Critical role for CXCR3 chemokine biology in the pathogenesis of bronchiolitis obliterans syndrome

Bronchiolitis obliterans syndrome (BOS) is the major limitation to survival post-lung transplantation and is characterized by a persistent peribronchiolar inflammation that eventually gives way to airway fibrosis/obliteration. Acute rejection is the main risk factor for the development of BOS and is characterized by a perivascular/bronchiolar leukocyte infiltration. The specific mechanism(s) by which these leukocytes are recruited have not been elucidated. The CXC chemokines (monokine induced by IFN-gamma (MIG)/CXC chemokine ligand (CXCL)9, IP-10/CXCL10, and IFN-inducible T cell alpha chemoattractant (ITAC)/CXCL11) act through their shared receptor, CXCR3. Because they are potent leukocyte chemoattractants and are involved in other inflammation/fibroproliferative diseases, we hypothesized that the expression of these chemokines during an allogeneic response promotes the persistent recruitment of mononuclear cells, leading to chronic lung rejection. We found that elevated levels of MIG/CXCL9, IFN-inducible protein 10 (IP-10)/CXCL10, and ITAC/CXCL11 in human bronchoalveolar lavage fluid were associated with the continuum from acute to chronic rejection. Translational studies in a murine model demonstrated increased expression of MIG/CXCL9, IP-10/CXCL10, and ITAC/CXCL11 paralleling the recruitment of CXCR3-expressing mononuclear cells. In vivo neutralization of CXCR3 or its ligands MIG/CXCL9 and IP-10/CXCL10 decreased intragraft recruitment of CXCR3-expressing mononuclear cells and attenuated BOS. This supports the notion that ligand/CXCR3 biology plays an important role in the recruitment of mononuclear cells, a pivotal event in the pathogenesis of BOS.     

IL-13 signaling through the IL-13alpha2 receptor is involved in induction of TGF-beta1 production and fibrosis

Interleukin (IL)-13 is a major inducer of fibrosis in many chronic infectious and autoimmune diseases. In studies of the mechanisms underlying such induction, we found that IL-13 induces transforming growth factor (TGF)-beta(1) in macrophages through a two-stage process involving, first, the induction of a receptor formerly considered to function only as a decoy receptor, IL-13Ralpha(2). Such induction requires IL-13 (or IL-4) and tumor necrosis factor (TNF)-alpha. Second, it involves IL-13 signaling through IL-13Ralpha(2) to activate an AP-1 variant containing c-jun and Fra-2, which then activates the TGFB1 promoter. In vivo, we found that prevention of IL-13Ralpha(2) expression reduced production of TGF-beta(1) in oxazolone-induced colitis and that prevention of IL-13Ralpha(2) expression, Il13ra2 gene silencing or blockade of IL-13Ralpha(2) signaling led to marked downregulation of TGF-beta(1) production and collagen deposition in bleomycin-induced lung fibrosis. These data suggest that IL-13Ralpha(2) signaling during prolonged inflammation is an important therapeutic target for the prevention of TGF-beta(1)-mediated fibrosis.     

IL-9 and IL-13 production by activated mast cells is strongly enhanced in the presence of lipopolysaccharide: NF-kappa B is decisively involved in the expression of IL-9

Mast cells, due to their ability to produce a large panel of mediators and cytokines, participate in a variety of processes in adaptive and innate immunity. Herein we report that in primary murine bone marrow-derived mast cells activated with ionomycin or IgE-Ag the bacterial endotoxin LPS strongly enhances the expression of IL-9 and IL-13, but not IL-4. This costimulatory effect of LPS is absent in activated mast cells derived from the LPS-hyporesponsive mouse strain BALB/c-LPS(d), although in these cells the proinflammatory cytokine IL-1 can still substitute for LPS. The enhanced production of mast cell-derived IL-13 in the presence of IL-1 is a novel observation. Coactivation of mast cells with LPS leads to a synergistic activation of NF-kappa B, which is shown by an NF-kappa B-driven reporter gene construct. In the presence of an inhibitor of NF-kappa B activation, the production of IL-9 is strongly decreased, whereas the expression of IL-13 is hardly reduced, and that of IL-4 is not affected at all. NF-kappa B drives the expression of IL-9 via three NF-kappa B binding sites within the IL-9 promoter, which we characterize using gel shift analyses and reporter gene assays. In the light of recent reports that strongly support critical roles for IL-9 and IL-13 in allergic lung inflammation, our results emphasize the potential clinical importance of LPS as an enhancer of mast cell-derived IL-9 and IL-13 production in the course of inflammatory reactions and allergic diseases.     

Schistosome infection of transgenic mice defines distinct and contrasting pathogenic roles for IL-4 and IL-13: IL-13 is a profibrotic agent

Experimental Schistosoma mansoni infections of mice lead to a dynamic type 2 cytokine-mediated pathological process. We have used IL-4-deficient, IL-13-deficient, and IL-4/13-deficient mice to dissect the role of these cytokines in the development of immune response and pathology following S. mansoni infection. We demonstrate that while both of these cytokines are necessary to develop a robust Th2 cell-driven, eosinophil-rich granuloma response, they also perform disparate functions that identify novel sites for therapeutic intervention. IL-13-deficient mice demonstrated significantly enhanced survival following infection, which correlated with reduced hepatic fibrosis. In contrast, increased mortality was manifest in IL-4-deficient and IL-4/13-deficient mice, and this correlated with hepatocyte damage and intestinal pathology. Therefore, we demonstrate that during a dynamic type 2 cytokine disease process IL-13 is detrimental to survival following infection, whereas IL-4 is beneficial.     

Implications of serum TNF-beta and IL-13 in the treatment response of childhood nephrotic syndrome

The cause of childhood nephrotic syndrome (NS) is unknown and whether it responds to steroid therapy remains unpredictable. In the present study, we measured the Th1/Th2 cytokines, serum tumor necrosis factor-beta (TNF-beta) and interleukin-13 (IL-13), levels in children with NS before and after prednisolone (60 mg/m(2)/day) treatment for 4 weeks, to evaluate their relationships with disease activity and treatment response. Patients with acute NS had higher serum TNF-beta and IL-13 levels than normal controls. After 4 weeks of prednisolone treatment, patients with steroid-resistant NS (SRNS) presented a higher serum TNF-beta level than that before treatment (p=0.008). In contrast, patients with steroid-sensitive NS (SSNS) presented a higher serum IL-13 level than that before treatment (p=0.027). This study demonstrates the significance of serum TNF-beta and IL-13 levels in relation to the disease activity and treatment response of childhood NS. Patients with SRNS appeared to have elevated TNF-beta after steroid therapy, while patients with SSNS tended to have elevated IL-13 after steroid therapy. Thus, an altered Th1/Th2 reaction as demonstrated by TNF-beta/IL-13 imbalance may play a pathophysiologic role in childhood NS.     

IL-13 induces a bronchial epithelial phenotype that is profibrotic

Background

Inflammatory cytokines (e.g. IL-13) and mechanical perturbations (e.g. scrape injury) to the epithelium release profibrotic factors such as TGF-β₂, which may, in turn, stimulate subepithelial fibrosis in asthma. We hypothesized that prolonged IL-13 exposure creates a plastic epithelial phenotype that is profibrotic through continuous secretion of soluble mediators at levels that stimulate subepithelial fibrosis.

Methods

Normal human bronchial epithelial cells (NHBE) were treated with IL-13 (0, 0.1, 1, or 10 ng/ml) for 14 days (day 7 to day 21 following seeding) at an air-liquid interface during differentiation, and then withdrawn for 1 or 7 days. Pre-treated and untreated NHBE were co-cultured for 3 days with normal human lung fibroblasts (NHLF) embedded in rat-tail collagen gels during days 22–25 or days 28–31.

Results

IL-13 induced increasing levels of MUC5AC protein, and TGF-β₂, while decreasing β-Tubulin IV at day 22 and 28 in the NHBE. TGF-β₂, soluble collagen in the media, salt soluble collagen in the matrix, and second harmonic generation (SHG) signal from fibrillar collagen in the matrix were elevated in the IL-13 pre-treated NHBE co-cultures at day 25, but not at day 31. A TGF-β₂ neutralizing antibody reversed the increase in collagen content and SHG signal.

Conclusion

Prolonged IL-13 exposure followed by withdrawal creates an epithelial phenotype, which continuously secretes TGF-β₂ at levels that increase collagen secretion and alters the bulk optical properties of an underlying fibroblast-embedded collagen matrix. Extended withdrawal of IL-13 from the epithelium followed by co-culture does not stimulate fibrosis, indicating plasticity of the cultured airway epithelium and an ability to return to a baseline. Hence, IL-13 may contribute to subepithelial fibrosis in asthma by stimulating biologically significant TGF-β₂ secretion from the airway epithelium.     

IL-13 is a susceptibility factor for Leishmania major infection

Leishmania major infection is useful as an experimental model to define factors responsible for the development and maintenance of Th cell immune responses. Studies using inbred mouse strains have identified that the Th1 response characteristic of C57BL/6 mice results in healing, whereas BALB/c mice fail to control the infection due to the generation of an inappropriate Th2 response. We now demonstrate that IL-13 is a key factor in determining susceptibility to L. major infection. Overexpression of IL-13 in transgenic mice makes the normally resistant C57BL/6 mouse strain susceptible to L. major infection even in the absence of IL-4 expression. This susceptibility correlates with a suppression of IL-12 and IFN-gamma expression. Furthermore, using BALB/c mice deficient in the expression of IL-4, IL-13, or both IL-13 and IL-4, we demonstrate that IL-13-deficient mice are resistant to infection and that there is an additive effect of deleting both IL-4 and IL-13.     

IL-13 receptor alpha2 selectively inhibits IL-13-induced responses in the murine lung

IL-13 is a critical cytokine at sites of Th2 inflammation. In these locations it mediates its effects via a receptor complex, which contains IL-4Ralpha and IL-13Ralpha1. A third, high-affinity IL-13 receptor, IL-13Ralpha2, also exists. Although it was initially felt to be a decoy receptor, this has not been formally demonstrated and the role(s) of this receptor has recently become controversial. To define the role(s) of IL-13Ralpha2 in IL-13-induced pulmonary inflammation and remodeling, we compared the effects of lung-targeted transgenic IL-13 in mice with wild-type and null IL-13Ralpha2 loci. We also investigated the effect of IL-13Ralpha2 deficiency on the OVA-induced inflammatory response. In this study, we show that in the absence of IL-13Ralpha2, IL-13-induced pulmonary inflammation, mucus metaplasia, subepithelial fibrosis, and airway remodeling are significantly augmented. These changes were accompanied by increased expression and production of chemokines, proteases, mucin genes, and TGF-beta1. Similarly, an enhanced inflammatory response was observed in an OVA-induced phenotype. In contrast, disruption of IL-13Ralpha2 had no effect on the tissue effects of lung-targeted transgenic IL-4. Thus, IL-13Ralpha2 is a selective and powerful inhibitor of IL-13-induced inflammatory, remodeling, and physiologic responses in the murine lung.     

Accelerated regeneration of the skeletal muscle in RNF13-knockout mice is mediated by macrophage-secreted IL-4/IL-6

RING finger protein 13 (RNF13) is a newly identified E3 ligase reported to be functionally significant in the regulation of cancer development, muscle cell growth, and neuronal development. In this study, the function of RNF13 in cardiotoxin-induced skeletal muscle regeneration was investigated using RNF13-knockout mice. RNF13^-/- mice exhibited enhanced muscle regeneration —characterized by accelerated satellite cell proliferation —compared with wild-type mice. The expression of RNF13 was remarkably induced in macrophages rather than in the satellite cells of wild-type mice at the very early stage of muscle damage. This result indicated that inflammatory cells are important in RNF13-mediated satellite cell functions. The cytokine levels in skeletal muscles were further analyzed and showed that RNF13^-/- mice produced greater amounts of various cytokines than wild-type mice. Among these, IL-4 and IL-6 levels significantly increased in RNF13^-/- mice. The accelerated muscle regeneration phenotype was abrogated by inhibiting IL-4/IL-6 action in RNF13^-/- mice with blocking antibodies. These results indicate that RNF13 deficiency promotes skeletal muscle regeneration via the effects on satellite cell niche mediated by IL-4 and IL-6.     

Structural model of human IL-13 defines the spatial interactions with the IL-13Ralpha/IL-4Ralpha receptor

Interleukin-13 (IL-13) plays a key role in immune responses and inflammation. A structural model of human IL-13 (HuIL-13) based on the nuclear magnetic resonance and X-ray structure of IL-4 is put forward. Unlike previous models, this model is based on new sequence alignments that take into account the formation of the two disulfide linkages that have been determined experimentally. The proposed structure of human IL-13 is similar to IL-4, consisting of a four helix bundle with hydrophobic residues lining the core of the molecule and surface polar residues showing a high degree of solvent accessibility. Regions of HuIL-13 that are critical for the interaction with its receptors are explored and discussed in relation to existing mutagenic studies. From these studies we predict that helices A and C of HuIL-13 interact with the IL-4 receptor alpha (IL-4Ralpha) region and helix D is responsible for the interaction with the IL-13 receptor alpha 1 (IL-13Ralpha1) receptor.     

IL-4 receptor alpha is an important modulator of IL-4 and IL-13 receptor binding: implications for the development of therapeutic targets

IL-4 is a key cytokine associated with allergy and asthma. Induction of cell signaling by IL-4 involves interaction with its cognate receptors, a complex of IL-4Ralpha with either the common gamma-chain or the IL-13R chain alpha1 (IL-13Ralpha1). We found that IL-4 bound to the extracellular domain of IL-4Ralpha (soluble human (sh)IL-4Ralpha) with high affinity and specificity. In contrast with the sequential mechanism of binding and stabilization afforded by IL-4Ralpha to the binding of IL-13 to IL-13Ralpha1, neither common gamma-chain nor IL-13Ralpha1 contributed significantly to the stabilization of the IL-4:IL-4Ralpha complex. Based on the different mechanisms of binding and stabilization of the IL-4R and IL-13R complexes, we compared the effects of shIL-4Ralpha and an IL-4 double mutein (R121D/Y124D, IL-4R antagonist) on IL-4- and IL-13-mediated responses. Whereas IL-4R antagonist blocked responses to both cytokines, shIL-4Ralpha only blocked IL-4. However, shIL-4Ralpha stabilized and augmented IL-13-mediated STAT6 activation and eotaxin production by primary human bronchial fibroblasts at suboptimal doses of IL-13. These data demonstrate that IL-4Ralpha plays a key role in the binding affinity of both IL-13R and IL-4R complexes. Under certain conditions, shIL-4Ralpha has the potential to stabilize binding IL-13 to its receptor to augment IL-13-mediated responses. Thus, complete understanding of the binding interactions between IL-4 and IL-13 and their cognate receptors may facilitate development of novel treatments for asthma that selectively target these cytokines without unpredicted or detrimental side effects.     

IL-18 is a potent coinducer of IL-13 in NK and T cells: a new potential role for IL-18 in modulating the immune response

IL-13 and IL-4 have similar biological activities and are characteristic of cytokines expressed by Th2 cells. In contrast, IL-12 and IL-18 have been shown to be strong cofactors for Th1 cell development. In this study, we found strong induction of IL-13 mRNA and protein by IL-2 + IL-18 in NK and T cells. In contrast, IL-12 did not enhance the IL-13 production induced by IL-2 alone. Moreover, IL-13 mRNA and protein expression induced by IL-2 + IL-18 in purified NK and T cells obtained from IFN-gamma knockout (-/-) mice were greater than seen in purified cells from normal controls. In contrast, IL-10 production induced by IL-2 and/or IL-12 was not significantly different in IFN-gamma (-/-) mice and normal controls. These results suggest IL-13 expression induced by IL-2 + IL-18 may be regulated by IFN-gamma in vivo, while IL-10 expression may be IFN-gamma-independent. Thus, depending upon the cell type, IL-18 may act as a strong coinducer of Th1 or Th2 cytokines. Our findings suggest that IL-12 and IL-18 have different roles in the regulation of gene expression in NK and T cells.     

IL-13-mediated worm expulsion is B7 independent and IFN-gamma sensitive

B7 costimulation is a required component of many type 2 immune responses, including allergy and protective immunity to many nematode parasites. This response includes elevations in Th2 cytokines and associated effector functions including elevations in serum IgG1 and IgE and parasite expulsion. In studies of mice infected with Trichuris muris, blocking B7 ligand interactions inhibited protective immunity, suppressed IL-4 production, and enhanced IFN-gamma production, but unexpectedly did not inhibit production of the Th2 cytokine, IL-13. Blocking both IFN-gamma and B7 restored protective immunity, which was IL-13 dependent, but did not restore IL-4 or associated IgE responses. Although IL-13 was required for worm expulsion in mice in which both IFN-gamma and B7 were blocked, IL-4 could mediate expulsion in the absence of both IL-13 and IFN-gamma. These studies demonstrate that 1) B7 costimulation is required to induce IL-4, but not IL-13 responses; 2) IL-13 is elevated in association with the IFN-gamma response that occurs following inhibition of B7 interactions, but can only mediate IL-4-independent protection when IFN-gamma is also inhibited; and 3) increased IL-13 production, in the absence of increased IL-4 production, is not associated with an IgE response, even in the absence of IFN-gamma.     

The negative-feedback regulation of the IL-13 signal by the IL-13 receptor alpha2 chain in bronchial epithelial cells

Bronchial asthma is a complex disease characterized by airway inflammation involving Th2 cytokines. Among Th2 cytokines, the significance of IL-13 in the pathogenesis of bronchial asthma has recently emerged. Particularly, the direct action of IL-13 on bronchial epithelial cells (BECs) is critical for generation of airway hyperresponsiveness. IL-13 has two binding units; the IL-13 receptor alpha1 chain transduces the IL-13 signal comprising a heterodimer with the IL-4R alpha chain, whereas the IL-13 receptor alpha2 chain (IL-13Ralpha2) is thought to act as a decoy receptor. However, it remains obscure how expression of these molecules is regulated in each cell. In this article, we analyzed the expression of these components in BECs. Either IL-4 or IL-13 induced intracellular expression of IL-13Ralpha2 in BECs, which was STAT6-dependent and required de novo protein synthesis. IL-13Ralpha2 expressed on the cell surface as a monomer inhibited the STAT6-dependent IL-13 signal. Furthermore, expression of IL-13Ralpha2 was induced in lung tissues of ovalbumin-induced asthma model mice. Taken together, our results suggested the possibility that IL-13Ralpha2 induced by its ligand is transferred to the cell surface by an unknown mechanism, and it down-regulates the IL-13 signal in BECs, which functions as a unique negative-feedback system for the cytokine signal.     

Severe airway epithelial injury, aberrant repair and bronchiolitis obliterans develops after diacetyl instillation in rats

Background

Bronchiolitis obliterans (BO) is a fibrotic lung disease that occurs in a variety of clinical settings, including toxin exposures, autoimmunity and lung or bone marrow transplant. Despite its increasing clinical importance, little is known regarding the underlying disease mechanisms due to a lack of adequate small animal BO models. Recent epidemiological studies have implicated exposure to diacetyl (DA), a volatile component of artificial butter flavoring, as a cause of BO in otherwise healthy factory workers. Our overall hypothesis is that DA induces severe epithelial injury and aberrant repair that leads to the development of BO. Therefore, the objectives of this study were 1) to determine if DA, delivered by intratracheal instillation (ITI), would lead to the development of BO in rats and 2) to characterize epithelial regeneration and matrix repair after ITI of DA.

Methods and Main Results

Male Sprague-Dawley rats were treated with a single dose of DA (125 mg/kg) or sterile water (vehicle control) by ITI. Instilled DA resulted in airway specific injury, followed by rapid epithelial regeneration, and extensive intraluminal airway fibrosis characteristic of BO. Increased airway resistance and lung fluid neutrophilia occurred with the development of BO, similar to human disease. Despite rapid epithelial regeneration after DA treatment, expression of the normal phenotypic markers, Clara cell secretory protein and acetylated tubulin, were diminished. In contrast, expression of the matrix component Tenascin C was significantly increased, particularly evident within the BO lesions.

Conclusions

We have established that ITI of DA results in BO, creating a novel chemical-induced animal model that replicates histological, biological and physiological features of the human disease. Furthermore, we demonstrate that dysregulated epithelial repair and excessive matrix Tenacin C deposition occur in BO, providing new insights into potential disease mechanisms and therapeutic targets.     

IL-13 activates a mechanism of tissue fibrosis that is completely TGF-beta independent

Fibrosis is a characteristic feature in the pathogenesis of a wide spectrum of diseases. Recently, it was suggested that IL-13-dependent fibrosis develops through a TGF-beta1 and matrix metalloproteinase-9-dependent (MMP-9) mechanism. However, the significance of this pathway in a natural disorder of fibrosis was not investigated. In this study, we examined the role of TGF-beta in IL-13-dependent liver fibrosis caused by Schistosoma mansoni infection. Infected IL-13-/- mice showed an almost complete abrogation of fibrosis despite continued and undiminished production of TGF-beta1. Although MMP-9 activity was implicated in the IL-13 pathway, MMP-9-/- mice displayed no reduction in fibrosis, even when chronically infected. To directly test the requirement for TGF-beta, studies were also performed with neutralizing anti-TGF-beta Abs, soluble antagonists (soluble TGF-betaR-Fc), and Tg mice (Smad3-/- and TGF-betaRII-Fc Tg) that have disruptions in all or part of the TGF-beta signaling cascade. In all cases, fibrosis developed normally and with kinetics similar to wild-type mice. Production of IL-13 was also unaffected. Finally, several genes, including interstitial collagens, several MMPs, and tissue inhibitors of metalloprotease-1 were up-regulated in TGF-beta1-/- mice by IL-13, demonstrating that IL-13 activates the fibrogenic machinery directly. Together, these studies provide unequivocal evidence of a pathway of fibrogenesis that is IL-13 dependent but TGF-beta1 independent, illustrating the importance of targeting IL-13 directly in the treatment of infection-induced fibrosis.     

Allergen-induced airway hyperresponsiveness mediated by cyclooxygenase inhibition is not dependent on 5-lipoxygenase or IL-5, but is IL-13 dependent

Cyclooxygenase (COX) inhibition during allergic sensitization and allergen airway challenge results in augmented allergic inflammation. We hypothesized that this increase in allergic inflammation was dependent on increased generation of leukotrienes that results from COX inhibition, as leukotrienes are important proinflammatory mediators of allergic disease. To test this hypothesis, we allergically sensitized and challenged mice deficient in 5-lipoxygenase (5-LO). We found that 5-LO knockout mice that were treated with a COX inhibitor during allergic sensitization and challenge had significantly increased airway hyperresponsiveness (AHR) (p < 0.01) and airway eosinophilia (p < 0.01) compared with 5-LO knockout mice that were treated with vehicle. The proinflammatory cytokines have also been hypothesized to be critical regulators of airway inflammation and AHR. We found that the increase in airway eosinophilia seen with COX inhibition is dependent on IL-5, whereas the increase in AHR is not dependent on this cytokine. In contrast, the COX inhibition-mediated increase in AHR is dependent on IL-13, but airway eosinophilia is not. These results elucidate the pathways by which COX inhibition exerts a critical effect of the pulmonary allergen-induced inflammatory response and confirm that COX products are important regulators of allergic inflammation.     

Bacterial infectious process in patients with chronic obliterating bronchiolitis

Chronic obliterating bronchiolitis in children is characterized by the presence of chronic multifactor inflammatory infectious process with Streptococcus pneumoniae and Haemophilus influenzae being the leading causative agents. The inflammatory process is induced and maintained by S. pneumoniae and H. influenzae or by the association of these two pathogens, but the severity of the clinical course of obliterating bronchiolitis is determined by H. influenzae, which is confirmed by its high detection rate at the phase of exacerbation in patients with pronounced bronchial obstruction.