitated ragweedextract and the other member to receive placebo.F ig. 1 showsthe concentrationof ragweed andgrassextractsrequiredfor 5 0 % histaminerelease in the 42 patientsand demonstrates the similarity between the two groups. In May, 1970,symptomdiarieswere distributedto the patientsand injectionswere also initiated. Symptom reporting during the 1970 grass pollen season (Figs. 2 and 3) showedthat the symptomscoreswere quite similar with the samem e a n(6.3) in both groups and a very similar spread(1 patient failed to furnish adequatediariesin tk’eplacebogroup). In May, June, July, and August, both groupsreceivedweekly injections, the numberof injectionsvarying from 11 to 17 with a m e a nof 15injections(1 patientwho startedon aIum precipitate moved away from Baltimore and could not finish the course).In the treatedgroup the cumulativepreseasonal doserangedfrom 4,800 PNU to 30,800PNU with a m e a ndoseof 13,746PNU (26 pg antigenE equivalent).Dosagesfor the threeyears of study are shown in F ig. 4.
too
90
80
70
60
50
4c l-
3c ,-
2c I-
ICI-
CI-ho INJECTKINS AVERAGE
II to I8
II to34
15
21
l5to22
FIG. 4. Cumulative annual dose of alum-pre&p&&e6l weed administered before the rmeed segi 1971, and 1972. Bars indicate mean for each vex.
19 4
r@g-
374
Norman
J. ALLERGY
and Lichtenstein
CLIN. IMMUNOL. JUNE 1978
2400
-
Treated
----
Untreated F’oilenCounl 2000
1600
‘I i3 ”
1200
b 3 B
800
400
0 7
I
I
17 20
24
I
.,I
28
I
I
3
4
8
1
I
1
9
1’1
12
16
20
24
28
I
4
OCTOBER
SEPTEMBER
MJGUST
1
1970
FIG. 5. Daily average symptom
/i :
I i !
Ii
16
20
24
28
scores and ragweed
pollen counts
in the fall of 1970.
/
/
i
“\J h
2
6
IO
APRIL
18
I4
22
26
50
2
6
IO
14
18
JUNE
MAY
22
26
30
4 JULY
1971
FIG. 6. Average
daily symptom
scores and tree and grass pollen counts in the spring of 1971.
During the subsequentragweedseason,symptom scores (Figs. 2 and 5) showed that the specifically treated patients fared significantly better than the placebo-treatedpatients(p = 0.0006, Mann-Whitney U test). Post-treatmentmeasurementsof blocking antibodies (IgG) for ragweedshowedthat all nineteenof the treated patients tested (one not done) showed a rise in titer. Despite theserises none of thesepatientsshoweda
reductionin cell sensitivity or reactivity. For this reason we initiated a more vigorous and prolonged treatmentprogram following the 1970 ragweedseason. Patients received injections every two weeks from late October to the following August. With the exception of one patient who could not attend clinic regularly and receivedonly 11 injections, the number of injections rangedfrom 17 to 24 with an averageof 21. Cumulative doses ranged from 20,700 PNU to
VOLUME NUMBER
61 6
Specificity of immunotherapy
,/~.._,f 16
375
,’ \.
20 24 AUGUST
28
2
6
IO 14 If3 SEPTEMBER 1971
22
26
30
4 OCTOBER
FlG. 7. Average daily symptom scores and ragweed pollen counts in the fall of 1971
e---e
Treated Unkeoled
- 600
Pdltm CwJl GUM
-500
Trees
i if I ii
__*-’ I 24 26 APRIL
I
4
_-./ a
I2
16 MAY
24
\ 8’ \