SEARCH

SEARCH BY CITATION

Keywords:

  • Dendritic cell;
  • Allergy;
  • Th1/Th2;
  • Toll-like receptor;
  • Hygiene hypothesis

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

Our previous study has shown that Chlamydia lung infection can inhibit local eosinophilic inflammation induced by allergen sensitization and challenge, which is correlated with altered cytokine production. In the present study, we examined the role played by dendritic cells (DC) in chlamydial infection-mediated modulation of allergic responses. The results showed that DC freshlyisolated from Chlamydia-infected mice (iIDC), unlike those from naive control mice (iNDC), could efficiently modulate immune responses to ovalbumin in vitro and in vivo. Co-culture of freshly isolated DC with naive CD4 cells from T cell receptor transgenic mice (DO11.10) showed that iIDC directed Th1-dominant, while iNDC directed Th2-dominant, allergen-specific CD4 T cell responses. Moreover, adoptive transfer of iIDC, but not iNDC, could inhibit systemic and local eosinophilia induced by allergen exposure. The reduction of eosinophilia was associated with a decrease in IL-5 receptor expression on bone marrow cells and the production of IL-5 and IL-13 by T lymphocytes. Analysis of the DC showed that iIDC expressed significantly higher levels of mRNA for Toll-like receptor 9 and produced more IL-12 compared to iNDC. The data demonstrate a critical role played by DC in infection-mediated inhibition of allergic responses.

Abbreviations:
Alum:

Al(OH)3

BAL:

Bronchoalveolar lavage

iIDC:

DC isolated from Chlamydia-infected mice

iNDC:

DC isolated from naive mice

MoPn:

Chlamydia muridarum mouse pneumonitis

TLR:

Toll-like receptor

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

In most industrialized countries, the prevalence of atopic diseases and asthma has increased significantly over the past few decades, which is associated with a steady decline of infectious diseases and reduction in vaccination during this period 1, 2. Several epidemiological studies have revealed an inverse relationship between allergy and infection, leading to the "hygiene hypothesis", which states that the exposure to microbial infections especially in early life may prevent or inhibit allergic diseases 3, 4. There is strong evidence that, upon exposure to allergen, atopic hosts develop Th2-dominant cytokine responses, particularly IL-4, IL-5 and IL-13 over-production. In contrast, in response to intracellular bacterial infections, Th1 cells are activated and high levels of IFN-γ are produced. Our and other groups have demonstrated that infections may hamper the development of allergen-specific Th2 cells and subsequent allergic responses 510. However, the cellular and molecular basis for the modulating effect of infection on allergic responses remains largely unknown.

The activation and differentiation of naive T cells to effector cells require a set of combinational signals provided by antigenic peptides and antigen-presenting cells (APC). The most efficient APC for naive T cell priming is the dendritic cell (DC), which displays unique properties in inducing and modulating immune responses 11, 12. The signals provided by DC to naive T cells include peptide-MHC complexes, co-stimulatory molecules and polarizing cytokines. Based on the functional preference in directing different types of T cell responses, DC have been categorized into various subtypes, such as DC1- and DC2-like cells 13, 14. Recent studies suggest that DC are a very heterogeneous cell population with high plasticity, and their function may be determined by multiple parameters including, but not limited to, phenotypic makers, cytokine production, cellular lineage and stages of maturation 15.

A close linkage between innate and adaptive immune responses has been shown in recent studies 16, 17. Signaling through pattern recognition receptors, especially Toll-like receptors (TLR) 18, 19, on APC appears to play an important role in modulating the adaptive immune responses. In particular, TLR4 and CD14 are important for recognizing Gram-negative bacterial products, and TLR9 is specific for the CpG motif of bacterial DNA. The involvement of TLR in allergic responses, although the data are limited, has also been shown in recent studies. For example, polymorphisms in CD14 and TLR4 genes have been found to be correlated with IgE levels 20, 21.

Chlamydiae are obligate intracellular bacteria, which cause human ocular, respiratory and genital tract infections. Previous studies by our and other groups have shown that respiratory tract C. muridarum (MoPn) infection induces predominant Th1-like immune responses 22, 23. Recent studies showed that adoptively transferred DC pulsed with nonviable chlamydial organisms induced Chlamydia-specific Th1 immune responses, which were equally protective as live chlamydial immunization 24. On the other hand, adoptive transfer of allergen-pulsed DC induced Th2-type immune responses and asthma-like reaction in mice 25.

In previous studies, we have shown that prior MoPn and mycobacterial infections can inhibit allergic asthmatic responses in murine models, which is correlated with enhanced Th1-type cytokine (IFN-γ) production and reduced Th2-type cytokine (IL-4, IL-5 and IL-13) production 1416. However, the mechanism for the alteration in T cell cytokine production and allergic inflammation mediated by these infections remains unclear. Since DC play an important role in directing T cell differentiation and different types of immune responses, we examined the role played by DC in the chlamydial infection-mediated inhibition of allergic reaction. The results showed that naive allergen-specific CD4 T cells from DO11.10 mice that were co-cultured with iIDC, in comparison with those co-cultured with iNDC, produced significantly less Th2 (IL-4) and more Th1 (IFN-γ) cytokine following specific allergen or peptide stimulation. More interestingly, adoptive transfer of iIDC, but not iNDC, significantly reduced allergen-driven IL-5 and IL-13 production in vivo and inhibited local eosinophilic inflammation. Analysis of the isolated DC showed significantly higher TLR9 message expression and IL-12 production by iIDC than iNDC. The data demonstrate a critical role played by DC in chlamydial infection-mediated inhibition of allergic responses.

2 Results

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

2.1 iIDC substantially reduce Th2-like OVA-specific CD4+ T cell differentiation

To determine whether iIDC and iNDC are functionally different in directing naive T cell differentiation, we co-cultured them, respectively, with naive CD4 T cells from syngeneic OVA peptide-specific TCR transgenic mice (BALB/c DO11.10) stimulated with specific OVA323–339 peptide or whole OVA protein. The results showed that, following 72-h co-culture with iIDC, the CD4+ T cells produced significantly higher levels of IFN-γ, but lower levels of IL-4, in comparison with those co-cultured with iNDC (Fig. 1A, B). The difference appears to be more dramatic in IL-4 (4–5-fold) than in IFN-γ (1.2–2.2-fold) production in the four repeated experiments (p<0.01). Similarly, stimulation with OVA peptide323–339 also showed higher IFN-γ and lower IL-4 production in iIDC-CD4 T co-cultures compared to iNDC-CD4 T co-cultures (Fig. 1C, D). The results suggest that chlamydial infection can modulate the function of DC, which then inhibit the development of Th2-like allergen-specific CD4 T cells.

thumbnail image

Figure 1. Co-culture of iIDC with OVA peptide-specific CD4 T cells reduces allergen-driven Th2 cytokine production. Naive CD4+ T cells (5×105 cells/well) from OVA peptide-specific TCR-transgenic mice (DO11.10, I-Ad) were co-cultured for 72 h with DC isolated from the spleen of naive BALB/c (H-2d) mice (iNDC) (empty bars) or Chlamydia-infected BALB/c mice (iIDC) (hatched bars). Culture supernatants were analyzed for IL-4 and IFN-γ concentrations by ELISA. (A, B) DC and naive CD4+ T cells at various DC:T ratios were co-cultured in the presence of intact OVA protein (0.1 mg/ml); (C, D) DC and CD4 T cells (DC:T=1:5) were co-cultured in the presence of various concentrations of the specific OVA323–339 peptide. Four replicate wells were set up for each culture condition. Data are presented as mean ± SD. One representative of three independent experiments with similar results is shown. ***, p<0.001; **, p<0.01, *, p<0.05.

Download figure to PowerPoint

2.2 Adoptive transfer of iIDC inhibits systemic and local eosinophilia induced by OVA exposure

Having demonstrated the ability of iIDC to preferentially promote a Th1-like allergen-specific CD4 T cell response in vitro, we further analyzed the role of these DC in the allergic response induced by OVA exposure in vivo. Freshly isolated iIDC and iNDC were adoptively transferred, respectively, to syngeneic naive mice (5×106 cells/mouse) by intravenous injection. Two hours after the adoptive transfer, the groups of DC-recipient mice and the group of naive mice without cell transfer were all sensitized intraperitoneally (i.p.) with OVA in Al(OH)3 (alum). Seven days post-sensitization, peripheral blood leukocyte differentials were counted, and the effect of DC transfer on systemic eosinophilia induced by allergen exposure was examined. It was found that OVA exposure induced a significant increase in peripheral blood eosinophils (from 13.5±5.2/mm3 in naive mice to 84.6±8.2/mm3 in sensitized mice without DC transfer). However, the same OVA sensitization induced a much lower degree of eosinophil increase in mice receiving iIDC (33.0±5.9/mm3 eosinophils in peripheral blood). This difference between recipients of iIDC and mice without DC transfer was statistically significant (p<0.01). As a control, transfer of iNDC had no significant effect on the allergen-induced eosinophil increase (69.8±6.9/mm3 eosinophil in peripheral blood). The difference between the levels of eosinophil in iNDC recipients and mice without DC transfer was not statistically significant (p>0.05). The data suggest that adoptive transfer of iIDC can significantly inhibit systemic eosinophilia caused by allergen exposure.

To test the basis for the reduced increase in circulating eosinophils in recipients of iIDC following OVA exposure, bone marrow (BM) cells from the different groups of mice were stained for IL-5Rα expression post OVA sensitization. As shown in Fig. 2A–D, following OVA sensitization, 20–25% BM cells from mice without cell transfer or from recipients of iNDC expressed IL-5Rα, the level being six to ten times higher than those from naive mice (2–4%) without OVA sensitization. In contrast, pooled data of four independent experiments showed that the recipients of iIDC exhibited significantly less IL-5Rα+ cells (5–8%) in their BM than the recipients of iNDC and the mice without cell transfer (p<0.01), following the same OVA treatment (Fig. 2C). Since IL-5Rα+ cells are largely eosinophil precursors in BM, the results suggest that adoptive transfer of iIDC can significantly inhibit the production of eosinophils in BM.

To further examine the role of DC in modulating allergic reaction in vivo, we tested local eosinophilic inflammation to allergen challenge in OVA-sensitized mice. As shown in Fig. 2E, cutaneous allergen challenge of the sensitized mice without DC transfer caused heavy intradermal edema and eosinophilic inflammatory cell infiltration. In contrast, the intensity of local inflammation was very mild in recipients of iIDC (Fig. 3G). The eosinophil infiltration in the skin of iNDC recipients was comparable to that in the control mice without DC transfer (Fig. 3F). Fig. 3H, which displays the quantitation of the percentage of eosinophils in infiltrating inflammatory cells of different groups of mice, shows that the recipients of iIDC exhibited statistically lower percentage of local eosinophil infiltration compared to mice without DC transfer or those receiving iNDC (p<0.01). The results demonstrate that transfer of iIDC, but not iNDC, has a significant inhibitory effect on local eosinophilic inflammation. Taken together, the data suggest that DC in Chlamydia-infected mice play a critical role in the inhibition of systemic and local eosinophilia induced by allergen exposure.

thumbnail image

Figure 2. Adoptive transfer of iIDC inhibits IL-5Rα expression on BM cells and local eosinophilic inflammation induced by OVA exposure. (A-D) C57BL/6 mice receiving adoptively transferred iNDC or iIDC from syngeneic mice and control mice (C57BL/6) without cell transfer (n=4/group) were sensitized i.p. with OVA (4 μg in alum). At 7 days following sensitization, BM cells were stained for IL-5Rα+ cells as described in Sect. 4. (A) Control mouse without cell transfer, (B) recipient of iNDC, (C) recipient of iIDC, (D) percentage of IL-5R+ cells (mean ± SD) in total BM cells of the different groups of mice (iNDC recipients, iIDC recipients and control mice without cell transfer). One representative of four independent experiments with similar results is shown. (E-H) C57BL/6 mice receiving adoptively transferred iIDC or iNDC isolated from syngeneic mice and the C57BL/6 control mice without cell transfer (n=4/group) were sensitized i.p. with OVA (4 μg in alum). At 14 days following sensitization, mice were challenged with 20 μl OVA (0.5 mg/ml in PBS) into the ventral surface. Local skin tissues were isolated at 72 h following challenge injection, and the sections were stained by H&E. Ten continuous tissue sections were analyzed in each mouse. The arrows indicate eosinophils infiltrating the skin. (E) Control mice without cell transfer, (F) recipients of iNDC, (G) recipient of iIDC, (H) percentage of eosinophils of local inflammatory cells (mean ± SD) in the recipients of various DC and control mice. One representative of two independent experiments with similar results is shown. (A–C, E–G) ×200; insets (e–g) ×400. *, p<0.05; iIDC recipients vs. control.

Download figure to PowerPoint

thumbnail image

Figure 3. Effect of DC transfer on allergen-driven cytokine production by splenic lymphocytes. Recipient mice (C57BL/6, n=4/group) of adoptively transferred iNDC or iIDC from syngeneic mice and control C57BL/6 mice without cell transfer were sensitized i.p. with OVA (4 μg in alum). At 7 days following sensitization, spleen cells from different groups of mice were cultured in the presence of OVA. Cytokines in the supernatants were analyzed by ELISA. Data are presented as mean ± SD. One representative of four independent experiments with similar results is shown *, p<0.05; iIDC recipients vs. control.

Download figure to PowerPoint

2.3 Inhibition of allergic response mediated by iIDC transfer is associated with decreased allergen-driven Th2 cytokine production

To test the effect of transferred DC on cytokine responses in vivo, we examined the allergen-driven cytokine profiles of spleen cells from mice with or without iIDC transfer. The results showed that spleen cells from iIDC recipients produced higher levels of IL-12 and significantly lower levels of Th2-related cytokines, IL-5 and IL-13, in comparison to those from iNDC recipients or mice without DC transfer (p<0.05) (Fig. 3). The levels of IL-4 and IL-10 also showed a decreased trend in iIDC recipients, although the differences were not statistically significant. The data demonstrate that adoptive transfer of iIDC, similar to naturally infected mice, can modulate T cell cytokine profiles, especially Th2-related cytokines to allergen exposure.

2.4 iIDC produce high levels of IL-12 and express high levels of TLR mRNA

To study the mechanisms by which iIDC modulate T cell cytokine production and allergic responses, we further examined the cytokine production and TLR expression of isolated DC. As shown in Fig. 4A, the production of IL-12 by ex vivo iIDC was twofold higher than iNDC (p<0.01). There was no measurable IL-10 in the cultured ex vivo DC. To further test the role of IL-12 produced by iIDC in modulating allergen-specific CD4 T cell responses, we used anti-IL-12 mAb to block endogenous IL-12 in co-cultures of iIDC and naive CD4 T cells. The results showed that blockage of IL-12 activity led to a significantly decreased IFN-γ production by CD4 T cells, but had no significant impact on IL-4 production (Fig. 4B, C). In addition, analysis of TLR message expression by isolated DC showed that iIDC expressed significantly higher TLR9 messages compared to iNDC (p<0.05) (Fig. 5). The expression of other pattern recognition receptors (TLR2, TLR4 and CD14) also showed a trend of higher expression in iIDC, although the differences were not statistically significant. The data suggest that iIDC have altered patterns of cytokine production and TLR expression. These alterations may provide a molecular basis for the inhibitory role of the iIDC in allergic responses.

To test whether the iIDC are directly infected with Chlamydia, we used PCR to detect the existence of the MoPn major outer membrane protein gene in the iIDC preparation. The results showed that there is no measurable chlamydial DNA in the iIDC, suggesting that the DC isolated from the spleen of lung-infected mice are not directly infected by Chlamydia.

thumbnail image

Figure 4.  IL-12 production by isolated DC and its role in modulating CD4 T cell cytokine production. (A) Fresh isolated iIDC and iNDC from BALB/c mice were cultured separately with complete culture medium at 5×105 cells/well for 72 h. IL-12 levels in the culture supernatants were tested using ELISA. (B, C) iIDC from BALB/c mice were co-cultured with naive CD4 T cells from DO11.10 mice (BALB/c background) at a 1:5 DC:T ratio with OVA (0.1 mg/ml) in the presence or absence of anti-IL-12 mAb (5 μg/ml). Supernatants from 72-h cultures were tested for IFN-γ and IL-4 levels by ELISA. Four replicate wells were set up for each culture condition. Data are presented as mean ± SD. One representative of two independent experiments with similar results is shown.

Download figure to PowerPoint

thumbnail image

Figure 5. iIDC express higher levels of TLR9 mRNA than iNDC. DC were isolated using CD11c column (MACS) from the spleen of naive (iNDC) or MoPn-infected mice (iIDC). Total cellular RNA was extracted and RT-PCR for measuring mRNA of TLR2, TLR4, TLR9 and CD14 were performed. (A) Electrophoretic visualization of the amplicons in one representative of three independent experiments with similar results. (B) β-Actin-normalized quantification of the PCR products from iNDC (empty bars) and iIDC (hatched bars). Pooled data from the three independent experiments are shown. Data are presented as mean ± SE.

Download figure to PowerPoint

3 Discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

Several recent studies have demonstrated the modulating effect of prior microbial infections or microbial products on T cell cytokine-producing patterns to allergen in animal models. We previously reported that intracellular bacterial infection (Mycobacteria and Chlamydia) given before allergen exposure is capable of inhibiting airway eosinophilic inflammation and mucus production induced by allergen sensitization and local challenge 810. In the present study, using both in vitro and in vivo approaches, we demonstrated a crucial role of DC in the infection-mediated modulation of allergen-specific CD4+ T cell responses and allergic inflammation. We have shown that, similar to observations in natural infections, adoptive transfer of DC isolated from infected mice is capable of reducing the development of Th2-like cytokine responses and eosinophilic inflammation induced by allergen. The data provide clear evidence that DC educated by chlamydial infection in vivo can efficiently modulate T cell responses to allergen. This finding is novel because, although previous studies have shown the modulating effect of microbial infection and microbial products on DC function, direct evidence was lacking for the modulating effect of DC educated by microbial infection on naive allergen-specific CD4 T cells. The results suggest that intracellular bacterial infection may prevent the development of Th2-like allergic responses by shifting the immune responses towards a dominant Th1 profile through the modulation of DC function. This finding also supports a role of missing immune deviation, due to lack of infection, in the increase of allergic diseases in developed areas. There are at least two mechanisms that may account for the modulating effect of transferred iIDC on CD4+ T cell response to allergen in vivo. Firstly, iIDC that are educated through microbial infection can directly present allergen to T cells in recipient mice, as shown in the in vitro experiments in this study, and secondly, transferred iIDC can further influence the function of DC in the recipient mice, leading to decreased Th2-like cell development.

A novel finding in the present study is the high TLR message expression on iIDC. TLR have been shown to be important in linking innate and adaptive immune responses 16, 17. Several studies comparing farm and non-farm children in the development of allergy and asthma have demonstrated significantly fewer allergies in the later group, who arguably have higher exposure to microbial products 26, 27. A recent study showed that the expression of TLR on peripheral blood cells of farmer' and non-farmer' children are different, suggesting the involvement of altered innate immunity caused by microbial exposure in the modulation of allergic reaction 28. The finding of high TLR9 expression in the present study is particularly interesting because TLR9 is the receptor for bacterial CpG motifs. Numerous studies have shown that CpG can inhibit de novo and established allergic responses in animal and human models 2931. Therefore, it is logical to speculate that chlamydial infection, via modulation of TLR expression, can change the function of DC, leading to decreased allergen-specific Th2 cell and, to a lesser degree, enhanced Th1 cell development. By examining the effect of DC with different TLR expression on allergic responses in vivo, our present study provides a closer link between the changes in innate immunity and the alteration of adaptive immune responses to allergen.

Another interesting finding is the reduced IL-5Rα expression on BM cells in recipients of iIDC following allergen exposure. Previous studies in human and mouse systems have shown that local allergen exposure results in enhanced eosinophilopoiesis in BM. This prominently contributes to local eosinophilia, possibly via a feedback mechanism existing between the local tissue and BM, which triggers allergic inflammatory reaction 3234. Recent studies have shown that eosinophil precursors in BM are identifiable as CD34+/IL-5Rα+ cells [35. CD34 is an O-sialylated glycoprotein, whose expression within the hemopoietic system is restricted to primitive progenitor cells of all lineages 36,while IL-5R expression is essentially limited to eosinophil and basophil lineages 37, 38. We found that most of the IL-5Rα+ cells in BM were CD34+ cells in our model (data not shown). The small percentage of IL-5Rα+ cells (10–15%), which are CD34, may be mature eosinophils and late lineage-committed CD34CD33+ cells 38. The dramatic decrease of IL-5Rα+ cells in BM of iIDC recipients, suggest that iIDC may inhibit the development of eosinophils. The direct mechanism y which adoptively transferred iIDC inhibit eosinophil production in BM is not clear. However, it is likely to be related to the inhibitory effect of iIDC on IL-5 production by T cells. IL-5 has been found to play a central role in eosinophil biology, by means of promoting the differentiation and maturation of eosinophil-basophil lineage-committed progenitors 39, 40, as well as the circulation, recruitment and survival of mature eosinophils 41. Recent studies have shown that IL-5 can up-regulate IL-5Rα on BM CD34+ progenitors 42. Our present data showing lower IL-5 production by T cells and lower IL-5Rα expression on BM cells in iIDC recipients, not only confirmed the critical role of IL-5 in the development of eosinophilia, but also provided a link between DC subset, IL-5 production and IL-5R expression.

In summary, the present study has demonstrated a key role played by DC in infection-mediated modulation of allergic reaction. The data suggest that the function of DC in initiating T cell responses to allergen is substantially influenced by the changes in microenvironment caused by microbial infection. The alteration in DC for cytokine production and pattern recognition receptors expression may contribute to the role of DC in inhibiting allergic reaction.

4 Materials and methods

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

4.1 Animals

Female C57BL/6 mice and BALB/c mice (7–10 weeks old) were bred at the University of Manitoba breeding facility (Winnipeg, Manitoba, Canada). I-Ad-restricted DO11.10 TCR-α β-transgenic mice (TCR recognizes OVA323–339 peptide), the breeding pairs purchased from the Jackson Laboratory (Bar Harbor, ME), were bred and maintained at the University of Manitoba. Animalswere used in accordance with the guidelines issued by the Canadian Council on Animal Care.

4.2 Medium and peptide

Complete RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 1% L-glutamine, 25 μg/ml gentamycin and 5×10–5 M 2-mercaptoethanol (Kodak, Rochester, NY) was used for cell culture. Peptide corresponding to residues 323–339 of OVA (ISQAVHAAHAEINEAGR) was synthesized by the GenomeBC Proteomics Centre at University of Victoria (Victoria, British Columbia, Canada).

4.3 Infection of mice and transfer of DC

Mice were inoculated intranasally with 2×103 inclusion-forming units (IFU) of C. muridarum (MoPn) to generate respiratory tract infection as previously described 39. Seven days post-infection, spleens were aseptically collected and DC were isolated using an MACS (Miltenyi Biotec, Auburn, CA) CD11c column according to manufacturer's instructions. The purity of the isolated CD11+ DC was 96–98% based on flow cytometry. To test the spontaneous production of cytokines by the freshly isolated DC, the cells were cultured with complete medium using 96-well culture plates at 5×105 cells/well for 72–120 h. The levels of IL-12 and IL-10 in culture supernatants were measured by ELISA. For adoptive transfer, isolated CD11c+ cells were first washed in protein-free PBS, and 5×106 DC were injected intravenously to syngeneic recipient mice. The adoptive transfer experiments were performed using C57BL/6 mice to be consistent with our previous studies 1416.

4.4 Purification of CD4 T cells and the set up of DC-T cell co-culture

Naive CD4+ T cells were isolated from the spleen of DO11.10 OVA peptide-specific TCR-α β-transgenic mice (BALB/c background) using an MACS-positive selection column. The naiveCD4+ T cells (5×105 cells/well) were co-cultured with isolated DC from infected (iIDC) or naive BALB/c (iNDC) mice at varying DC:T ratios in the presence of either OVA (100 μg/ml) or OVA323–339 peptide (5–50 μg/ml). Cell culture supernatants were collected at 72 h for cytokine analysis. As indicated in particular experiments, anti-IL-12 mAb (PharMingen,San Diego, CA) at 5 μg/ml were added to the co-culture wells to block endogenous IL-12 activity.

4.5 Allergen sensitization, spleen cell culture and cytokine measurement

Mice with or without DC transfer were sensitized i.p. with 4 μg OVA in 2 mg alum adjuvant. For DC recipients, the sensitization was performed at 2 h after adoptive transfer of DC. Mice were killed 7–14 days post OVA sensitization, and spleen cells were cultured at a concentration of 7.5×106 cells/ml (2 ml/well) in the presence or absence of OVA (1 mg/ml) stimulation. Culture supernatants were harvested at 72 h (for IL-4, IL-5, IL-13, IFN-γ and IL-12) and 120 h (for IL-10) for the measurement of cytokines by sandwich ELISA using paired antibodies as described 15, 16.

4.6 Peripheral blood leukocyte differentials

Mouse peripheral blood smears were prepared and stained for leukocytes using Hema 3 Stain Set (Fisher Scientific, Ontario, Canada). Slides were fixed, stained, and air-dried using the reagents. The numbers of monocytes, lymphocytes, neutrophils and eosinophils were counted based on cellular morphology and staining characteristics.

4.7 Skin test and histological analysis

Skin tests were performed at day 11 post OVA sensitization. The ventral surface of the mouse was shaved, followed by intradermal injection of 20 μl OVA (0.5 mg/ml) in PBS. Skin tissue samples of about 0.5 cm2 were collected from injection sites at 72 h post-injection for histological analysis. The tissues were fixed in 10% buffered formalin, embedded in paraffin and continuously sectioned using a microtome. The sections were stained by hematoxylin and eosin (H&E) and examined under a light microscope. Infiltrating inflammatory cells were identified based on cellular morphology and staining characteristics. Infiltrating cells in ten continuous skin sections were counted, and the percentage of eosinophils in total infiltrating cells was calculated.

4.8 Immunohistochemical analysis

IL-5 receptor expression on BM cells was determined by immunohistochemical analysis. Femora were collected from mice at 7 days post OVA (alum) sensitization. BM cell smears were air-dried and stained for IL-5Rα. Rabbit anti-mouse IL-5Rα antibody was purchased from RDI (Flanders, NJ). The blocking reagent, secondary antibody and AEC substrate were purchased from DAKO (Carpinteria,CA) as a peroxidase kit. The percentage of IL-5R+ cells was calculated based on the counting of total cells (200 cells) and IL-5R+ cells on the smear.

4.9 PCR and RT-PCR analysis

To test whether MoPn exists in the iIDC, total DNA from iIDC was isolated using DNAzol reagent according to the manufacturer's instruction (Invitrogen, Carlsbad, CA). Major outer membraneprotein genes of MoPn were determined by PCR using the following primer: 5′-TGGTGTGATGCCATCAGCCTACG-3′ (forward) and 5′-CAAGCGTGTCTCAACAGTAACTGC-3′ (reverse).

For analysis of TLR and CD14 gene expression in the isolated DC, total cellular RNA was extracted from isolated DC using the phenol-guanidinium method (TRIzol reagents, Invitrogen), followed by ethanol precipitation. The first-strand cDNA was generated from 1.2 μg total RNA in a final volume of 15 μl using M-MLV reverse transcriptase (Invitrogen) and oligo(dT) primer. One microliter of cDNA was used for each PCR reaction. The presence of RNA specific for TLR2, TLR4, TLR9 and CD14 was determined by semiquantitative PCR. The PCR primer sets used in this study were as follows:TLR2, 5′-GCTCCAGGTCTTTCACCTCTATTC-3′ (sense) and 5′-TCCAGCAGGAAAGCAGACTCGCTTA-3′ (antisense); TLR4, 5′-GGAAGCTTGAATCCCTGCATAGAG-3′ (sense) and 5′-TCCACATGTACTAGGTTCGTCAG-3′ (antisense); TLR9, 5′-AACCTGCGGCAGCTGAACCTCAA-3′ (sense) and 5′-GAGTTCAGTGTATGGAGAGAGCTG-3′ (antisense); CD14, 5′-TGTTCCTGCAACTTCTCAGATCC-3′ (sense) and 5′-CAATTCAGGATTGTCAGACAGGTC-3′(antisense); β-actin, 5′-GTGGGCCGCCCTAGGCACCA-3′ (sense) and 5′-CTCTTTGATGTCACGCACGATTTC-3′ (antisense). The reaction condition for PCR were as follows: 1 cycle at 95°C for 5 min; 31 cycles (for TLR2 and CD14) or 34 cycles (for TLR4 and TLR9) at 95°C for 1 min, at 55°C for 1 min, and 72°C for 1 min. β-actin was used as a loading control. PCR products were run on a 1% agarose gel containing 0.1 mg/ml ethidium bromide. Image analysis was performed using Gel Doc 2000 gel documentation system (Bio-Rad, Hercules, CA) and quantified using Scion Image software (Scion Corporation, Frederick, MD).

4.10 Statistical analysis

Cytokine production and blood cell differentials were analyzed by unpaired Student's t-test.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

This work was supported by an operating grant to X.Y. from Canadian Institutes of Health Research (CIHR). X.H. and L.B. are trainees in the CIHR National Training Program in Allergy and Asthma, and recipients of Manitoba Health Research Council Graduate Studentship. X.Y. holds a Canada Research Chair in Infection and Immunity.

  • 1

    WILEY-VCH

  • 2

    WILEY-VCH

  • 3

    WILEY-VCH

  • 4

    WILEY-VCH

  • 5

    WILEY-VCH

  • 1
    Cookson, W. O. and Moffatt, M. F., Asthma: an epidemic in the absence of infection? Science 1997.275: 4142.
  • 2
    Ring, J., Krämer, U., Schäfer, T. and Behrendt, H., Why are allergies increasing? Curr. Opin. Immunol .2001. 13: 701708.
  • 3
    Strachan, D. P., Hay fever, hygiene, and household size. BMJ 1989. 299: 12591260.
  • 4
    Strachan, D. P., Family size, infection and atopy: the first decade of the "hygiene hypothesis". Thorax 2000. 55: S210.
  • 5
    Krb, K. J., Atopic disorders: a default pathway in the absence of infection? Immnol. Today 1999. 20: 317320.
  • 6
    Shirakawa, T., Enomoto, T., Shimazu, S. and Hopkins, J. M., The inverse association between tuberculin responses and atopic disorder. Science 1997. 275: 7779.
  • 7
    Erb, K. J., Holloway, J. W., Sobeck, A., Moll, H. and Le Gros, G., Infection of mice with Mycobacterium bovis-bacille Calmette-Guérin (BCG) suppresses allergen-induced airway eosinophilia. J. Exp. Med .1998. 187: 561569.
  • 8
    Yang, X., Wang, S., Fan, Y. and Zhu, L., Systemic mycobacterial infection inhibits antigen-specific immunoglobulin E production, bronchial mucus production and eosinophilic inflammation induced by allergen. Immunology 1999. 98: 329337.
  • 9
    Yang, X., Fan, Y., Wang, S., Han, X., Yang, J., Bilenki, L. and Chen, L., Mycobacterial infection inhibits established allergic inflammatory responses via alteration of cytokine production and vascular cell adhesion molecule-1 expression. Immunology 2002. 105: 336343.
  • 10
    Bilenki, L., Wang, S., Fan, Y., Yang, J., Han, X. and Yang, X., Chlamydia trachomatis infection inhibits airway eosinophilic inflammation induced by ragweed. Clin. Immunol .2002. 102: 2836.
  • 11
    Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque,S., Liu, Y., Pulendran, B. and Palucka, K., Immunobiology of dendritic cells. Annu. Rev. Immunol .2000. 18: 767811.
  • 12
    Pulendran, B., Maraskovsky, E., Banchereau, J. and Maliszewski, C., Modulating the immune responses with dendritic cells and their growth factors. Trends Immunol .2001. 22: 4147.
  • 13
    Moser, M. and Murphy, K. M., Dendritic cell regulation of Th1-Th2 development. Nat. Immunol .2000. 1: 199205.
  • 14
    Lanzavecchia, A. and Sallusto, F., Regulation of T cell immunity by dendritic cells. Cell 2001. 106: 263266.
  • 15
    Liu, Y. J., Kanzler, H., Soumelis, V. and Gilliet, M., Dendritic cell lineage, plasticity and cross-regulation. Nat. Immunol .2001. 2: 585589.
  • 16
    Akira, S., Takeda, K. and Kaisho, T., Toll-like receptors: critical proteins linking innate andacquired immunity. Nat. Immunol .2001. 2: 675680.
  • 17
    O'Neill, L. A., Toll-like receptor signal transduction and the tailoring of innate immunity: a role for Mal? Trends Immunol .2002. 23: 296300.
  • 18
    Hallman, M., Ramet, M. and Ezekowitz, R. A., Toll-like receptors as sensors of pathogens. Pediatr. Res .2001. 50: 315321.
  • 19
    Kaisho, T. and Akira, S., Dendritic cell function in Toll-like receptor- and MyD88-knockout mice. Trends Immunol .2001. 22: 7883.
  • 20
    Baldini, M., Lohman, I. C., Halonen, M., Erickson, R. P., Holt, P. G. and Martinez, F. D., A polymorphism in the 5′-flanking region of the CD14 gene is associated with circulating soluble CD14 levels and with total serum immunoglobulin E. Am. J. Respir. Cell Mol. Biol .1999. 20: 976983.
  • 21
    Arbour, N. C., Lorenz, E., Schutte, B. C., Zabner, J., Kline, J. N., Jones, M., Frees, K., Watt, J. L. and Schwartz, D. A., TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nat. Genet .2000. 25: 187191.
  • 22
    Wang, S., Fan, Y., Brunham, R. C. and Yang, X., IFN-gamma knockout ice show Th2-associated delayed-type hypersensitivity and the inflammatory cells fail to localize and control chlamydial infection . Eur. J. Immunol. 1999. 29: 37823792.
  • 23
    Yang, X., Role of cytokines in Chlamydia trachomatis protective immunity and immunopathology. Curr. Pharm. Des. 2003. 9: 6773.
  • 24
    Su, H., Messer, R., Whitmire, W., Fischer, E., Portis, J. C. and Caldwell, H. D., Vaccination against chlamydial genital tract infection after immunization with dendritic cells pulsed ex vivo with nonviable Chlamydiae. J. Exp. Med .1998. 188: 809818.
  • 25
    Draffi, S. J., Dekan, G., Stingl, G. and Epstein, M. M., Systemic administration of antigen-pulsed dendritic cells induces experimental allergic asthma in mice upon aerosol antigen rechallenge. Clin. Immunol .2002. 103: 176184.
  • 26
    Riedler, J., Braun-Fahrlander, C., Eder, W., Schreuer, M., Waser, M., Maisch, S., Carr, D., Schierl, R., Nowak, D., von Mutius, E. and ALEX Study Team, Exposure to farming in early life and development of asthma and allergy: a cross-sectional survey. Lancet 2001. 358: 11291133.
  • 27
    von Mutius, E., Braun-Fahrlander, C., Schierl, R., Riedler, J., Ehlermann, S., Maisch, S., Waser, M. and Nowak, D., Exposure to endotoxin or other bacterial components might protect against the development of atopy. Clin. Exp. Allergy .2000. 30: 12301234.
  • 28
    Lauener, R. P., Birchler, T., Adamski, J., Braun-Fahrlander, C., Bufe, A., Herz, U., von Mutius, E., Nowak, D., Riedler, J., Waser, M., Sennhauser, F. H. and ALEX study group, Expression of CD14 and Toll-like receptor 2 in farmers' and non-farmers' children. Lancet 2002. 360: 465466.
  • 29
    Jain, V. V., Kitagaki, K., Businga, T., Hussain, I., George,C., O'Shaughnessy, P. and Kline, J. N., CpG-oligodeoxynucleotides inhibit airway remodeling in a murine model of chronic asthma. J. Allergy Clin. Immunol .2002. 110: 867872.
  • 30
    Bohle, B., CpG motifs as possible adjuvants for the treatment of allergic diseases. Int. Arch. Allergy Immunol .2002. 129: 198203.
  • 31
    Krieg, A. M., CpG motifs in bacterial DNA and their immune effects. Annu. Rev. Immunol .2002. 20: 709760.
  • 32
    Wood, L. J., Inman, M. D., Watson, R. M., Foley, R., Denburg, J. A. and O'Byrne, P. M., Changes in bone marrow inflammatory cell progenitors after inhaled allergen in asthmatic subjects. Am. J. Respir. Crit. Care Med .1998. 157: 99105.
  • 33
    Gaspar Elsas, M. I. C., Joseph, D., Elsas, P. X. and Vargaftig, B. B., Rapid increase in bone marrow eosinophil production and responses to eosinopoietic interleukins triggered by intranasal allergen challenge. Am. J. Respir. Cell Mol. Biol. 1997. 17: 404413.
  • 34
    Tomaki, M., Zhao, L.-L., Lundahl, J., Sjöstrand, M., Jordana, M., Lindén, A., O'Byrne, P. and Lötvall, J., Eosinophilopoiesis in a murine model of allergic airway eosinophilia: involvement of bone marrow IL-5 and IL-5 receptor α. J. Immunol .2000. 165: 40404050.
  • 35
    Robinson, D. S., Damia, R., Zeibecoglou, K., Molet, S., North, J., Yamada, T., Kay, A. B. and Hamid, Q., CD34+/interleukin-5Rα messenger RNA+ cells in the bronchial mucosa in asthma: potential airway eosinophil progenitors. Am. J. Respir. Cell Mol. Biol .1999. 20: 913.
  • 36
    Sehmi, R., Wood, L. J., Watson, R., Foley, R., Hamid, Q., O'Byrne, P. M. and Denburg, J. A., Allergen-induced increases in IL-5 receptor α-subunit expression on bone marrow-derived CD34+ cells from asthmatic subjects. J. Clin. Invest. 1997. 100: 24662475.
  • 37
    Takagi, M., Hara, T., Ichihara, M., Takatsu, K. and Miyajima, A., Multi-colony stimulating activity of interleukin 5 (IL-5) on hematopoietic progenitors from transgenic mice that express IL-5 receptor α subunit constitutively . J. Exp. Med .1995. 181: 889899.
  • 38
    Upham, J. W., Sehmi, R., Hayses, L. M., Howie, K., Lundahl, J. and Denburg, J. A., Retinoic acid modulates IL-5 receptor expression and selectively inhibits eosinophil-basophil differentiation of hemopoietic progenitor cells. J. Allergy Clin. Immunol .2002. 109: 307313.
  • 39
    Lach-Trifilieff, E., McKay, R. A., Monia, B. P., Karras, J. G. and Walker, C., In vitro and in vivo inhibition of interleukin (IL)-5-mediated eosinopoiesis by murine IL-5Ralpha antisense oligonucleotide. Am.J. Respir. Cell Mol. Biol .2001. 24: 116122.
  • 40
    Saito, H., Matsumoto, K., Denburg, A. E., Crawford, L., Ellis, R., Inman, M. D., Sehmi, R., Takatsu, K., Matthaei, K. I. and Denburg, J. A., Pathogenesis of murine experimental allergic rhinitis: a study of local and systemic consequences of IL-5 deficiency. J. Immunol .2002. 168: 30173023.
  • 41
    Van Rijt, L. S. and Lambrecht, B. N., Role of dendritic cells and Th2 lymphocytes in asthma: lessons from eosinophilic airway inflammation in the mouse . Microsc. Res. Tech .2001. 53: 256272.
  • 42
    Denburg, J. A., Sehmi, R. and Upham, J., Regulation of IL-5 receptor on eosinophil progenitors in allergic inflammation: role of retinoic acid. Int. Arch. Allergy Immunol. 2001. 124: 246248.