Suppression of IgE responses by passive antigen inhalation: Dissociation of local (mucosal) and systemic immunity

CELLULAR

IMMUNOLOGY

l&4,434-439

(1987)

SHORT COMMUNICATIONS Suppression of IgE Responses by Passive Antigen Inhalation: Dissociation of Local (Mucosal) and Systemic Immunity PATRICK

G.HOLT,**‘MURRAY REID,*DESLEYBRITTEN,*JONSEDGWICK,*~* ANDHERVEBAZIN-~

*Clinical Immunology Research Unit, Princess Margaret Hospital, Subiaco, 6008, WesternAustralia and iExperimental Immunology Unit, University of Louvain, Brussels, Belgium Received March 18, 1986; accepted September 17.1986 Animals from hi- and low-IgEresponder rat strains were preexposed to antigen-containing aerosols of different droplet sixes, prior to parenteral antigenic challenge. Depending upon the type of aerosol employed, systemic immunological tolerance developed in high-IgE-responder animals in the IgE antibody class either with or without concommitant production of salivary IgA, indicating that the two antibody isotypes were under independent control, and further that &A-mediated immune exclusion was not central to the development of tolerance in the IgE class. Low-@-responder rats exhibited biphasic salivary IgA responsesduring exposure, which could not be recalled by subsequent parenteral challenge, suggestingthat secretory immunity in the respiratory tract may also be down regulated by repeated exposure to airborne antigens. @ 1987 Academic Press,Inc.

INTRODUCTION Recent studies have shown that passive inhalation of antigen induces systemic immunological tolerance, which is antigen specific, and preferentially expressedagainst the IgE antibody isotype ( l-4). Tolerant animals in these experiments contained IgEisotype-specific suppressor cells in central lymphoid organs ( 1,4), which in the mouse express surface Thy 1.2 (2), and in the rat carry the MRC OX8 marker (5). These cells developed initially in the lymph nodes draining the oronasal cavity and the pharynx (5). In experiments with high- and low-&E-responder rat strains, up to lOOO-fold differences in sensitivity to tolerogenesis were demonstrated, where high responders required microgram antigen dosages in contrast to low responders, which became tolerant following inhalation of nanogram levels of the antigen (4). These finding are consistent with the view (6, 7) that the magnitude of the IgE response in individual animal strains is primarily dependent upon the inducibility of their respective IgE homeostatic mechanisms following antigenic challenge. However, a sign&ant body of evidence also implicates &A-mediated immune ’ To whom correspondence should be addressed. ’ present address:MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, United Kingdom. 434 0008-8749187$3.00 Copyri&t Q 1987 by Academic Press,Inc. All rights of reprodwtion in any form reserved.

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exclusion mechanisms in down regulation of IgE responses,in particular those initiated at mucosal surfaces(8), and accordingly the possible role of specific IgA antibody in suppression of IgE responsesto inhaled antigen was explored in the present experiments. In this communication, we report on the status of local (salivary) IgA responsesin high- and low-@responder rats, during and after exposure to aerosols of ovalbumin (OA)3 at dose levels which induce systemic tolerance in the IgE antibody class.Our data suggestfirst that the effectson local immunity resulting from antigenic stimulation of the respiratory mucosa are dissociable from those occurring systemically, and second that specific IgA-mediated immune exclusion mechanisms are not central to tolerance induction in response to antigen inhalation. MATERIALS AND METHODS Animals. SPF rats of the Brown Norway (BN) and WAG strains, which, respectively, express high- and low-&E-responder phenotype (9), were obtained from the Animal Resource Centre, Murdoch University. SPF Sprague-Dawley rats were used for passive cutaneous anaphylaxis (PCA) reactions (seebelow). Antigen exposure and immunization. Two aerosol exposure systems were used. The first employed standard clinical nebulizers which generate a mist comprising droplets which have a mean diameter of 7 pm at the point of generation. The mist was piped into a plexiglass exposure chamber (volume 27 liters) asdetailed previously (l), forming a visible cloud within which droplet association would be likely to occur, further increasing mean droplet size (1). The animals were exposed in this chamber once weekly for 7 min, over a period of 8 weeks. The second exposure protocol employed the Tri-R Airborne Infection Apparatus (Tri-R Instruments, N.Y.), which comprises an inhalation chamber with a volume of 135 liters, into which was piped an aerosol mist consisting of droplets within the “respirable” range, i.e., d 1.O-pm diameter; the mist forms a stable nonvisible cloud over the exposure period (10, 11). These rats were exposed to the aerosol for 30-min intervals. Exposure was again once weekly, for 8 weeks. The solution employed for aerosol generation in both systems comprised varying concentrations of OA (Grade V, Sigma Chemicals) in phosphate-buffered saline (PBS). Parenteral challenge of rats was via intraperitoneal (ip) immunization with 10.0 mg aluminium hydroxide (AH; amphogel, Wyeth Parmaceuticals, Australia) plus 100 pg OA (AH-OA), in 0.5 ml PBS. Sample collection for antibody assays. Serum was collected from blood samples taken from the tail vein. For saliva collection, the rats were lightly anesthetized with ether, and then slowly injected intravenously with OS-O.6 ml of a one in five dilution of the short-term acting anesthetic Sat-fan(Alphaxalone; Glaxo, Australia Ltd.). They were then inoculated subcutaneously in the nape of the neck with 0.2 ml 2% pilocarpine (Alcon Labs, Australia) to induce salivation, and saliva was collected over the ensuing 5 min. All samples were clarified by centrifugation and stored frozen at -20°C prior to assay. Antibody assays. Serum IgE-anti-OA and IgG-anti-OA levels were measured, respectively, by the PCA method of Ovary (12) and hemagglutination (HA), as detailed 3Abbreviations used: DTH, delayed type hypersensitivity; OA, ovalbumin; PCA, passive cutaneous anaphylaxis; PBS, phosphate-buffered saline; AH, aluminium hydroxide; ip, intraperitoneal; OD, optical density.

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SHORT COMMUNICATIONS WAG-1

-BN

T 12

IO

B

+ I I e I I L

2 3

W

l E

I50

25

PBS

PiS Aoroool

6

2

4

2

c

2

SiO%

(%OA)

FIG. 1. Salivary and serum IgA responses in rats repeatedly exposed to an antigen-containing aerosol comprising droplets in the respirable range. Data shown are j2 f SD derived from groups of five rats, 10 (serum) or 2 1 (saliva) days after parenteral challenge. (m), serum IgE, as logz reciprocal PCA dilution; (m) serum IgA, and (8) salivary IgA, as ELISA units.

previously ( 1,5). IgA-anti-OA titers in serum and saliva were determined in serially diluted samples by ELISA (13), employing affinity-purified goat-anti-rat-&A (provided by Dr. Herve Bazin), and rabbit-anti-goat-&G conjugated to alkaline phosphatase (a gift from Dr. G. A. Stewart). The specificity of both reagents was confirmed by ELISA, against purified immunoglobulins. Internal standards (known positive and negative serum pools) were included in all assaysto control for interassay variability. All data were initially graphed as reciprocal dilution against optical density (OD), and linearity was observed between reciprocal dilutions of 4 and 16. The data were finally expressedas ELISA units, being 100 X OD at a reciprocal dilution of 4, after subtraction of relevant background values (mean background OD of 0.08 and 0.24 for nonimmune saliva and serum, respectively). The significance of differences between groups was assessedby t test. RESULTS In the experiments of Fig. 1, high-&E-responder BN rats were exposed via the TriR inhalation apparatus to a submicronic aerosol mist containing varying concentrations of OA. One week after the last exposure, they were challenged ip with AH-OA, together with PBSexposed controls. As shown in earlier experiments employing large droplet aerosols (3), the efficiency of tolerance induction in the IgE isotype is related to antigen concentration in the aerosol. It can be seenthat preexposure of animals to 3.0% OA completely eliminated their capacity to synthesize OA-specific IgE in response to systemic challenge, while significantly reduced responses (P < 0.001) 00 curred in the groups preexposed to lower OA concentrations. Salivary IgA responses

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SHORT COMMUNICATIONS TABLE 1 Salivary IgA and Serum IgE ResponsesResulting from Repeated Exposure to a Large Droplet Aerosol Containing OA Antigen Salivary IgA during exposure

Postparenteral challenge

Aerosol

Week 3

Week 6

Salivary IgAd

Serum IgE

BN

PBS 0.01% OA 0.1% OA 1.O%OA

<5 <5 <5 46+ 15

15 31& 1 lo+ 7 37+ 16

<5 18+9 <5 <5

12.4 + 6.1 11.8 + 4.6 8.4 + 4.7’ 2.2 + 1.4”

WAG

PBS 0.0001% OA 0.001% OA 0.01% OA 0.1% OA 1.O%OA

<5 32+ 16 73* 17 47?23 12* 7 16f 9

<5 <5 15 15 is <5

<5 15 <5 <5 <5 <5

4.5 + 1.9 2.1 + 0.4b 0” 0” 0” 0”

Strain

Note. Groups of rats (n = 5-10) were exposed to OA aerosols on eight occasions. Data shown are 2 + SD of ELISA units for IgA or PCA units (1% reciprocal PCA dilution) for IgE. Postchallenge blood samples were taken 10 days after immunization (peak of primary response), and salivary samples were collected on Day 2 1 (IgA samples on Day 10 were comparable). ’
in the aerosol-exposed animals were, in contrast, sigticantly greater than in PBS controls (P < 0.01-0.02). IgA responses appeared greatest in the group exposed to the highest concentration of aerosolized OA although this trend was not significant; this same group were the only animals to display OA-specific IgA titers in serum which were above background (P < 0.05). A group of low-&E-responder WAG rats, a strain previously shown by us to be 1OOO-foldmore sensitive to tolerance induction via antigen inhalation than the highresponder BNs (4), were also included in this trial (Fig. 1). At the single OA-aerosol concentration tested, these animals displayed complete tolerance in the &E-antibody class,but unlike the BN rats exhibited no evidence of enhanced salivary IgA production resulting from antigen inhalation. The data of Table 1 came from experiments employing the second (larger droplet) aerosol system described above, which hasbeen employed in previously reported studies with this model (l-5). The dose levels employed for exposure of the two strains were determined in earlier trials (4), and bracket their respective biologically active dose ranges. Employing this antigen exposure protocol, BN rats again developed tolerance with respect to IgE as shown by their reduced capacity to respond to parenteral challenge (see 1.O%OA, data column 4), but displayed enhanced IgA responses(column 3) only at the lowest exposure level (0.0 1%OA), which was below the threshold for tolerance induction. These variations in IgA responsivenessin BN rats resulting from different aerosol-exposure conditions were confirmed in subsequent experiments. Low-h&responder WAG rats become tolerant in the IgE class

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to parenteral challenge following repeated aerosol exposure throughout the antigen dose range tested, again without evidence of IgA priming. Saliva samples were also collected from these animals during the course of the aerosol regime employed in Table 1. Transient salivary IgA responseswere observed in all animals exposed to OA, after either the third or sixth exposure, but antigenspecific IgA was not detected in the postchallenge samples. The self-limiting nature of these local IgA responsesare similar to that observed with systemic IgE responses during prolonged aerosol exposure ( 1,3). DISCUSSION The salient features of this study are as follows. In confirmation of earlier experiments (4), repeated exposure of high- and low-&E-responder rats to an antigen-containing aerosol produced immunological tolerance in the IgE antibody class which was dose dependent, and the process was up to lOOO-foldmore efficient (on an antigen dosagebasis) in the low-&E-responder WAG strain (Table 1). In animals of the high-@E-responder BN strain, the physical nature of the aerosol mist clearly influenced qualitative aspects of tolerance. Thus, exposure to the small droplet, respirable aerosol which is deposited mainly in the lower respiratory tract ( 14), suppressedsubsequent IgE responsivenessin a dose-dependent fashion and concommitantly primed salivary IgA responses,over the same antigen dose range (Fig. 1). This finding is consistent with a recent report on the concommitant induction of local (gut) secretory immunity and systemic tolerance, in response to food antigens(15). In contrast, repeated inhalation of the large droplet aerosol, which is initially deposited in the oronasal cavity and the upper airways (14), induced tolerance in the IgE class in the absence of detectable salivary IgA synthesis. Collectively, these findings indicate that local IgA responses and systemic IgE responses in the high-IgE-responder animals are under independent control, and they additionally suggestthat &A-mediated exclusion barriers at the level of the respiratory mucosa do not play a central role in acquired suppression of the IgE response to inhaled antigens. In support of these conclusions, rats of the low-@responder WAG strain produced consistent results in these experiments regardless of the type of aerosol employed, and developed tolerance in the IgE isotype (data column 4, Table 1) without priming for salivary IgA production (column 3; seealso Fig. 1). However, examination of antigen-specific salivary IgA levels in these animals during the course of aerosol exposure (Table l), shows the situation to be somewhat more complex. It can be seen that WAG rats displayed large salivary IgA responses during exposure which were spontaneously switched off by exposure Week 6. The biphasic nature of the IgA response in these animals during aerosol exposure, and its failure to reemerge following parenteral challenge, is identical to the situation reported earlier for serum IgE responsesin both rats and mice ( 1,3), and suggeststhat mucosal immunity in these animals may also be susceptible to tolerance induction under appropriate conditions of prolonged antigenic exposure. Similar conclusions follow from the data on high-&$-responder BN rats in Table 1. The disparity between IgA responsivenessin BN rats here and in Fig. 1 may reflect quantitative differences in antigenic stimulation of IgA regulatory T cells associated with the upper versus lower respiratory tract, in response to inhalation of the two types of aerosols.

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ACKNOWLEDGMENTS This work was supported by the Asthma Foundation of Western Australia, and the Princess Margaret Children’s Medical Research Foundation (PMCMRF). This is Publication No. 262 of the Clinical Immunology Research Unit of the PMCMRF. We thank Dr. G. A. Stewart for help and advice with the ELISA assay.

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