Introduction

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Epidemiologic links between the increase of allergic respiratory disease and environmental air pollution have been well established (1). Diesel exhaust particles (DEP) are major air components of inhaled particulate pollution in the industrialized world. DEP and their polycyclic aromatic hydrocarbon components have been demonstrated to enhance IgE production in animals and humans both in vivo and in vitro (4). Combined intranasal challenge with DEP plus allergen induced a large increase in allergen-specific IgE in allergic subjects associated with a Th2-type cytokine profile including enhanced interleukin (IL)-4 and IL-13 production (8, 9). Human B cells have been shown to produce IgE following stimulation with either IL-4 or IL-13 plus CD40 or CD58 ligation (10) with both IL-4 and IL-13 acting as switch factors. We investigated whether in humans, mucosal DEP exposure could be shown to be involved in in vivo Ig isotype switching to IgE. Although earlier in vitro studies employing DEP alone failed to find evidence for induction of IgE isotype switching, the magnitude of the in vivo increase in allergen IgE suggested that allergen-responsive B cells, in the presence of DEP plus allergen, were undergoing isotype switching to IgE.

Ig isotype switching is the process whereby B cells initially expressing IgM and/or IgD on their surface rearrange the active encoding variable-diversity joint region to other Ig heavy chain loci and thereby provide antibodies with different effector functions but the same antigen specificity (15, 16). Molecular analyses of Ig isotype switching strongly supports a model of alignment of two switch (S) region sequences with looping out and deletion of the intervening DNA region (17). This model has been directly demonstrated by the identification of circular Ig switch DNA isolated from in vivo and in vitro stimulated human and mouse B cells (18).

Definitive proof of cells having been driven to undergo isotype switching requires demonstration of the ability to induce the appropriate deleted switch circular DNA. Other measures of isotype switched cells, such as Ig producing cells, IgE protein levels, and even the presence of isotype switched (rearranged) DNA cannot distinguish between recruitment of previously switched cells and cells having just undergone isotype switching. Thus, in this study we tested whether nasal challenge with DEP plus ragweed allergen was able to induce in vivo isotype switching to IgE in patients with ragweed allergy by determining the production of circular switch DNA representing µ to  switching.

	Material and Methods

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Subjects

Eight healthy, nonsmoking volunteers (four men and four women), ranging in age from 21 to 36 yr, were recruited in Los Angeles, CA. All had a positive intracutaneous skin test to short ragweed (over 10 mm) and a history consistent with allergic rhinitis. While two of the eight subjects showed positive skin tests for other allergens, all were asymptomatic during the course of the study. The subjects did not take any medication for 3 d prior to or at any time during the duration of the study. The allergen used in this study, short ragweed, is not present in the Los Angeles area. No subject had been previously treated with immunotherapy for ragweed. Although the local Western ragweed cross-reacts with short ragweed, Western ragweed is a minor allergen in Los Angeles and was not pollinating during the time of this study. All studies were approved by the Human Subject Protection Committee of the University of California, Los Angeles.

All subjects in this study who were treated with intranasal challenge of the ragweed allergen Amb a I displayed an immediate allergic reaction. Challenge with both DEP and ragweed did not result in a significant increase of the symptom as compared with challenge with ragweed alone. Challenge with DEP alone did not result in symptoms (6).

Nasal Challenge of DEP and Allergen

All subjects were challenged on three different occasions with either ragweed alone, DEP alone, or DEP plus ragweed. At least 8 wk passed between challenges, which were performed in a random order. The method of nasal challenge with ragweed allergen alone has been described previously (6). Briefly, subjects were then challenged by spraying the nose with increasing doses of short ragweed Amb a I starting at 10 AU and increasing in 10-fold steps until immediate allergic symptoms were apparent or 1,000 AU had been administered. In two subsequent visits, each a minimum of 8 wk apart, subjects were randomly challenged with both DEP (0.3 mg) and ragweed allergen or with DEP alone. The doses of DEP were chosen according to previous reports (6, 9). DEP (0.15 mg) in 100 µl of saline was sprayed into each nostril for a total of 0.3 mg DEP.

For each subject, three nasal lavages were performed prior to challenge on three different days; then three consecutive nasal lavages were performed 4 d following each challenge. Thus, six nasal lavages were performed on each subject. The methods for nasal lavage and challange have previously been described in detail (6, 8, 9). Briefly, 5 ml of normal saline was delivered into each nostril of the subjects and after 10 s, the wash fluid was collected into a tube. The subjects performed four subsequent nasal washes at 10-min intervals. After the tubes were centrifuged at 350 × g for 10 min, the supernatants were transferred to other tubes and stored at 20°C. The pellets of cells were stored at 20°C.

Isolation of Circular DNA

The cell pellets from nasal lavages were stored until all subjects had finished all their nasal challenges. After the frozen cells were thawed, all eight samples from the different subjects from the same kind and time of nasal challenge were combined into one tube on ice. This was necessary in order to achieve sufficient quantities of B cells and B-cell DNA. Initial attempts to isolate circular DNA from a cell pellet from a single patient obtained 4 d after a DEP-plus-ragweed challenge were unsuccessful because the amount of total circular DNA fraction recovered from a single challenge was extremely small (< 0.02 µg), such that amplified polymerase chain reaction (PCR) bands could not be seen. Therefore, we pooled the samples from the allergic subjects (n = 8) challenged under identical conditions so as to have adequate amounts of circular DNA fraction for each type of challenge. Samples were pooled because it was not possible to challenge the same patient multiple times; the challenge itself serves as an allergen exposure and would alter ("prime") subsequent challenges. Pooling different patient samples increased the amount of DNA assayed for switch circular DNA. There is no evidence that pooling samples would alter the performance or interpretation of the PCR data.

Circular DNA was prepared from the alkaline lysate of the total cell pellets, as described previously (23). To exclude the highly unlikely possibility of our findings resulting from DNA recombination via some undefined recombinase activity acting in vitro, the cell pellets were boiled for 2 min before treatment with lysis buffer and isolation of circular DNA. The precipitated circular DNA was digested with EcoR I, a critical step to linearize circular DNA, and then treated with ribonuclease for 2 h at 37°C to remove RNA. The Sµ and S regions have no internal EcoR I sites while EcoR I sites are present in the 5' and 3' sequences flanking the S regions (30, 31). Linearization of circular DNA provides for improved amplification compared with undigested supercoiled DNA (23). After linearization, the treated circular DNA was used as PCR templates.

PCR Strategy to Detect Recombined S /Sµ Sequences

Nested primer PCR runs for recombined S /Sµ switch fragments were performed on prepared linearized DNA as described (23). Briefly, PCR was run in a 50-µl/reaction with 50 mM KCl, 20 mM Tris-HCl (pH 8.4), 1.5 mM MgCl₂, 5% dimethyl sulfoxide, 50 pmoles primer/reaction, and 2.5 U of Taq polymerase. The PCR assays were carried out as 1 min denaturing (94°C), 1 min annealing (65°C on the first PCR, 68°C on the second), and 2 min extension (72°C), for 40 cycles. The primer sequences have been published elsewhere (23). The µ primers were located just 3' to the end of Sµ (31) while the  primers were located in the I region (32), which is just 5' to the S sequence.

Cloning, Screening, and DNA Sequencing

The PCR products of switch circular DNA were cloned by TA cloning (Invitrogen, San Diego, CA). Positive clones were selected by hybridization with a 5'S probe labeled with [³²P]-cytidine triphosphate by random priming (23). Hybridization was conducted according to the standard methods, with hybridization in 50% formamide being performed at 42°C and the washing temperature for blots being 65°C. Nucleotide sequences were determined by the standard dideoxyl chain termination method using a DNA sequencing kit (Sequenase version 2.0; Amersham, Arlington Heights, IL).

Ig Measurements

Total IgE levels of nasal lavage were measured by enzyme-linked immunosorbent assays (ELISAs) as previously described (6, 11, 33), with minor modifications. The sensitivity of the assay for total IgE was 0.1 ng/ml. The samples were repeated if there was more than a 5% variation between them.

Ragweed-specific IgE were determined using the same procedure as for total Ig isotypes, except that an amplification system previously described (9) was used with minor modifications. The plates were cultured and coated with 10 µg/ml of ragweed extract standardized for its Amb a I content (Hollister Stier/Baxter, Spokane, WA), and alkaline phosphatase-labeled anti-IgE (Tago, Burlingame, CA) at 1/3,000 was used.

	Results

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Combined Nasal Challenge with DEP and Allergen Induced S /Sµ Switch Circles

Multiple DNA fragments were amplified by nested PCR using linearized DNA from the DNA representing the eight samples of nasal cells obtained 4 d following combined nasal challenge with DEP plus ragweed allergen (Figure 1). No such DNA fragments could be amplified from nasal cells obtained prior to challenge or from cells obtained following challenge with DEP or ragweed alone. A number of the amplified fragments shown in Figure 1(I) from DEP-plus-ragweed-stimulated cells hybridized with an S probe. No hybridizing bands were observed with the S probe in any of the pre-challenge samples or in samples collected after challenge with ragweed alone or DEP alone (Figure 1[II]). This result was not simply a reflection of increased cell recovery in the combined challenge pellets because the total amount of DNA in the "circular DNA" preparation before PCR assay was not significantly different among the three challenge protocols (DEP and ragweed = 0.489 and 0.568 µg; ragweed alone = 0.510 and 0.581 µg; DEP alone = 0.412 and 0.612 µg; before and after nasal challenge, respectively). The DNA in our circular DNA fraction may have contained some genomic DNA because it is not possible to exclude genomic DNA completely in the process of circular DNA isolation. Peripheral blood B lymphocytes were obtained from three of the study subjects on Day 4 following combined nasal challenge with DEP plus ragweed allergen. No S /Sµ circular DNA fragment could be amplified by nested PCR (data not shown).

View larger version (33K): [in this window] [in a new window]   Figure 1.   Identification of PCR products representing S /Sµ switch circular DNA in nasal lavage samples. (I) Agarose gel electrophoresis of PCR-amplified switch circle fragments derived from cells obtained before (B) and 4 d after (A) various nasal challenges. Multiple amplified bands were detected only in cells after nasal challenge of DEP plus ragweed allergen (lane 3). Subjects were challenged intranasally with DEP alone (DEP), Amb a I alone (RW), and DEP plus Amb a I (DEP + RW). Nasal lavages were collected before and 4 d after challenge, and cell pellets were obtained. Circular DNA was isolated from nasal pellets and used as templates for nested PCR to amplify deleted switch circles as detailed in MATERIALS AND METHODS. The difference in the background among the lanes is due to the exposure time when the gel was photographed. There is no difference among the groups in the total amount of DNA in the circular DNA preparation before PCR. Lane M represents markers of multiples of 123 bp. (II) Southern blot analysis of PCR products shown in (I). The sample was transferred to a nylon membrane and hybridized with the S probe as discussed in MATERIALS AND METHODS. Multiple bands in lane 3 showed positive hybridization. These were demonstrated to be chimeric S /Sµ fragments by sequencing, as shown in Figure 3.

View larger version (14K): [in this window] [in a new window]   View larger version (14K): [in this window] [in a new window]   Figure 3.   (A) The effect of intranasal challenge with DEP, ragweed (RW), or DEP + RW on total IgE levels in nasal lavage samples. Subjects were intranasally challenged with DEP (0.3 mg) alone, Amb a I alone (ragweed allergen), and DEP plus Amb a I. The mean level (+1 standard deviation) of total IgE in nasal lavage samples obtained before and 4 d after challenge from the eight individual subjects is shown. There was a significant increase in IgE following each type of challenge (*P < 0.005), whereas there was no significant difference in the levels of IgE between the post-challenge samples. IgE levels were determined by ELISA. (B) The effect of intranasal challenge with DEP, ragweed (RW), or DEP + RW on ragweed-specific IgE levels in nasal lavage samples. The mean level (+1 standard deviation) of ragweed-specific IgE in nasal lavage samples before and 4 d after challenge is shown from the eight individual subjects. There was a marked increase in ragweed-specific IgE 4 d after challenge with RW (*P < 0.005) or RW plus DEP (**P < 0.001). One unit of ragweed-specific IgE was determined by employing the patient serum with ragweed allergy.

The PCR products from the cells following challenge with DEP plus ragweed allergen were cloned. A total of six independent clones were isolated and sequenced (Figure 2). The structures of all six clones showed the predicted general structure of 5' S sequence directly joined to 3' Sµ sequence. As expected for isotype switching (19- 28), no consensus sequence was found at the S /Sµ breakpoints.

View larger version (39K): [in this window] [in a new window]   Figure 2.   Nucleotide sequences surrounding the breakpoints in six cloned switch circular DNA fragments from cells recovered following nasal challenge of DEP plus ragweed allergen (Amb a I). The clones represent the direct joining of 5'S sequence to 3'Sµ sequence as part of the deleted switch circular DNA. The sequence in the middle of each set of sequences is the chimeric sequence found; the S and Sµ genomic sequences are shown on the top and bottom, respectively. The arrows indicate the breakpoint site of the S part (top) and Sµ part (bottom).

In order to obtain an upper limit for the amount of S / Sµ cells that would have gone undetected, the ability of our nested PCR assay to detect switch circular DNA was assessed using a cloned plasmid containing an S /Sµ fragment as previously described (25, 27). The S /Sµ plasmid was serially diluted and then used as a template for the nested PCR assay. A band was detected on gel electrophoresis following PCR when there were between one and 10 copies of template present per reaction (data not shown).

Effect of Nasal Challenge on Total and Ragweed-Specific IgE Levels

Consistent with our previous results, total IgE levels in nasal lavage were significantly increased following nasal challenge with DEP alone (before = 0.70 ± 0.46 ng/ml, after = 3.36 ± 1.60 ng/ml, P < 0.005, Figure 3A) (6, 8) or ragweed allergen alone (before = 0.88 ± 0.58 ng/ml, after = 6.04 ± 2.18 ng/ml, P < 0.005) (9). Combined challenge with ragweed allergen plus DEP also increased total IgE (before = 1.01 ± 0.56 ng/ml, after = 5.09 ± 2.63 ng/ml, P < 0.005). There was no significant difference between the total IgE levels among the three types of challenge. No enhancement in IgE was observed after saline challenge (data not shown; see Diaz-Sanchez and associates [6]).

Combined intranasal challenge with DEP and ragweed allergen induced a marked increase in the ragweed-specific IgE levels of nasal lavage (from 0.08 ± 0.06 U/ml to 4.01 ± 2.01 U/ml, P < 0.001) (Figure 3B). Challenge with ragweed alone also caused a significant increase in ragweed-specific IgE (before = 0.07 ± 0.06 U/ml, after = 2.34 ± 1.30 U/ml, P < 0.005). The increase in allergen-specific IgE levels after challenge with ragweed alone was significantly less than seen with combined DEP plus ragweed challenge (P < 0.05), reproducing previous findings (9). Nasal challenge with DEP alone had no effect on production of ragweed-specific IgE in the nose (before = 0.03 ± 0.05 U/ml, after = 0.03 ± 0.05 U/ml). There were no significant elevations of albumin levels over baseline values after all three forms of nasal challenge at Day 4 (data not shown).

	Discussion

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In this study we demonstrate for the first time the induction of in vivo isotype switching to IgE in humans. Following challenge of ragweed-allergic subjects with DEP plus ragweed allergen, local in vivo production of circular switch DNA, representing switching from µ to  , was detected. We isolated, cloned, and sequenced S /Sµ switch circular DNA fragments as a direct demonstration that isotype switching occurred in the nose. While we cannot completely exclude the possibility that IgE-switching B cells containing switch circles migrated into the nasal mucosa following the combined challenge, no switch circles were detected in four independent experiments using freshly isolated, unstimulated peripheral blood mononuclear cells from subjects following combined DEP-plus-ragweed challenge (data not shown).

Our laboratory has previously established an in vivo human model of a local allergic response induced by challenge with either allergen or DEP. We have shown that challenge with an 0.3-mg dose of DEP, approximately equivalent to breathing the outdoor air in Los Angeles for a 24-h period on an average day, alters the regulation of local IgE production in the human nose (6, 9). Isotype switching to IgE in human B cells requires at least two signals (10). One signal, provided by IL-4 or IL-13, induces  germ-line transcripts from Ig heavy chain loci (11, 12, 32), which precede the occurrence of  isotype switching. Combined nasal challenge with DEP plus allergen has been shown to result in a local Th2-type cytokine pattern characterized by increased levels of messenger RNA (mRNA) for IL-4, IL-5, IL-10, and IL-13, and decreased interferon- (8, 9). The second signal required for isotype switching is a cell contact activation signal that can be provided by CD40-CD40L or CD58-CD2 interactions. Intratracheal injection of DEP and allergen in mice has been shown to induce local T-cell activation which can account for an increased CD40 ligand expression on T cells (34). Thus, we tested whether nasal challenge with DEP plus allergen would result in suitable local conditions for the induction of isotype switching to IgE.

Numerous animal models have been established whereby initial injection of adjuvant plus allergen, followed by repeated allergen exposure, induce  isotype switching in vivo. Challenge of atopic subjects with allergen induces preferential activation of cells having a Th2-type cytokine mRNA expression pattern characterized by the production of IL-3, IL-4, IL-5, and granulocyte/macrophage colony-stimulating factor (35). However, in our system, nasal challenge with ragweed alone generated no switch circles in nasal lavage cells, showing that the exposure of allergen alone did not induce local isotype switching for IgE in the nose. This result is likely due to the low level of allergen challenge employed. We used a single exposure to ragweed in a dose that induced a minimum clinical reaction and little induction of local cytokines in order to investigate the effect of DEP in vivo (9). Repeated intranasal allergen challenge has been shown to induce local in vivo  isotype switching in mice models (38). It is possible that the minimum nasal allergen challenge employed triggered antigen-specific B cells via cognate recognition and resulted in expansion of antigen-specific IgE cells. However, this challenge was inadequate to drive the Th2-type cytokine dominant milieu necessary for generation of new isotype switching to IgE. In contrast, DEP challenge alone, a potent non-cognate stimulus, enhanced a whole range of cytokines (a Th0-type reaction) and caused an increase in total IgE in the nasal lavage (8) but did not induce switch circular DNA in nasal B cells. Together, DEP plus allergen likely provided the required cognate plus non-cognate signals necessary to drive local B cells to undergo  isotype switching in vivo. These results further support the concept that increasing environmental DEP with unchanged levels of allergen could be a factor in the increasing clinical sensitization and prevalence of allergic respiratory disease observed over the past two centuries. It is also possible that DEP may not only enhance isotype switching and IgE production but, in conjunction with an antigen, it may also help to induce a de novo specific IgE mucosal response. Research is currently underway to support this hypothesis.