PERSPECTIVE
Food for Thought
Can Immunological Tolerance Be Induced to Treat Asthma?

Lauren Cohn

Department of Internal Medicine, Section of Pulmonary and Critical Care, Yale University School of Medicine, New Haven, CT

The immune system has the onerous responsibility of recognizing when to fight infectious pathogens and when to temper responses that might cause harm. T lymphocytes have evolved mechanisms to regulate both of these tasks, relying on interactions with other cells to transmit appropriate instructions. Activation of T cells requires two signals, one from the T cell receptor (TCR) interacting with a specific antigen in the context of MHC on an antigen presenting cell (APC) and one from costimulatory interactions between cells, like the CD28-CD80/86 interaction and/or LFA-1-ICAM-1. If both of these signals are present, T cells produce interleukin (IL)-2, proliferate, and become effector cells in an immune response. The classical doctrine is that CD4 T cells can be stimulated to become T helper (Th)1 cells producing interferon (IFN)- and lymphotoxin or Th2 cells producing IL-4, IL-5, IL-10, and IL-13. Recent evidence also suggests that regulatory subsets of CD4 T cells can be generated from naïve CD4 T cells. If TCR or costimulatory signals are absent or inadequate, T cell activation will be thwarted, resulting in tolerance.

Tolerance is a state of nonresponsiveness to an antigen that develops by one of three mechanisms: deletion of antigen responsive cells (clonal deletion), inactivation of responsive cells (anergy), or activation of regulatory cells. Self-reactive lymphocytes are deleted in the thymus at an early stage in lymphoid development and are absent from the mature lymphoid repertoire. But, not all self antigens are present in the thymus, and mature lymphocytes that interact with self proteins in peripheral tissue are either deleted or rendered unresponsive. Inactivation of lymphocytes may be an active process (suppression) or may be the result of disarming lymphocytes, rendering them incapable of causing harm (anergy) (Figure 1).

View larger version (25K): [in this window] [in a new window]   Figure 1.   Induction of peripheral tolerance at mucosal surfaces.

The peripheral mechanisms that guard us from self- reactivity also protect us from immune responses to nonharmful proteins. Mucosal surfaces encounter foreign antigens continually, yet under normal circumstances, an active immune response does not develop to nontoxic foods or inhaled particles. Such a response would be detrimental to the host. In 1911, Wells showed that proteins absorbed in the small intestine induce a state of immunological tolerance, protecting guinea pigs from systemic anaphylaxis (1). Since then, many studies have shown that ingestion of an antigen leads to unresponsiveness to subsequent challenge with that antigen. In addition to the gut, both the respiratory tract and vaginal mucosa have been shown to be sites of tolerance induction (2, 3).

Oral tolerance has been used in animal models to block development of different diseases. In Th1-mediated autoimmune disease models including experimental autoimmune encephalomyelitis (EAE, a model of multiple sclerosis (MS)), diabetes, autoimmune uveitis, and collagen-induced arthritis, antigens that were ingested prior to induction of disease led to reduced pathology (4). In Th2-induced diseases like allergic airway inflammation, oral tolerance also blocks development of disease (5, 6). In this issue, Russo and colleagues show that feeding ovalbumin (OVA) to mice has potent inhibitory effects on the development of acute allergic airway inflammation, mucus production, and airway hyperresponsiveness (AHR) (7).

Oral tolerance is easily induced in animals by feeding protein antigens over a period of days to weeks. The mechanisms of oral tolerance have now been well characterized, especially in response to OVA. The OVA-specific TCR transgenic mouse, DO11.10, and now other TCR transgenic mice, have allowed investigators to characterize and track the antigen-specific CD4 T cells in vivo (8). The dose of antigen and duration of exposure may determine the predominant mechanism(s) of tolerance (9). High dose of antigen generally leads to anergy and deletion, while low doses of antigen stimulate active suppression. In general, early after any oral dose of antigen there is expansion of antigen-specific CD4 T cells, with increased production of both Th1 and Th2 cytokines and expression of early activation markers like CD69, indicating activation of naïve CD4 T cells. When high doses of antigen are ingested, there is down-regulation of cell surface expression of the TCR followed by deletion of antigen-specific T cells in the gut-associated lymphoid tissue. The CD4 T cells that persist have low expression of IL-2R, a reduced response to antigen, and do not secrete cytokines (IL-2, IL-4, IL-10, or TGF-) when activated with specific antigen. After ingestion of low doses of antigen, the activated, antigen-specific CD4 T cell population persists, but the cytokine pattern shifts to production of IL-4, IL-10, and TGF-. Mice exposed to inhaled antigens for short time intervals over a period of days to weeks also develop tolerance (13). Inhaled antigens have been shown to stimulate tolerance by both anergy and active suppression (16, 17). Both Th1 and Th2 responses can be suppressed by mucosal antigen exposure (18, 19). Some studies suggest that low dose/less frequent dosing leads to Th1-predominant suppression and high dose/continuous antigen administration results in Th2-predominant suppression (20). Tolerance induced at different mucosal sites appears to result from similar immunologic mechanisms, leading to antigen-specific, systemic down-regulation of the response to immunization with antigen and these effects can persist for many months.

Until recently, characterization of the regulatory cells responsible for mucosal tolerance was elusive. In different animal species, at different sites of tolerance induction, and in different models of tolerance, CD4+, CD8+, and / + cells were shown to be responsible for suppression (9, 22, 24), but characterization of the TCR usage, the epitope specificity, and individual cell cytokine secretion was not possible from bulk cultures of lymphocytes. By cloning from populations of cells or using T cells from TCR transgenic mice, active suppressor cells have been identified. These technologies have allowed investigators to generate well defined populations of cells in vitro and later test them in vivo for suppressor functions. CD4 T cells that differentiate at mucosal surfaces in response to soluble proteins secrete the immunosuppressive cytokines, TGF- and IL-10. Two classes of antigen-specific CD4 T suppressor cells have been identified, Th3 and T regulatory 1 (Tr1) cells. Th3 cells were cloned from mesenteric lymph nodes of mice fed myelin basic protein (MBP) and then intraperitoneally immunized with MBP (9). When activated with MBP, these clones each produced high levels of TGF- and sometimes IL-4 and IL-10, but never IFN-. When the cells were adoptively transferred into mice at the time of induction of EAE, disease severity was markedly reduced. The suppressive effects of Th3 cells were blocked when an anti-TGF- antibody was administered. At nonmucosal surfaces, TGF--producing cells were rarely generated. Tr1 cells were generated in vitro from naïve TCR transgenic CD4 T cells when activated with antigen in the presence of IL-10 (27). This cell population produces high levels of IL-10, but low levels of TGF-, IFN-, IL-4, and IL-2. They proliferate poorly and, when activated, inhibit proliferation of bystander cells. When murine Tr1 cells were adoptively transferred into mice and activated with specific antigen, they protected from inflammatory bowel disease. These two subsets of CD4 regulatory cells received both TCR and costimulatory signals from APCs during development, and when activated by antigen, they secrete cytokines (Figure 1). Yet, they blur the line between traditional effector cells and anergic cells because they proliferate poorly and secrete minimal IL-2.

APCs at mucosal surfaces have unique features that stimulate the development of tolerance. The primary APC at mucosal surfaces is the dendritic cell (DC). Mucosal DCs have an immature phenotype that promotes uptake and processing of antigens which are presented to specific T cells by DC in the local lymphoid tissue (28, 29). Unlike splenic DC, both Peyer's patch and respiratory tract DCs produce IL-10 which promotes IL-4- and IL-10-producing CD4 T cell populations. In vitro, DCs isolated from the respiratory tract and the gut skew T cell differentiation toward Th2/Th3, while DCs isolated from spleen tend to stimulate Th1-like responses. It should be noted that some studies looking at Th1/Th2 generation at mucosal sites identified populations of IL-4 producing cells and called them Th2. In fact, these populations might also include TGF- producing cells. This distinction becomes important since Th3 cells can suppress both Th1 and Th2 cell effects (4). Once a population of effector/suppressor T cells producing IL-10, IL-4, and TFG- is established in the lymphoid tissue, the cytokine environment will influence the cell-cell interactions that lead to immune tolerance. IL-4 is critical for Th2 cell differentiation (30) and, in some autoimmune diseases, immune deviation toward Th2 leads to protection from disease (20, 31). TFG- suppresses IFN- and IL-12 production, explaining its effects in vivo in shifting naïve T cell responses toward Th2/Th3 (32). Low expression of Class II MHC and costimulatory molecules on DCs at mucosal surfaces might be expected to play a role in the deviation of mucosal immune responses toward Th2/Th3 or anergy (33, 34). IL-10 decreases MHC Class II and CD80 expression (35). Current evidence, though, does not show a significant difference in the surface expression of CD80/86 on mucosal DCs compared to splenic DCs (28, 29). In addition to immune deviation, mucosal DCs have other methods to induce tolerance. DCs expressing CD8 stimulate apoptosis of CD4 T cells by fas-fasL interaction, thus deleting reactive T cells (36), and suppress CD8 T cell activation and cytokine production (37).

In the most widely studied model of acute allergic airway inflammation, mice immunized with OVA in alum intraperitoneally (i.p.) and later exposed to inhaled OVA develop airway inflammation with CD4 T cells and eosinophils. If the animals are fed OVA prior to the immunization, tolerance is induced. Russo and coworkers, in earlier work, showed that oral tolerance could suppress the development of airway eosinophilia (6). Now Russo and coworkers report that in multiple different strains of mice fed OVA for five days and then immunized and aerosol challenged with OVA, eosinophilia, AHR, and mucus production are all reduced (7). Nakao and coworkers showed that OVA feeding inhibits airway inflammation by markedly reducing splenic responses to immunization with OVA (5). Thus, oral tolerance inhibits T cell priming to OVA, few OVA responsive CD4 T cells are generated, and on subsequent exposure to inhaled OVA, there are few antigen-specific cells to recruit to the respiratory tract. The effects of oral tolerance are profound if the feeding regimen precedes immunization and the establishment of active CD4 effector T cells. When the mice are fed antigen subsequent to the i.p. immunization, inhibition of airway inflammation is less effective. There is progressively less inhibition of inflammation, the longer the delay in feeding the antigen after immunization. Thus, as more CD4 effector T cells are generated, the ability to tolerize becomes more difficult. If CD4 effector T cells are generated and then recruited to the respiratory tract with inhaled antigen, oral feeding increases airway inflammation. These are similar to findings by Holt and colleagues, who showed that in presensitized animals, aerosol exposures enhanced immune responses (38). In models of Th1-mediated autoimmune diseases, when oral tolerance is initiated after the induction of disease, disease pathology is also enhanced (4).

In chronic diseases, persistent, localized inflammation leads to disease pathology. Allergic asthmatics have established antigen-responsive CD4 T cells in the airways and local lymphoid tissue. Children predisposed to develop asthma often have antibodies to common allergens (39), indicating their immune system is already primed to the allergen. Based on animal models of asthma, one might speculate that feeding allergens to allergic asthmatics would increase inflammation and be dangerous. Thus, animal models might convince us that attempts to stimulate mucosal tolerance should not be used to prevent or treat asthma.

Yet patients with both autoimmune and allergic inflammatory diseases have tolerated the mucosal administration of antigens without flares in disease, and some have shown symptomatic improvement. In patients who have MS or rheumatoid arthritis that were fed bovine myelin or chicken collagen, respectively, disease pathology was not increased (40, 41). A subset of patients in each study showed improvement in symptoms. In a double-blind, placebo-controlled, multicenter study, long-term feeding of type II collagen at low dose led to a significant reduction in symptoms (42). In another trial, a single dose of myelin had no effect on the course of disease in MS (20). In allergic diseases, immunotherapy using mucosal routes of administration have also proven safe and, in some cases, effective. In atopic patients, oral, sublingual, or inhaled administration of mite or pollen antigens for one to two years led to a reduction in symptoms (43) and decreased local inflammation (48). In asthmatics, these therapies led to a reduction in dyspnea and reduced AHR (43).

Why do animal models fail to predict this clinical outcome in asthma? One explanation is that the animal model used to test oral tolerance in asthma does not closely mimic human disease. While airway inflammation is present in both the animal model and in asthmatic patients with quiescent disease (the group that would receive immunotherapy), the characteristics of the inflammatory response are quite different. The acute airway inflammation induced in mice is characterized by a high density of antigen-specific CD4 effector cells and eosinophils surrounding the airways and extending to the blood vessels (49, 50). In comparison, in chronic asthma there is less airway inflammation, including fewer effector cells and eosinophils, and in local lymphoid tissues reside memory T cells responsible for recall responses (51, 52). One possibility is that the state of activation and the subsets of CD4 T cells present in the respiratory tract of asthmatics allow tolerance to be induced in response to administration of mucosal antigens. It is well known that it is difficult to tolerize CD4 effector cells and this may be related to their hair-trigger activation response (53). Memory cells require more stringent activation signals and may allow tolerance induction (54, 55). In addition, there are other cell components in the chronic inflammatory milieu in asthmatics that may affect the local immune response and/or the recruitment of antigen-experienced T cells to the respiratory tract after mucosal antigen administration, thus allowing tolerance to be induced. These may include effects of regulatory cells, anti-inflammatory cytokines and chemokines, and the number and activity of APCs and their costimulatory molecules. It is also possible that airway remodeling in asthma affects lung inflammation. While these concepts are presently speculative, it will be important to determine if tolerance can be induced in asthmatics, what mechanisms control tolerance induction, and how long lasting the effects are. Future studies in humans, for now, will provide us with this information. As only limited analyses can be performed on patients, research should be directed toward developing animal models with chronic inflammatory airway disease to aid in testing immunomodulatory therapies for asthma.

	Footnotes

Address correspondence to: Lauren Cohn, M.D., Yale University School of Medicine, P.O. Box 208057, New Haven, CT 06520-8057. E-mail: lauren. cohn{at}yale.edu

(Received in original form April 6, 2001).

Acknowledgments: The author thanks Dr. Donna Farber for critical reading of this manuscript.

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