Atopic Allergy: A Failure of Antigen-Presenting Cells to Properly Polarize Helper T Cells?

MARTIEN L. KAPSENBERG, CATHARIEN M. U. HILKENS, TINEKE C. M. T. van der POUW KRAAN, EDDY A. WIERENGA, and PAWEL KALINSKI

Department of Cell Biology and Histology, Department of Dermatology, and Laboratory of Clinical Experimental Immunology, Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands

	INTRODUCTION

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The immunological hallmark of adult atopic allergy is an aberrant production of helper T cell type 2 (Th2) cytokines interleukin 4 (IL-4), IL-5, IL-9, and IL-13 by Th cells reacting to common environmental antigens (1, 2). Mouse model experiments suggest strongly that airway hyperreactivity depends on IL-13 (3) and airway inflammation depends on IL-5 (4). In fact, the bias to type 2 cytokine production in atopic allergy is generalized (5), and an important issue is why these cells have developed in atopic allergic individuals. Th2 cells are effector Th cells that develop from naive Th cells after activation in the presence of certain polarizing signals. A first activation signal (signal 1) is the recognition by the T cell receptor of antigen-derived peptides presented in association with MHC molecules expressed by antigen-presenting cells (APCs). An obligatory second signal (signal 2) for these primary T cells is costimulation through the ligation of CD28 by B7.1 or B7.2, expressed by APCs as well. Primary Th cells do produce low levels of Th1 (e.g., interferon  [IFN-], tumor necrosis factor  [TNF-]) and Th2 cytokines, but these cytokines will be produced after one or more cell cycles and the concomitant maturation into effector Th cells. Polarization into either Th1 or Th2 cytokine production can be induced by various soluble or membrane-bound molecules, constituting a set of polarizing activities, acting as a third signal (signal 3) during the initial activation of naive Th cells. The process of polarization can occur early, since already within a few days after the initiation of the immune response lymph nodes contain polarized T cells (6). Here we discuss the perspective that the aberrant Th2 cell development in atopic allergy follows from a failure in the Th cell polarization by APCs.

	EARLY POLARIZATION BY IL-12 IS A CRUCIAL COMPONENT OF SIGNAL 3

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Various soluble and membrane-bound molecules may contribute to Th cell polarization and some are definitively not associated with APCs. There is little doubt about the view that IL-4 is critical for the development of Th2 cells (7). Although various cell types, including mast cells and eosinophils, can produce IL-4, mouse models indicate that under normal conditions it is the endogenous production of IL-4 by T cells themselves that plays a major role. For instance, BALB/c mice mount strong Th2 responses to Leishmania major, because prior to immunization these mice already harbor IL-4-producing memory Th cells that cross-react to a Leishmania-derived protein sequence (8). In this and other experimental models, IL-4 acts in a positive feedback loop. However, in most cases Th cells must first become memory T cells producing sufficiently high levels of IL-4. The level of this autocrine IL-4 production is strongly influenced by factors affecting T cells during the early stages after T cell stimulation, and these factors are often associated with APCs. At this early phase, an essential Th cell-polarizing molecule is IL-12. This cytokine is produced mainly by various types of APC and selectively upregulates IFN- production (9, 10). In high concentrations of IL-12, T cells polarize into Th1 cells. In low concentrations of IL-12, T cells will polarize into Th2 cells because of the lack of induction of IFN-. IL-18 is another Th1-polarizing factor, acting mainly by perpetuating IFN- production in IL-12-prepolarized Th1 cells (11). Alternatively, early-stage Th2 polarization may be induced directly (12, 13) or indirectly (14) by prostaglandin E₂ (PGE₂) and nitric oxide (15), which are produced by APCs or bystander cells and act mainly through inhibition of IL-12 production by APCs. The cell surface expression by APCs of OX40 ligand (OX40L) (16) and B7.1/2 (17) may provide further Th2-promoting pathways and the cell surface expression of intercellular adhesion molecule 1 (ICAM-1) (18), another Th1-promoting pathway, but the physiological significance of these membrane-bound molecules is less well established to date. Other non-APC-derived polarizing factors are the antigen dose (19) and hormones such as progesterone (20), of which the modes of action still must be determined, and it is possible that their effect in fact is exerted via APCs. As numerous mouse models for infectious diseases, autoimmunity, or allergy stress that production of IL-12 and the expression of its receptor in particular play an important and probably central role in the polarization of Th cells (9, 10), it is crucial, therefore, to understand how IL-12 production and IL-12 responsiveness are regulated.

	IL-12 PRODUCTION AND IL-12 RESPONSIVENESS ARE SUBJECT TO EXTENSIVE REGULATION

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IL-12 responsiveness is determined by the expression level of the signaling ₂ subunit of the IL-12 receptor (IL-12R) heterodimer, which is absent in unstimulated naive T cells but is rapidly induced to moderate levels after antigen-specific stimulation. IL-12R ₂ expression is strongly upregulated by IL-12 and type 1 IFNs and downregulated by IL-4, IL-10, and transforming growth factor  (TGF-). Consequently, highly polarized Th2 cells do not respond to IL-12 (21), as the expression of the IL-12R ₂ subunit is suppressed (22). For instance, the high levels of early IL-4 production after L. major infection in BALB/c mice prevents the upregulation of the expression of the IL-12R ₂ chain (23). IL-12 production by APCs can be induced by bacterial or viral infection or by incubation with microbial compounds, for example, lipopolysaccharide (LPS), DNA, or double-stranded RNA (dsRNA) (9, 10). Alternatively, IL-12 production by APCs can be induced in APCs through ligation of CD40 by CD40 ligand (CD40L) expressed by the T cells after initial activation by antigen. Like IL-12R ₂, the level of IL-12 production is extensively regulated (9, 10). It is upregulated by IFN- and downregulated by IL-10, TGF-, corticoids and by cAMP-inducing agents such as PGE₂,  agonists, and histamine (24). More importantly, dendritic APCs (DCs) can be committed to produce high or low levels of IL-12 (25). Immature DCs reside as antigen-sampling cells in the peripheral tissues and, on receiving an activating signal (signal 0), they migrate to draining lymph nodes and mature into efficient T cell stimulatory cells expressing the required costimulatory signals. The IL-12 production in mature DCs is committed when compounds that regulate IL-12 production are present during their initial stimulation at their immature stage. In this way, exposure of immature DCs to IFN- or viral RNA during their initial activation induces mature type 1 DCs, defined as APCs that on subsequent CD40-CD40L-mediated contact with T cells in the lymph nodes, produce high levels of IL-12 and induce Th1 cell development (26; and P. Vieira and coworkers, unpublished observations, 1999). In contrast, PGE₂ pretreatment induces mature type 2 DCs with low levels of IL-12 and the potential to induce Th2 cell development (27). It is to be expected that several more such DC-polarizing factors will be identified in the future. The data discussed above imply that they not only provide T cells with stimulatory (signal 1) and costimulatory (signal 2) signals, but also with a polarizing third signal. On the basis of their experience in inflamed peripheral tissues, DCs thus can inform T cells of the type of immune response they should mount. The composition of this signal 3 largely depends on the type of inflammatory agent and the type of affected tissue. In the case of viruses, the development of type 1 DCs with high IL-12 levels may be induced via a direct pathway (injection of double-stranded RNA) or an indirect pathway (IFN- production in infected nonspecific bystander immune cells). Similarly, certain helminths may induce type 2 DCs as a result of their PGE₂ production. Interestingly, although there are molecules (e.g., dsRNA) that may both start the specific immune response by activating DCs (signal 0) and contribute to the polarization of this response (signal 3), most molecules tested either activate DCs (i.e., TNF- and IL-1, LPS, and CD40L) or polarize these DCs (IFN- and PGE₂).

	ABERRANT THIRD SIGNALS IN ATOPIC ALLERGY

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IL-12 is also crucial in airway responses in the mouse. Normally occurring Th2 cell development, airway hyperreactivity, and eosinophil-mediated inflammation are abolished when rIL-12 is coadministered with allergen, either during the priming or in the challenging phase (28). The predisposition to develop eosinophilia and hyperreactivity after challenge dramatically varies between inbred mouse strains, depending on the ability to produce endogenous IL-12 in the airway tissues (29). In patients with atopic allergy Th2 cell development may indeed be causally related to inadequate APC-derived IL-12 production, as the IL-12 production in whole blood cultures of patients with allergic asthma (30) and in peripheral blood monocytes of patients with atopic dermatitis (31) is significantly lower compared with control subjects, and is associated with decreased IFN- production in the Th cells (30). Also, the expression of IL-12 mRNA in biopsies of allergen-challenged patients with allergic asthma is increased after steroid therapy (32). IL-12 may not be the only important factor in this respect, as simultaneously with the decreased IL-12 production, PGE₂ production is increased in atopic dermatitis monocytes (31, 33), in association with indomethacin-dependently decreased IFN- production (33). These data suggest that atopic allergy is causally related to the occurrence of type 2-biased APCs with decreased IL-12 and increased PGE₂ production and the ability to induce Th2 cell development. These studies reflect the properties of peripheral blood APCs, whereas the most relevant APCs, during both the sensitization and inflammatory phases of allergic asthma, are dendritic cells. Unfortunately, these cells are difficult to study in vitro, because of the laborious isolation procedures, the low purity, and the low cell yields.

	TYPE 1 AND TYPE 2 APCs: CAUSE OR RESULT OF ABERRANT Th CELL POLARIZATION?

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An interesting question is whether the occurrence of these type 2-biased APCs in atopic allergy reflects an intrinsic aberrancy of APCs, or follows from commitment imposed by extrinsic factors. As implied by the concept that APCs, especially DCs, become functionally polarized according to the signals they experience in affected peripheral tissues, an attractive hypothesis is that atopic APCs are type 2 polarized by aberrant microenvironmental signals. In the airways, DCs are located within, or close to, the epithelium of the trachea. In allergic asthma, the airway epithelium shows enhanced levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) and inflammatory cytokines (34) and an impaired repair mechanism ([35], this issue). Similarly, skin epithelial cells from nonlesional skin of patients with atopic dermatitis produce much higher GM-CSF levels compared with control subjects (36) and have an impaired water barrier function. It is, therefore, tempting to speculate that the epithelial cells in these groups of allergic patients also express aberrant factors that bias the functional phenotype of DCs. As the high GM-CSF production in skin epithelial cells can be induced by IFN- (37), it remains to be established whether the epithelial cell abnormalities are not simply the result of subclinical, chronic inflammatory conditions, such as in nonlesional atopic dermatitic skin (38).

	CONCLUSION

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Here we have discussed the evidence that, as for effector B and T cells, effector APCs can also be functionally polarized and that the occurrence of type 2-biased Th cells in atopic allergy may result from the activity of type 2-biased APCs. As for T and B cells, polarization of DCs is imposed by cells that are in close contact with these DCs at the moment of their initial activation in their early immunocompetent ("immature") stage. The generalized aberrant APC phenotype in atopic allergy thus may follow from an aberrant function of cells resident in peripheral tissues, such as epithelial cells.

	DISCUSSION

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Holgate: I have a question on the importance of PGE₂, which probably is originating through the induction of the inducible form of cyclooxygenase Cox2 by keratinocytes and, in the airways, by bronchial epithelial cells. Recent work has shown that that is not the primary reponse, it is a secondary response, in the lower airways at least. This response is secondary to the generation of epidermal growth factors, probably from injury to the epithelium. Is it possible that the PGE₂ you measure from keratinocytes is secondary to keratinocyte activation by EGF ligands released as a consequence of epithelial injury and that the defect lies in the inability of the epithelial cells to initiate an adequate repair response, once it is injured in some way?

Kapsenberg: I fully agree. It may be that this whole business of Th2 cells is the result of this primary aberrancy of epithelial cells, either to respond, or an intrinsic failure to produce certain compounds. I mentioned here PGE₂ and IFN-, but I think these are just examples showing how dendritic cells can be committed to a certain functional phenotype.

Holt: In relation to the range of factors that can be produced locally, as far as the airways are concerned the important one we are thinking about is GM-CSF, which is made almost constitutively in the airways and it certainly upregulates quite spectacularly. We also know from some of the experimental systems that there is a range of molecules which are also made quite readily in that same microenvironment, which seem to regulate the capacity of these dendritic cells to read out GM-CSF signals. In the experimental animal systems one of these happens to be NO. The real question I have goes back to the skin. Have you had a chance yet to look at the difference between keratinocytes from lesional sites and nonlesional sites, because that is addressing the same issue in a different way?

Kapsenberg: Dr. Girolomoni provided us with supernatants of keratinocytes from lesional and nonlesional sites. There was not much difference in terms of GM production. Still, it is uncertain if keratinocytes from the nonlesional site were nonactivated. Dr. Bruynzeel-Koomen showed that endothelial cells at the nonlesional site show an activated profile, so, even at nonlesional sites something is going on.

Aarden: There are two papers from Lille, one on the NO synthase knockout mice, that get a Th1-dominated response. They suggested that NO might inhibit IL-12 production and this has been confirmed in vitro for a murine cell line. We have data that IL-12 production by human monocytes is very strongly inhibited by NO.

Björkstén: Back in the Seventies there were several papers about a dysfunction in monocytes in relation to atopy. My other comment is a question: Have you looked whether there is a difference in the propensity for IL-12 production in young APCs or old or established APCs? The reason is, of course, the Th2 skewing in early infancy.

Kapsenberg: There are several papers showing that the IL-12 production is relatively low in cord blood. It will be intriguing to see when IL-12 production and IL-12 responsiveness come up.

Aarden: You need probably IFN- to get decent production and you need IL-12 to get IFN-. There is a way out, and that is the NK cell. I know that NK cell function in cord blood is inhibited. There seems to be a T cell population that inhibits at least the lytic capacity of NK cells, maybe also the IFN--producing activity. Is that related to this Th2 skewing of the response?

Kapsenberg: So far, there are no hard data that IFN- derived from NK cells is a major polarizing factor. NK cells work in in vitro models, where you have a lot of cells packed together.

Aarden: The problem might be to activate the NK cells in a proper way. In vivo, it might be a virus.

Lamers: Is there anything known about IL-12 production by dendritic cells in the skin after UV irradiation under these conditions?

Kapsenberg: There are two reports about IL-12 production by Langerhans cells. However, we could not reproduce these data. The IL-12 p70 production by Langerhans cells was extremely low.

Aalberse: I would like to discuss the danger signal. Two questions. Dr. Holt mentioned that mycobacteria were surprisingly good in inducing Th2 and IgE. That is different from what I thought. The other issue is: To what extent are allergens dangerous, in Dr. Matzinger's sense?

Kapsenberg: I have some doubts about their intrinsic ability to have a biological activity to turn on the immune system. Dr. Platts-Mills is probably going to tell us that we are exposed to 1 ng of these compounds per year. Probably, the number of molecules is not enough to have any effect at all. I think we are dealing with a default pathway of Th2 cells at the mucosa. There is no danger, it is just the route where they come in. If you put in another route, the immunological effect will be different.

Holt: Let me clarify the thing about mycobacteria. The comment we were talking about was in fact Freund's complete adjuvant, which is different from mycobacterial infection. Freund's adjuvant in the mouse is really good for getting IgE responses, notwithstanding some publications.

Aalberse: How does that relate to the danger model?

Kapsenberg: It depends on the type of danger and on the type of tissue where the danger occurs. There are defaults in tissues for a certain danger. But also the type of molecule which induces the danger. If it is a very potent mycobacterium, it overrules what the tissue tells. So, you may have a Th1-biased response to infection to herpes zoster in the eye, which may damage the eye; it overrules the default pathway, which is some regulatory T cell or Th2 cell.

Kauffman: You have nicely shown that when you preincubate the cells with PGE₂ you will have a polarization into the Th2 direction. Dr. Borger in our laboratory has shown, however, that both Th1 and Th2 cells in their final stage are effectively inhibited by PGE₂, since in both cells you will get an elevation of cAMP. You can interpret the enhanced PGE₂ production in atopic dermatitis not only as a signal that will polarize your system in a Th2 direction, but also as a mechanism that will inhibit the intense inflammatory response.

Kapsenberg: If T cells are stimulated in the absence of adequate levels of IL-2, for example when costimulation is not optimal, PGE₂ inhibits all cytokines. Selective regulation, when only IFN- goes down, requires high levels of IL-2. But dendritic cells are 100 times more sensitive to PGE₂ than T cells, as shown by van der Pouw Kraan and coworkers. Whatever happens on the level of the T cell, it may not be as important as the effect of PGE₂ on DC, fitting the concept that the APC is the director of the T cell response.

Weiss: I want to make some comments about the epidemiology. I am not sure how it bears on the actual Th1/Th2 paradigm. The question is: How does it have relevance in terms of trying to understand what is going on in human disease. I do a large-scale asthma genetic study in rural central China. Certain patterns are emerging that have relevance to Western disease. First of all, the prevalence of asthma is very low, a fifth to a twentieth of what we see in Western industrialized countries. The second point is that clinical asthma is the same, except for one outstanding feature. Unlike childhood asthma in Western industrialized countries, which is almost universally associated with hay fever, there is no hay fever in China whatsoever. When you out migrate these people to live in a Western industrialized country, they develop hay fever, but they certainly don't have hay fever in the environment they are in. The third important point is with regard to the atopy phenotype. Skin test positivity is present in both asthmatic and nonasthmatic subjects, so it does not really bear any relationship to disease. The fourth important thing about the phenotype is that these people have very high IgE levels. The average total IgE level is about 500. That is the same in asthmatic and nonasthmatic people. We measured parasite infections in these people and that is clearly not the explanation for the difference. I don't know how it is that the tissue specificity is important or the nature of the antigenic signal, or what is doing this. I think that those of you who are molecular immunologists need to help me.

Holt: How do you know it is not the parasite?

Weiss: The IgE levels in the parasitized and nonparasitized asthmatics and nonasthmatics are not terribly different. They don't all have parasites. It is just like any other environmental exposure, the attack rate is 30 or 40%.

Aarden: Do you think it is an environmental or a genetic effect? Have you looked at recent immigrants?

Weiss: I can take children of nonasthmatic parents or one asthmatic parent and one sibling in the family has asthma and the other sibling has not, so you can control for the genetics. They will have similar IgE levels, they will both be very high, and yet one child will be asthmatic and the other child will not be.

Martinez: There are 30-fold differences in the prevalence of asthma between people who are in mainland China and people who are living, for example, in Hong Kong or Malaysia. These are people with the same ethnic background as the people in mainland China. So, in answer to your question, most probably this has something to do with environmental factors.

Weiss: Yes, I would agree. Other studies have shown that there is a gradient of asthma prevalence, depending on how urbanized or Westernized the population is.

Yazdanbaksh: I am not sure whether you can just wipe away the idea that maybe helminths play a role. Helminth parasites are extremely chronic and they lead to strong downmodulation of inflammatory responses. Have you compared populations before and after treatment?

Weiss: No, we have not done that. Our study is observational. Lynch published data that suggest that if you take people that have been parasitized and have asthma, that you can make the asthma worse by treating their disease. I am not saying that.

Martinez: It was an uncontrolled study.

Weiss: Yes, but as the IgE level drops these people develop more skin test positivity and more asthma symptoms.

Martinez: I am insisting that it was an uncontrolled study, because I have been reviewing the literature on the use of medicines for asthma and, as you know, in most cases 60-70% of any effect is placebo effect. So you have to be very careful if studies are uncontrolled.

Weiss: I would go back and say: It is difficult to explain the findings in China on the basis of the presence or absence of parasitic infection. The lack of a difference in total IgE between people who have active parasites and those people who have no evidence of parasitic infections whatsoever is really quite remarkable.

Holgate: I would like to come back to this danger thing again, whatever it means. One of the interesting features that seem to be coming out of the allergy literature now is the fact that many allergens are biologically active: the cysteine proteases and the serine proteases in dust mites and so on and on. We have recently done an immunostaining study of some bronchial biopsies from patients with active asthma with one of Dr. Chapman's monoclonal antibodies to Der p 1, and the bronchial epithelium just lights up. Just in ordinary people walking around the street. With antigen not only on the surface, but actually right through the epithelium. These allergens can activate chemokine and cytokine responses in epithelial cells (and I suspect in other cells as well). The papers by Steward and Thompson describe epithelial cells responses, but there will be other responses.

Kapsenberg: It is a very interesting observation. I would not expect many molecules to be present and they are easily degraded at a very early stage. It is surprising.

Platts-Mills: Not only would I not expect that many, but I think that if you know how the allergen is being inhaled in terms of Der p 1, it is extraordinary surprising that it would be diffusely spread out. I would worry about that. The other thing is that if you ever mention that these allergens are enzymes, you have got a list of ones that are and ones that are not. Der p 2 is not, Fel d 1 is not, all the lipocalins are not.

Kapsenberg: We should realize that there are antigens and adjuvants. Within one allergenic entity you may find not only allergen molecules but also other components that may have crucial biological activities.

Holt: I have had this debate with Dr. Steward for years, about doing something to the active site of Der p 1 and rendering it nonenzymatic, to see if it is immunogenic in various models. I think he might have done it.

	Footnotes

Correspondence and requests for reprints should be addressed to M. L. Kapsenberg, M.D., Academic Medical Center of the University of Amsterdam, Laboratory of Cell Biology and Histology, Meibergdreef 15, 1105AZ Amsterdam, The Netherlands. E-mail: m.l.kapsenberg{at}amc.uva.nl

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