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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 28, 2003 as doi:10.1096/fj.02-0985fje. |
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,
* Department of Pharmacology and Pathophysiology, Utrecht Institute for Pharmaceutical Sciences and
¶ Faculty of Biology, Utrecht University, Utrecht;
Laboratory for Pathology and Immunobiology, National Institute of Public Health and the Environment, Bilthoven;
Department of Health Risk Analysis and Toxicology, Maastricht University, Maastricht; and
Department of General Pediatrics, Wilhelmina Children’s Hospital, UMC, Utrecht, The Netherlands
2Correspondence: Department of Pharmacology and Pathophysiology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands. E-mail: M.A.Bloksma{at}pharm.uu.nl
SPECIFIC AIMS
Based on the hygiene hypothesis that states that lack of early childhood bacterial infections would favor development of allergic disease, we hypothesize that genes controlling antibacterial resistance can be of significance in the pathogenesis of allergic disease as well. We therefore studied whether Nramp1 (Slc11a1), a gene that strongly determines resistance (Nramp1r) or susceptibility (Nramp1s) to intracellular bacteria at the macrophage level in the mouse, affects sensitivity to induction of allergic asthma in a mouse model.
PRINCIPAL FINDINGS
1. Nramp1 does not influence airway hyperresponsiveness and inflammation
Airway hyperresponsiveness to methacholine and eosinophilic airway inflammation, both hallmarks of asthma, were assessed after airway challenge of ovalbumin-sensitized mice with ovalbumin aerosols, using challenge with saline aerosols for the controls. Increasing doses of aerosolized methacholine caused similar (P>0.10), shallow increases in airway responses, assessed as Penh, in saline-challenged Nramp1s and Nramp1r mice and significantly higher responses in the ovalbumin-challenged mice. However, the degree of hyperresponsiveness was the same in both strains. Determination of types and numbers of leukocytes in the lung lavage fluid as a measure of airway inflammation showed that ovalbumin challenge induced a significant increase in the number of eosinophils compared with saline challenge, but the increase was similar in Nramp1s and Nramp1r mice.
2. Nramp1 affects the IgE response
The serum antibody levels before (data not shown) and after saline challenge were similar in both strains. Compared with these levels, ovalbumin challenge increased levels of ovalbumin-specific IgG1 and IgG2a, particularly of total and ovalbumin-specific IgE. Whereas the increases in IgG1 and IgG2a levels were similar in both strains, increases in IgE levels were much less prominent in Nramp1r mice than Nramp1s mice (Fig. 1
).
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3. Nramp1 affects the development of Th2 cell responses
Saline-challenged mice of both strains had no detectable levels of IL-4, IL-5, or IL-13 in their lung lavage fluid and low levels of IL-10 (Fig. 2
A–D). Ovalbumin challenge increased the levels of these cytokines significantly in both strains, but cytokine levels in the lung lavage fluid of Nramp1r mice were only half to one-third those found in the lavage fluid of Nramp1s mice. IFN-
was not detectable in the lavage fluid (detection limit ELISA: 10 pg/mL), irrespective of treatment and mouse strain.
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4. Nramp1 affects mast cell degranulation
Levels of mucosal mast cell protease-1 (MMCP-1) in the lung lavage fluid, a measure in vivo mast cell degranulation, were low in saline-challenged mice but significantly increased after ovalbumin challenge (Fig. 2E
). This increase was markedly lower in the Nramp1r mice. To exclude a direct role of Nramp1 in the degranulation, Nramp1 expression was assessed by RT-PCR. PMA-stimulated bone marrow-derived mast cells and CFTL-12 mast cells did not express Nramp1 RNA, whereas positive control cells, i.e., macrophages stimulated with IFN- and LPS, did.
CONCLUSIONS
In the present study we investigated whether the Nramp1 gene, which controls the resistance to intracellular bacteria, influences sensitivity to induction of allergic asthma-like disease in mice. We demonstrated that ovalbumin sensitization and challenge of Nramp1s and Nramp1r mice caused comparable cholinergic airway hyperreactivity, airway eosinophilia, and a similar increase in serum levels of ovalbumin-specific IgG1 and IgG2a. Ovalbumin challenge, however, induced significantly lower serum levels of total and ovalbumin-specific IgE in Nramp1r mice and markedly less mast cell degranulation in the airways of these mice compared with Nramp1s mice. Ovalbumin challenge of Nramp1r mice led to significantly less release of IL-4, IL-5, IL-10, and IL-13 into the airways than similar treatment of Nramp1s mice.
The lower IgE response in the Nramp1r mice in the absence of differences in the specific IgG1 and IgG2a responses is in line with previous experiments by other investigators. In Nramp1 congenic B10 mice, it was demonstrated that Nramp1r mice mounted a lower IgE response than Nramp1s mice after infection with an attenuated strain of Salmonella typhimurium, but similar responses of total immunoglobulins (IgG1, IgG2a, and IgM). Thus, the antibody response to a soluble protein antigen, like ovalbumin, and to viable salmonella bacteria apparently is similarly influenced by Nramp1, although the role of macrophages in both responses is likely to be different.
How Nramp1 determines the height of the specific IgE response is not known, but macrophage activity as controlled by Nramp1 probably plays a role. Airway macrophages of ovalbumin-sensitized BALB/c mice reportedly are activated upon respiratory ovalbumin challenge, whereas the degree of macrophage activity is known to control the height of antibody responses. Notably, macrophages of Biozzi high antibody responder mice were found to display less catabolic activity, antigen degradation, and bactericidal activity than macrophages of Biozzi low antibody responder mice. Likewise, Nramp1r macrophages on B10 or BALB/c background were shown to display faster and/or more activation in reaction to various stimuli than Nramp1s macrophages. Hence, the lower IgE production by the Nramp1r strain vs. the Nramp1s strain can be related to more prominent pulmonary macrophage activation, including more NO production, upon ovalbumin challenge of the Nramp1r strain.
Elevated NO production may be relevant, because NO was found to be the mediator by which alveolar macrophages down-regulate the presentation of protein antigens by pulmonary dendritic cells to primed rat T cells in vitro. Moreover, NO was shown to be a potent inhibitor of T cell proliferation irrespective of their cytokine profile. So, elevated NO production by sensitized Nramp1r mice after ovalbumin challenge is likely to result in inhibition of T cell restimulation in these mice. This notion is compatible with our observation that the Nramp1r mice had lower IL-4, IL-5, IL-10, and IL-13 levels in the lung lavage fluid after respiratory ovalbumin challenge than the Nramp1s mice. The decreased IL-4 and IL-13 production in turn conceivably would have led to reduced production of IgE, as actually observed. However, why ovalbumin-specific serum levels of IgG1 were similar to those of Nramp1s mice is unknown, since IgG1 production, like IgE production by ovalbumin/alum sensitized BALB/c mice, was found to be highly dependent on IL-4. Another issue is that the significantly lower IL-5 levels in the airways of the Nramp1r mice compared with the Nramp1s mice were not paralleled by lower numbers of eosinophils. This may be explained by the significantly lower IL-10 levels in the airways of Nramp1r mice, since IL-10 is a potent inhibitor of allergen challenge-induced eosinophilic inflammation in the airways of BALB/c mice. Besides having anti-inflammatory activity, IL-10 was reported to increase cholinergic responsiveness of allergen-sensitized and -challenged BALB/c mice. We, however, observed the same degree of cholinergic responsiveness in both strains, although Nramp1r mice produced significantly less IL-10 in the airways than the Nramp1s mice. Likewise, although IL-13 may be closely related to airway hyperresponsiveness, the significantly lower IL-13 levels in the airways of the Nramp1r than the Nramp1s mice were not paralleled by a decrease in airway hyperresponsiveness. These data stress the complexity of development of nonspecific airway hyperresponsiveness and confirm that there are various mechanisms leading to this phenomenon.
Besides reduced IgE production, Nramp1r mice displayed reduced mast cell degranulation. The reduced serum IgE levels may conceivably have led to reduced mast cell-bound IgE in the lungs and hence to less allergen-induced IgE cross-linking and degranulation. Moreover, circulating IgE was found to increase Fc
RI expression by mast cells in mice and thus the sensitivity to and intensity of allergen-induced mast cell degranulation in a concentration dependent way. IL-4 and IL-10 are shown to be able to promote the proliferation of mast cells and allergen-induced degranulation of these cells. Therefore, the reduced levels of IL-4 and IL-10 in the airways of Nramp1r mice may have led to the reduced mast cell degranulation as well. Decreased mast cell degranulation in Nramp1r mice may also be explained by the presumed increased NO production by Nramp1r macrophages in response to ovalbumin challenge as stated above, since NO was found to cause a concentration dependent inhibition of mast cell degranulation in vitro as well as in vivo. Finally, since mast cells belong to the myeloid lineage and Nramp1 is expressed almost exclusively by myeloid cells, it is possible that Nramp1 is expressed in mast cells and so is able to affect mast cell degranulation directly. PCR analysis, however, showed that Nramp1 RNA is not expressed in stimulated cultures of mast cells. Thus, a direct effect of Nramp1 on mast cell degranulation is unlikely.
We conclude that Nramp1 can affect the development of IgE-mediated allergy but not the development of airway hyperresponsiveness, an untoward symptom of asthma, in the mouse. Allergy is considered a risk factor for the development of asthma in humans. In our mouse model, components of allergy in Nramp1r were lower, including levels of IgE, IL-4, and IL-13. Eosinophilic inflammation, which has been related to airway hyperresponsiveness, however, was not affected by Nramp1, which might explain the lack of effect of Nramp1 on development of airway hyperresponsiveness. Effects of Nramp1 on macrophage function probably mediate its selective effects on Th2-mediated immunity (Fig. 3
). Since Nramp1 controls the nonspecific defense of macrophages against intracellular microorganisms, it is likely to affect the effects of intracellular bacteria on development of allergic diseases. This is a subject of current studies.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0985fje; to cite this article, use FASEB J. (March 28, 2003) 10.1096/fj.02-0985fje 
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, macrophage; Th2, T helper 2