- Email: [email protected]
Effects of established allergen sensitization on immune and airway responses after secondary allergen sensitization
Katharina Blumchen, MD,a Kerstin Gerhold, MD,a Marcus Schwede, MD,a Bodo Niggemann, MD,a Anzhela Avagyan, MD,a Anna-Maria Dittrich, MD,a Birgit Wagner, PhD,b Heimo Breiteneder, PhD,b and Eckard Hamelmann, MDa Berlin, Germany, and Vienna, Austria
Background: Spreading of sensitization with clinical manifestation of allergy is often observed in atopic individuals. Objective: To investigate the effects of an established primary allergen sensitization on immune responses and airway inflammation/reactivity on secondary allergen sensitization and airway challenges in a murine model. Methods: Balb/c mice were primarily sensitized intraperitoneally with ovalbumin or PBS, followed by systemic sensitization and airway challenges with latex extract as a secondary, unrelated allergen. Purely shamsensitized animals were included as controls. In a second set of experiments, the primary and secondary allergens were switched. Results: Sensitization with ovalbumin before sensitization with latex resulted in increased production of total and latexspecific (Hev b 3–specific) IgE and IgG1, and enhanced secretion of TH2-cytokines by spleen mononuclear cells cultured with mitogen compared with single latex-sensitized mice. Furthermore, airway challenges of double-sensitized mice (ovalbumin 1 latex) with latex caused a significant increase in airway reactivity compared with purely latexsensitized and challenged animals. These effects were dependent on dosing and timing of the primary sensitization in relation to the secondary sensitization and independent of the primary allergen used. Conclusion: Primary sensitization boosted systemic TH2 immune responses and enhanced the development of airway reactivity after sensitization and airway challenges with a secondary, unrelated allergen. This effect of consecutive
From athe Department of Pediatric Pneumology and Immunology, University Hospital Charite´, Berlin; and bthe Center of Physiology and Pathophysiology, Medical University of Vienna. Supported in part by grants of the German Research Council Ha2162/2-1, NBL3 01ZZ0101, and the Austrian Science Fund grants P12838-GEN and SFB F01802. Disclosure of potential conflict of interest: K. Blumchen and E. Hamelmann have received grant support from German Research Council Ha2162/2-1, NBL-3 01ZZ0101. The rest of the authors have declared that they have no conflict of interest. Received for publication July 19, 2005; revised April 9, 2006; accepted for publication April 17, 2006. Available online July 28, 2006. Reprint requests: Katharina Blumchen, MD, Department of Pediatric Pneumology and Immunology, University Hospital Charite´, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail: [email protected]. 0091-6749/$32.00 Ó 2006 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2006.04.054
priming was dependent on the strength of the primary sensitization but independent of the allergen used. The results explain the increased susceptibility toward sensitization spreading in atopic individuals. Clinical implications: Because sensitization spreading is facilitated by primary sensitization, early prevention measurements or immunotherapy should be considered at this stage of monosensitization. (J Allergy Clin Immunol 2006;118:615-21.) Key words: Sensitization, mice, TH1/TH2 cytokines, airway reactivity, airway inflammation
During priming with antigen, naive helper T cells differentiate into activated, proliferating TH cells with either a predominant TH1 or TH2 cell phenotype.1 Different signals like certain master cytokines and transcription factors have been identified that may be responsible for this differentiation. IL-4–induced signaling via GATA-3 was identified as the driving force for in vitro differentiation of TH2 cells,2,3 the central players of allergic immune responses.4 Therefore, it is intriguing to speculate that an established TH2 milieu with increased numbers of IL-4– producing TH2 cells as a consequence of primary allergen sensitization may influence subsequent immune responses of naive T cells and enhance differentiation toward TH2type cells with specificity against other allergens. This hypothesis is supported by epidemiologic data in children showing that sensitization increases not only the risk of developing subsequent sensitizations but also clinical symptoms like bronchial asthma.5-8 Similarly, specific immunotherapy in patients with allergic rhinitis prevents sensitization to other allergens and development of asthma.9-12 To delineate further the hypothesis of spreading of sensitization, we established a mouse model to investigate the effects of established allergen sensitization on systemic immune responses, airway inflammation, and airway reactivity toward a secondary allergen sensitization and airway challenge. We used 2 unrelated allergens, ovalbumin (OVA) and latex extract, applied without adjuvant to mimic natural sensitization more closely (protocols previously published13,14 or modified after15,16). We show here for the first time that active in vivo sensitization with allergen in a murine model not only boosted systemic TH2 immune responses but also enhanced the development of airway hyperreactivity (AHR) after sensitization and 615
Mechanisms of asthma and allergic inflammation
Effects of established allergen sensitization on immune and airway responses after secondary allergen sensitization
616 Blumchen et al
J ALLERGY CLIN IMMUNOL SEPTEMBER 2006
Mechanisms of asthma and allergic inflammation
FIG 1. Experimental protocol. Mice were immunized with either ovalbumin or latex as a primary sensitization followed by a secondary systemic sensitization with the other allergen (OVA/Latex, Latex/OVA). Singlesensitized mice received a sham sensitization followed by sensitization with either latex (PBS/Latex) or ovalbumin (PBS/OVA). A control group was purely sham-sensitized (PBS/PBS). All animals received airway challenges with the second allergen. in, Intranasal; ip, intraperitoneal; Neb, via nebulization.
Abbreviations used AHR: Airway hyperreactivity AI: Airway inflammation AR: Airway reactivity MNC: Mononuclear cell OVA: Ovalbumin
airway challenges with a secondary allergen. This effect was dependent on the strength of the primary sensitization and was unrelated to the kind of primary allergen used.
METHODS Animals Female Balb/c mice, 6 to 8 weeks old, were obtained from the Bundesinstitut fu¨r Risikobewertung, Berlin, Germany. Mice were kept under pathogen-and latex-free conditions, and maintained on an ovalbumin-free diet. All experiments were approved by the animal care committee.
Experimental protocol Primary systemic sensitization. See Fig 1. Mice (3-5 mice/group/ experiment) were systemically sensitized by 6 intraperitoneal injections over a period of 3 weeks of (1) 10 mg ovalbumin (grade VI; Sigma, St Louis, Mo), (2) 30 mg total protein of a latex extract of Hevea brasiliensis (preparation as described17), or (3) PBS for primary sham sensitization (Seromed; Biochrom, Berlin, Germany). To analyze effects of dosing, another group of mice received half of the ovalbumin dose for primary sensitization: 10 mg ovalbumin intraperitoneally 3 times within 3 weeks (days 23-43), resulting in a weaker primary allergic sensitization compared with the other primary allergen sensitization protocols (data not shown).
Secondary systemic sensitization. See Fig 1. For secondary sensitization, animals were injected on days 50, 54, and 61 with (1) 30 mg latex extract intraperitoneally (PBS/Latex or OVA/Latex group), (2) 20 mg ovalbumin intraperitoneally (PBS/OVA or Latex/OVA group), or (3) PBS for secondary sham sensitization (PBS/PBS). Analyzing the timing effect of the introduction of the secondary sensitization, 1 murine group received ovalbumin as a primary sensitization within the first 3 weeks (days 1-21) followed by a 31-day pause and secondary sensitization starting on day 50 (OVAlong/Latex group), whereas another group received ovalbumin from day 23 to 43 followed by only a 7-day pause and secondary sensitization on day 50 (OVAshort/Latex group). Kinetic studies of immunoglobulin production showed that 7 days after the last primary immunization (day 36), total immunoglobulin production peaked and then slowly declined, whereas ovalbuminspecific IgE production reached a maximum on day 43 and was stable from then on through day 50 (data not shown). Airway challenges. See Fig 1. All mice were airway challenged with the secondary allergen on days 68, 69, and 70 by (1) intranasal application of 30 mg latex extract in 50 mL PBS or (2) ultrasonic nebulization (LS2000; SYSTAM, Villeneuve-sur-Lot, France) of 1% ovalbumin (grade V; Sigma) for 10 minutes daily. For safety reasons, nebulization of latex extract was not allowed in the animal facility, so that the intranasal route had to be used.
Serum levels of total and specific immunoglobulins Total and allergen-specific immunoglobulin serum levels were measured on days 50 and 72 by ELISA as previously described.17-19 To monitor latex-specific immunoglobulin production, levels of immunoglobulin specific for Hev b 317 were measured and expressed as OD.
Cell preparation and culture On day 72, spleen mononuclear cells (MNCs) were isolated by density gradient centrifugation (Lympholyte-M; Cedarline Laboratories, Hornby, British Columbia, Canada) and cultured (106 cells/ 200 mL) in 96-well round-bottom plates at 37°C, 5% CO2 with (1) RPMI 1640, (2) concanavalin A (2.5 mg/mL; Sigma), (3) ovalbumin (50 mg/mL), or (3) latex extract (100 mg/mL). After 48 hours (IFN-g)
Blumchen et al 617
FIG 2. Total IgE. Experimental protocol as indicated in Fig 1. Mice were bled on day 72, and serum total IgE was determined by ELISA. Double-sensitized vs single-sensitized mice: PBS/Latex vs OVA/Latex, PBS/OVA vs Latex/OVA, **P < .01, *P < .05. Sham-sensitized vs allergen-sensitized mice: PBS/PBS vs PBS/Latex or PBS/OVA, 11P < .01.
or 96 hours (IL-5, IL-4), respectively, supernatants were harvested and stored at 220°C until further analysis.
Cytokine measurements Levels of IL-5, IL-4 and IFN-g in cell culture supernatants were assessed by ELISA, as previously described.14 Detection limits were 16 pg/mL for IL-5 and IL-4 and 39 pg/mL for IFN-g.
Bronchoalveolar lavage On day 72, lungs were lavaged twice with 0.8 mL cold PBS via a tracheal tube. Cells were counted, stained with Diff-Quik (Dade Behring AG, Du¨dingen, Switzerland), and differentiated according to morphologic criteria by counting at least 200 cells under light microscopy.
Measurement of in vivo airway reactivity On day 71, 24 hours after the last airway challenge, airway reactivity (AR) to inhaled provocation with rising doses (6-100 mg/mL) of methacholine (Sigma) was measured by whole-bodyplethysmography (Buxco; EMKA Technologies, Paris, France) as described.14 Methacholine doses resulting in 200% (PC 200) and 500% (PC 500) increase of baseline values for enhanced pause were calculated for each animal.14
Statistical analysis Animals of similar groups of 2 to 3 independent experiments were pooled (n 5 12 for each pooled group). Values for all measurements were expressed as means 6 SEMs. Pairs of groups were compared by Mann-Whitney U test. Values of P for significance were set at .05 (*) and .01 (**).
RESULTS Immune responses after primary sensitization with ovalbumin or latex extract Immunizing Balb/c mice 6 times within 3 weeks (Fig 1) intraperitoneally with low doses of ovalbumin (10 mg ovalbumin/mouse/dose) without adjuvant resulted in a robust systemic immune response (n 5 12; see this article’s Table E1 in the Online Repository at www.jacionline.org). On day 50, 31 days after the last intraperitoneal injection,
levels of total IgE and IgG2a were significantly higher in ovalbumin-sensitized compared with sham-sensitized mice (OVAlong/PBS vs PBS/PBS or PBS/Latex; P < .05). More profound, allergen-specific immune responses revealed a strong TH2-type response in ovalbumin-sensitized mice with high production of ovalbumin-specific IgE and low IgG2a levels, whereas sham-sensitized animals showed no allergen-specific immunoglobulin production. Analyzing immunoglobulin levels 7 days after the last immunization (day 50, OVAshort/Latex), we also observed increased TH2-type immunoglobulin production but lower levels of ovalbumin-specific IgE compared with analysis after the longer interval (see this article’s Table E1 in the Online Repository at www.jacionline.org). Repeated latex sensitization (6 3 30 mg latex extract intraperitoneally, Latex/OVA) without adjuvant also induced increased systemic TH2-type responses with significant increase in total IgE and IgG2a levels (see this article’s Table E1 in the Online Repository at www.jacionline.org). Hev b 3–specific IgE (see this article’s Table E1 in the Online Repository at www.jacionline.org) levels were markedly raised in latex-sensitized mice, whereas PBS– sham-sensitized animals (PBS/OVA) showed no detectable specific immunoglobulin production.
Immune responses after secondary sensitization and airway challenges with latex extract Mice were primarily sensitized with ovalbumin as described, followed by a secondary sensitization with latex extract, a noncross-reactive allergen, 31 days after the last ovalbumin injection (Fig 1). These double-sensitized mice (OVAlong/Latex) showed significantly stronger unspecific systemic TH2-type responses compared with single-sensitized animals (PBS/Latex). On day 72, total IgE levels were significantly increased in double-sensitized (OVAlong/Latex) compared with single-sensitized (PBS/ Latex) or sham-sensitized animals (PBS/PBS, Fig 2).
Mechanisms of asthma and allergic inflammation
J ALLERGY CLIN IMMUNOL VOLUME 118, NUMBER 3
618 Blumchen et al
J ALLERGY CLIN IMMUNOL SEPTEMBER 2006
Mechanisms of asthma and allergic inflammation
FIG 3. Mitogen-induced cytokine production by spleen MNCs. Experimental protocol as indicated in Fig 1. Splenic MNCs were obtained on day 72 and restimulated in vitro with concanavalin A. Cytokine production in supernatants was measured by ELISA: IL-4 (A), IL-5 (B), and IFN-g (C). Single-sensitized vs double-sensitized mice: PBS/Latex vs OVA/Latex, PBS/OVA vs Latex/OVA, **P < .01, *P < .05. Sham-sensitized vs allergensensitized mice: PBS/PBS vs PBS/Latex or PBS/OVA, 11P < .01.
FIG 4. Latex-specific immunoglobulin production. Experimental protocol as indicated in Fig 1. Mice were bled on day 72. Latex-specific IgE (A), IgG1 (B), and IgG2a (C) were evaluated by ELISA in measuring immunoglobulin production specific for Hev b 3, a major latex allergen. Levels are documented in OD units. Singlesensitized vs double-sensitized mice: PBS/Latex vs OVA/Latex, *P < .05. Sham-sensitized vs single-sensitized mice: PBS/PBS vs PBS/Latex, 11P < .01, 1P < .05.
Similarly, IL-4 and IL-5 production in response to mitogen was significantly higher in double-sensitized (OVAlong/ Latex) compared with single-sensitized (PBS/Latex) or sham-sensitized mice (PBS/PBS, Fig 3, A and B). Production of total IgG2a (OVAlong/Latex: 987 6 144 ng/mL vs PBS/Latex: 671 6 130 ng/mL) and IFN-g (Fig 3, C) was low and not significantly different between double-sensitized and single-sensitized animals (Fig 3, C). Next, allergen-specific immune responses to the secondary allergen were analyzed. On day 72, double-sensitized animals (OVAlong/Latex) produced significantly higher levels of allergen (Hev b 3)–specific IgE (Fig 4, A) and IgG1 (Fig 4, B) compared with single-sensitized mice (PBS/Latex). There was no difference in Hev b 3– specific IgG2a levels between double-sensitized or single-sensitized mice (Fig 4, C). Stimulation of spleen MNCs with secondary allergen, latex extract, resulted in high in vitro cytokine production of IL-4 and IL-5 and no measurable IFN-g. However, we found no significant difference between single-sensitized and double-sensitized animals (data not shown).
Airway responses after secondary sensitization and airway challenges with latex extract Airway latex challenges of latex-sensitized mice (PBS/ Latex) induced a robust eosinophilic airway inflammation (AI) with a significant increase in numbers of total cells, lymphocytes, and eosinophils in bronchoalveolar lavage fluids (Fig 6) and a trend toward development of increased AHR (Fig 7, A) compared with sham-sensitized, airwaychallenged mice (PBS/PBS).
In contrast, latex sensitization and airway challenges of mice that had been primarily sensitized with OVA (OVAlong/Latex) resulted in significantly increased AR compared with single-sensitized animals (PBS/Latex; Fig 7, A). PC 200 and, even more pronounced, PC 500 levels were significantly reduced in double-sensitized animals in comparison with both single-treated and sham-treated mice. In contrast, eosinophilic AI was not significantly different between double-sensitized and single-sensitized animals (Fig 6).
Effects of timing of the primary on secondary sensitization To analyze whether the interval between primary and secondary sensitization was relevant for the enhancing effect on immune responses after secondary sensitization and airway challenges, we compared groups of mice with a 7-day interval between primary and secondary sensitization (OVAshort/Latex) with the group with a 31-day interval between primary and secondary sensitization (OVAlong/Latex; Fig 1). Both OVAlong/Latex and OVAshort/Latex groups developed significantly increased total IgE (Fig 2) and mitogen-induced IL-4 and IL-5 production by spleen MNCs (Fig 3, A and B) compared with single-sensitized animals (PBS/Latex). There were no differences in total IgG2a (data not shown) or in IFN-g levels (Fig 3, C) in both double-sensitized groups (OVAlong/ Latex and OVAshort/Latex) compared with control mice (PBS/Latex). Generally, Hev b 3 specific IgE and IgG1 production in double-sensitized mice was increased compared with controls, but this increase was significant only in OVAlong/Latex animals compared with PBS/Latex
Blumchen et al 619
FIG 5. Ovalbumin-specific immunoglobulin production. Experimental protocol as indicated in Fig 1. Mice were bled on day 72. Ovalbumin-specific IgE (A), IgG1 (B), and IgG2a (C) were measured by ELISA and compared with known hyperimmune standards (EU/mL). Single-sensitized vs double-sensitized mice: PBS/OVA vs Latex/OVA, **P < .01. Purely sham-sensitized animals (PBS/PBS) showed OVA-IgE, IgG1, and IgG2a below detection limits.
mice (Fig 4, A and B). However, similar to the long protocol, OVAshort/Latex animals showed significant increases in AR compared with single-sensitized animals (PBS/ Latex, Fig 7, A), although eosinophilic AI was not significantly different between these 2 groups (Fig 6).
Effects of allergen sequence of primary and secondary sensitization To analyze whether the sequence of allergens for primary and secondary sensitization was important for the enhancing effects observed in double-sensitized animals, we switched the order of allergens, now immunizing mice 6 times intraperitoneally with latex extract as a primary sensitization followed, after a 7-day interval, by systemic ovalbumin sensitization and ovalbumin airway challenges (Fig 1). Similar to the results of the previous experiments, total IgE serum levels were significantly increased in double-sensitized mice (Latex/OVA) compared with single-sensitized (PBS/OVA) or sham-treated controls (PBS/PBS; Fig 2). However, there was no significant difference between double-sensitized and single-sensitized mice regarding mitogen-induced IL-4 (Latex/OVA: 183 6 31 pg/mL vs PBS/OVA: 137 6 33 pg/mL), IL-5 (Latex/OVA: 521 6 62 pg/mL vs PBS/OVA: 652 6 94 pg/mL), or IFN-g production (Latex/OVA: 1200 6 270 pg/mL vs PBS/OVA: 1058 6 288 pg/mL) by splenic MNCs. More importantly, double-sensitized animals produced significantly higher amounts of ovalbumin-specific IgE than single-sensitized mice (Fig 5, A), whereas levels of ovalbumin-specific IgG1 and IgG2a were similar between the 2 groups (Fig 5, B and C). Single-sensitized mice that were purely sensitized and airway-challenged with ovalbumin (PBS/OVA) showed a robust eosinophilic airway inflammation (data not shown) as well as increased AR (Fig 7, B) compared with shamsensitized and ovalbumin airway-challenged mice (PBS/ PBS). Once again, eosinophilic inflammation was not significantly different between double-sensitized and single-sensitized mice, but there was a trend toward increased AR in double-sensitized compared with single-sensitized ones (Fig 7, B, Latex/OVA vs PBS/OVA). DISCUSSION We have shown in a murine model that primary sensitization enhanced systemic immune and airway
FIG 6. Bronchoalveolar lavage. Experimental design as indicated in Fig 1. On day 72, two days after last airway challenge, lungs were lavaged, and differential cell counts in bronchoalveolar lavage fluids were obtained. Total cell counts, eosinophil counts, and lymphocyte counts are presented. Single-sensitized vs double-sensitized mice: PBS/Latex vs OVA/Latex, *P < .05. Sham-sensitized vs allergen-sensitized mice: PBS/PBS vs PBS/Latex or OVA/Latex, 11P < .01.
responses on subsequent sensitization and airway challenges with an unrelated, not cross-reactive, secondary allergen (consecutive priming). Experiments with ovalbumin as the primary allergen followed by latex extract as the secondary allergen (OVA/Latex) resulted in an enhanced TH2 response and increased development of airway hyperreactivity on secondary sensitization. This was most probably a result of an unspecific amplification of TH2 immune responses against the secondary allergens by a predominant systemic TH2 milieu, reflected by a TH2-skewed immune status at the time point when secondary sensitization was initiated (see this article’s Table E1 in the Online Repository at www.jacionline.org). The magnitude of the enhancement of secondary sensitization was decreased when we shortened the interval between primary and secondary sensitization from 31 to 7 days (OVAlong/Latex vs OVAshort/Latex). Although a similar increase in unspecific TH2-type immune responses was noted, the increase in allergen-specific IgE and IgG1 production was lower in the OVAshort/Latex group than in the OVAlong/Latex group. This difference can be explained by differences in the level of the primary sensitization between the OVAlong/Latex vs OVAshort/Latex protocols, where we found higher TH2 intensity in mice of the long protocol at the time point when the secondary sensitization
Mechanisms of asthma and allergic inflammation
J ALLERGY CLIN IMMUNOL VOLUME 118, NUMBER 3
620 Blumchen et al
J ALLERGY CLIN IMMUNOL SEPTEMBER 2006
Mechanisms of asthma and allergic inflammation
FIG 7. Airway reactivity. Experimental design as indicated in Fig 1. On day 71, lung function to inhaled methacholine was measured by whole-body plethysmography. The dosage of methacholine increasing AR by 200% (PC 200) or 500% (PC 500) was calculated for each animal. A, AR of mice that received OVA as primary and Latex as secondary sensitization agent (OVA/Latex). B, AR of mice that received the reversed protocol (Latex/OVA). Single-sensitized vs double-sensitized mice: PBS/Latex vs OVA/Latex, PBS/OVA vs Latex/OVA, **P < .01, *P < .05. Sham-sensitized vs allergen-sensitized mice: PBS/PBS vs PBS/Latex, PBS/OVA or double-sensitized mice, 1P < .05. MCh, Methacholine.
was established (day 50; see this article’s Table E1 in the Online Repository at www.jacionline.org). In kinetic studies with ovalbumin-sensitized mice, we found a maximum, robust, and stable TH2-skewed immunoglobulin production 31 days after the last ovalbumin immunization. We conclude that introducing the secondary sensitization into a comparable weak primary sensitization process might result in only a moderate priming of allergen-specific TH2 immune responses and development of AHR after secondary sensitization and airway challenges. This conclusion was strongly supported by data from mice sensitized with ovalbumin 3 times instead of 6 times, resulting in weaker primary immune responses compared with ovalbumin injected 6 times (data not shown). Using this weak primary ovalbumin sensitization protocol, the enhancement of specific and unspecific TH2 and airway responses after secondary sensitization and airway challenges with latex extract was no longer significant (data not presented). Importantly, the effects of consecutive priming were also found by using the reversed protocol (Latex/OVA). We observed a robust sensitization against the primary allergen (latex extract) on day 50, and found highly and significantly enhanced TH2-type immune responses against the secondary allergen (ovalbumin) even with the short 7-day interval between primary and secondary sensitization. This shows that the enhancement of TH2 responses after the secondary allergen sensitization was not dependent on 1 specific allergen, a notion supported by clinical data showing increased risk of secondary allergen sensitizations in children primarily sensitized against hen’s egg protein5 or latex allergens.20,21 There may be various reasons for the differences we see between the outcomes of Latex/OVA and OVA/Latex animals regarding the strength of the immune responses as well as the development of enhanced AR after consecutive priming. Although a direct comparison of the allergenicity of the 2 allergens used seems rather impracticable because of differences in concentration and preparation of the compounds (single protein suspension for ovalbumin vs protein mixture in latex extract), we established 2 quite similar but weak secondary sensitization protocols (PBS/ Latex vs PBS/OVA). Therefore, the differences in the magnitude of enhancement of allergen specific IgE
production after secondary sensitization (OVAshort/Latex vs Latex/OVA) might be explained by the strength of the primary sensitization with latex extract resulting in higher total IgE and TH2 cytokine production than primary sensitization with ovalbumin (day 50; see this article’s Table E1 in the Online Repository at www.jacionline.org). However, differences in the effects on AR between the Latex/OVA and OVA/Latex group might be a result of differences either in allergenicity or to the routes of airway challenges applied. It was obvious that aerosolized ovalbumin induced a stronger development of AHR in ovalbumin-sensitized and airway-challenged mice (PBS/OVA) compared with mice that were latex-sensitized and airwaychallenged with latex extract intranasally (PBS/Latex). This stronger airway response in the former group may have obscured any subtle differences provoked by consecutive priming after primary allergen sensitization with latex extract. Another reason could be that OVA/Latex mice showed significantly enhanced production of mitogen-induced IL-4 and IL-5 by spleen MNCs at the time of airway allergen challenges compared with single-sensitized mice (PBS/Latex). This enhancing effect on unspecific TH2 cytokine production was not detected in Latex/ OVA mice. Although the difference in cytokine production did not lead to measurable variation of eosinophilic airway infiltration, it may have contributed toward the development of significantly enhanced AR in the OVA/Latex vs PBS/Latex group. This is supported by convincing evidence that differences in eosinophilic AI may, in some experimental settings, be too subtle to be detected, and are not mandatory for the development of enhanced AR.22-24 Other recent data from murine models underscore the effect of cytokine milieu on T-cell development at the time of initial priming.25,26 Adoptive transfer of TH2-polarized OVA-T cell receptor–transgenic T cells (DO11.10) into naive Balb/c mice enhanced specific airway responses of endogenous naive T cells after airway challenges with a secondary allergen.25 Further, Schipf et al26 showed that adoptively transferred TH2-skewed cells were even capable of overruling an adjuvant-induced TH1 shift toward a secondary antigen. In both experiments,25,26 the effects were dependent on (1) the presence of IL-4 and (2) the
simultaneous introduction of the first and second antigen (collateral priming). We have extended these observations here, showing that the amplification of secondary TH2 immune responses was not dependent on the simultaneous application of both allergens, but occurred as the consequence of 2 subsequent individual and active sensitization procedures (consecutive priming). Furthermore, for the first time, we have demonstrated the effect of enhanced TH2 immune responses for allergen-induced airway inflammation and development of AHR, emphasizing the clinical finding that sensitization spreading is also associated with an increased risk of developing asthma.7,8 As the most likely mechanism underlying this phenomenon, we suggest that primary sensitization systemically and unspecifically increased the susceptibility toward a secondary sensitization, even with unrelated allergen. This could be achieved by inhibition of regulatory T-cell development via enhanced GATA-3 expression (Schmidt-Weber CB, January 2006, personal communication27) or by impaired IL-12 production by dendritic cells28 as the result of enhanced TH2 immune responses after primary sensitization. In conclusion, established primary sensitization leads to enhanced allergen-mediated TH2-type immune and airway responses after subsequent sensitization and airway challenges with a secondary unrelated allergen in vivo. Importantly, this was not specific or dependent on a certain type of allergen but related to the strength of the primary sensitization protocol. The careful tuning between cytokines in the initial phase of priming seemed to be the most important factor.29 Avoiding early and strong allergen sensitization may therefore be a very relevant means for primary prevention of subsequent allergen-induced airway diseases. Furthermore, the concept of consecutive priming indirectly supports the notion and requirement for early immunotherapy as an important way to avoid allergen sensitization spreading and disease aggravation. We thank C. Seib for her helpful technical assistance and H. G. Hoymann from the Frauenhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany, for his calculation of ovalbumin deposition in the lung after airway challenges with ovalbumin in mice.
REFERENCES 1. O’Garra A, Arai N. The molecular basis of T helper 1 and T helper 2 cell differentiation. Trends Cell Biol 2000;10:542-50. 2. Paul WE, Seder RA. Lymphocyte responses and cytokines. Cell 1994; 76:241-51. 3. Grogan JL, Locksley RM. T helper cell differentiation: on again, off again. Curr Opin Immunol 2002;14:366-72. 4. Ngoc PL, Gold DR, Tzianabos AO, Weiss ST, Celedon JC. Cytokines, allergy, and asthma. Curr Opin Allergy Clin Immunol 2005;5:161-6. 5. Nickel R, Kulig M, Forster J, Bergmann R, Bauer CP, Lau S, et al. Sensitization to hen’s egg at the age of twelve months is predictive for allergic sensitization to common indoor and outdoor allergens at the age of three years. J Allergy Clin Immunol 1997;99:613-7. 6. Illi S, von Mutius E, Lau S, Nickel R, Niggemann B, Sommerfeld C, et al. The pattern of atopic sensitization is associated with the development of asthma in childhood. J Allergy Clin Immunol 2001;108:709-14. 7. Sherrill D, Stein R, Kurzius-Spencer M, Martinez F. On early sensitization to allergens and development of respiratory symptoms. Clin Exp Allergy 1999;29:905-11.
Blumchen et al 621
8. Rhodes HL, Sporik R, Thomas P, Holgate ST, Cogswell JJ. Early life risk factors for adult asthma: a birth cohort study of subjects at risk. J Allergy Clin Immunol 2001;108:720-5. 9. Purello-D’Ambrosio F, Gangemi S, Merendino RA, Isola S, Puccinelli P, Parmiani S, et al. Prevention of new sensitizations in monosensitized subjects submitted to specific immunotherapy or not: a retrospective study. Clin Exp Allergy 2001;31:1295-302. 10. Pajno GB, Barberio G, De Luca F, Morabito L, Parmiani S. Prevention of new sensitizations in asthmatic children monosensitized to house dust mite by specific immunotherapy: a six-year follow-up study. Clin Exp Allergy 2001;31:1392-7. 11. Des Roches A, Paradis L, Menardo JL, Bouges S, Daures JP, Bousquet J. Immunotherapy with a standardized Dermatophagoides pteronyssinus extract, VI: specific immunotherapy prevents the onset of new sensitizations in children. J Allergy Clin Immunol 1997;99:450-3. 12. Moller C, Dreborg S, Ferdousi HA, Halken S, Host A, Jacobsen L, et al. Pollen immunotherapy reduces the development of asthma in children with seasonal rhinoconjunctivitis (the PAT-study). J Allergy Clin Immunol 2002;109:251-6. 13. Gerhold K, Bluemchen K, Franke A, Stock P, Hamelmann E. Exposure to endotoxin and allergen in early life and its effect on allergen sensitization in mice. J Allergy Clin Immunol 2003;112:389-96. 14. Blumchen K, Gerhold K, Thorade I, Seib C, Wahn U, Hamelmann E. Oral administration of desloratadine prior to sensitization prevents allergen-induced airway inflammation and hyper-reactivity in mice. Clin Exp Allergy 2004;34:1124-30. 15. Kurup VP, Kumar A, Choi H, Murali PS, Resnick A, Kelly KJ, et al. Latex antigens induce IgE and eosinophils in mice. Int Arch Allergy Immunol 1994;103:370-7. 16. Woolhiser MR, Munson AE, Meade BJ. Immunological responses of mice following administration of natural rubber latex proteins by different routes of exposure. Toxicol Sci 2000;55:343-51. 17. Wagner B, Krebitz M, Buck D, Niggemann B, Yeang HY, Han KH, et al. Cloning, expression, and characterization of recombinant Hev b 3, a Hevea brasiliensis protein associated with latex allergy in patients with spina bifida. J Allergy Clin Immunol 1999;104:1084-92. 18. Hamelmann E, Oshiba A, Loader J, Larsen GL, Gleich G, Lee J, et al. Antiinterleukin-5 antibody prevents airway hyperresponsiveness in a murine model of airway sensitization. Am J Respir Crit Care Med 1997;155:819-25. 19. Hufnagl K, Wagner B, Winkler B, Baier K, Hochreiter R, Thalhamer J, et al. Induction of mucosal tolerance with recombinant Hev b 1 and recombinant Hev b 3 for prevention of latex allergy in BALB/c mice. Clin Exp Immunol 2003;133:170-6. 20. Niggemann B, Buck D, Michael T, Wahn U. Latex provocation tests in patients with spina bifida: who is at risk of becoming symptomatic? J Allergy Clin Immunol 1998;102:665-70. 21. Liebke C, Niggemann B, Wahn U. Sensitivity and allergy to latex in atopic and non-atopic children. Pediatr Allergy Immunol 1996;7:103-7. 22. Takeda K, Haczku A, Lee JJ, Irvin CG, Gelfand EW. Strain dependence of airway hyperresponsiveness reflects differences in eosinophil localization in the lung. Am J Physiol Lung Cell Mol Physiol 2001;281:L394-402. 23. Bochner BS. Verdict in the case of therapies versus eosinophils: the jury is still out. J Allergy Clin Immunol 2004;113:3-9, quiz 10. 24. Alam R, Busse WW. The eosinophil: quo vadis? J Allergy Clin Immunol 2004;113:38-42. 25. Eisenbarth SC, Zhadkevich A, Ranney P, Herrick CA, Bottomly K. IL-4dependent Th2 collateral priming to inhaled antigens independent of Toll-like receptor 4 and myeloid differentiation factor 88. J Immunol 2004;172:4527-34. 26. Schipf A, Heilmann A, Boue L, Mossmann H, Brocker T, Rocken M. Th2 cells shape the differentiation of developing T cell responses during interactions with dendritic cells in vivo. Eur J Immunol 2003;33:1697-706. 27. Mantel RZ KH, Onakqd N, Seckez B, Karagiannidis C, Lambrecht B, Blaser K, et al. Inhibition of T regulatory cell development by GATA 3. Swiss Med Wkly 2006;136:149:4s. 28. Traidl-Hoffmann C, Mariani V, Hochrein H, Karg K, Wagner H, Ring J, et al. Pollen-associated phytoprostanes inhibit dendritic cell interleukin12 production and augment T helper type 2 cell polarization. J Exp Med 2005;201:627-36. 29. Constant SL, Bottomly K. Induction of Th1 and Th2 CD41 T cell responses: the alternative approaches. Annu Rev Immunol 1997;15: 297-322.
Mechanisms of asthma and allergic inflammation
J ALLERGY CLIN IMMUNOL VOLUME 118, NUMBER 3
Background: Spreading of sensitization with clinical manifestation of allergy is often observed in atopic individuals. Objective: To investigate the effects of an established primary allergen sensitization on immune responses and airway inflammation/reactivity on secondary allergen sensitization and airway challenges in a murine model. Methods: Balb/c mice were primarily sensitized intraperitoneally with ovalbumin or PBS, followed by systemic sensitization and airway challenges with latex extract as a secondary, unrelated allergen. Purely shamsensitized animals were included as controls. In a second set of experiments, the primary and secondary allergens were switched. Results: Sensitization with ovalbumin before sensitization with latex resulted in increased production of total and latexspecific (Hev b 3–specific) IgE and IgG1, and enhanced secretion of TH2-cytokines by spleen mononuclear cells cultured with mitogen compared with single latex-sensitized mice. Furthermore, airway challenges of double-sensitized mice (ovalbumin 1 latex) with latex caused a significant increase in airway reactivity compared with purely latexsensitized and challenged animals. These effects were dependent on dosing and timing of the primary sensitization in relation to the secondary sensitization and independent of the primary allergen used. Conclusion: Primary sensitization boosted systemic TH2 immune responses and enhanced the development of airway reactivity after sensitization and airway challenges with a secondary, unrelated allergen. This effect of consecutive
From athe Department of Pediatric Pneumology and Immunology, University Hospital Charite´, Berlin; and bthe Center of Physiology and Pathophysiology, Medical University of Vienna. Supported in part by grants of the German Research Council Ha2162/2-1, NBL3 01ZZ0101, and the Austrian Science Fund grants P12838-GEN and SFB F01802. Disclosure of potential conflict of interest: K. Blumchen and E. Hamelmann have received grant support from German Research Council Ha2162/2-1, NBL-3 01ZZ0101. The rest of the authors have declared that they have no conflict of interest. Received for publication July 19, 2005; revised April 9, 2006; accepted for publication April 17, 2006. Available online July 28, 2006. Reprint requests: Katharina Blumchen, MD, Department of Pediatric Pneumology and Immunology, University Hospital Charite´, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail: [email protected]. 0091-6749/$32.00 Ó 2006 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2006.04.054
priming was dependent on the strength of the primary sensitization but independent of the allergen used. The results explain the increased susceptibility toward sensitization spreading in atopic individuals. Clinical implications: Because sensitization spreading is facilitated by primary sensitization, early prevention measurements or immunotherapy should be considered at this stage of monosensitization. (J Allergy Clin Immunol 2006;118:615-21.) Key words: Sensitization, mice, TH1/TH2 cytokines, airway reactivity, airway inflammation
During priming with antigen, naive helper T cells differentiate into activated, proliferating TH cells with either a predominant TH1 or TH2 cell phenotype.1 Different signals like certain master cytokines and transcription factors have been identified that may be responsible for this differentiation. IL-4–induced signaling via GATA-3 was identified as the driving force for in vitro differentiation of TH2 cells,2,3 the central players of allergic immune responses.4 Therefore, it is intriguing to speculate that an established TH2 milieu with increased numbers of IL-4– producing TH2 cells as a consequence of primary allergen sensitization may influence subsequent immune responses of naive T cells and enhance differentiation toward TH2type cells with specificity against other allergens. This hypothesis is supported by epidemiologic data in children showing that sensitization increases not only the risk of developing subsequent sensitizations but also clinical symptoms like bronchial asthma.5-8 Similarly, specific immunotherapy in patients with allergic rhinitis prevents sensitization to other allergens and development of asthma.9-12 To delineate further the hypothesis of spreading of sensitization, we established a mouse model to investigate the effects of established allergen sensitization on systemic immune responses, airway inflammation, and airway reactivity toward a secondary allergen sensitization and airway challenge. We used 2 unrelated allergens, ovalbumin (OVA) and latex extract, applied without adjuvant to mimic natural sensitization more closely (protocols previously published13,14 or modified after15,16). We show here for the first time that active in vivo sensitization with allergen in a murine model not only boosted systemic TH2 immune responses but also enhanced the development of airway hyperreactivity (AHR) after sensitization and 615
Mechanisms of asthma and allergic inflammation
Effects of established allergen sensitization on immune and airway responses after secondary allergen sensitization
616 Blumchen et al
J ALLERGY CLIN IMMUNOL SEPTEMBER 2006
Mechanisms of asthma and allergic inflammation
FIG 1. Experimental protocol. Mice were immunized with either ovalbumin or latex as a primary sensitization followed by a secondary systemic sensitization with the other allergen (OVA/Latex, Latex/OVA). Singlesensitized mice received a sham sensitization followed by sensitization with either latex (PBS/Latex) or ovalbumin (PBS/OVA). A control group was purely sham-sensitized (PBS/PBS). All animals received airway challenges with the second allergen. in, Intranasal; ip, intraperitoneal; Neb, via nebulization.
Abbreviations used AHR: Airway hyperreactivity AI: Airway inflammation AR: Airway reactivity MNC: Mononuclear cell OVA: Ovalbumin
airway challenges with a secondary allergen. This effect was dependent on the strength of the primary sensitization and was unrelated to the kind of primary allergen used.
METHODS Animals Female Balb/c mice, 6 to 8 weeks old, were obtained from the Bundesinstitut fu¨r Risikobewertung, Berlin, Germany. Mice were kept under pathogen-and latex-free conditions, and maintained on an ovalbumin-free diet. All experiments were approved by the animal care committee.
Experimental protocol Primary systemic sensitization. See Fig 1. Mice (3-5 mice/group/ experiment) were systemically sensitized by 6 intraperitoneal injections over a period of 3 weeks of (1) 10 mg ovalbumin (grade VI; Sigma, St Louis, Mo), (2) 30 mg total protein of a latex extract of Hevea brasiliensis (preparation as described17), or (3) PBS for primary sham sensitization (Seromed; Biochrom, Berlin, Germany). To analyze effects of dosing, another group of mice received half of the ovalbumin dose for primary sensitization: 10 mg ovalbumin intraperitoneally 3 times within 3 weeks (days 23-43), resulting in a weaker primary allergic sensitization compared with the other primary allergen sensitization protocols (data not shown).
Secondary systemic sensitization. See Fig 1. For secondary sensitization, animals were injected on days 50, 54, and 61 with (1) 30 mg latex extract intraperitoneally (PBS/Latex or OVA/Latex group), (2) 20 mg ovalbumin intraperitoneally (PBS/OVA or Latex/OVA group), or (3) PBS for secondary sham sensitization (PBS/PBS). Analyzing the timing effect of the introduction of the secondary sensitization, 1 murine group received ovalbumin as a primary sensitization within the first 3 weeks (days 1-21) followed by a 31-day pause and secondary sensitization starting on day 50 (OVAlong/Latex group), whereas another group received ovalbumin from day 23 to 43 followed by only a 7-day pause and secondary sensitization on day 50 (OVAshort/Latex group). Kinetic studies of immunoglobulin production showed that 7 days after the last primary immunization (day 36), total immunoglobulin production peaked and then slowly declined, whereas ovalbuminspecific IgE production reached a maximum on day 43 and was stable from then on through day 50 (data not shown). Airway challenges. See Fig 1. All mice were airway challenged with the secondary allergen on days 68, 69, and 70 by (1) intranasal application of 30 mg latex extract in 50 mL PBS or (2) ultrasonic nebulization (LS2000; SYSTAM, Villeneuve-sur-Lot, France) of 1% ovalbumin (grade V; Sigma) for 10 minutes daily. For safety reasons, nebulization of latex extract was not allowed in the animal facility, so that the intranasal route had to be used.
Serum levels of total and specific immunoglobulins Total and allergen-specific immunoglobulin serum levels were measured on days 50 and 72 by ELISA as previously described.17-19 To monitor latex-specific immunoglobulin production, levels of immunoglobulin specific for Hev b 317 were measured and expressed as OD.
Cell preparation and culture On day 72, spleen mononuclear cells (MNCs) were isolated by density gradient centrifugation (Lympholyte-M; Cedarline Laboratories, Hornby, British Columbia, Canada) and cultured (106 cells/ 200 mL) in 96-well round-bottom plates at 37°C, 5% CO2 with (1) RPMI 1640, (2) concanavalin A (2.5 mg/mL; Sigma), (3) ovalbumin (50 mg/mL), or (3) latex extract (100 mg/mL). After 48 hours (IFN-g)
Blumchen et al 617
FIG 2. Total IgE. Experimental protocol as indicated in Fig 1. Mice were bled on day 72, and serum total IgE was determined by ELISA. Double-sensitized vs single-sensitized mice: PBS/Latex vs OVA/Latex, PBS/OVA vs Latex/OVA, **P < .01, *P < .05. Sham-sensitized vs allergen-sensitized mice: PBS/PBS vs PBS/Latex or PBS/OVA, 11P < .01.
or 96 hours (IL-5, IL-4), respectively, supernatants were harvested and stored at 220°C until further analysis.
Cytokine measurements Levels of IL-5, IL-4 and IFN-g in cell culture supernatants were assessed by ELISA, as previously described.14 Detection limits were 16 pg/mL for IL-5 and IL-4 and 39 pg/mL for IFN-g.
Bronchoalveolar lavage On day 72, lungs were lavaged twice with 0.8 mL cold PBS via a tracheal tube. Cells were counted, stained with Diff-Quik (Dade Behring AG, Du¨dingen, Switzerland), and differentiated according to morphologic criteria by counting at least 200 cells under light microscopy.
Measurement of in vivo airway reactivity On day 71, 24 hours after the last airway challenge, airway reactivity (AR) to inhaled provocation with rising doses (6-100 mg/mL) of methacholine (Sigma) was measured by whole-bodyplethysmography (Buxco; EMKA Technologies, Paris, France) as described.14 Methacholine doses resulting in 200% (PC 200) and 500% (PC 500) increase of baseline values for enhanced pause were calculated for each animal.14
Statistical analysis Animals of similar groups of 2 to 3 independent experiments were pooled (n 5 12 for each pooled group). Values for all measurements were expressed as means 6 SEMs. Pairs of groups were compared by Mann-Whitney U test. Values of P for significance were set at .05 (*) and .01 (**).
RESULTS Immune responses after primary sensitization with ovalbumin or latex extract Immunizing Balb/c mice 6 times within 3 weeks (Fig 1) intraperitoneally with low doses of ovalbumin (10 mg ovalbumin/mouse/dose) without adjuvant resulted in a robust systemic immune response (n 5 12; see this article’s Table E1 in the Online Repository at www.jacionline.org). On day 50, 31 days after the last intraperitoneal injection,
levels of total IgE and IgG2a were significantly higher in ovalbumin-sensitized compared with sham-sensitized mice (OVAlong/PBS vs PBS/PBS or PBS/Latex; P < .05). More profound, allergen-specific immune responses revealed a strong TH2-type response in ovalbumin-sensitized mice with high production of ovalbumin-specific IgE and low IgG2a levels, whereas sham-sensitized animals showed no allergen-specific immunoglobulin production. Analyzing immunoglobulin levels 7 days after the last immunization (day 50, OVAshort/Latex), we also observed increased TH2-type immunoglobulin production but lower levels of ovalbumin-specific IgE compared with analysis after the longer interval (see this article’s Table E1 in the Online Repository at www.jacionline.org). Repeated latex sensitization (6 3 30 mg latex extract intraperitoneally, Latex/OVA) without adjuvant also induced increased systemic TH2-type responses with significant increase in total IgE and IgG2a levels (see this article’s Table E1 in the Online Repository at www.jacionline.org). Hev b 3–specific IgE (see this article’s Table E1 in the Online Repository at www.jacionline.org) levels were markedly raised in latex-sensitized mice, whereas PBS– sham-sensitized animals (PBS/OVA) showed no detectable specific immunoglobulin production.
Immune responses after secondary sensitization and airway challenges with latex extract Mice were primarily sensitized with ovalbumin as described, followed by a secondary sensitization with latex extract, a noncross-reactive allergen, 31 days after the last ovalbumin injection (Fig 1). These double-sensitized mice (OVAlong/Latex) showed significantly stronger unspecific systemic TH2-type responses compared with single-sensitized animals (PBS/Latex). On day 72, total IgE levels were significantly increased in double-sensitized (OVAlong/Latex) compared with single-sensitized (PBS/ Latex) or sham-sensitized animals (PBS/PBS, Fig 2).
Mechanisms of asthma and allergic inflammation
J ALLERGY CLIN IMMUNOL VOLUME 118, NUMBER 3
618 Blumchen et al
J ALLERGY CLIN IMMUNOL SEPTEMBER 2006
Mechanisms of asthma and allergic inflammation
FIG 3. Mitogen-induced cytokine production by spleen MNCs. Experimental protocol as indicated in Fig 1. Splenic MNCs were obtained on day 72 and restimulated in vitro with concanavalin A. Cytokine production in supernatants was measured by ELISA: IL-4 (A), IL-5 (B), and IFN-g (C). Single-sensitized vs double-sensitized mice: PBS/Latex vs OVA/Latex, PBS/OVA vs Latex/OVA, **P < .01, *P < .05. Sham-sensitized vs allergensensitized mice: PBS/PBS vs PBS/Latex or PBS/OVA, 11P < .01.
FIG 4. Latex-specific immunoglobulin production. Experimental protocol as indicated in Fig 1. Mice were bled on day 72. Latex-specific IgE (A), IgG1 (B), and IgG2a (C) were evaluated by ELISA in measuring immunoglobulin production specific for Hev b 3, a major latex allergen. Levels are documented in OD units. Singlesensitized vs double-sensitized mice: PBS/Latex vs OVA/Latex, *P < .05. Sham-sensitized vs single-sensitized mice: PBS/PBS vs PBS/Latex, 11P < .01, 1P < .05.
Similarly, IL-4 and IL-5 production in response to mitogen was significantly higher in double-sensitized (OVAlong/ Latex) compared with single-sensitized (PBS/Latex) or sham-sensitized mice (PBS/PBS, Fig 3, A and B). Production of total IgG2a (OVAlong/Latex: 987 6 144 ng/mL vs PBS/Latex: 671 6 130 ng/mL) and IFN-g (Fig 3, C) was low and not significantly different between double-sensitized and single-sensitized animals (Fig 3, C). Next, allergen-specific immune responses to the secondary allergen were analyzed. On day 72, double-sensitized animals (OVAlong/Latex) produced significantly higher levels of allergen (Hev b 3)–specific IgE (Fig 4, A) and IgG1 (Fig 4, B) compared with single-sensitized mice (PBS/Latex). There was no difference in Hev b 3– specific IgG2a levels between double-sensitized or single-sensitized mice (Fig 4, C). Stimulation of spleen MNCs with secondary allergen, latex extract, resulted in high in vitro cytokine production of IL-4 and IL-5 and no measurable IFN-g. However, we found no significant difference between single-sensitized and double-sensitized animals (data not shown).
Airway responses after secondary sensitization and airway challenges with latex extract Airway latex challenges of latex-sensitized mice (PBS/ Latex) induced a robust eosinophilic airway inflammation (AI) with a significant increase in numbers of total cells, lymphocytes, and eosinophils in bronchoalveolar lavage fluids (Fig 6) and a trend toward development of increased AHR (Fig 7, A) compared with sham-sensitized, airwaychallenged mice (PBS/PBS).
In contrast, latex sensitization and airway challenges of mice that had been primarily sensitized with OVA (OVAlong/Latex) resulted in significantly increased AR compared with single-sensitized animals (PBS/Latex; Fig 7, A). PC 200 and, even more pronounced, PC 500 levels were significantly reduced in double-sensitized animals in comparison with both single-treated and sham-treated mice. In contrast, eosinophilic AI was not significantly different between double-sensitized and single-sensitized animals (Fig 6).
Effects of timing of the primary on secondary sensitization To analyze whether the interval between primary and secondary sensitization was relevant for the enhancing effect on immune responses after secondary sensitization and airway challenges, we compared groups of mice with a 7-day interval between primary and secondary sensitization (OVAshort/Latex) with the group with a 31-day interval between primary and secondary sensitization (OVAlong/Latex; Fig 1). Both OVAlong/Latex and OVAshort/Latex groups developed significantly increased total IgE (Fig 2) and mitogen-induced IL-4 and IL-5 production by spleen MNCs (Fig 3, A and B) compared with single-sensitized animals (PBS/Latex). There were no differences in total IgG2a (data not shown) or in IFN-g levels (Fig 3, C) in both double-sensitized groups (OVAlong/ Latex and OVAshort/Latex) compared with control mice (PBS/Latex). Generally, Hev b 3 specific IgE and IgG1 production in double-sensitized mice was increased compared with controls, but this increase was significant only in OVAlong/Latex animals compared with PBS/Latex
Blumchen et al 619
FIG 5. Ovalbumin-specific immunoglobulin production. Experimental protocol as indicated in Fig 1. Mice were bled on day 72. Ovalbumin-specific IgE (A), IgG1 (B), and IgG2a (C) were measured by ELISA and compared with known hyperimmune standards (EU/mL). Single-sensitized vs double-sensitized mice: PBS/OVA vs Latex/OVA, **P < .01. Purely sham-sensitized animals (PBS/PBS) showed OVA-IgE, IgG1, and IgG2a below detection limits.
mice (Fig 4, A and B). However, similar to the long protocol, OVAshort/Latex animals showed significant increases in AR compared with single-sensitized animals (PBS/ Latex, Fig 7, A), although eosinophilic AI was not significantly different between these 2 groups (Fig 6).
Effects of allergen sequence of primary and secondary sensitization To analyze whether the sequence of allergens for primary and secondary sensitization was important for the enhancing effects observed in double-sensitized animals, we switched the order of allergens, now immunizing mice 6 times intraperitoneally with latex extract as a primary sensitization followed, after a 7-day interval, by systemic ovalbumin sensitization and ovalbumin airway challenges (Fig 1). Similar to the results of the previous experiments, total IgE serum levels were significantly increased in double-sensitized mice (Latex/OVA) compared with single-sensitized (PBS/OVA) or sham-treated controls (PBS/PBS; Fig 2). However, there was no significant difference between double-sensitized and single-sensitized mice regarding mitogen-induced IL-4 (Latex/OVA: 183 6 31 pg/mL vs PBS/OVA: 137 6 33 pg/mL), IL-5 (Latex/OVA: 521 6 62 pg/mL vs PBS/OVA: 652 6 94 pg/mL), or IFN-g production (Latex/OVA: 1200 6 270 pg/mL vs PBS/OVA: 1058 6 288 pg/mL) by splenic MNCs. More importantly, double-sensitized animals produced significantly higher amounts of ovalbumin-specific IgE than single-sensitized mice (Fig 5, A), whereas levels of ovalbumin-specific IgG1 and IgG2a were similar between the 2 groups (Fig 5, B and C). Single-sensitized mice that were purely sensitized and airway-challenged with ovalbumin (PBS/OVA) showed a robust eosinophilic airway inflammation (data not shown) as well as increased AR (Fig 7, B) compared with shamsensitized and ovalbumin airway-challenged mice (PBS/ PBS). Once again, eosinophilic inflammation was not significantly different between double-sensitized and single-sensitized mice, but there was a trend toward increased AR in double-sensitized compared with single-sensitized ones (Fig 7, B, Latex/OVA vs PBS/OVA). DISCUSSION We have shown in a murine model that primary sensitization enhanced systemic immune and airway
FIG 6. Bronchoalveolar lavage. Experimental design as indicated in Fig 1. On day 72, two days after last airway challenge, lungs were lavaged, and differential cell counts in bronchoalveolar lavage fluids were obtained. Total cell counts, eosinophil counts, and lymphocyte counts are presented. Single-sensitized vs double-sensitized mice: PBS/Latex vs OVA/Latex, *P < .05. Sham-sensitized vs allergen-sensitized mice: PBS/PBS vs PBS/Latex or OVA/Latex, 11P < .01.
responses on subsequent sensitization and airway challenges with an unrelated, not cross-reactive, secondary allergen (consecutive priming). Experiments with ovalbumin as the primary allergen followed by latex extract as the secondary allergen (OVA/Latex) resulted in an enhanced TH2 response and increased development of airway hyperreactivity on secondary sensitization. This was most probably a result of an unspecific amplification of TH2 immune responses against the secondary allergens by a predominant systemic TH2 milieu, reflected by a TH2-skewed immune status at the time point when secondary sensitization was initiated (see this article’s Table E1 in the Online Repository at www.jacionline.org). The magnitude of the enhancement of secondary sensitization was decreased when we shortened the interval between primary and secondary sensitization from 31 to 7 days (OVAlong/Latex vs OVAshort/Latex). Although a similar increase in unspecific TH2-type immune responses was noted, the increase in allergen-specific IgE and IgG1 production was lower in the OVAshort/Latex group than in the OVAlong/Latex group. This difference can be explained by differences in the level of the primary sensitization between the OVAlong/Latex vs OVAshort/Latex protocols, where we found higher TH2 intensity in mice of the long protocol at the time point when the secondary sensitization
Mechanisms of asthma and allergic inflammation
J ALLERGY CLIN IMMUNOL VOLUME 118, NUMBER 3
620 Blumchen et al
J ALLERGY CLIN IMMUNOL SEPTEMBER 2006
Mechanisms of asthma and allergic inflammation
FIG 7. Airway reactivity. Experimental design as indicated in Fig 1. On day 71, lung function to inhaled methacholine was measured by whole-body plethysmography. The dosage of methacholine increasing AR by 200% (PC 200) or 500% (PC 500) was calculated for each animal. A, AR of mice that received OVA as primary and Latex as secondary sensitization agent (OVA/Latex). B, AR of mice that received the reversed protocol (Latex/OVA). Single-sensitized vs double-sensitized mice: PBS/Latex vs OVA/Latex, PBS/OVA vs Latex/OVA, **P < .01, *P < .05. Sham-sensitized vs allergen-sensitized mice: PBS/PBS vs PBS/Latex, PBS/OVA or double-sensitized mice, 1P < .05. MCh, Methacholine.
was established (day 50; see this article’s Table E1 in the Online Repository at www.jacionline.org). In kinetic studies with ovalbumin-sensitized mice, we found a maximum, robust, and stable TH2-skewed immunoglobulin production 31 days after the last ovalbumin immunization. We conclude that introducing the secondary sensitization into a comparable weak primary sensitization process might result in only a moderate priming of allergen-specific TH2 immune responses and development of AHR after secondary sensitization and airway challenges. This conclusion was strongly supported by data from mice sensitized with ovalbumin 3 times instead of 6 times, resulting in weaker primary immune responses compared with ovalbumin injected 6 times (data not shown). Using this weak primary ovalbumin sensitization protocol, the enhancement of specific and unspecific TH2 and airway responses after secondary sensitization and airway challenges with latex extract was no longer significant (data not presented). Importantly, the effects of consecutive priming were also found by using the reversed protocol (Latex/OVA). We observed a robust sensitization against the primary allergen (latex extract) on day 50, and found highly and significantly enhanced TH2-type immune responses against the secondary allergen (ovalbumin) even with the short 7-day interval between primary and secondary sensitization. This shows that the enhancement of TH2 responses after the secondary allergen sensitization was not dependent on 1 specific allergen, a notion supported by clinical data showing increased risk of secondary allergen sensitizations in children primarily sensitized against hen’s egg protein5 or latex allergens.20,21 There may be various reasons for the differences we see between the outcomes of Latex/OVA and OVA/Latex animals regarding the strength of the immune responses as well as the development of enhanced AR after consecutive priming. Although a direct comparison of the allergenicity of the 2 allergens used seems rather impracticable because of differences in concentration and preparation of the compounds (single protein suspension for ovalbumin vs protein mixture in latex extract), we established 2 quite similar but weak secondary sensitization protocols (PBS/ Latex vs PBS/OVA). Therefore, the differences in the magnitude of enhancement of allergen specific IgE
production after secondary sensitization (OVAshort/Latex vs Latex/OVA) might be explained by the strength of the primary sensitization with latex extract resulting in higher total IgE and TH2 cytokine production than primary sensitization with ovalbumin (day 50; see this article’s Table E1 in the Online Repository at www.jacionline.org). However, differences in the effects on AR between the Latex/OVA and OVA/Latex group might be a result of differences either in allergenicity or to the routes of airway challenges applied. It was obvious that aerosolized ovalbumin induced a stronger development of AHR in ovalbumin-sensitized and airway-challenged mice (PBS/OVA) compared with mice that were latex-sensitized and airwaychallenged with latex extract intranasally (PBS/Latex). This stronger airway response in the former group may have obscured any subtle differences provoked by consecutive priming after primary allergen sensitization with latex extract. Another reason could be that OVA/Latex mice showed significantly enhanced production of mitogen-induced IL-4 and IL-5 by spleen MNCs at the time of airway allergen challenges compared with single-sensitized mice (PBS/Latex). This enhancing effect on unspecific TH2 cytokine production was not detected in Latex/ OVA mice. Although the difference in cytokine production did not lead to measurable variation of eosinophilic airway infiltration, it may have contributed toward the development of significantly enhanced AR in the OVA/Latex vs PBS/Latex group. This is supported by convincing evidence that differences in eosinophilic AI may, in some experimental settings, be too subtle to be detected, and are not mandatory for the development of enhanced AR.22-24 Other recent data from murine models underscore the effect of cytokine milieu on T-cell development at the time of initial priming.25,26 Adoptive transfer of TH2-polarized OVA-T cell receptor–transgenic T cells (DO11.10) into naive Balb/c mice enhanced specific airway responses of endogenous naive T cells after airway challenges with a secondary allergen.25 Further, Schipf et al26 showed that adoptively transferred TH2-skewed cells were even capable of overruling an adjuvant-induced TH1 shift toward a secondary antigen. In both experiments,25,26 the effects were dependent on (1) the presence of IL-4 and (2) the
simultaneous introduction of the first and second antigen (collateral priming). We have extended these observations here, showing that the amplification of secondary TH2 immune responses was not dependent on the simultaneous application of both allergens, but occurred as the consequence of 2 subsequent individual and active sensitization procedures (consecutive priming). Furthermore, for the first time, we have demonstrated the effect of enhanced TH2 immune responses for allergen-induced airway inflammation and development of AHR, emphasizing the clinical finding that sensitization spreading is also associated with an increased risk of developing asthma.7,8 As the most likely mechanism underlying this phenomenon, we suggest that primary sensitization systemically and unspecifically increased the susceptibility toward a secondary sensitization, even with unrelated allergen. This could be achieved by inhibition of regulatory T-cell development via enhanced GATA-3 expression (Schmidt-Weber CB, January 2006, personal communication27) or by impaired IL-12 production by dendritic cells28 as the result of enhanced TH2 immune responses after primary sensitization. In conclusion, established primary sensitization leads to enhanced allergen-mediated TH2-type immune and airway responses after subsequent sensitization and airway challenges with a secondary unrelated allergen in vivo. Importantly, this was not specific or dependent on a certain type of allergen but related to the strength of the primary sensitization protocol. The careful tuning between cytokines in the initial phase of priming seemed to be the most important factor.29 Avoiding early and strong allergen sensitization may therefore be a very relevant means for primary prevention of subsequent allergen-induced airway diseases. Furthermore, the concept of consecutive priming indirectly supports the notion and requirement for early immunotherapy as an important way to avoid allergen sensitization spreading and disease aggravation. We thank C. Seib for her helpful technical assistance and H. G. Hoymann from the Frauenhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany, for his calculation of ovalbumin deposition in the lung after airway challenges with ovalbumin in mice.
REFERENCES 1. O’Garra A, Arai N. The molecular basis of T helper 1 and T helper 2 cell differentiation. Trends Cell Biol 2000;10:542-50. 2. Paul WE, Seder RA. Lymphocyte responses and cytokines. Cell 1994; 76:241-51. 3. Grogan JL, Locksley RM. T helper cell differentiation: on again, off again. Curr Opin Immunol 2002;14:366-72. 4. Ngoc PL, Gold DR, Tzianabos AO, Weiss ST, Celedon JC. Cytokines, allergy, and asthma. Curr Opin Allergy Clin Immunol 2005;5:161-6. 5. Nickel R, Kulig M, Forster J, Bergmann R, Bauer CP, Lau S, et al. Sensitization to hen’s egg at the age of twelve months is predictive for allergic sensitization to common indoor and outdoor allergens at the age of three years. J Allergy Clin Immunol 1997;99:613-7. 6. Illi S, von Mutius E, Lau S, Nickel R, Niggemann B, Sommerfeld C, et al. The pattern of atopic sensitization is associated with the development of asthma in childhood. J Allergy Clin Immunol 2001;108:709-14. 7. Sherrill D, Stein R, Kurzius-Spencer M, Martinez F. On early sensitization to allergens and development of respiratory symptoms. Clin Exp Allergy 1999;29:905-11.
Blumchen et al 621
8. Rhodes HL, Sporik R, Thomas P, Holgate ST, Cogswell JJ. Early life risk factors for adult asthma: a birth cohort study of subjects at risk. J Allergy Clin Immunol 2001;108:720-5. 9. Purello-D’Ambrosio F, Gangemi S, Merendino RA, Isola S, Puccinelli P, Parmiani S, et al. Prevention of new sensitizations in monosensitized subjects submitted to specific immunotherapy or not: a retrospective study. Clin Exp Allergy 2001;31:1295-302. 10. Pajno GB, Barberio G, De Luca F, Morabito L, Parmiani S. Prevention of new sensitizations in asthmatic children monosensitized to house dust mite by specific immunotherapy: a six-year follow-up study. Clin Exp Allergy 2001;31:1392-7. 11. Des Roches A, Paradis L, Menardo JL, Bouges S, Daures JP, Bousquet J. Immunotherapy with a standardized Dermatophagoides pteronyssinus extract, VI: specific immunotherapy prevents the onset of new sensitizations in children. J Allergy Clin Immunol 1997;99:450-3. 12. Moller C, Dreborg S, Ferdousi HA, Halken S, Host A, Jacobsen L, et al. Pollen immunotherapy reduces the development of asthma in children with seasonal rhinoconjunctivitis (the PAT-study). J Allergy Clin Immunol 2002;109:251-6. 13. Gerhold K, Bluemchen K, Franke A, Stock P, Hamelmann E. Exposure to endotoxin and allergen in early life and its effect on allergen sensitization in mice. J Allergy Clin Immunol 2003;112:389-96. 14. Blumchen K, Gerhold K, Thorade I, Seib C, Wahn U, Hamelmann E. Oral administration of desloratadine prior to sensitization prevents allergen-induced airway inflammation and hyper-reactivity in mice. Clin Exp Allergy 2004;34:1124-30. 15. Kurup VP, Kumar A, Choi H, Murali PS, Resnick A, Kelly KJ, et al. Latex antigens induce IgE and eosinophils in mice. Int Arch Allergy Immunol 1994;103:370-7. 16. Woolhiser MR, Munson AE, Meade BJ. Immunological responses of mice following administration of natural rubber latex proteins by different routes of exposure. Toxicol Sci 2000;55:343-51. 17. Wagner B, Krebitz M, Buck D, Niggemann B, Yeang HY, Han KH, et al. Cloning, expression, and characterization of recombinant Hev b 3, a Hevea brasiliensis protein associated with latex allergy in patients with spina bifida. J Allergy Clin Immunol 1999;104:1084-92. 18. Hamelmann E, Oshiba A, Loader J, Larsen GL, Gleich G, Lee J, et al. Antiinterleukin-5 antibody prevents airway hyperresponsiveness in a murine model of airway sensitization. Am J Respir Crit Care Med 1997;155:819-25. 19. Hufnagl K, Wagner B, Winkler B, Baier K, Hochreiter R, Thalhamer J, et al. Induction of mucosal tolerance with recombinant Hev b 1 and recombinant Hev b 3 for prevention of latex allergy in BALB/c mice. Clin Exp Immunol 2003;133:170-6. 20. Niggemann B, Buck D, Michael T, Wahn U. Latex provocation tests in patients with spina bifida: who is at risk of becoming symptomatic? J Allergy Clin Immunol 1998;102:665-70. 21. Liebke C, Niggemann B, Wahn U. Sensitivity and allergy to latex in atopic and non-atopic children. Pediatr Allergy Immunol 1996;7:103-7. 22. Takeda K, Haczku A, Lee JJ, Irvin CG, Gelfand EW. Strain dependence of airway hyperresponsiveness reflects differences in eosinophil localization in the lung. Am J Physiol Lung Cell Mol Physiol 2001;281:L394-402. 23. Bochner BS. Verdict in the case of therapies versus eosinophils: the jury is still out. J Allergy Clin Immunol 2004;113:3-9, quiz 10. 24. Alam R, Busse WW. The eosinophil: quo vadis? J Allergy Clin Immunol 2004;113:38-42. 25. Eisenbarth SC, Zhadkevich A, Ranney P, Herrick CA, Bottomly K. IL-4dependent Th2 collateral priming to inhaled antigens independent of Toll-like receptor 4 and myeloid differentiation factor 88. J Immunol 2004;172:4527-34. 26. Schipf A, Heilmann A, Boue L, Mossmann H, Brocker T, Rocken M. Th2 cells shape the differentiation of developing T cell responses during interactions with dendritic cells in vivo. Eur J Immunol 2003;33:1697-706. 27. Mantel RZ KH, Onakqd N, Seckez B, Karagiannidis C, Lambrecht B, Blaser K, et al. Inhibition of T regulatory cell development by GATA 3. Swiss Med Wkly 2006;136:149:4s. 28. Traidl-Hoffmann C, Mariani V, Hochrein H, Karg K, Wagner H, Ring J, et al. Pollen-associated phytoprostanes inhibit dendritic cell interleukin12 production and augment T helper type 2 cell polarization. J Exp Med 2005;201:627-36. 29. Constant SL, Bottomly K. Induction of Th1 and Th2 CD41 T cell responses: the alternative approaches. Annu Rev Immunol 1997;15: 297-322.
Mechanisms of asthma and allergic inflammation
J ALLERGY CLIN IMMUNOL VOLUME 118, NUMBER 3