Cytokines Involved in Promoting Allergic or Asthmatic Inflammation

Inflammation is often described in the context of TH1 responses, particularly with regard to classical TH1-derived inflammatory cytokines such as TNFa and IFNg. However, asthmatic inflammation involves cytokines whose actions typically oppose the actions of cytokines involved in the inflammation of other inflammatory diseases. Inflammation of the airways in asthma, although not exclusively so, is primarily a TH2-mediated phenomenon. Overproduction of GM-CSF in the airway mucosa of patients with asthma enhances MHC class II-restricted Ag presentation by dendritic cells and increases the accumulation of bronchoalveolar macrophages (Holt et al. 1999; Kay 2001), which further present allergen to CD4+ T cells and stimulate the production of TH2-type cytokines (Larché et al. 1998). Reports of TH2 cytokines involved in asthmatic inflammation have implicated IL-4 and IL-13 in stimulating the production of IgE and vascular-cell adhesion molecule 1 (VCAM-1), IL-5 and IL-9 in the development of eosinophils, IL-4 and IL-9 in the development of mast cells, IL-9 and IL-13 in airway hyperresponsiveness, and IL-4, IL-9, and IL-13 in the overproduction of mucus (Wills-Karp et al. 1998). A role for IL-13 in asthmatic inflammation has been especially emphasized in recent literature. Using mice transgenic for and overexpressing IL-13, researchers have demonstrated phe-notypic changes typical of asthma as a result of allergen exposure, including pulmonary fibrosis, goblet cell hyperplasia, elevated TH2 cytokines and IgE levels, eosinophilia, and airways occluded by mucus and Charcot-Leyden crystals (Fallon et al. 2001). Moreover, secondary exposure to Ag in these mice induces fatal ana-phylaxis as a result of mast cell degranulation and histamine release, as well as marked pulmonary eosinophilia associated with hypertrophic bronchial epithelium.

The regulatory effects of cytokines alone or in combination in the development of atopic disease are even more complicated than presented above, however. Hahn and coworkers recently demonstrated some of this complexity using a mutated IL-4 variant as the basis for an IL-13/IL-4 inhibitor during allergic sensitization and in established disease in a murine model of asthma (Hahn et al. 2003a). This study indicates that while inhibition of the IL-4/IL-13 system efficiently prevents the development of the allergic phenotype, these cytokines play only a minor role in established allergy. Perhaps the disparity observed in the effects of an IL-4/IL-13 inhibitor on sensitization vs. established asthma lies in differences between signaling pathways not yet completely understood. In support of this idea, Zimmerman and colleagues used microarray analysis to conduct expression profiling of lung tissue from mice with experimental asthma (Zimmerman et al. 2003). The authors identified

Overproduction of mucus Overproduction of GM-CSF in airway mucosa of Pts w/asthma

APC costimulatory molecules B7-1 & B7-2 are over expressed in BAM of asthmatics

Both IL-4 & IL-13 strongly induce lung arginase and mRNA, but lung arginase activity and mRNA induction differentially depend on STAT-6


•|HLA CI II-restricted Ag presentation by DCs

Required for Switch to lgE in B cells

■ • | Accumulation of BAM —j Presentation of Ag to CD4+ T cells

Figure 14.9 The role of the antigen-presenting cell in asthmagenesis. Ag presentation to TH2 cells causes the release of the cytokines IL-4, IL-9, and IL-13, all of which are associated with the overproduction of mucus and GM-CSF found in the airway mucosa of patients with asthma. These cytokines increase HLA class II-restricted Ag presentation by dendritic cells (DCs) to CD4+ T cells and also increase the accumulation of bronchoalveolar macrophages (BAM) that, in turn, lead to increased Ag presentation to CD4+ T cells. In addition, the BAM from asthmatics have been shown to overexpress the APC co-stimulatory molecules B7-1 and B7-2. IL-4 and IL-13 stimulate the APC through receptors that share the IL-4R alpha chain in a process requiring activation of the STAT-6 transcription factor. However, while both IL-4 and IL-13 strongly induce lung arginase and mRNA, lung arginase activity and mRNA induction appear to differentially depend on STAT-6 activation in the APC. If the APC is a B cell, STAT-6 activation is required for isotype switching to allergy- and asthma-associated IgE production, but this process also requires NF-kB activation secondary to CD40 ligation by the interacting T cell.

6.5% of the tested genome whose expression was altered in asthmatic lung, and prominent among the asthma signature genes were those related to metabolism of basic amino acids, specifically the cationic amino acid transporter 2, arginase I, and arginase II. In particular, lung arginase activity and mRNA expression were strongly induced by IL-4 and IL-13 but were differentially dependent on signal transducer and activator of transcription 6 (STAT-6) (Figure 14.9).

Elucidating the differences between IL-4 and IL-13 signaling pathways as they relate to the pathogenesis of asthma may not be enough, however, to explain the differences in outcomes observed between phenotypically similar asthma patients treated under similar circumstances. Faffe and coworkers determined that human airway smooth muscle cells (HASMC) produce the chemokine thymus and activation regulated chemokine (TARC), known to induce selective migration of TH2, but not TH1 lymphocytes and known to be upregulated in the airways of asthmatic patients (Faffe et al. 2003) (Figure 14.7). These authors found that, alone, none of the cytokines IL-4, IL-13, IL-1p, IFNg, or TNFa stimulated TARC release into the supernatant of cultured HASMC, whereas both IL-4 and IL-13 increased TARC protein and messenger RNA expression when administered in combination with

TNFa but not IL-1p or IFNg. Of particular relevance to asthma patients is that the authors determined that polymorphisms of the IL-4Ra chain impact the ability of IL-4 or IL-13 to induce TARC release from HASMC. Genotype studies indicated that cells expressing the Val50/Pro478/Arg551 haplotype had significantly greater IL-4- or IL-13-induced TARC release than cells with other IL-4Ra genotypes. TH2 cytokines appear to enhance TARC expression in these cells, resulting in a positive feedback loop for the recruitment of TH2 cells to asthmatic airways in an IL-4Ra genotype-dependent fashion. Thus, research approaches that inhibit IL-13/IL-4 pathways with receptor inhibitors, as discussed earlier, are likely to have variable results depending on the receptor genotype of the patient (Figure 14.7).

IL-13 and G Protein-Coupled Receptor Systems

Interestingly, Faffe and coworkers also determined that agents known to activate protein kinase A (PKA), including the beta-adrenergic receptor agonist isoproterenol, inhibited TARC release induced by TNFa plus IL-13, or TNFa plus IL-4 (Figure 14.7). In other words, biochemical signal transduction pathways involved in asthma pathogenesis could be inhibited by stimulation of cAMP-elevating G protein-coupled receptors. Particularly regarding IL-13 signaling, this concept is supported by another recent study that may go a long way toward helping us understand the potential effects of perinatal environmental toxicant exposure on asthma sensitization. Blackburn and colleagues recently demonstrated that adenosine and adenosine signaling contribute to and influence the severity of IL-13-induced tissue responses (Blackburn et al. 2003). Of particular relevance to this chapter is the work of Sitkovsky and colleagues at the National Institutes of Health, who have explored the causes and effects of the phenomenon that thymic T cells (thymocytes) are bathed in adenosine during the process of thymic education (reviewed in: Sitkovsky 1998). Although the effects of adenosine depend on the type of adenosine receptor expressed on different T cell subtypes, manipulation of this environment by altering the physiologic mechanisms for lowering extracellular adenosine levels can have dramatic results. In support of the findings of Sitkovsky's group, the findings of Faffe and colleagues indicate that IL-13 causes a progressive increase in adenosine accumulation, inhibits the activity and mRNA accumulation of adenosine deaminase (which is responsible in part for lowering extracellular adenosine levels), and augments the expression of the A1, A2B, and A3 but not the anti-inflammatory A2A adenosine receptors in mice. While the murine adenosine A2A receptor is associated with cAMP elevation due to signaling through Gs protein-coupled receptors, the other murine adenosine receptors mentioned signal through cAMP-lowering Gi-protein coupled receptors. Since PKA is dependent upon cAMP for its activation, the findings presented by Blackburn and colleagues that adenosine mediates IL-13-induced inflammation and remodeling in the lung and interacts in an IL-13-adenosine amplification pathway are in agreement with Faffe's findings that TARC release is inhibited by PKA-activating pathways. Together, these reports suggest a novel asthmagenic pathway associated with IL-13 signaling. In this paradigm, asthma-promoting TARC release is indirectly facilitated by IL-13-induced, adenosine-mediated inhibition of cAMP and PKA. In support of these findings, it is interesting to note that our own experiments using developing murine thymocytes have shown that elevated cAMP levels resulting from A2A adenosine receptor-mediated signaling resulted in a specific downregulation of the TCR-triggered MAPK pathway (Armstrong et al. 1988). It may be particularly relevant that agents that inhibit cAMP levels in pathogenic T cells are known to facilitate and amplify the effects of IL-13 by recruiting TH2 cells to asthmatic airways, turning on these T cells in a self-amplifying proliferation cycle in response to antigen. As Blackburn and colleagues noted, the human A3 adenosine receptor is known to be expressed in an exaggerated fashion in tissues from patients with asthma and COPD. This same group has recently demonstrated that lung mast cells are signaled to degranulate by activation through the murine adenosine A3 receptor (Zhong et al. 2003). Because the human adenosine A3 receptor is known to be Gi-linked, it is reasonable to speculate that the IL-13-mediated increase in expression of the Gi-linked murine adenosine receptors reported in this study may explain some of the adverse effects associated with IL-13 in human airway disorders such as asthma and COPD.

All chemokine receptors are GPCR and, as such, are known to be regulated by a protein called p-arrestin. A particularly interesting report by Walker and colleagues demonstrated a requirement for p-arrestin-2 in the manifestation of allergic asthma (Walker et al. 2003). In that study, allergen-sensitized mice with a targeted deletion of their p-arrestin-2 gene were incapable of accumulating T lymphocytes in their airways. In addition, these mice failed to demonstrate other physiological and inflammatory features characteristic of asthma, thus emphasizing a pivotal role for p-arrestin-2 in the regulation of the development of allergic inflammation at a proximal step in the inflammatory cascade (Figure 14.7).

The presentation by APC of cognate Ag directs the development of nascent T cells into either TH1 cells in the presence of IL-12, or IL-4/13-producing TH2 cells in the presence of IL-4 (Figure 14.1). The initial source of IL-4 is obscure but appears to be dependent on the presence of a cell type expressing an invariant TCR. NKT cells producing IL-4 and IL-13 have recently been shown to play an essential role in the development of allergen-induced AHR. Using NKT cell-deficient mice, Akbari and colleagues found that the inability to develop allergen-induced AHR in the absence of Va14i NKT cells was not due to an inability to produce TH2 responses (Akbari et al. 2003). Instead, the authors demonstrated that the failure to develop AHR was reversible either by the adoptive transfer of tetramer-purified NKT cells producing IL-4 and IL-13 or by the administration of recombinant IL-13, emphasizing that regulation of the development of asthma and TH2-biased respiratory immunity against nominal exogenous antigens is dependent on pulmonary Va14i NKT cells, and presumably their production of the cytokines IL-4 and IL-13.

Other cells known to express an invariant TCR have also been shown to regulate airway hyperreactivity in allergen-sensitized and allergen-challenged mice. In a recent report by Hahn and colleagues, the investigators used ovalbumin (OVA)-sensitized mice to demonstrate that Vg4+ gd T cells act as negative regulators of AHR and that their regulatory effect allows much of the allergic inflammatory response coincident with AHR, including eosinophilic inflammation of the lung and airways, to be bypassed (Hahn et al. 2003b). Thus, invariant chain-expressing T cells, of both the a/p and g/d types, appear to be involved in regulating allergen-induced AHR in mice. It will be interesting to see if these results are confirmed in humans.

Noninterleukin cytokines have also been implicated in asthmatic inflammation, and these include neurotrophins secreted by macrophages, T cells, eosinophils, and mast cells (Braun et al. 2000). Aspects of allergic inflammation, including vasodilation, increased vascular permeability, contraction of the smooth muscles of the airway and hypersecretion of mucus in the lung, have been attributed to several neuropeptides, particularly including substance P, calcitonin-gene-related peptide, and neurokinin A (Braun et al. 2000) (Figure 14.6). Although these neuronal cytok-ines are all primarily of sensory neuron origin, they are also expressed in inflammatory cells (Belvisi and Fox 1997), and they have been reported to cause histamine release from mast cells in the lungs (Forsythe et al. 2000).

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