Int. Archs Allergy appl. Immun. 57: 554-559 (1978)
Enhancement of IgE Antibody Production in AKR M ice1* Naohiro Watanabe and Zoltán Ovary Department of Parasitology, Jikei University School of Medicine, Tokyo, and Department of Pathology, New York University Medical Center, New York, N.Y.
Abstract. When dinitrophenylated keyhole limpet hemocyanin (DNP-KLH) primed AKR mice were injected with dinitrophenylated Nippostrongylus brasiliensis extract (DNP-Nb), an enhancement of IgE antibody production was obtained by nematode parasite, N. brasi liensis, infection or by Nb antigen injection 2 weeks before DNP-Nb injection. A prefe rential enhancement of IgE production was observed in infected mice compared with Nbinjected mice. Anti-hapten IgE antibody response was suppressed in AKR mice after appro priate immunization. No suppression of IgE response was observed after whole body irra diation (180-540 R) at an early stage of immunization. High and persistent IgE response was observed in (AKRXDBA/1)F! mice of both sexes, therefore, the suppressive effect on IgE response in AKR mice was inherited as an autosomal recessive trait. The similarity of IgE class specific suppression in AKR and SJL mice is discussed.
IgE response is extremely dependent upon the regulatory influences of T cells [6, 15, 18]. Helper and suppressor cells have important roles on IgE antibody production. We report that enhancement of IgE anti body production was obtained by priming helper cells with parasite infection and that X-ray irradiation eliminated suppressor cells in AKR mice.
1 Supported by National Institutes of Health grant AI-03075-19 and National Cancer Institute grant 5-POl CA 16247-03.
Materials and Methods Antigen DNP13KLH, DNP,,Nb and Nb were used for immunization. Dinitrophenylated bovine serum al bumin (DNP3;BSA) and dinitrophenylated ovalbu min (DNPu Ov) were used as challenging antigens for passive cutaneous anaphylaxis (PCA) reac tions. The subscripts refer to the average number of DNP groups per molecule of protein except for Nb and KLH where they refer to 105 daltons. DNP conjugation to protein was done according to Eisen et al. [3]. Nippostrongylus brasiliensis adult worm extract was prepared as described [7], Animals The following strains of 8- to 12-week-old fe male mice were used: AKR/J, C3H/J, CBA/J, DBA/1, and SJL/J. All mice were purchased from
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Introduction
Anti-DNP IgE in AKR Mice
Immunization Immunization schedules, infections, and boosters were done as published [7], Briefly, 5 mice in each group were immunized intraperitoneally with 1 fig DNP-KLH mixed with 1 mg Al(OH)s on day 0. On day 21, the mice were infected subcutaneously by 750 third stage N. brasiliensis larvae. Mice were reinjected (challenged) intraperitoneally with 1 fig DNP-Nb mixed with 1 mg Al(OH)3 on day 35. Mice were bled weekly beginning 7 days after in jection of DNP-Nb from the retro-orbital sinus. 0.2 ml of blood was added to 0.9 ml heparinized saline (10 U/ml), then centrifuged for 10 min at 1,000 g. The supernate was considered to be a 1/10 dilution. Irradiation Immunized, infected and challenged mice re ceived appropriate doses of X-ray irradiation from a Gammator M (Radiation Machinery Corp., Parsippany, N.Y.). Titration of Antibody Titers of IgE and IgG, antibody of pooled sera from each group were determined by PCA reac tions [11, 17]. For IgE antibody titration, male Sprague-Dawley rats were used [10, 13, 17]. Shaved rats were injected intradermally with 0.1 ml of serial dilution of antiserum. The rats were challenged in the tail vein with 1 mg of DNP-BSA in 1 ml of 0.5°/o Evans blue 2 h after sensitization [13]. 30 min after challenge, the reac tion was read from the outside of the skin. The re action spots larger than 5 mm in diameter were considered as PCA reactions. Each experiment was performed in 2 rats. For IgG, antibody titration, female SJL mice were used. Shaved mice were intradermally inject ed with 0.03 ml of serial dilution of antiserum. The mice were challenged by an intravenous injec tion into the tail vein of 500 ug of DNP-OA in 0.2 ml of 0.5°/o Evans blue 1*/2 h after the intradermal injection of the antiserum dilution [13]. 30 min after challenge, the reaction was graded
from 0 to 4+ according to the diameter [12]. The end point was taken when at least 1+ reaction was produced in 2 of 4 mice. In order to titrate the antihapten complement fixing antibody, namely, IgM and IgG2, passive lysis tests were performed [1]. Serial dilution of antiserum was mixed with DNP-BSA coated sheep erytrocytes and guinea pig complement. The hemolysis was examined after incubation at 37 °C for 45 min. The titer was taken at the highest dilu tion giving hemolysis.
Results Effect of N. brasiliensis Infection on IgE Antibody Response Three strains of mice, which had H-2k haplotype, were injected with 1 fig DNP-KLH and 1 mg A!(OH)3 as adjuvant on day 0 to prime DNP-specific B cells (ta ble I). On day 21, groups of mice (group 1, 4, 7) were infected with N. brasiliensis and other groups of mice (group 2, 5, 8) were injected intraperitoneally with 1 fig Nb in 1 mg Al(OH)3. A s controls, groups of mice (group 3, 6, 9) had no treatment. On day 35, all groups of mice were challenged with 1 pg DNP-Nb in 1 mg Al(OH)3. Antihapten IgE antibody titers were higher in infected mice and in Nb-injected mice than in the control groups on day 42 and on day 56. The magnitude of the TgF. response was higher in infected mice than in Nb injected mice. It should be noted that antihapten IgE titer in AKR mice on day 56 was relatively lower than in the other groups. IgE antibody response in AKR mice were transient in contrast to CBA and C3H mice. Antihapten IgG, antibody titers were also higher in mice infected or in mice in jected with Nb than in control mice on day 42. On day 56, there were some variations
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the Jackson Laboratory, Bar Harbor, Me., (AKR X DBA/1)F, mice were bred in our labora tory. Male Sprague-Dawley rats, weighing 250300 g, were obtained from Blue Spruce Farms, Altamont, N.Y.
555
Watanabe/Ovary
556
Table I. Effect of N. brasiliensis infection on IgE antibody response Group Strain
Antihapten PCA titer1
Immunization day 0
day 21
day 35
day 42 IgE
1
AKR
2
AKR
3
AKR
4
C3H
5
C3H
6
C3H
7
CBA
8
CBA
9
CBA
1 us DNP-KLH + 1 mg Al(OH)3 1 us DNP-KLH + 1 mg Al(OH)3 1 //g DNP-KLH + 1 mg Al(OH)3 1 US DNP-KLH + 1 mgAl(OH)3 1 us DNP-KLH + 1 mg Al(OH)3 1 US DNP-KLH + 1 mg Al(OH)3 1 us DNP-KLH + 1 mg Al(OH)3 1 us DNP-KLH + 1 mg Al(OH)3 1 US DNP-KLH + 1 mg Al(OH)3
750 N. brasiliensis 1 us DNP-Nb + 1 mg Al(OH)3 infection 1 US Nb + I mg Al(OH)3 1 /¡S DNP-Nb + 1 mg Al(OH)3 no treatment 1 uS DNP-Nb + 1 mg Al(OH)3 750 N. brasiliensis 1 us DNP-Nb infection + 1 mg Al(OH)3 1 US Nb + 1 mg Al(OH)3 1us DNP-Nb + 1 mg Al(OH)3 no treatment 1 us DNP-Nb + 1 mg Al(OH)3 750 N. brasiliensis 1US DNP-Nb + 1 mg Al(OH)3 infection 1 uS Nb + 1 mg Al(OH)3 1 US DNP-Nb + 1 mg Al(OH)3 no treatment 1 US DNP-Nb + 1 mg Al(OH)3
day 56 IgGi IgE
800 400
160
80
10 160
100 600 10
IgGi
0
50
20
3,200 800
1,600 160
1,200 400
320 160
400
50
3,200 200
160
40
3,200 300
600
80
640
20
100
40
160
40
1 Titer determined by pooled serum from five mice of each group.
Effect of Irradiation on IgE Antibody Response Antihapten IgE antibody response was transient in AKR mice (table I). Since the transient IgE response was the effect of sup pressor cells in SJL mice and since suppres sor cells are irradiation sensitive, the effect of irradiation on antibody responses was tested. AKR mice were immunized, infected and challenged. 1 day after the challenge, mice received different doses of irradiation
Table II. Effect of irradiation on IgE antibody response in AKR mice Dose of irradiation, R 1
0 180 360 540
Antihapten PCA titer2 day 423
day 63 3
IgE
IgGi
IgE
IgGi
800 3,200 3,200 800
400 400 400 100
200 800 1,600 800
400 200 200 50
1 Immunized, infected and challenged AKR mice were irradiated on day 36. 2 Titer determined by pooled serum from 5 mice of each group. 3 Days after DNP-KLH injection.
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in the titers; however, the IgGi titers in AKR mice were not lower than in the other respective groups. No elevation of antihap ten IgGt titers was observed in infected mice compared with Nb-injected mice.
Anti-DNP IgE in AKR Mice
557
The timing of irradiation was examined in AKR mice (fig. 1). A dose of 180 R of ir radiation was given to immunized mice on day 34, 36, or 43 (1 day before, 1 day or 8 days after challenge, respectively). When the irradiation was done 1 day before or after challenge, the antihapten IgE antibody response persisted. IgE antibody response in mice irradiated 8 days after challenge was of the same magnitude as the nonirradiated control and diminished gradually.
(180, 360, or 540 R). As shown in table II, antihapten IgE antibody titers were higher in mice irradiated with 180 R or 360 R than in nonirradiated control mice on day 42. The titers persisted at a higher level than in the controls. Irradiation with 540 R induced somewhat lower titers on day 42 than 180 or 360 R. However, on day 63, the titer in the sera of mice irradiated with 540 R was about the same as the titers in the sera of mice irradiated with 180 or 360 R and was higher than that of the controls. Antihapten IgG, antibody response was comparable in mice receiving 180 R, 360 R and nonirradiated controls. Mice irradiated with 540 R produced lower IgGj response than the mice in other groups.
Discussion IgE antibody is dependent on T helper cells since congenitally athymic (nu/nu) mice [9] do not produce IgE antibody and as no IgE antibody is produced in other strains with ‘T independent’ antigens such as DNP-Salmonella [15] and DNP-Ficoll [19]. The AKR strain of mice is a low IgE re sponder [16] and this led us to investigate the possibility of the existence of suppressor
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/8/2018 1:10:40 AM
Fig. 1. Antihapten IgE antibody response in irradiated AKR mice. AKR mice were immunized with 1 /
Enhancement of IgE Antibody Production in AKR M ice1* Naohiro Watanabe and Zoltán Ovary Department of Parasitology, Jikei University School of Medicine, Tokyo, and Department of Pathology, New York University Medical Center, New York, N.Y.
Abstract. When dinitrophenylated keyhole limpet hemocyanin (DNP-KLH) primed AKR mice were injected with dinitrophenylated Nippostrongylus brasiliensis extract (DNP-Nb), an enhancement of IgE antibody production was obtained by nematode parasite, N. brasi liensis, infection or by Nb antigen injection 2 weeks before DNP-Nb injection. A prefe rential enhancement of IgE production was observed in infected mice compared with Nbinjected mice. Anti-hapten IgE antibody response was suppressed in AKR mice after appro priate immunization. No suppression of IgE response was observed after whole body irra diation (180-540 R) at an early stage of immunization. High and persistent IgE response was observed in (AKRXDBA/1)F! mice of both sexes, therefore, the suppressive effect on IgE response in AKR mice was inherited as an autosomal recessive trait. The similarity of IgE class specific suppression in AKR and SJL mice is discussed.
IgE response is extremely dependent upon the regulatory influences of T cells [6, 15, 18]. Helper and suppressor cells have important roles on IgE antibody production. We report that enhancement of IgE anti body production was obtained by priming helper cells with parasite infection and that X-ray irradiation eliminated suppressor cells in AKR mice.
1 Supported by National Institutes of Health grant AI-03075-19 and National Cancer Institute grant 5-POl CA 16247-03.
Materials and Methods Antigen DNP13KLH, DNP,,Nb and Nb were used for immunization. Dinitrophenylated bovine serum al bumin (DNP3;BSA) and dinitrophenylated ovalbu min (DNPu Ov) were used as challenging antigens for passive cutaneous anaphylaxis (PCA) reac tions. The subscripts refer to the average number of DNP groups per molecule of protein except for Nb and KLH where they refer to 105 daltons. DNP conjugation to protein was done according to Eisen et al. [3]. Nippostrongylus brasiliensis adult worm extract was prepared as described [7], Animals The following strains of 8- to 12-week-old fe male mice were used: AKR/J, C3H/J, CBA/J, DBA/1, and SJL/J. All mice were purchased from
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/8/2018 1:10:40 AM
Introduction
Anti-DNP IgE in AKR Mice
Immunization Immunization schedules, infections, and boosters were done as published [7], Briefly, 5 mice in each group were immunized intraperitoneally with 1 fig DNP-KLH mixed with 1 mg Al(OH)s on day 0. On day 21, the mice were infected subcutaneously by 750 third stage N. brasiliensis larvae. Mice were reinjected (challenged) intraperitoneally with 1 fig DNP-Nb mixed with 1 mg Al(OH)3 on day 35. Mice were bled weekly beginning 7 days after in jection of DNP-Nb from the retro-orbital sinus. 0.2 ml of blood was added to 0.9 ml heparinized saline (10 U/ml), then centrifuged for 10 min at 1,000 g. The supernate was considered to be a 1/10 dilution. Irradiation Immunized, infected and challenged mice re ceived appropriate doses of X-ray irradiation from a Gammator M (Radiation Machinery Corp., Parsippany, N.Y.). Titration of Antibody Titers of IgE and IgG, antibody of pooled sera from each group were determined by PCA reac tions [11, 17]. For IgE antibody titration, male Sprague-Dawley rats were used [10, 13, 17]. Shaved rats were injected intradermally with 0.1 ml of serial dilution of antiserum. The rats were challenged in the tail vein with 1 mg of DNP-BSA in 1 ml of 0.5°/o Evans blue 2 h after sensitization [13]. 30 min after challenge, the reac tion was read from the outside of the skin. The re action spots larger than 5 mm in diameter were considered as PCA reactions. Each experiment was performed in 2 rats. For IgG, antibody titration, female SJL mice were used. Shaved mice were intradermally inject ed with 0.03 ml of serial dilution of antiserum. The mice were challenged by an intravenous injec tion into the tail vein of 500 ug of DNP-OA in 0.2 ml of 0.5°/o Evans blue 1*/2 h after the intradermal injection of the antiserum dilution [13]. 30 min after challenge, the reaction was graded
from 0 to 4+ according to the diameter [12]. The end point was taken when at least 1+ reaction was produced in 2 of 4 mice. In order to titrate the antihapten complement fixing antibody, namely, IgM and IgG2, passive lysis tests were performed [1]. Serial dilution of antiserum was mixed with DNP-BSA coated sheep erytrocytes and guinea pig complement. The hemolysis was examined after incubation at 37 °C for 45 min. The titer was taken at the highest dilu tion giving hemolysis.
Results Effect of N. brasiliensis Infection on IgE Antibody Response Three strains of mice, which had H-2k haplotype, were injected with 1 fig DNP-KLH and 1 mg A!(OH)3 as adjuvant on day 0 to prime DNP-specific B cells (ta ble I). On day 21, groups of mice (group 1, 4, 7) were infected with N. brasiliensis and other groups of mice (group 2, 5, 8) were injected intraperitoneally with 1 fig Nb in 1 mg Al(OH)3. A s controls, groups of mice (group 3, 6, 9) had no treatment. On day 35, all groups of mice were challenged with 1 pg DNP-Nb in 1 mg Al(OH)3. Antihapten IgE antibody titers were higher in infected mice and in Nb-injected mice than in the control groups on day 42 and on day 56. The magnitude of the TgF. response was higher in infected mice than in Nb injected mice. It should be noted that antihapten IgE titer in AKR mice on day 56 was relatively lower than in the other groups. IgE antibody response in AKR mice were transient in contrast to CBA and C3H mice. Antihapten IgG, antibody titers were also higher in mice infected or in mice in jected with Nb than in control mice on day 42. On day 56, there were some variations
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/8/2018 1:10:40 AM
the Jackson Laboratory, Bar Harbor, Me., (AKR X DBA/1)F, mice were bred in our labora tory. Male Sprague-Dawley rats, weighing 250300 g, were obtained from Blue Spruce Farms, Altamont, N.Y.
555
Watanabe/Ovary
556
Table I. Effect of N. brasiliensis infection on IgE antibody response Group Strain
Antihapten PCA titer1
Immunization day 0
day 21
day 35
day 42 IgE
1
AKR
2
AKR
3
AKR
4
C3H
5
C3H
6
C3H
7
CBA
8
CBA
9
CBA
1 us DNP-KLH + 1 mg Al(OH)3 1 us DNP-KLH + 1 mg Al(OH)3 1 //g DNP-KLH + 1 mg Al(OH)3 1 US DNP-KLH + 1 mgAl(OH)3 1 us DNP-KLH + 1 mg Al(OH)3 1 US DNP-KLH + 1 mg Al(OH)3 1 us DNP-KLH + 1 mg Al(OH)3 1 us DNP-KLH + 1 mg Al(OH)3 1 US DNP-KLH + 1 mg Al(OH)3
750 N. brasiliensis 1 us DNP-Nb + 1 mg Al(OH)3 infection 1 US Nb + I mg Al(OH)3 1 /¡S DNP-Nb + 1 mg Al(OH)3 no treatment 1 uS DNP-Nb + 1 mg Al(OH)3 750 N. brasiliensis 1 us DNP-Nb infection + 1 mg Al(OH)3 1 US Nb + 1 mg Al(OH)3 1us DNP-Nb + 1 mg Al(OH)3 no treatment 1 us DNP-Nb + 1 mg Al(OH)3 750 N. brasiliensis 1US DNP-Nb + 1 mg Al(OH)3 infection 1 uS Nb + 1 mg Al(OH)3 1 US DNP-Nb + 1 mg Al(OH)3 no treatment 1 US DNP-Nb + 1 mg Al(OH)3
day 56 IgGi IgE
800 400
160
80
10 160
100 600 10
IgGi
0
50
20
3,200 800
1,600 160
1,200 400
320 160
400
50
3,200 200
160
40
3,200 300
600
80
640
20
100
40
160
40
1 Titer determined by pooled serum from five mice of each group.
Effect of Irradiation on IgE Antibody Response Antihapten IgE antibody response was transient in AKR mice (table I). Since the transient IgE response was the effect of sup pressor cells in SJL mice and since suppres sor cells are irradiation sensitive, the effect of irradiation on antibody responses was tested. AKR mice were immunized, infected and challenged. 1 day after the challenge, mice received different doses of irradiation
Table II. Effect of irradiation on IgE antibody response in AKR mice Dose of irradiation, R 1
0 180 360 540
Antihapten PCA titer2 day 423
day 63 3
IgE
IgGi
IgE
IgGi
800 3,200 3,200 800
400 400 400 100
200 800 1,600 800
400 200 200 50
1 Immunized, infected and challenged AKR mice were irradiated on day 36. 2 Titer determined by pooled serum from 5 mice of each group. 3 Days after DNP-KLH injection.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/8/2018 1:10:40 AM
in the titers; however, the IgGi titers in AKR mice were not lower than in the other respective groups. No elevation of antihap ten IgGt titers was observed in infected mice compared with Nb-injected mice.
Anti-DNP IgE in AKR Mice
557
The timing of irradiation was examined in AKR mice (fig. 1). A dose of 180 R of ir radiation was given to immunized mice on day 34, 36, or 43 (1 day before, 1 day or 8 days after challenge, respectively). When the irradiation was done 1 day before or after challenge, the antihapten IgE antibody response persisted. IgE antibody response in mice irradiated 8 days after challenge was of the same magnitude as the nonirradiated control and diminished gradually.
(180, 360, or 540 R). As shown in table II, antihapten IgE antibody titers were higher in mice irradiated with 180 R or 360 R than in nonirradiated control mice on day 42. The titers persisted at a higher level than in the controls. Irradiation with 540 R induced somewhat lower titers on day 42 than 180 or 360 R. However, on day 63, the titer in the sera of mice irradiated with 540 R was about the same as the titers in the sera of mice irradiated with 180 or 360 R and was higher than that of the controls. Antihapten IgG, antibody response was comparable in mice receiving 180 R, 360 R and nonirradiated controls. Mice irradiated with 540 R produced lower IgGj response than the mice in other groups.
Discussion IgE antibody is dependent on T helper cells since congenitally athymic (nu/nu) mice [9] do not produce IgE antibody and as no IgE antibody is produced in other strains with ‘T independent’ antigens such as DNP-Salmonella [15] and DNP-Ficoll [19]. The AKR strain of mice is a low IgE re sponder [16] and this led us to investigate the possibility of the existence of suppressor
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/8/2018 1:10:40 AM
Fig. 1. Antihapten IgE antibody response in irradiated AKR mice. AKR mice were immunized with 1 /
