INTRODUCTION

TOP ABSTRACT INTRODUCTION CYTOKINES AND CYTOKINE... ANTI-IgE MONOCLONAL ANTIBODIES CHEMOKINES AND CHEMOKINE... MEMBERS OF THE IL-1... COSTIMULATORY SIGNALS: CD28 AND... CONCLUSION REFERENCES

Naive T lymphocytes travel to T cell areas of secondary lymphoid organs in search of antigens presented by dendritic cells (Figure 1). On encountering specific antigen, T helper naive precursor (Thp) cells become activated, an event that is regulated not only by engagement of the T cell receptor (TCR) with peptide presented in the context of MHC class II molecules but by a number of costimulatory signals. Recently activated effector T (Th_e) cells subsequently migrate either to B cell areas in the germinal center to assist B cells to mature, or to inflamed tissues. Under the influence of cytokines in the microenvironment, T cells also differentiate into effector populations that differ on the basis of their cytokine profiles and functional properties. Helper T type 1 (Th1) cells characteristically produce interferon  (IFN-) and contribute to host defense against pathogens, whereas Th2 cells produce interleukin 4 (IL-4) and IL-5 and are associated with allergic reactions involving IgE, eosinophils, and basophils. It has become apparent that lymphocyte cells differ not only in the production of secreted factors, but also in the different usage of surface receptors, which function to guide effector populations to inflammatory sites. As many of these targets involve protein-protein interactions it is unlikely in the near future that small, low molecular weight inhibitors can be designed. This review highlights the potential use of biotherapeutic agents (monoclonal antibodies/Fc fusion proteins) against secreted molecules (cytokines) and surface receptors in allergic airway disease.

View larger version (33K): [in this window] [in a new window]   Figure 1.   T cells enter secondary lymph organs, where they encounter specific antigen presented by dendritic cells, which express the costimulatory molecules B7-1 and B7-2. Recently activated cells express CD40L and other member of the TNF family, such as OX40, and migrate to either B cell areas in the germinal center to provide help for B cell maturation or migrate to inflamed tissues. In a secondary response, the frequency of specific T cells is dramatically increased and other molecules such as ICOS expressed on either recently activated T cells or memory cells may play a more important role than CD28.

	CYTOKINES AND CYTOKINE RECEPTORS

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Interleukin 5

Human IL-5 is a disulfide-linked homodimer with 115 amino acid residues in each chain. The biological effects of IL-5 are mediated through the IL-5 receptor (IL-5R) complex, a heterodimer consisting of a unique  subunit (which is predominantly expressed on eosinophils) and a  subunit that is shared with the receptors for IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) and exhibits a wider pattern of expression. The  subunit is required for ligand-specific binding, whereas association with the  subunit results in increased binding affinity. It has now been well documented that IL-5 is highly expressed in the bronchial mucosa of subjects with atopic and intrinsic asthma and is predominantly T cell derived. IL-5R  mRNA is also expressed by cells in bronchial biopsies from subjects with atopic and nonatopic asthma, as well as by infiltrating eosinophils.

More than 20 years ago, the development of eosinophilia in nematode-infected rodents was demonstrated to be lymphocyte dependent. Subsequently, the responsible soluble factor from T cells was shown to be identical to B cell growth factor 2, now designated as IL-5. The advent of murine immunology and its application to the study of allergic lung disease has resulted in a greatly improved understanding of the contribution of cytokine to allergic inflammation. In vivo, the administration of exogenous IL-5 induces eosinophil recruitment, and transgenic mice overexpressing the IL-5 gene under a variety of different tissue-specific promoters (CD3 , metalloprotein) develop peripheral blood, bone marrow, and tissue eosinophilia, although this overexpression does not itself result in any overt disease (1, 2). These data contrast with more recent work in which IL-5 was overexpressed from a Clara cell-specific (CC10) promoter, resulting in high levels of IL-5 in the bronchoalveolar lavage (BAL) fluid. These transgenic mice exhibited evidence of airway remodeling (subepithelial fibrosis), the formation of bronchus-associated lymphoid tissue (BALT) and the induction of airway hyperresponsiveness (3). Administration of neutralizing anti-IL-5 monoclonal antibodies (MAbs) has been demonstrated to inhibit the eosinophilia induced by nematode infection or antigen exposure in sensitized animals (4, 5). Likewise, exposure of IL-5 gene knockout mice to aerosolized antigens caused an ablated eosinophil recruitment into the lungs and minimal change in airway responsiveness (6). In contrast, other workers have reported that administration of anti-IL-5 MAbs, which resulted in a complete inhibition of eosinophilic inflammation, did not affect the induction of bronchial hyperreactivity (BHR) (7). These same workers showed that anti-IL-4 MAbs, which fail to attenuate the eosinophilic inflammation of the airways, reduced the BHR (7). These differences can be explained in terms of the generic background of the mice used; thus IL-5 deletion on a C57/B6 background results in an eosinophil-dependent BHR, whereas the same deletion on a BALB/c background results in an eosinophil-independent BHR. In addition, the nature of the stimuli also plays an important role. Infection of C57/B6 mice with Nippostrongylus braziliensis results in eosinophil-dependent tissue damage characterized by edema, hemorrhage, and destruction of septal walls (8). In similarly infected IL-5-deficient mice, airway hyperresponsiveness was not affected despite the lack of widespread tissue damage (8). Taken together, these studies of murine models suggest that under some circumstances eosinophil activation plays an important role in airway pathology, but its contribution is greatly influenced by genetic factors and the nature of the allergen challenge. More recently, humanized IL-5 MAbs have been used in primate studies of allergic airway disease and have demonstrated that a single administration of antibody provided protection for up to 3 mo (9). Clinical studies are at present in progress, and it is anticipated that they will clarify the relationship between IL-5 and eosinophilic inflammation of the airway in the near future.

Interleukin 4 and Interleukin 13

IL-4 and IL-13 are pleiotropic cytokines produced in large quantities by activated CD4⁺ Th2 lymphocytes and mast cells. The IL-4 gene is situated in closed proximity to the IL-13 gene on mouse chromosome 11 and human chromosome 5. The amino acid sequence homology between IL-4 and IL-13 is low (30%), although there is a high level of conservation in their tertiary structures. IL-4 and IL-13 share a number of common biological functions including upregulation of MHC class II expression and suppression of inflammatory cytokine production from macrophages. Unlike IL-4, IL-13 is not a growth factor for T cells, although IL-13 may function to regulate T cell function. Analysis of the receptor usage suggests an explanation for these differences. Both IL-4 and IL-13 compete for the common interleukin 4 receptor  subunit (IL-4R), but only IL-4 binds directly to the receptor chain, whereas IL-13 interacts with its own specific chain, IL-13R, which then recruits the IL-4R into the receptor complex, resulting in high-affinity binding and signal transduction (10). Both IL-4R and IL-13R recruit the common IL-2 common  chain into the complex. A second IL-13-specific chain has been identified, IL-13RII. The function of this subunit in IL-13 signaling remains to be evaluated (10).

In vitro, IL-4 plays a central role in the differentiation of naive T cells into Th2 cells (11, 12). Furthermore, mice deficient in IL-4 exhibit impaired Th2 cytokine generation after either allergen challenge or nematode infection (5, 11). In addition, in murine models, IL-4 is essential for the induction of class switch to IgE production. Overexpression of IL-4 in the lungs elicits hypertrophy of epithelial cells of the trachea, bronchi, and bronchioles. Histologic examination of parenchyma revealed multinucleated macrophages and occasional islands of cells consisting largely of eosinophils or lymphocytes (13). Mice expressing IL-4 had greater baseline airway resistance but did not demonstrate hyperreactivity to methacholine (13). However, while IL-4 is critical for many other aspects of both Th2 function and allergic airway disease, studies of the common IL-4R  chain suggest an alternative route of Th2 cell induction. After infection with Schistosoma mansoni, Th2 cytokine production is abrogated only in the absence of both IL-4 and IL-13 (14). Likewise, while IgE and IgG₁ are moderately reduced in the absence of either IL-4 or IL-13, they are completely suppressed when both cytokines are deficient. Similar data obtained in IL-4R- and STAT6 (signal transducer and activator of transcription 6)-deficient mice demonstrate a greater reduction in Th2 cytokine production than that observed in mice deficient only in IL-4. Interestingly, while the response to infection with N. braziliensis is delayed in IL-4/IL-13 double-deficient mice, IL-5 production and eosinophilia can occur, suggesting alternative mechanisms for the recruitment of eosinophils (14).

Overexpression of IL-13 results in significant numbers of eosinophils in the airways, associated with epithelial cell hypertrophy, mucous cell metaplasia, the deposition of Charcot-Leyden-like crystals, and subepithelial airway fibrosis (15). Eotaxin protein and mRNA are also present in the lungs. Mice overexpressing IL-13 exhibit significant increases in baseline airway resistance and airway hyperresponsiveness. In models of airway inflammation, administration of IL-13R-Fc inhibited eosinophilic inflammation more effectively than IL-4 (16). Thus it now appears that IL-4 and IL-13 contribute to eosinophilic inflammation, production of mucus, and the development of airway hyperresponsiveness, and so strategies that inhibit signaling through the common receptor may be more effective than neutralization of either cytokine alone. In this context, it has been reported that in a double-blind, placebo-controlled trial of patients with moderate asthma a single nebulized dose of soluble IL-4R significantly improved the FEV₁ on Day 4 and stabilized symptoms despite abrupt withdrawal of corticosteroids (17).

Other Th2-derived Cytokines

While IL-5 clearly has an important role in eosinophil recruitment, other Th2 cytokines have also been implicated and merit discussion as potential antibody targets. Data obtained from genetic studies have implicated IL-9 as an important gene in asthma. To address this issue further, IL-9 transgenic mice have been generated, with IL-9 under the control of a lung epithelial cell-specific promoter. These mice develop eosinophilic inflammation, mucous cell hyperplasia, and airway hyperresponsiveness (18). IL-11 transgenic mice exhibit similar alterations in airway inflammation and increased basal airway resistance, but show no evidence of airway hyperresponsiveness (19). Further studies are required to establish the contribution of these cytokines to the pathogenesis of allergic inflammation.

	ANTI-IgE MONOCLONAL ANTIBODIES

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The association between elevated levels of antigen specific IgE and the pathogenesis of bronchial asthma has long been recognized. Allergen exposure of allergic individuals induces an acute bronchoconstriction, which is believed to be a consequence of IgE-dependent triggering of mast cells to produce bioactive amines and metabolites of arachidonic acid. In approximately 50% of allergic individuals, the acute bronchoconstriction is followed by a late asthmatic response (LAR), which develops some 3 h after challenge and resolves by 6-12 h later. In contrast to the acute response, the LAR is associated with airway inflammation, in particular an infiltration of eosinophils into the bronchial mucosa. The relationship between IgE and this eosinophilic inflammation of the airways is at present unknown. To investigate this further, we generated a rat anti-mouse IgE antibody that has the unique properties of neutralizing serum-free IgE, and binding to IgE on Fc R11 on B cells, while failing to bind to IgE on Fc R1 on mast cells. The antibody therefore does not induce mast cell degranulation and as such, is designated nonanaphylactogenic (20). Administration of this MAb attenuated eosinophilic inflammation of the airways and Th2 cytokine production in a murine asthma model (21). The precise mechanisms by which anti-IgE MAbs suppress eosinophilic inflammation are unclear, but several workers have demonstrated that the binding of antigen-IgE complex to CD23 on the surface of antigen-presenting cells facilitates antigen presentation to antigen-specific T cells, resulting in a greatly amplified T cell response (22). Likewise, serum from atopic individuals has also been demonstrated to enhance antigen presentation to antigen-specific CD4⁺ T cells via a CD23-dependent mechanism.

Clinical studies have revealed that a recombinant anti-human IgE MAb designated E25 can inhibit not only the early bronchoconstrictor response to inhaled antigen, but also the late-phase response and the associated infiltration of eosinophils into the airways (23). More recently, administration of the same MAb to subjects with asthma has been shown to cause a significantly greater reduction in the use of steroids and improvement in symptom score than did treatment with placebo alone (24). These data suggest that inhibition of IgE may be useful in patients with severe asthma who require high-dose inhaled or oral corticosteroid therapy.

It is worth discussing briefly the data generated in experimental animals versus clinical experience with the IgE MAb obtained to date. Although administration of anti-IgE MAbs in a mouse model revealed a positive outcome, a series of studies performed in either B-cell deficient (25) or IgE-deficient mice (26) showed that IgE is not required in experimental animal models of asthma. The precise reason for these differences are unclear and may be related to the strain of the mice or compensatory mechanisms in mice in which a particular process is deficient during development. Whatever the precise reason, it is critical to bear in mind that data obtained in genetically targeted animals may not always be predictive of the effects of antibody intervention in humans.

	CHEMOKINES AND CHEMOKINE RECEPTORS

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Transendothelial migration of leukocytes into inflammatory sites requires a coordinated multistep series of events, involving leukocyte tethering and rolling along the vascular endothelium, followed by integrin-dependent adhesion. Under the influence of directional signals provided by locally generated chemoattractants, these cells then undergo migration across the vascular endothelium, through the underlying extracellular matrix, and into the site of inflammation. There are now increasing data to suggest that members of the chemokine superfamily play an important role in this response. These proteins range in size from 8 to 10 kD and are classified into four families, namely C, CC, CXC, and CX₃C, on the basis of the variations of the amino-terminal shared cysteine motif. The different families are associated with more or less distinct biological activities. CXC chemokines, of which IL-8 is the best studied, are principally neutrophil chemoattractants. CC chemokines, of which RANTES (regulation on activation, normal T cell expressed and secreted), macrophage chemoattractant protein 1 (MCP-1), eotaxin, and macrophage inflammatory protein 1 (MIP-1) are members, exert their biological activities on lymphocytes, eosinophils, and monocytes. Two minor families, namely the C chemokines, of which lymphotactin is the prototype, and CX₃C chemokines, of which neurotactin is the only known member, have been described. Several groups have examined the profile of expression of chemokines in vivo during the induction of an allergic response both in animal models and in clinical studies (27, 28).

The importance of different chemokines in mediating eosinophil recruitment in vivo is at present unsettled, with reports suggesting critical roles for MCP-1, eotaxin, MCP-5, RANTES, and/or MIP-1 (29). It is important to note, however, that in each of these studies the degree of inhibition of eosinophilic inflammation was only partial, implying a significant redundancy with multiple chemokines involved in the process.

The effects of chemokines are mediated by a family of seven transmembrane G protein-coupled receptors, of which nine human CC receptors have been cloned and functionally characterized to date. CCR1 is expressed principally on monocytes and macrophages, as is CCR2, which is also expressed on T cells, the principal ligands being MIP-1 and MCP-1, respectively. Of particular interest in the context of allergic inflammation, CCR3, the only identified receptor for eotaxin, is expressed on eosinophils and basophils.

More recently, it has become apparent that populations of lymphocytes can be differentially expressed, opening up the possibility that selectively targeting these receptors may be able to modify discrete effector functions of T and B cells. Naive and memory T cells can be separated by their expression of different isoforms of CD45, naive being CD45RA⁺, and memory being CD45RO⁺. It has been shown that memory T cells can be further subdivided on the basis of the expression of CCR7. CCR7CD45RO⁺ cells also express receptors for migration to inflamed tissue and exhibit immediate effector function (32). In contrast, CCR7⁺CD45RO⁺ cells express lymph node homing receptors, do not display effector functions, but efficiently prime T cells. These two populations are referred to as central memory cells and effector memory cells (32). The importance of CCR7 has been described using CCR7-deficient mice, which exhibit impaired antibody responses and profound morphological alterations in secondary lymph organs (33).

CCR3, while originally described on eosinophils, is in addition expressed on Th2 cells, while it is virtually absent on Th1 cells (34). In addition, CCR-4, the receptor for monocyte-derived chemokine (MDC) and thymus-associated chemokine (TARC), is also expressed on Th2 cells, but not on Th1 cells (35). In contrast, CXCR3, which is activated by interferon -inducible protein 10 (IP-10), MIG (monokine induced by interferon ), and interferon-inducible T cell  chemoattractant (I-TAC), is preferentially expressed on Th1 cells (35). Thus targeting CCR3 or CCR4 may inhibit the accumulation of Th2 cells, whereas inhibition of CXCR3 may prevent the migration of Th1 cells to sites of inflammation. Interestingly, in vivo, a greater proportion of Th2 cells expressed CCR3 rather than CCR4 early in the immune response. The converse was true at later stages of allergic inflammation in a murine model, where the vast majority (> 95%) of Th2 cells found in the lung expressed CCR4, whereas only a small number (< 5%) were found to express CCR3 (36). Moreover, these pathways act in a coordinated cooperative manner, with the CCR3-eotaxin pathway being critical in the acute stages of a response after initial challenge. However, repeated antigen challenge results in an increased frequency of CCR4-expressing Th2 cells. Consequently, the CCR4-MDC pathway ultimately dominates in the recruitment of antigen-specific Th2 cells.

	MEMBERS OF THE IL-1 RECEPTOR SUPERFAMILY

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The IL-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily comprises a diverse family of cell surface receptors defined by a characteristic conserved sequence in their cytosolic regions termed the Toll/IL-1 receptor domain (TIR). These receptors function in inflammation and host defense against microbial pathogens. Two members of the IL-1R superfamily have been shown to be differentially expressed on T cell populations. The IL-18 receptor (originally identified as IL-1- related protein, IL-1Rp) was expressed on Th1 cells, whereas T1/ST2 was expressed on Th2 cells (37). The IL-18 receptor is expressed on activated Th1 cells and regulates IFN- secretion, IL-12R2 expression, and Th1-mediated inflammation in vivo. T1/ST2, originally identified as a gene induced by serum stimulation of fibroblasts, has more recently been demonstrated to be overexpressed on Th2 effector cells in vitro and in vivo. Inhibition of T1/ST2 attenuates Th2-driven responses in vivo and inhibits eosinophilic inflammation of the airways (40). The signaling events that occur after ligation of T1/ST2 on Th2 cells are unknown, however. Other members of the IL-1 receptor family have been shown to activate MyD88, the functional homolog of the Drosphila protein Tube, which in turn recruits interleukin 1 receptor-associated kinase 1 (IRAK 1) to intracellular residues in the IL-1 receptor complex, leading to activation of the stress-activated protein kinase (SAPK)/c-Jun NH₂-terminal kinase (JNK) cascade and the subsequent activation of NF-B and AP-1. Whether similar mechanisms for T1/ST2 signaling occur in Th2 cells remains to be determined, although it is interesting to note that AP-1-binding sites have been identified in the IL-4 promoter and demonstrated to be crucial for Th effector generation. Nevertheless, T1/ST2 is more than a useful stable marker for identifying Th2 cells and functions as an important receptor for optimal cytokine production from Th2 cells. Taken together, these data add to the growing appreciation that members of the IL-1 receptor superfamily, including the IL-18 receptor, TRL-2, TRL-4, and now T1/ST2, are critical regulators of innate and adaptive immunity.

	COSTIMULATORY SIGNALS: CD28 AND TNF RECEPTOR SUPERFAMILIES

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The most important costimulatory signal delivered to resting T cells occurs on CD28 engagement by B7. The absence of CD28 ligation results in either anergy or clonal deletion. A large number of studies have attempted to address the importance of CD28-mediated costimulation in the induction of lung inflammation. Clinical studies using CTLA4-Ig (cytotoxic T lymphocyte-associated antigen 4-immunoglobulin) to block T cell costimulation in individuals with psoriasis have demonstrated the importance of this pathway (41), but its involvement in the allergic airway disease of individuals with asthma remains to be examined.

In vitro experiments have suggested that the dependency on CD28/B7-mediated costimulation is greatly influenced by the antigenic experience of the T cell. Although naive CD4⁺ T cells require CD28-mediated signaling for IL-2 production, optimal activation of recently activated helper T subsets occurs independent of CD28 ligation (42), suggesting that other costimulatory signals are important in Th2 effector function. Members of the tumor necrosis factor (TNF):TNF receptor family, which includes OX40, OX40L, RANK:RANKL, and 4-1BB:4-1BBL, have been implicated as regulators of T cell function (Figure 1). OX40 is expressed on activated T cells whereas its ligand has a broader pattern of expression and is present on T cells, B cells, and dendritic cells. In vivo, administration of an OX40 antibody results in a severely impaired IgG response. However, mice deficient in OX40 generate normal antibody responses and cytotoxic T lymphocyte (CTL) response to lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus (VSV) infection. In contrast, OX40 is important for the induction of CD4⁺, but not CD8⁺, effector cells. Similar results were obtained using OX40 ligand-deficient antigen-presenting cells (APCs) which fail to prime CD4⁺ T cells (43). Thus OX40 is an important molecule for helper T function. Whether OX40 plays an important role in the mechanisms leading to the induction of allergic inflammation remains to be addressed. In contrast, 4-1BB:4-1BBL is currently believed to be more important for effective CTL than for Th cell responses (44).

The third member of the CD28 family has been identified and termed ICOS (inducible T cell costimulator) (45). ICOS, unlike CD28, is induced on activation of T cells. In situ analysis of lymph node cells supports this finding, revealing increased expression in the T cell paracortex region after oxazolone sensitization (46). In vitro, ICOS delivers a CD28- independent signal for IFN-, IL-4, and IL-10, but not IL-2, production. ICOS binds its own unique counterreceptor, B7RP-1, which has 20% homology to B71 and B7-2 and is expressed on macrophages and B cells, but not dendritic cells (46). Cross-linking ICOS upregulates CD40 ligand expression and facilitates T-B interactions (45). These data are consistent with observations of B7RP-1 transgenic mice, which develop B cell hyperplasia, plasmacytosis, and hypergammablobulinemia (46). Furthermore, administration of B7RP-1 augments the effector response during a cutaneous hypersensitivity reaction. The requirement of stimulation for ICOS induction and the expression of its ligand on B cells raise the possibility that ICOS plays a more important role than CD28 in secondary immune responses, where a clear CD28-independent response has been described.

	CONCLUSION

TOP ABSTRACT INTRODUCTION CYTOKINES AND CYTOKINE... ANTI-IgE MONOCLONAL ANTIBODIES CHEMOKINES AND CHEMOKINE... MEMBERS OF THE IL-1... COSTIMULATORY SIGNALS: CD28 AND... CONCLUSION REFERENCES

The mechanisms by which T cells migrate from the blood to secondary lymph organs, interact with B cells and dendritic cells, and acquire effector or memory function are now beginning to be understood. It is anticipated that as a consequence of our increasing appreciation of events that regulate this response, we will be able to design better therapeutic agents to selectively target T cells in diseases such as allergic asthma, without altering protective immune responses against pathogens. Clinical studies with anti-cytokine/cytokine receptor antibodies and with members of the TNF receptor and CD28 families are currently being evaluated in allergic asthma. If the clinical experience with these agents is as promising as in other inflammatory diseases such as rheumatoid arthritis and psoriasis, the way we approach management of the asthmatic condition may change in the future.