Imported fire ant (IFA) sensitivity: IFA-specific levels of serum IgG and IgG4

Imported fire ant (IFA) sensitivity: IFA-specific levels of serum IgG and lgG4

Margaret A. Reed, MS, and Brian T. Butcher, PhD New Orleans, La.

ImportedJire ant (IFA) whole body extract (WBE) and venom (V)-specific IgG and IgG4 antibodies and specific IgE antibodies were evaluated in sera from 56 IFA-sensitive individuals (18 undergoing immunotherapy with IFAWBE and 38 individuals not being treated) and 44 nonatopic and atopic control subjects with no history of IFA allergy. Although there was no difSerence in the level of IFAWBE- or IFAV-specific IgG between treated and untreated patients, both groups had higher levels of IFAWBE- and IFAV-specific IgG (p =C 0.05) than did control subjects. Patients receiving treatment tended to have higher levels of IFAWBE-speciJic IgG4 than did either untreated patients (0.05 < p CC 0.10) or control subjects (p < 0.051. Levels of IFAV-specijic IgG4 were higher in treated patients than in control subjects (p < 0.05) but were not dtflerent between treated and untreated patients. Levels of IFAWBE- and IFAV-specific IgE antibodies did not differ between the two patient populations but were higher in both groups than in control subjects (p < 0.05). The ratio of IgG4 to IgE (G4lE) for both IFAWBE and IFAV was calculated for all groups. The ratios of IFAWBE- and IFAV-specijk G4lE were higher in treated patients and in control subjects as compared to nontreated patients (p < 0.05). IFAV-speciJic G4lE ratios were lower in treated patients than in control subjects, but IFAWBE-specijc G4 I E ratios were the same as ratios for control subjects. These jindings demonstrated that immunotherapy with IFAWBE tends to raise the level of IFAWBE-spec$c IgG4 and thus influence the ratio of IFAWBE G4lE. The observation that IFAV G4lE ratios were higher in treated patients as compared to untreated patients may reject an interaction between a modest, but nonsignificant, rise in IFAV IgG4 together with a modest, but nonsignificant, decrease in IFAVIgE. (JALLERGYCLINIMMUNOL 1990;85:737-42.)

Allergic reactions to IFA are a significant medical problem in the southeastern United States. Of the two species of’ IFA, Solenopsis invicta and S. richteri, the former is more common and is responsible for most allergic reactions. Up to 58% of the population in infested areas are stung yearly.’ As many as 16% experience some type of hypersensitivity reaction with approximately 1% requiring medical treatment .* Re- actions range in severity from large local dermal le- sions, which persist for several days, to severe systemic anaphylaxis, manifested by hives, edema, short- ness of breath, fainting, and in some cases, death.3z 4 For individuals who are diagnosed as IFA sensitive, immunotherapy is often initiated. Because of the dif- ficulty in obtaining sufficient amounts of pure IFAV, IFAWBE is the only material currently available com-

From the Section of Clinical Immunology, Department of Medicine, Tulane Univesity Medical Center, New Orleans, La.

Supported by National Institutes of Health Grant AI 21552. Received for publication April 21, 1989. Revised Sept. 5, 1989. Accepted for publication Sept. 11, 1989. Reprint requests: Margaret A. Reed, MS, 1700 Perdido St.,

New Orleans, LA 70112. l/1/18029

Abbreviations used IFA: Imported fire ant

WBE: Whole body extract WBE-G,G4: Whole body extract IgG or IgG4 ratio

WBE-E: Whole body extract RAST percent binding

WBE-G4/E: Whole body extract IgG4 to IgE ratio V: Venom

V-G,G4: Venom IgG or IgG4 ratio V-E: Venom RAST percent binding

V-G4/E: Venom IgG4 to IgE ratio KWANOVA: Kruskal-Wallis analysis of variance

OD: Optical density PBS: Phosphate-buffered saline BSA: Bovine serum albumin

RT: Room temperature (approximately 22” C)

mercially for desensitization. In studies of hypersensitivity to other Hymenoptera, venom was demonstrated to contain the relevant allergens.5 The venom was also superior to WBE for desensitization of allergic patienk6s ’ Levels of Hymenoptera-specific serum IgG and IgG4 antibodies were demonstrated to

737

738 Reed and Butcher J. ALLERGY CLIN. IMMUNOL. APRIL 1990

TABLE I. Mean IFAWBE- and IFAV-specific IgG and lgG4 OD ratios + SEM

Group IFAWBE-G IFAV-G IFAWBE-G4 IFAV-G4 n

Treated 3.08 -+ 0.18 3.27 k 0.16 6.34 k 0.47 5.55 + 0.45 18 Untreated 2.58 k 0.15 2.90 k 0.15 3.41 + 0.42 4.13 +- 0.42 38 Control subjects 1.57 * 0.12 0.95 2 0.08 0.82 k 0.03 0.51 2 0.02 44

rise over the progression of immunotherapy.s-10 The increase in Hymenoptera-specific IgG and, in partic- ular, IgG4 antibodies, has been proposed to be important in conferring protection against Hymenoptera anaphylaxis. “-I3

No studies of class-specific antibody response to immunotherapy for IFA sensitivity have been reported. We have developed an ELISA to measure IFAWBE- and IFAV-specific IgG and IgG4 in sera from patients with lFA sensitivity, some of whom are undergoing immunotherapy. The results of these studies are reported here.

MATERIAL AND METHODS Study population

Sera were obtained from 56 individuals with documented reactions to IFA sting attending allergy clinics in New Or- leans and the surrounding area. Thirty-eight sera were from patients who had had reactions to IFA sting ranging in severity from large local to systemic allergic reactions, and, for various reasons, did not receive immunotherapy. Some, but not all, patients had been skin tested, and not all those patients skin tested had a positive skin test. Detailed infor- mation of skin tests for these patients is not available. Eigh- teen of these sera were from patients receiving immunotherapy for different lengths of time (mean, 4.8 years, ranging from 0.5 to 14.0 years), with IFAWBE extracts from commercial suppliers. All 18 patients had had at least one documented systemic allergic reaction to IFA sting. All but one patient were skin test positive. (This patient was a minor and the parents declined the skin test; RAST was performed for confirmation of sensitivity.) Allergic and demographic history were obtained by questionnaire. Control sera were from six individuals stung by IFA who denied reaction (five nonatopic and one atopic individual) and from 38 individuals residing in Ohio, where IFA are not indigenous, who had not been exposed to IFA (19 atopic and 19 nonatopic individuals) .

IFAWBE and IFAV reagents Reference IFAWBE was prepared by a method developed

in our laboratory.14 Dry weight and protein concentration were 35.0 mglrnl and 18 mg/ml, respectively. IFAWBE extracts were aliquoted and stored at -20” C until use. S. invictu IFAV, collected by an electroshock method, was a generous gift from Vespa Laboratories (Spring Hills, Pa.). IFAV was supplied in sterile glass vials as a freeze-dried powder, with reported weight of 0.4 mg per vial.

IFA-specific IgG ELISA ELISA was performed according to, and with the reagents

supplied in, the ELISAmate kit manufactured by Kirkegaard & Perry Laboratories, Gaithersburg, MD.,‘5 with modifi- cations, as follows: For IFA-specific total IgG (all subclasses) antibodies, wells of Immulon II polystyrene mi- crotiter plates (Dynatech Industries, Alexandria, Va.) were coated with 100 p.1 of a 50 kg/ml solution of either IFAWBE or IFAV in coating buffer (PBS, pH 8.6), and plates were incubated overnight at 4” C. Coating antigen was discarded, wells were filled with 300 ~1 of PBS, pH 7.4, containing 1% BSA to block unbound sites, and plates were incubated for 1 hour at RT. Blocking solution was discarded, and 100 p.1 of patient’s sera, diluted 1: 50 in PBS-BSA, was added to duplicate wells. A series of dilutions (1: 20, 1:50, 1:200, and 1: 500) of a pool of IFA RAST-positive (IFAWBE-E, 19.8; IFAV-E, 28.6) sera or a pool of IFA RAST-negative (IFAWBE-E, 0.8; IFAV-E, 0.9) sera were added to duplicate wells. Plates were incubated at RT for 1 hour. After removal of unreacted serum, plates were washed three times with PBS containing 0.02% Tween 20. One hundred microliters of affinity-purified, peroxidase- labeled goat antihuman IgG (1 Fg/ml) (Kirkegaard & Perry) in PBS-BSA was added to all wells, and plates were incubated 1 hour at RT. After removal of peroxidase conju- gate, plates were washed three times as above, but the last wash was left for 5 minutes. Color was developed by adding 100 p,l of 0.03% 2,2’-azinodi[3-ethylbenzthiazoline sulfo- nate] in citrate buffer containing 0.01% hydrogen peroxide. The reaction was stopped after 10 minutes by addition of 50 p.1 of 1% of sodium dodecyl sulfate, and plates were read at 690 em. The resulting ODs were recorded, and the ratios of test OD to the IFA-negative semm pool OD (IFAWBE-G and IFAV-G) were calculated. These ratios were used for statistical analyses.

IFA-specific lgG4 ELISA For IFA-specific IgG4 antibodies, Immulon II plates were

coated with IFAV or IFAWBE, incubated, and blocked as described above. Plates containing sera in duplicate of patients or control subjects were incubated overnight at 4” C. Sera were removed and plates were washed three times with PBS containing 0.02% Tween 20. One hundred microliters of mouse IgGl monoclonal antihuman IgG4 (1 Fgg/ ml in PBS-BSA) was added, and plates were incubated at RT for 2 hours. The monoclonal antibody was discarded, and plates were washed three times as described above. One hundred micmliters of peroxidase-conjugated, affinity-purified goat antimouse IgG (1 kg/ ml in PBS-BSA) was added to wells

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IFA IgG and lgG4 levels 739

TABLE II. Mean IFAWBE- and IFAV-specific IgE RAST percent binding -c SEM and G4/E

ratios * SIEM

Grwp IFAWBE-E IFAV-E IFAWBE-04/E IFAV-GQIE n

Treated 7.41 ? 1.24 14.54 + 2.03 1.41 -c 0.24 0.59 t 0.11 18 Untreated 9.22 * 1.34 19.37 r 1.72 0.89 rt 0.20 0.39 k 0.11 38 Control subjects 0.89 5 0.12 0.70 2 0.08 1.27 ” 0.08 0.89 ? 0.06 44

and incubated for 1 hour at RT. The remaining washing and color-development steps were performed as above. ODs were recorded, and ratios of test OD to IFA-negative serum pool (IFAWBE-G4 and IFAV-G4) were calculated. These ratios were used for statistical analyses.

IFA-specific IgE RAST

Sera were tested for IFAWBE and IFAV-specific IgE by RAST (Kallestad Laboratories, Austin, Texas).16 Briefly, IFAWBE (10 mg/ml of protein) or IFAV (0.1 mg/ml) was coupled to cyanogen bromide-activated paper disks. Disks were incubated overnight with 50 p.1 of sera plus 50 t.~l of PBS, in dul)licate, at RT, and then washed three times with 2.5 ml of physiologic saline for 10 minutes per wash to remove unreacted serum. One hundred microliters of “‘I- labeled anti-IgE diluted 1: 1 with physiologic saline was added, and disks were incubated overnight at RT. Disks were washed as above to remove unreacted isotope and then counted on a gamma counter to determine amount of bound radioactivity. Results were expressed as percent binding of total activity added.

Statistical analyses

Summary descriptive statistics and analysis of the data were performed with the Statistix program for the IBM.” Since most of the data were not normally distributed, nonparametric statistics, which are applicable to both normally and nonnormally distributed sample populations, were done. Analyses included the rank-sum test and one-way KWANOVA. I8 Multiple comparison testsI were performed on the ANOVA of the ranks of the three groups to determine which one of the three groups was different.

RESULTS Study population

Patient and control populations were divided into three groups: untreated patients (not currently receiving immunotherapy, n = 38), treated patients (receiving immunotherapy, n = 18), and all control subjects (n == 44). There were no statistical differences between nonexposed and exposed control subjects in any parameters analyzed.

IFA-specific IgG and lgG4

As an ndication of IgG and IgG4 levels relative to negative control pool, mean values for IFAWBE- and IFAV-specific IgG and IgG4 ratios were determined

(Table I). KWANOVA and multiple comparison of the ranks of the values indicated the following: levels of IFAWBE-G and IFAV-G did not differ between treated and untreated patients (p > 0.05), but both groups of patients had higher levels of both as compared to control subjects (p < 0.05). Treated patients tended to have higher levels of IFAWBE-G4 than did untreated patients (0.05 < p < O.lO), but both groups of patients had significantly higher levels than control subjects (p < 0.05). Levels of IFAV-G4 were not different between treated and untreated patients (p > 0.60) but were higher in untreated patients and treated patients than in control subjects (p < 0.05). Rank- sum test demonstrated that in both the treated and untreated patients, IFAV-G was not different from IFAWBE-G (p > 0.05); however, IFAWBE-G was higher than IFAV-G in control subjects (p < 0.001). Treated and untreated patients’ levels of IFAV-G4 did not differ from levels of IFAWBE-G4 (p > 0.10); whereas, in control subjects, IFAWBE-G4 antibody values were higher than IFAV-G4 (p -=c 0.001). Sum- maries of the statistical analyses are presented (see Tables III and IV).

IFA-specific IgE (RAST) and G4/E ratios

Mean values of IFAWBE- and IFAV-specific IgE (IFAWBE-E and IFAV-E) serum levels are presented in Table II). Untreated patients were not statistically different from treated patients for either IFAWBE-E or IFAV-E (p > 0.05), but both groups had higher levels than control subjects for both (p < 0.05). Levels of IFAV-E were higher than IFAWBE-E in both untreated and treated patients (p < O.Ol), but IFAWBE-E was higher than IFAV-E in control subjects (p < 0.05). A ratio of the IFA-specific IgG4 to IFA- specific IgE RAST percent binding (G4/E) was determined to establish a relationship between IgG4 and IgE antibody levels as a possible measure of efficacy of immunotherapy. Mean values of these ratios are also presented in Table II. Nonparameteric statistical analyses performed on these ratios indicated that treated patients had a significantly higher IFAWBE- G4/ E and IFAV-G4/E than did untreated patients (p < 0.05). Control values of IFAV-G4/E were statistically higher than either treated or untreated pa-


TABLE III. Summary of KWANOVA and multiple comparison tests*

Parameter Treated vs untreated

Treated vs control subjects

Untreated vs control subjects

IFAWBE-G IFAV-G IFAWBE-G4 IFAV-G4 IFAWBE-E IFAV-E IFAWBE-G4/ E IFAV-G4/E

*All significant p values, CO.05. to.05 < p < 0.10.

No difference No difference No difference? No difference No difference No difference Treated higher Treated higher

Treated higher Treated higher Treated higher Treated higher Treated higher Treated higher No difference Control subjects higher

Untreated higher Untreated higher Untreated higher Untreated higher Untreated higher Untreated higher Control subjects higher Control subjects higher

TABLE IV. Summary of rank-sum test of IFAWBE-specific as compared to IFAV-specific antibody levels among groups*

Control Antibody Treated Untreated subjects

IgG IgG4 W IgG4/E

No difference No difference IFAV higher IFAWBE higher

No difference No difference IFAV higher IFAWBE higher

IFAWBE higher IFAWBE higher IFAWBE higher IFAWBE higher

*See text p values.

tients (p < 0.05). Levels in control subjects of IFAWBE-G4 / E were higher than in untreated patients (p < 0.05) but were not different from levels in treated patients (p > 0.05). Levels of IFAWBE- G4/E was higher than IFAV-G4/E in all three groups (p < 0.05). Summaries of the statistical analyses of these parameters are found in Tables III and IV.

DISCUSSION

These studies demonstrate that patients being treated for IFA sensitivity did not have significantly higher levels of IFAWBE- or IFAV-specific IgG than patients not treated. However, both groups of patients had higher levels of these antibodies than did control subjects. Patients receiving immunotherapy tended to have higher levels of IFAWBE-specific IgG4 antibody levels than did patients not receiving immunotherapy, and IFAV-G4 levels were the same for both groups. IFAWBE- or IFAV-specific IgE levels did not differ between treated and untreated patients, but both groups had higher levels than did control subjects. In individuals receiving immunotherapy, IEAWBE IgG4/IgE ratios did not differ significantly from control subjects but were significantly higher than those of IFA-allergic patients not receiving immunotherapy. Treated patients had higher ratios of IFAV-G4/E than

untreated patients, but control subjects had higher levels of IFAV-G4/E than either treated or untreated patients.

Our findings that IFAWBE-G levels did not differ between treated and untreated patients and that sera from control subjects also had significant IFAWBE- specific IgG antibodies may reflect cross-reactivity between IFAWBE components and other commonly encountered antigens. IFAWBE have been demonstrated to contain as many as 29 antigenic components,14 many of which may share epitopes with other antigens. Our inability to demonstrate a significant increase in IFAV-specific IgG antibodies of treated patients over patients who were not treated may be related to the IFAV content of IFAWBE. In other studies we have demonstrated that some commercial IFAWBEs have significant deficiencies in IFAV content and therefore may lack potency.19

The finding that IFAWBE-G4 levels tended to increase in patients undergoing immunotherapy is sim- ilar to results observed with other allergens, that is, house dust mite,” ryegrass pollen,” and bee venom.‘* These studies indicated that high levels of allergen- specific IgG4 have been demonstrated to be associated with immunotherapy and may be important in conferring protection. It is interesting to note that levels

VOLUME 85 NUMBER 4

of IFAV-G4. did not differ between the treated and the untreated patients. This, too, may be due to the low or absent levels of IFAV in commercially available IFAWBE used for treatment.

We have not yet evaluated the time course of the IgG4 response to immunotherapy nor of other IgG antibody subclasses. In studies of patients undergoing immunotherapy for other Hymenoptera, such as bees, wasps, and hornets, a rise in venom-specific IgG1 occurred early in the course of immunotherapy, reached a plateau, and then decreased, whereas IgG4 rose more slowly but stayed elevated with time, even after the maintenance dose was reached. Levels of venom-specific IgG2 and IgG3 were virtually unde- tectable.*‘. 24 It has been proposed that this initial rise in IgGl , b’efore the development of IgG4 antibodies, is important in conferring protection and that this early IgGl response may modulate venom-specific IgE production.25 Subsequently, only IgG4, which acts as blocking antibody, is necessary to maintain protection.

In previous studies we have indicated that level of IFAV-specific IgE is a better indicator of sensitivity than IFAWBE-specific IgE.26.27 Thus, it would follow that production of IgG blocking antibodies against IFAV would be more useful in conferring protection against IFA stings than IFAWBE IgG antibodies. This is in keepmg with findings concerning other Hyme- noptera antibody measurements in which it has been demonstrated that venom-specific rather than whole body-specific antibody measurements provide a better indication of the protection conferred by immunotherapy.‘, ’

We calculated the ratio of IFA-specific IgG4 to specific IgE based on the premise that immunotherapy increases allergen-specific IgG4 over time while it concurrently inhibits allergen-specific IgE production.24. 25 The significant increase in IFAWBE and IFAV G4 / E ratios in treated patients over untreated patients could be attributed to immunotherapy, indicating that there is IFAV present in some commercial prepara- tions. However, the fact that control subjects had significantly higher IFAV G4/E ratios than treated patients, whereas IFAWBE G4/E levels did not differ between these two groups, may again indicate that the amount of IFAV contained in commercially available extracts may not be sufficient to increase levels of IFAV IgG4 and/or inhibit IFAV IgE antibody production.

Further studies relating antibody subclass responses to immunotherapy and to outcome of controlled IFA sting challenge or field sting will be necessary to determine whether increased levels of IFAWBE-specific IgG or IgG4 antibodies are sufficient for protection

IFA IgG and lgG4 levels 741

or whether it is necessary to insure that levels of IFAV-specific IgG and/or IgG4 antibodies are also increased for patients to be successfully immunized. It might also be useful to evaluate the use of IFAWBE- and/or IFAV-specific G4/E ratios as indicators of efficacy of immunotherapy.

We thank Mr. M. Guralnick, Vespa Laboratories, Spring Mills, Pa., for the generous donation of IFAV; Mr. S. Sackett, New Orleans Mosquito Control, New Orleans, La., for identification of IFA species; and Ms. Dane11 Wat- kins for excellent technical assistance.

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Imported fire ant (IFA) sensitivity: IFA-specific levels of serum IgG and IgG4