Endogenous Inhibitory Mechanisms in Asthma

PETER J. BARNES

Department of Thoracic Medicine, National Heart and Lung Institute, Imperial College School of Medicine, London, United Kingdom

	INTRODUCTION

TOP INTRODUCTION ENDOGENOUS INHIBITORY... IMPORTANT QUESTIONS REFERENCES

Much of the research in asthma has focused on the multiple proinflammatory mechanisms involved in this complex inflammatory disease (1). Much less attention has been paid to endogenous antiinflammatory mechanisms that may counteract or limit the inflammatory process in asthma. It is possible that these inhibitory mechanisms might be defective, resulting in increased or more prolonged inflammation. Such mechanisms, if deficient, might also be important in determining the severity of asthma, which differs markedly and inexplicably between patients. Little is understood about the factors that determine severity in chronic inflammatory diseases, such as asthma, but it is possible that endogenous inhibitory mechanisms may be of critical importance. There has been relatively little research into endogenous inhibitory mechanisms in asthma, but it is likely that understanding these processes may give a better understanding of disease severity. This may also lead to novel therapeutic approaches, such as drugs that increase the release of endogenous inhibitors or mimic their effects. Such therapeutic approaches are attractive since they may restore antiinflammatory mechanisms to normal and thus carry a lower risk of adverse effects in the long term.

	ENDOGENOUS INHIBITORY MECHANISMS IN ASTHMA

TOP INTRODUCTION ENDOGENOUS INHIBITORY... IMPORTANT QUESTIONS REFERENCES

Although multiple proinflammatory mediators have been implicated in asthma, relatively few mechanisms that inhibit the inflammatory process have been identified. Potential endogenous mechanisms in asthma include cortisol, prostaglandin E₂ (PGE₂), vasoactive intestinal peptide (VIP), and adrenomedullin (Table 1). There is increasing evidence that certain cytokines have antiinflammatory or immunomodulatory effects and that their secretion may be defective in patients with asthma (2).

Cortisol

Despite the fact that corticosteroids are the most effective therapy in asthma there is little information about the role of endogenous cortisol in asthma. Blocking the synthesis of endogenous corticosteroids with metyrapone results in increased late response to allergen in sensitized dogs (3) and increases the late skin response to allergen in humans (4). It is possible that local inactivation of cortisol in the airways by the enzyme 11-hydroxysteroid dehydrogenase may regulate the local inflammatory response, so that increased expression of this enzyme may enhance inflammation (5).

Prostaglandin E₂

PGE₂ has several inhibitory effects in asthma, and in addition to its bronchodilator effect inhibits the release of inflammatory mediators from mast cells, eosinophils, and macrophages (6). It may account for the refractory period in exercise- induced asthma (7). Inhaled PGE₂ inhibits both early and late responses to inhaled allergen, indicating its bronchodilator and antiinflammatory actions (8). PGE₂ is generated in several cells in the airways, including epithelial cells and airway smooth muscle cells via inducible cyclooxygenase (COX-2) (9, 10). Whether defective synthesis of PGE₂ is a determinant of asthma severity is uncertain, but it might be of particular relevance in aspirin-sensitive asthma, which is often a more severe form of asthma.

Lipoxins

Lipoxins (LxA₄, LxB₄) are formed from an interaction of 5-lipoxygenase and 15-lipoxygenase products. Lipoxins have some antiinflammatory effects and inhibit neutrophil and eosinophil activation and may counteract the action of leukotrienes (11, 12).

Adrenomedullin

Adrenomedullin has a structure similar to calcitonin gene- related peptide and is highly expressed in lung (13). It is protective against bronchoconstriction (14). It may be secreted by macrophages in response to inflammatory stimuli (15) and has an antiinflammatory action via an increase in intracellular cyclic AMP (16). Nothing is know about the role of adrenomedullin in asthma, however.

Vasoactive Intestinal Polypeptide

VIP is a neuropeptide localized to cholinergic and sensory nerves in the airways. It is a bronchodilator of human airways, but may also have antiinflammatory actions (17). In the absence of a specific antagonist of VIP receptor, the potential antiinflammatory role of VIP in asthma is uncertain. There is some evidence that VIP is deficient in asthma (18), but this has not subsequently been confirmed (19, 20).

Nitric Oxide

Endogenous NO has contrasting effects in the airways and may increase or decrease inflammation (21). Most evidence suggests that NO generated by the constitutive NO synthases (nNOS and eNOS) are antiinflammatory or protective, whereas NO generated by inducible NO synthase (iNOS) is proinflammatory (22). Thus, plasma exudation in the airways in increased by the NO synthase inhibitor L-NAME under basal conditions, but after induction of iNOS by endotoxin L-NAME reduces plasma exudation (23). Inhibition of NO synthase by inhaled L-NAME has no effect on resting airway tone in patients with asthma (24), but increases the bronchoconstrictor response to histamine (25). This suggests that there is a protective effect of NO against bronchoconstriction, but whether endogenous NO has an anti- or proinflammatory effect in asthma is uncertain. Although exhaled NO is increased during the late response to allergen, inhaled L-NAME has no effect on the late response to allergen, suggesting that endogenous NO does not have any major effect, at least in allergic inflammation (26).

Carbon Monoxide

There is increased production of carbon monoxide (CO) in asthma, presumably as a result of increased hemeoxygenase 1 (HO-1) expression (27, 28). CO, like NO, activates guanylyl cyclase to increase cyclic GMP and may be protective against cellular stress (29, 30), but whether endogenous CO production in the airways in antiinflammatory is not yet determined.

Interleukin 10

Interleukin 10 (IL-10) is a 36-kD homodimeric cytokine that was originally identified as a product of murine helper T cell type 2 (Th2) lymphocyte clones that suppressed the synthesis of cytokines from Th1 cells and was termed cytokine synthesis inhibitory factor. IL-10 is produced by several cell types, including Th1 and Th2 cells, mast cells, and dendritic cells, but in lungs the major cellular source is the macrophage (31). IL-10 has a broad spectrum of immunosuppressive and antiinflammatory effects (Table 2). It inhibits the synthesis of proinflammatory cytokines (IL-1, tumor necrosis factor  [TNF-], IL-6), chemokines (macrophage inflammatory protein 1 [MIP-1], RANTES, IL-8), IL-4, and IL-5 (32). In addition, it inhibits the expression of the inflammatory enzymes inducible nitric oxide synthase (iNOS) and COX-2 in macrophages. Thus IL-10 has the capacity to inhibit the expression of many of the inflammatory genes that are abnormally expressed in asthmatic airways. It also inhibits the proliferation of CD4⁺ T lymphocytes by inhibiting IL-2 release and reduces expression of major histocompatibility (MHC) Class II molecules, the costimulatory molecules B7-1 and B7-2, and low-affinity IgE receptors (CD23) in antigen-presenting cells, thus effectively blocking allergen presentation by mononuclear cells and dendritic cells to T cells (31). IL-10 upregulates the expression of IL-1 receptor antagonist (IL-1ra) (33) and also decreases the expression of IL-1 receptors, thereby blocking the inflammatory action of IL-1. IL-10 inhibits the release of cytokines from several cell types, including mast cells (34) and proliferating airway smooth muscle cells (35). It is effective in inhibiting eosinophilic inflammation and this may be through its combined inhibitory effects on IL-5 synthesis, the release of eosinophil chemotactic chemokines (such as eotaxin and RANTES), and through a reduction in eosinophil survival (32, 36).

The molecular mechanisms by which IL-10 exerts these antiinflammatory and immunomodulatory effects are still not well understood, and although the IL-10 receptor has been cloned, it is not established how it signals uniquely. In common with other interferon-like receptors, the transcription factors Stat-1 and Stat-3 have been implicated, but are not unique (31), together with another interferon-like receptor (CRF 2- 4), which appears to be an essential component of the IL-10 receptor, at least in mice (37). Some of the antiinflammatory effects of IL-10 are mediated through inhibition of nuclear factor B (NF-B) (38), although this cannot account for all the actions of IL-10, such as inhibition of IL-5 synthesis, which is independent of NF-B. A blocking antibody to IL-10 increases the release of cytokines from monocytes and macrophages, suggesting that IL-10 may serve as an endogenous feedback inhibitory mechanism to damp down the inflammatory response. This has also been demonstrated in vivo in a murine model of allergic inflammation, where an IL-10-blocking antibody increases the airway inflammatory response to allergen (39). The kinetics of IL-10 production show a late secretion, which is not maximal until 24 h after stimulation, whereas the inflammatory genes suppressed by IL-10 are much more rapidly expressed (6-12 h). This suggests that IL-10 may function as a late "braking mechanism" that prevents persistence of the inflammatory response (Figure 1).

View larger version (77K): [in this window] [in a new window]   Figure 1.   Macrophage release of interleukin 10 (IL-10) in response to inflammatory stimuli. Inflammatory stimuli activate the transcription factor nuclear factor B (NF-B), resulting in the increased transcription of many inflammatory genes and the release of inflammatory mediators from macrophages. The same stimuli give a delayed synthesis of IL-10, which then inhibits the expression of these inflammatory genes, thus terminating the inflammatory response. In patients with asthma the IL-10 signal is reduced, leading to increased and more prolonged inflammation, but IL-10 secretion can be restored by treatment with steroids or theophylline. Abbreviations: IL-1 = interleukin 1; TNF- = tumor necrosis factor ; GM-CSF = granulocyte-macrophage colony-stimulating factor; iNOS = inducible nitric oxide synthase; COX-2 = inducible cyclooxygenase.

Defective production in asthma. There is increasing evidence that IL-10 secretion may be defective in patients with asthma. Lower concentrations of IL-10 are found in bronchoalveolar lavage fluid from subjects with asthma than from health control subjects (40). There is a reduced secretion of IL-10 from alveolar macrophages obtained by bronchoalveolar lavage from patients with asthma compared with healthy controls, and this is at the level of gene expression (41). This reduced expression of IL-10 is correlated with an increased production of proinflammatory cytokines such as TNF- and granulocyte-macrophage colony-stimulating factor (GM-CSF) and the chemokine MIP-1. This suggests that a defect in IL-10 synthesis may result in exaggerated and more prolonged inflammatory responses in asthmatic airways. Furthermore, since IL-10 appears to act as an inhibitor of antigen presentation by mononuclear cells, this may also account for the previous observation that macrophages from patients with asthma are less effective at inhibiting T cell-proliferative responses (42).

The gene for IL-10 has been mapped to chromosome 1 and polymorphisms in the 5' promoter region of the IL-10 gene have been identified that are associated with altered synthesis of IL-10 in response to inflammatory stimuli (43). These polymorphisms are not associated with the prevalence of asthma, but a polymorphism that results in reduced IL-10 synthesis is found significantly more often in patients with severe asthma, who require high doses of inhaled or oral corticosteroids for control (44). This suggests that IL-10 may play a key role in determining disease severity and that this may be genetically determined.

Interleukin 1 Receptor Antagonist

IL-1ra is produced by monocytes, macrophages, and epithelial cells in response to inflammatory stimuli (45). It binds to the IL-1 receptor (IL-1R) without signaling and thus inhibits the effects of IL-1 and IL-1. IL-1ra inhibits the synthesis of IgE and inflammatory cytokines in human peripheral blood mononuclear cells stimulated with lipopolysaccharide (46). Increased expression of IL-1ra has been reported in asthmatic airways (47). In ovalbumin-sensitized guinea pigs aerosolized IL-1ra protects against the development of airway hyperresponsiveness and reduces eosinophil infiltration and activation (48). This has suggested that human recombinant IL-1ra may be useful in asthma, but clinical trials have apparently been unsuccessful.

Interferon

IFN- production is restricted to T lymphocytes and natural killer (NK) cells. It is produces by Th1 cells and has an inhibitory effect on Th2 cells, thus reducing the synthesis of IL-4 and IL-5. In mice aerosolized FIN- inhibits allergen-induced eosinophil inflammation in the lungs, whereas targeted disruption of the IFN- receptor gene results in a prolonged airway eosinophilia in response to allergen (49). Some of the effects of IFN- may be mediated via induction of the IL-10 gene. IFN- also inhibits IL-4-induced synthesis in B cells. On the other hand, IFN- potentiates the effects of other proinflammatory mediators and induces expression of MHC Class II molecules on monocytes, macrophages, and dendritic cells and of adhesion molecules on endothelial and epithelial cells, suggesting that it could worsen the ongoing inflammatory process.

Several studies have demonstrated reduced production of IFN- by T cells of patients with asthma and this correlates with disease severity, but this appears to correlate with atopy rather than with asthma itself (50, 51). Defective production of IFN- may be important in asthma, although no polymorphisms of the IFN- gene have so far been associated with the disease (52).

Recombinant IFN- inhibits eosinophilic inflammation in animal models of asthma, suggesting therapeutic potential in asthma. Nebulized IFN- reduces the number of eosinophils in bronchoalveolar lavage fluid of patients with asthma, although the effects are small (53). This might be because access of the inhaled protein to target cells in the airways is difficult to achieve. Allergen immunotherapy, which is not effective in the treatment of asthma, increases the in vitro production of IFN- in circulating helper T cells (54) and increases the number of IFN--expressing cells in the nasal mucosa of patients with allergic rhinitis (55).

Interleukin 12

IL-12 is a heterodimer composed of two covalently linked proteins (p40 and p35) that are encoded by separate genes (56). It acts on specific receptors that are expressed on T cells and NK cells. It is produced by antigen-presenting cells, including monocytes, macrophages, and dendritic cells, and is upregulated by IFN-, TNF-, and GM-CSF. IL-12 plays a pivotal role in cell-mediated immunity. A major action of IL-12 is to induce the development of Th1 cells, while suppressing Th2 cells (Figure 2). These effects are largely but not completely mediated via the release of IFN-. It is likely that IL-12 plays a critical role in determining the balance between Th1 and Th2 cells, thereby inhibiting IgE synthesis and allergic inflammation. In mice recombinant IL-12 treatment during active sensitization reduces allergen-induced eosinophil inflammation, and inhibits the development of Th2 responses and IgE synthesis (57, 58). Once the animal is sensitized to allergen, IL-12 is effective at inhibiting allergen-induced hyperresponsiveness and inflammation if given before allergen challenge, but is less effective if given after the allergen (59). The effects of IL-12 are dependent on IFN- during the initial sensitization to allergen, since they are not seen in mice with targeted disruption of the IFN- gene, but are independent of IL-12 once allergic inflammation is established (60).

View larger version (41K): [in this window] [in a new window]   Figure 2.   Helper T (CD4+) cells in allergic diseases. Allergen is processed by antigen-presenting cells (dendritic cells and macrophages) and presented via major histocompatibility Type II molecules (MHCII) to T cell receptors (TCR) on uncommitted helper T cells (Thp). Accessory molecules B7-2 and CD28 amplify this interaction. Thp cells differentiate in response to IL-12 into Th1 cells, which under the influence of IL-12 and IL-18 release interferon  (IFN-). Thp cells under the influence of IL-4 differentiate into Th2 cells, which release IL-4, IL-13, and IL-5. IFN- inhibits Th2 cell differentiation and in this system IL-12, IL-18, and IFN- all result in inhibition of Th2 cells and the release of Th2 cytokines.

The production of IL-12 and IL-12-induced IFN- release is reduced in whole blood cultures from patients with asthma compared with healthy controls (61). There is also a reduction of IL-12 mRNA expression in airway biopsies from patients with asthma compared with healthy subjects, and an increase after treatment with inhaled corticosteroids (62). This contrasts with an inhibitory effect of corticosteroids on IL-12 secretion from human blood monocytes (63). ₂-Agonists decrease IL-12 production by human monocytes and this might provide an explanation for the possible worsening of asthma by high doses of inhaled ₂-agonists (64).

Interleukin 18

IL-18 is an 18-kD cytokine formerly known as IFN--inducing factor, as it releases IFN- from T cells. It is structurally related to IL-1, but acts like IL-12 to promote Th1 cell development and to suppress Th2 cells, although it appears to act via distinct cell-signaling pathways (65). IL-18 is synthesized as a precursor that requires IL-1-converting enzyme to release the active cytokine. IL-18 has biological effects similar to those of IL-12 and acts synergistically with it to increase IFN- secretion from Th1 cells (66). However, unlike IL-12, it does not induce Th1 cell development. IL-18 acts synergistically with IL-12 to inhibit IgE production from activated B cells (67). This suggests that IL-18 may be of potential value in asthma therapy, but may not be as useful as IL-12 if it does not induce Th1 cell development. A combination of IL-12 and IL-18 may be of potential benefit and may reduce the toxicity of IL-12. However, IL-18 may also have proinflammatory effects and increases the expression of proinflammatory cytokines and chemokines from human monocytes, probably via activation of NF-B (68).

Interleukin 4 and Interleukin 13

IL-4 is normally regarded as proinflammatory in asthma, since it is critical for the development of Th2 cells and for IgE synthesis from B cells. Indeed, IL-4 antibodies and IL-4 soluble receptors are in development as potential antiasthma treatments. However, IL-4, and the related cytokine IL-13, also have antiinflammatory effects. Both cytokines inhibit the gene expression of inflammatory cytokines, such as chemokines, TNF-, and IL-1. For example, both cytokines inhibit the expression of iNOS from human airway epithelial cells, and chemokines from human macrophages and airway smooth muscle cells (35, 69, 70). However, any potential antiinflammatory effect of these cytokines may be counteracted by their amplifying effects on allergic inflammation. Furthermore, both of these cytokines have been implicated in corticosteroid resistance in asthma (71).

Transforming Growth Factor

TGF- has highly complex effects, depending on the presence of other cytokines. TGF- has multiple proinflammatory effects and promotes remodeling, but is also a potent immunosuppressant owing to a direct inhibitor effect in helper T cells. However, it is unlikely to have therapeutic potential in asthma, as the balance of its action may favor fibrosis in the airways.

	IMPORTANT QUESTIONS

TOP INTRODUCTION ENDOGENOUS INHIBITORY... IMPORTANT QUESTIONS REFERENCES

• What are the key endogenous antiinflammatory mechanisms in the airways? Although several mediators have been identified that have antiinflammatory effects, little is known about the role of these mediators in limiting asthma.
• Does a deficiency in antiinflammatory mechanisms account for the severity of asthma? Some indication of this is the association between asthma severity and polymorphisms of the IL-10 gene associated with reduced production.
• Can endogenous antiinflammatory mechanisms be exploited in the development of new antiinflammatory treatments for asthma? Although therapy with the cytokines IL-10, IL-12, IFN-, and IL-1ra is possible, drugs that enhance these antiinflammatory cytokines may be of value in future.

	Footnotes

Correspondence and requests for reprints should be addressed to P. J. Barnes, M.D., National Heart and Lung Institute, Imperial College, Dovehouse Street, London SW3 6LY, UK. E-mail: p.j.barnes{at}ic.ac.uk

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