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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 244-249, 2002
© 2002 American Thoracic Society

The Effect of Bacillus Calmette-Guérin Immunization Depends on the Genetic Predisposition to Th2-Type Responsiveness

Machteld N. Hylkema, Wim Timens, Marjan Luinge, Nienke van der Werf and Maarten O. Hoekstra

Department of Pathology and Laboratory Medicine, University Hospital Groningen, Groningen; and Department of Pediatrics, Wilhelmina Children's Hospital, Utrecht, The Netherlands

Address correspondence to: Prof. Dr. W. Timens, Department of Pathology and Laboratory Medicine, University Hospital Groningen, P.O. Box 30.001, 9700 RB, Groningen, The Netherlands. E-mail: w.timens{at}path.azg.nl


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The aim of this study was to investigate whether the effect of bacillus Calmette-Guérin (BCG) immunization on ovalbumin-induced allergic inflammation in a rat model depends on the genetic predisposition to react with a T helper cell (Th) 2-type cytokine response. This study was performed in an inbred Th2-predisposed "asthma prone" rat strain (brown Norway [BN]) and in an outbred nonpredisposed strain (Sprague Dawley [SD]), to differentiate between genetic and environmental factors. BCG decreased numbers of lung eosinophils and macrophages in the SD rat. This effect was not seen in the BN rat. In the BN rat, but not in the SD rat, BCG downregulated levels of total serum IgE. No significant differences were found with respect to frequencies of IFN{gamma}– or interleukin-4–producing cells in the lung in both rat strains. These results indicate that the degree and pathway of immunomodulatory effect of BCG in two genetically different rat strains is dependent on the genetic predisposition to develop a Th2-type response. Therefore, differences in genotype in relation to environment may result in difference in involvement of contributing pathogenic factors and thus different responsiveness to therapeutic strategies.

Abbreviations: Antibody, Ab • bacillus Calmette-Guérin, BCG • brown Norway, BN • 3,3'Diaminobenzidine, DAB • interleukin, IL • monoclonal antibody, mAb • ovalbumin, OVA • reverse transcription polymerase chain reaction, RT-PCR • Sprague Dawley, SD • T helper cell, Th


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Both the incidence and the prevalence of asthma among children have increased dramatically in the past three decades (1–3). This increase in allergic asthma is speculated to be partly due to the decline of many infectious diseases in developed countries as the result of improved living standards and immunization programs (4–6). Childhood respiratory infections that might strongly modify the developing immune system, both systemically and in the lung, include measles, whooping cough, and tuberculosis. Mycobacterium tuberculosis is among the most potent inducers of a T helper cell (Th) 1-type response (7, 8). It is hypothesized that these Th1-type stimuli early in life are needed to skew the Th2-type immune response, obligatory at birth, to a Th1-type immune response. Lack of appropriate numbers of Th1-like infections early in life or living in a clean environment is suggested to contribute to the increased prevalence of asthma. This ‘hygiene’ hypothesis is supported by epidemiologic data showing that the prevalence of hayfever and asthma was inversely associated with childhood infections such as measles (9) and hepatitis A (10), a higher number of (older) siblings, an early entry in daycare (11, 12), self-reported childhood infections (13), anthroposophic lifestyle and living on a farm (6).

Still, environmental factors like respiratory infectious diseases and allergen exposure (14–16) are not the only factors important in development of allergic asthma. There is also significant genetic predisposition to the development of asthma in humans (17–19).

Recently, a Japanese study in a population of 867 school children over the age of 12 years showed an inverse association between responses to tuberculin and development of atopic disorders (20). However, these data do not confirm a causal relationship in that mycobacterial infection can reduce development of atopy; genetic factors contributing to susceptibility for atopy or tuberculosis could be mutually exclusive. In most species the capacity to respond in a Th1- or Th2-type fashion is to a large extent genetically determined.

Our aim was to study whether the effect of bacillus Calmette-Guérin (BCG) immunization on ovalbumin (OVA)-induced allegric inflammation in a rat model depends on the genetic predisposition to react with a Th2-type cytokine response. For this purpose the Brown Norway (BN) rat and the Sprague Dawley (SD) rat were used to differentiate between genetic and environmental factors. In particular, in this study we used very young (3 wk old) rats to mimic respiratory infections in early childhood. The study hypothesis is that BCG would have a greater effect on reducing the allergic inflammatory airway response in the SD rat strain than in the BN rat strain because the BN rat is considered to be a Th2-type responder and the SD rat, being an outbred strain, is not predisposed to having a predictable Th1-like or Th2-like response (10, 21).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male, 3 wk old BN/RIJ HSD (RT1n) and SD/HSD (outbred) rats were obtained from Harlan (Zeist, The Netherlands) and maintained under specific pathogen-free conditions in the Central Animal Facility of the University of Groningen.

BCG Immunization
Rats were inoculated intranasally with 50 µL (107 CFU in PBS) of an attenuated Mycobacterium bovis-BCG strain which is commonly used for vaccination in infants. Control mice received PBS only.

Sensitization Procedure
OVA grade V (Sigma-Aldrich, Zwijndrecht, The Netherlands) was prepared at 2 mg/ml in pyrogen-free PBS and precipitated at a 1:1 ratio with Al(OH)3 (45 mg/ml, Imject Alum; Pierce, Rockford, IL), following the instructions of the manufacturer. Rats were sensitized with 1 mg of OVA (1 ml of OVA/Al(OH)3 suspension) given intradermally on the back. Sham immunizations were done with PBS.

Airway Allergen Challenge
Four weeks after sensitization, rats were either challenged with OVA intratracheally (1.7 mg/100 µL PBS) or were placed in a perspex exposure chamber (9 liters) and challenged with an aerosol of 1% OVA in saline for 30 min on 2 consecutive days. The aerosol was delivered by a De Vilbiss nebulizer (type 646; De Vilbiss, Somerset, PA) driven by an airflow of 8 L/min, providing aerosol with an output of 0.33 ml/min.

Experimental Design
Assessment of BCG immunization. To investigate whether BCG alone induced a Th1-type immune response in the lung, numbers of IFN{gamma} producing cells were assessed 7, 14, 21 and 28 d after BCG immunization by ELISPOT assay (n = 5). Control rats (n = 3) were sham immunized with PBS. Data were expressed as percentages of control to correct for inter-assay variation.

BCG immunization in combination with OVA-induced airway inflammation. As indicated in Figure 1 , rats (n = 6–9) were sensitized with OVA either 21 d after BCG immunization (protocol I), or simultaneously with BCG immunization (protocol II).



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  		Figure 1. BCG immunization (A) prior to OVA protocol and (B) simultaneously with OVA protocol.

 
Cell Preparation And Lung Digest
Single-cell suspensions from lungs were obtained as previously described (22). In brief, rats were killed and the lung vascular bed was flushed in situ via the right cardiac ventricle with 20 ml of ice-cold PBS to remove all blood including intravasculair leukocytes. Minced lungs were incubated on a rocker for 90 min at 37°C in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum, DNase I (100 U/ml; Boehringer, Mannheim, Germany), and collagenase I (250 U/ml; C9891; Sigma-Aldrich, Zwijndrecht, The Netherlands). Purified vital lung cells were obtained by passing the digested lung tissue through a stainless steel mesh and subsequently performing discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradient centrifugation (20 to 55%).

Cells were counted using a Coulter Counter Z1 (Coulter, Hialeah, FL).

Antibodies
Monoclonal antibodies (mAbs) used in this study were DB-1 (anti-IFN{gamma}; Biosource, Etten Leur, The Netherlands) and MRC OX81 (anti–IL-4; MRC, Oxford, UK). The polyclonal rabbit anti-rat antibodies to IFN{gamma} and IL-4 were a kind gift of Dr. P. van der Meide (BPRC, Rijswijk, the Netherlands). ED1 (CD68) was a kind gift of Dr. C. Dijkstra (VU Medical Center, Amsterdam, The Netherlands).

Determination of Cytokine Producing Cells by ELISPOT Assay
IFN{gamma}. For the detection of numbers of IFN{gamma} producing cells the ELISPOT assay was used as described previously (10). Briefly, flatbottom 96-well microtiter plates (Nunc Maxisorp; Life Technologies, Merelbeeke, Belgium) were coated overnight with DB-1 in a concentration of 10 µg/ml in PBS. For detection of IFN{gamma} producers 4 x 104 cells in 100 µL were tested. Cells were stimulated with 4ß-phorbol 12ß-myristate 13{alpha}-acetate (PMA, 20 ng/ml; Sigma) and ionomycin (1 µM, Sigma) for 18 h at 37°C in a humidified atmosphere with 5% CO2. After 18 h, cells were lysed with ice-cold distilled water and incubated for 1.5 h with a polyclonal rabbit anti-rat IFN{gamma} antibody (Ab) in PBS, containing 1% vol/vol BSA (RIA-grade BSA A-7888, Sigma). The plates were washed five times with PBS containing 0.05% Tween 20 and incubated with goat anti-rabbit Ab conjugated with alkaline phosphatase (1 h in PBS, containing 4% vol/vol BSA). The plates were then washed with PBS containing 0.01% Tween 20 and refilled with a solution of 1 mg/ml of 5-bromo-4-chloro-3-indolylphosphate dissolved at 40°C in 0.6% (wt/vol) low gelling agarose containing 0.1 M 2-amino-2-methyl-1-propanol, 0.02% (vol/vol) Triton X-405 (BDH, Poole, England) and 5.0 mM MgCl2. Blue spots developed during a 4 h incubation period at 37°C in a humidified chamber and were counted using an inverted microscope. As a negative control, unstimulated cells were used. No spots were found in control wells.

IL-4. For the detection of numbers of IL-4–producing cells, the mAb OX81 was used as capture Ab, and a polyclonal rabbit anti–IL-4 Ab was used for detection. A total of 4 x 105 cells were tested for detection of numbers of IL-4–producing cells. Ratios of IFN{gamma}/IL-4 were calculated.

Total IgE Sandwich ELISA
Serum was collected just before sensitization (Day 0) and 7, 14, 21 and 28 d after sensitization. Samples were stored at -20°C until analyzed. Total serum IgE was determined using a sandwich ELISA. All 96-well plates were coated overnight at 4°C with a mouse mAb to rat IgE (B41–1, PharMingen, San Diego, CA) at 2 µg/ml, 100 µl/well. After washing the plates three times with PBS/0.05%Tween-20, plates were blocked with 4% BSA/PBS for 2 h (100 µl/well). Then serum samples, diluted 1: 25 in 2% BSA/PBS, were added to the plates (100 µl/well) and were titrated. Plates were incubated for 1 h at 37°C and subsequently washed three times with PBS/0.05% Tween-20, after which biotinylated mouse anti-rat IgE mAb was added (B41–3, PharMingen) for 1 h at 37°C. After washing, the plates were incubated with horseradish peroxidase-conjugated streptavidin (1:4000 in PBS Catalog no. P0397; Dako, Denmark) for 15 min at 37°C. Plates were developed for 15 min at room temperature after addition of 3,5,3',5'-tetramethylbenzidin (Merck) at 100 µg/ml in 0.11 M sodium acetate (pH 6.0) containing 0.003% H2O2. Color development was stopped with 2 M H2SO4 and absorption was measured in a Titrek multiscan (Titrek, Salzburg, Austria) at 405 nm. On each plate serial dilutions of a hyperimmune rat serum sample were run as a positive control and a normal serum sample was run as a negative control to correct for interassay variation.

Optical densities were converted to arbitrary units (ELISA units) according to the standard curve obtained from serial dilutions of the hyperimmune rat serum sample.

Measurement of OVA-specific IgE
OVA-specific IgE was measured as described by Schneider and colleagues (23) with some modifications. Wells were coated overnight at 4°C with a mouse mAb to rat IgE (B41–1, PharMingen) at 2 µg/ml, 100 µl/well. After washing the plates three times with PBS/0.05%Tween-20, plates were blocked with 1% milk in PBS for 2 h and serum samples were added to the wells at a 1:5 dilution in 2% BSA/PBS. Serum samples were titrated and plates were incubated for 1 h at 37°C. After washing the plates three times with PBS/0.05% Tween-20 biotinylated OVA (10 µg/ml in 2% BSA/PBS) was added for 1 h and plates were washed again. As a second step reagent horseradish peroxidase-conjugated streptavidin (Dako) was added for 15 min in a 1:4000 dilution in 1% milk. Plates were developed, stopped and read as described for the total IgE ELISA. Optical densities were converted to arbitrary units (ELISA units) according to the standard curve obtained from serial diutions of a hyperimmune rat serum sample.

Analysis of Numbers of Eosinophils, Neutrophils and Macrophages
Eosinophils were determined in frozen lung tissue stained for endogenous eosinophil peroxidase. Peroxidase activity was visualized using 3,3'Diaminobenzidine (DAB) and sections were not counterstained. Measurement of the number of eosinophils in lung tissue was done by morphometric analysis. Whole lung tissue sections were screened using a light microscope equipped with a camera connected to a video screen. Because it was not possible to reliably detect individual cells, the total surface area with stained cells was measured and divided by the total surface of the lung section to express numbers of eosinophils as volume percentages, enabling comparison between groups.

The presence of neutrophils was analyzed in frozen lung sections by staining for neutrophil alkaline phosphatase. The presence of macrophages was analyzed in frozen lung sections by staining with the mAb ED1 (CD68).

Slides were counted by one observer in a blind fashion in coded, random order using an Olympus BX40 microscope (Olympus Optical, Tokyo, Japan). Counts were expressed as means of 14 microscopic fields per tissue section.

Statistical Analysis
For statistical analysis, the nonparametric Mann-Whitney test was used for group-wise comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Increased Frequencies of IFN{gamma}-Producing Cells After BCG Immunization in Both Rat Strains
To study the Th1-like response to BCG alone in the two genetically different rat strains, 3 wk old rats were inoculated intranasally with BCG. At 7, 14, 21, and 28 d after infection, numbers of IFN{gamma}-producing cells isolated from the lung were determined by ELISPOT assay.

As shown in Figure 2 , both rat strains showed an increased IFN{gamma} response when compared with PBS-inoculated control animals. However, when the IFN{gamma} response of both rat strains was compared, the SD rat showed a significantly higher response to BCG than the BN rat (P > 0.05) 21 d after BCG immunization.



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  		Figure 2. IFN{gamma}-producing cells in isolated lung cells of BN (solid bars) and SD (hatched bars) rat strains 7, 14, 21 and 28 d after intranasal immunization with BCG or PBS. Results are expressed as percentages of control animals. Numbers of IFN{gamma}-producing cells were determined by ELISPOT assay after 18 h of stimulation with 4ß-phorbol 12ß-myristate 13{alpha}-acetate and ionomycin. Numbers of IFN{gamma}-producing cells varied from 30–250 spots per 4 x 105 cells in BCG-inoculated animals to 20–150 spots per 4 x 105 cells in PBS-inoculated animals. Means ± SD are presented for each group, with three to five mice in each group. *Value significantly different from BN rats at Day 21 (P < 0.05).

 
BCG Immunization Prior to OVA Protocol
In a first set of experiments, 3 wk old rats were inoculated with BCG 3 wk prior to OVA sensitization. The effect of BCG inoculation on OVA-induced allergic airway inflammation was determined by measurement of frequencies of lung eosinophils (expressed as volume percentages), frequencies of isolated lung IFN{gamma}– and IL-4–producing cells, and levels of total and OVA-specific serum IgE. As shown in Figure 3 , BCG reduced the number of eosinophils in the lung only in the SD rat strain. BCG had no effect on numbers of IFN{gamma}– and IL-4–producing cells from the lung or on OVA-specific IgE levels in serum of either rat strain (Figures 4 and 5) .



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  		Figure 3. Eosinophils, expressed as volume percentages in frozen lung sections of two different rat strains treated with BCG prior to OVA sensitization (solid bars, n = 6–9), rats treated with OVA alone (gray bars, n = 5), and rats treated with PBS (white bars, n = 3), 18 h after allergen/PBS challenge. Means ± SD are presented. *Value significantly different from SD rats treated with OVA alone (P < 0.05).

 


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  		Figure 4. IFN{gamma}– (A) and IL-4– (B) producing cells in isolated lung cells of two different rat strains treated with BCG prior to OVA sensitization (solid bars, n = 6–9), rats treated with OVA alone (gray bars, n = 5), and rats treated with PBS (white bars, n = 3), 18 h after allergen/PBS challenge. Numbers of cytokine-producing cells were determined by ELISPOT assays after 18 h of stimulation with 4ß-phorbol 12ß-myristate 13{alpha}-acetate and ionomycin. Ratios of IL-4/IFN{gamma} producing cells (C) were calculated by dividing numbers of IL-4–producing cells by numbers of IFN{gamma}-producing cells. Means ± SD are presented for each group. *Values significantly different from BN of SD rats treated with OVA alone (P < 0.05).

 



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  		Figure 5. Total serum IgE in BN rat (A) and SD rat (C), and OVA-specific serum IgE in BN rat (B) and SD rat (D) 7, 14, 21 and 28 d after OVA sensitization. Rats were treated with BCG prior to OVA sensitization (closed squares, n = 6–9), with OVA alone (open squares, n = 5) or with PBS (inverted triangles, n = 3). Data are presented as the mean ± SD.

 
BCG Immunization Administered Simultaneously with OVA Sensitization
In a second set of experiments, 3 wk old rats were simultaneously inoculated with BCG and OVA sensitized. In these experiments, the same parameters were analyzed as those described for the first set of experiments (see above) to study the effect of BCG on OVA-induced allergic airway inflammation. In addition, the effect was studied on numbers of neutrophils and macrophages. Again, SD rats treated with BCG simultaneously with OVA had reduced numbers of eosinophils in the lung when compared with animals treated with OVA alone (not shown). BCG did not have such an effect in the BN rat. In the SD rat, BCG also reduced the number of macrophages but not the number of neutrophils. This effect, again, was not seen in the BN rat (Figures 6A and 6B) . However, at 21 d and 28 d after BCG inoculation and OVA sensitization, BCG did significantly reduce the level of serum IgE in the BN rat when compared with animals treated with OVA alone (P < 0.05) (Figure 7A) . This effect was not seen in serum OVA-specific IgE levels (Figure 7B). In the SD rat serum IgE levels, if any, were too low to be measured.



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  		Figure 6. Presence of macrophages (A) or neutrophils (B) in the lung of BN rats and SD rats treated with BCG simultaneously with OVA (solid bars, n = 6–9), treated with OVA alone (gray bars, n = 5), or treated with PBS (white bars, n = 3) 18 h after allergen/PBS challenge. Data are presented as the mean ± SD. *Value significantly different from SD rats treated with OVA alone (P < 0.05).

 


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  		Figure 7. Total serum IgE in BN rats (A) and OVA-specific serum IgE in BN rats (B) 7, 14, 21 and 28 d after OVA sensitization. Rats were treated with BCG simultaneously with OVA sensitization (closed squares, n = 6–9), with OVA alone (open squares, n = 5), or with PBS (inverted triangles, n = 3). Data are presented as the mean ± SD. *Value significantly different from BN rats treated with BCG (P < 0.05) or PBS (P < 0.001).

 
In addition, BCG had no effect on the number of IFN{gamma} or IL-4–producing cells in the lung in either rat strain when compared with animals treated with OVA alone.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The study hypothesis was that BCG would have a greater effect on reducing the allergic inflammatory airway response in the SD rat strain than in the BN rat strain. The results of the present study supported our hypothesis. The first experiment showed that in response to BCG alone, the BN rat had a lower Th1-like response 21 and 28 d after BCG inoculation when compared with the SD rat (Figure 2). The second experiment to support our hypothesis was that in which BCG was given prior to OVA sensitization, and no effect was seen in the BN rat with respect to eosinophilia, frequencies of IL-4– or IFN{gamma}–producing cells, or serum IgE. In contrast, BCG downregulated the number of eosinophils in the lungs of the SD rat. This is in line with the only article in which the effect of BCG was studied in a rat model for asthma (24). In that study, Koh and colleagues also used SD rats.

In a third set of experiments, BCG was inoculated simultaneously with OVA sensitization when the rats were 3 wk old. This protocol was used because it was shown that concomitant administration of BCG (or CpG, oligonucleotides containing CpG motifs found in bacterial DNA) with OVA strongly downregulated airway eosinophilia and Th2 cell activation in a mouse model (25–27). Using this protocol, BCG downregulated the IgE response in the BN rat 3 wk after BCG and OVA administration. Apparently, coadministration of both antigens is needed to downregulate the Th2 response in this genetically asthma-prone rat strain. The observation that this effect is seen 3 wk after BCG/OVA administration is in line with our observation that the peak response to BCG alone in this rat was 3 wk after inoculation (Figure 2). BCG again had no effect on the number of lung eosinophils in the BN rat nor on the number of neutrophils or macrophages. In contrast, BCG did downregulate the number of eosinophils and the number of macrophages in the SD rat. No IgE was present in the SD rat, probably because of the very young age. This is likely different in young BN rats because of the extreme shift to Th2-type reaction already occurring at that point in time. In small children it is also often difficult to measure IgE production (28). The assumption that the BN rat strain is predisposed to be a Th2-type responder is shown clearly when the Th1-/Th2-like responsiveness to OVA treatment alone is compared in both rat strains. In this study, both strains exhibited significant development of asthma-like pulmonary eosinophilia, increased ratios of IL-4–/IFN{gamma}–producing cells in the lung, and elevated, antigen-specific serum IgE levels due to airway antigen exposure after sensitization. However, the BN rat showed a more pronounced eosinophil response in the lung and higher serum (OVA-specific) IgE levels after the OVA protocol than the SD rat (Figures 3, 5 and 7). This is in line with the finding that the BN rat has the highest expression of RT6 (the so-called Th2-like marker on Th cells in the rat) when compared with ten other rat strains (10, 21).

In this study, in both rat strains administration of BCG prior or simultaneous to OVA had no effect on the number of IFN{gamma}– or IL-4–producing cells isolated from the lung. This seems in contrast with the studies performed in mouse asthma models (29, 30). One of the reasons could be that in our study the number of cytokine-producing cells was analyzed (by ELISPOT assay) instead of the total amount of cytokine production in culture supernatant of stimulated lung cell isolates, as was done in mice. Studying frequencies of cytokine-producing cells only indicates how many cells have the potency to produce cytokines. It does not provide information on how much cytokine is actually produced by one cell. However, in the rat, the ELISPOT assay provides the only opportunity to detect small amounts of rat IL-4 at the protein level (10). So far, the limited data available for IL-4 were exclusively based on semiquantitative reverse transcription polymerase chain reaction (RT-PCR) analysis or in situ hybridization performed on cytospin preparations obtained from bronchoalveolar lavage (31).

In summary, we have demonstrated that the degree of immunomodulatory effect of BCG in two genetically different rat strains was dependent on the genetic predisposition to develop a Th2-type response. In addition, we found variability in the manner and extent to which both rat strains respond to BCG after being subjected to OVA sensitization and exposure protocols. Whereas BCG downregulated IgE in the BN rat, which speculatively may be considered an IL-4 effect, it downregulated eosinophilia in the SD rat, which may be considered an IL-5 effect. This, together with our finding of unchanged Th1/Th2 cell numbers in the lung, could suggest that in the context of allergic sensitization, infections in subjects with different genotypes likely induces differential regulation with cellular production of different, selective cytokines. Altogether, our study indicates that differences in genotype in relation to environment may result in differences in involvement of contributing pathogenic factors and thus different responsiveness to therapeutic strategies.


   Acknowledgments
 
The authors thank Pieter Klok for excellent technical assistance. This work was supported by a grant of the Netherlands Asthma Foundation (97.42).

Received in original form October 1, 2001

Received in final form March 7, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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