Claudia Jakubzick

Therapeutic Effect of IL-13 Immunoneutralization During Chronic Experimental Fungal Asthma

IL-13 and IL-4 are key contributors to the asthmatic phenotype. The temporal role of these cytokines in airway function, inflammation, and remodeling were assessed in a chronic murine model of Asperigillus fumigatus-induced allergic asthma. IL-13 and IL-4 protein levels were significantly elevated by 30 days after conidia challenge in A. fumigatus-sensitized mice. Furthermore, IL-13Rα1 mRNA expression was significantly elevated 7 days after conidia challenge and remained elevated until day 21. In contrast, IL-13Rα2 mRNA expression, although constitutively expressed in naive lung, was absent in the lungs of A. fumigatus-sensitized mice both before and after conidia challenge. Membrane-bound IL-4R mRNA expression was significantly elevated 7 days after conidia challenge; however, soluble IL-4R mRNA expression was increased 30 days after conidia challenge. Immunoneutralization of IL-13 between days 14 and 30 or days 30 and 38 after fungal sensitization and challenge significantly attenuated airway hyperresponsiveness, collagen deposition, and goblet cell hyperplasia at day 38 after conidia challenge; however, the effects of IL-4 immunoneutralization during the same time periods were not as marked. IFN-γ and IL-12 release after Aspergillus Ag restimulation was elevated from spleen cells isolated from mice treated with IL-4 anti-serum compared with IL-13 anti-serum or normal rabbit serum-treated mice. This study demonstrates a pronounced therapeutic effect of IL-13-immunoneutralization at extended time points following the induction of chronic asthma. Most importantly, these therapeutic effects were not reversed following cessation of treatment, and IL-13 anti-serum treatment did not alter the systemic immune response to Ag restimulation, unlike IL-4 immunoneutralization. Therefore, IL-13 provides an attractive therapeutic target in allergic asthma.

Therapeutic Attenuation of Pulmonary Fibrosis Via Targeting of IL-4- and IL-13-Responsive Cells

Severe forms of idiopathic interstitial pneumonia (IIP), such as usual interstitial pneumonia, can be impervious to modern steroid and immunosuppressive treatment regimens, thereby emphasizing the need for novel effective therapies. Consequently, research attention has been directed toward understanding the cytokine networks that may affect fibroblast activation and, hence, the progression of certain IIPs. This led us to investigate whether the specific targeting of resident lung cells responsive to IL-4 and IL-13 exerted a therapeutic effect in an experimental model of IIP, namely the bleomycin-induced model of pulmonary fibrosis. IL-4, IL-13, and their corresponding receptor subunits, IL-4Rα, IL-13Rα1, and IL-13Rα2, were maximally expressed at the mRNA and protein levels in whole lung samples on day 21 or 28 after an intratracheal bleomycin challenge. The intranasal administration of an IL-13 immunotoxin chimeric molecule (IL13-PE) from days 21–28, but not for 1-wk periods at earlier times, after bleomycin challenge had a significant therapeutic effect on histological and biochemical parameters of bleomycin-induced pulmonary fibrosis compared with the control group. The intranasal IL13-PE therapy significantly reduced the numbers of IL-4 and IL-13 receptor-positive mononuclear cells and macrophages and the levels of profibrotic cytokine and chemokine in the lungs of bleomycin-challenged mice on day 28. Thus, this study demonstrates that IL-4- and/or IL-13-binding cells are required for the maintenance of pulmonary fibrosis induced by bleomycin and highlights the importance of further investigation of antifibrotic therapeutics that target these cells during pulmonary fibrosis.

Stat6-Deficient Mice Develop Airway Hyperresponsiveness and Peribronchial Fibrosis during Chronic Fungal Asthma

Signal transducer and activator of transcription 6 (Stat6) is critical for Th2-mediated responses during allergic airway disease. To investigate the role of Stat6 in fungus-induced airway hyperresponsiveness and remodeling, Stat6-deficient (Stat6-/-) and Stat6-wildtype (Stat6+/+) mice were sensitized to Aspergillus fumigatus and airway disease was subsequently assessed in both groups at days 21, 30, 38, and 44 after an intratracheal challenge with live A. fumigatus conidia. At all times after conidia, histological analysis revealed an absence of goblet cell hyperplasia and markedly diminished peribronchial inflammation in Stat6-/- mice in contrast to Stat6+/+ mice. Airway hyperresponsiveness and peribronchial fibrosis in Stat6-/- mice were significantly reduced at day 21 after conidia compared with Stat6+/+ mice, but both groups exhibited significant, similar increases in these parameters at all subsequent times after conidia. In separate experiments, IL-13-responsive cells in Stat6-/- mice were targeted via the daily intranasal administration of 200 ng of IL-13-PE38QQR (IL13-PE), comprised of human IL-13 and a derivative of Pseudomonas exotoxin, from days 38 to 44 after the conidia challenge. IL13-PE treatment abolished airway hyperresponsiveness, but not peribronchial fibrosis in Stat6-/- mice. Taken together, these data demonstrate that the chronic development of airway hyperresponsiveness during fungal asthma is IL-13-dependent but Stat6-independent.

Therapeutic targeting of IL-4-and IL-13-responsive cells in pulmonary fibrosis

Severe forms of idiopathic interstitial pneumonia (IIP), such as usual interstitial pneumonia (UIP), can be impervious to modern steroid and immunosuppressive treatment regimens, thereby emphasizing the need for novel effective therapies. Understanding the cytokine networks that may affect immune and structural cell activation and, hence, the progression of these fatal fibrotic diseases, has been a focus in our research. In this regard, we have examined the role of interleukin (IL)-4 and IL-13 and their respective receptor subunits in this process. Examination of clinical surgical lung biopsies (SLBs) showed that IIP is characterized by the abnormal, heightened expression of the receptor subunits that bind IL-4 and IL-13. Specifically, IL-4Rα and IL-13Rα2 (the high-affinity IL-13 receptor subunit) was present in greater abundance in SLBs and fibroblasts from IIP patients compared with normal patients, who exhibited no evidence of pulmonary fibrosis. These clinical findings prompted us to investigate whether the targeting of pulmonary cell types that were highly responsive to IL-4 and IL-13 was a viable therapeutic option in IIP. Using a chimeric protein comprised of human IL-13 and a truncated version of an exotoxin from Pseudomonas (abbreviated IL13-PE), we observed that IL13-PE selectively targeted human pulmonary fibroblasts grown from IIP SLBs, whereas it had a minimal effect on fibroblasts grown from biopsies from normal patients. In murine models characterized by abnormal airway or interstitial fibrotic responses, the intranasal administration of IL13-PE significantly attenuated the fibrotic response through the targeting of IL-4Rα-and IL-13Rα2-expressing pulmonary cells, including monocytes, macrophages, and pulmonary fibroblasts. Together, these data demonstrate that IL-4 and IL-13 are required for the initiation and maintenance of pulmonary fibrosis, and highlight the importance of further investigation of anti-fibrotic therapeutics that prevent the action of both cytokines during clinical pulmonary fibrosis.

Human Pulmonary Fibroblasts Exhibit Altered Interleukin-4 and Interleukin-13 Receptor Subunit Expression in Idiopathic Interstitial Pneumonia

Abnormal proliferation of pulmonary fibroblasts is a prominent feature of chronic pulmonary fibrotic diseases such as idiopathic interstitial pneumonia (IIP), but it is not presently clear how this proliferative response by lung fibroblasts can be therapeutically modulated. In the present study, we examined whether it was possible to selectively target primary human pulmonary fibroblasts grown out of surgical lung biopsies (SLBs) from IIP patients based on their expression of interleukin-4 receptor (IL-4R) and IL-13R subunits. Pulmonary fibroblast lines cultured from patients with the severest form of IIP, namely usual interstitial pneumonia, exhibited the greatest gene and protein expression of IL-4Ralpha, IL-13Ralpha1, and IL-13Ralpha2 compared with primary pulmonary fibroblast lines grown from other IIP SLBs and normal SLBs. When exposed to increasing concentrations of a chimeric protein comprised of human IL-13 and a truncated version of Pseudomonas exotoxin (IL13-PE), the proliferation of primary usual interstitial pneumonia fibroblasts was inhibited to a much greater extent compared with fibroblast lines from nonspecific interstitial pneumonia and respiratory bronchiolitis/interstitial lung disease patient groups. Fibroblasts from normal patients exhibited minimal susceptibility to the cytotoxic effect of IL13-PE. IL13-PE-mediated targeting of IIP fibroblasts was dependent on their expression of IL-4Ralpha and IL-13Ralpha2. Thus, these data suggest that the abnormal proliferative properties of human lung fibroblasts from certain IIP patient groups can be modulated in a manner that is dependent on the IL-4 and IL-13 receptor subunit expression by these cells.

IL-13 Fusion Cytotoxin Ameliorates Chronic Fungal-Induced Allergic Airway Disease in Mice

IL-13 has emerged as a major contributor to allergic and asthmatic responses, and as such it represents an attractive target in these diseases. In this study, IL-13-responsive cells in the lung were targeted via the intranasal administration of IL-13-PE38QQR (IL-13-PE), comprised of human IL-13 and a derivative of Pseudomonas exotoxin, to Aspergillus fumigatus-sensitized mice challenged with A. fumigatus spores, or conidia. Mice received 50, 100, or 200 ng of IL-13-PE or diluent alone (i.e., control group) on alternate days from day 14 to day 28 after the conidia challenge. The control group of mice exhibited significant airway hyperreactivity, goblet cell hyperplasia, and peribronchial fibrosis at day 28 after conidia. Although the two lower doses of IL-13-PE had limited therapeutic effects in mice with fungal-induced allergic airway disease, the highest dose of IL-13-PE tested significantly reduced all features of airway disease compared with the control group. Whole lung mRNA expression of IL-4Rα and IL-13Rα1 was markedly reduced, whereas bronchoalveolar lavage and whole lung levels of IFN-γ were significantly elevated in mice treated with 200 ng of IL-13-PE compared with the control group. This study demonstrates that a therapy designed to target IL-13-responsive cells in the lung ameliorates established fungal-induced allergic airway disease in mice.

Role of CCR4 ligands, CCL17 and CCL22, during Schistosoma mansoni egg-induced pulmonary granuloma formation in mice.

Controversy persists pertaining to the role of CCR4 ligands, namely CCL17 (or thymus and activation regulated chemokine; TARC) and CCL22 (or macrophage-derived chemokine; MDC), in Th2-type cytokine-dominated responses in the lung. Accordingly, the present study addressed the relative role of each of these CC chemokines during an evolving pulmonary granulomatous response elicited by the intrapulmonary embolization of live Schistosoma mansoni eggs into S. mansoni-sensitized mice. CCL22 protein expression peaked at day 4, but CCL17 levels were not increased significantly at any time after egg challenge. CCR4 transcript and protein expression were highest at day 8 after egg embolization and CCR4 protein was prominently expressed in macrophages surrounding S. mansoni eggs. Systemic immunoneutralization of CCL22 from the time of egg injection into S. mansoni-sensitized mice for 8 days significantly decreased CCR4 protein expression, the eosinophil content, the overall size of the egg granuloma, and its hydroxyproline content. Whole lung levels of interferon-gamma were also significantly increased at day 8 in anti-CCL22-treated mice. The systemic immunoneutralization of CCL17 had a lesser effect on all of the granuloma parameters listed above, but this antibody treatment significantly decreased granuloma hydroxyproline content to a greater extent than the anti-CCL22 antibody treatment. In addition, the immunoneutralization of CCL17 significantly increased whole lung levels of interleukin (IL)-4, IL-5, IL-13, transforming growth factor-beta, IL-12, and tumor necrosis factor-alpha at day 8 after egg infusion. Thus, these studies demonstrate a major role for CCL22 and a lesser role for CCL17 during an evolving S. mansoni egg granuloma in the lung.

Impact of Interleukin-13 Responsiveness on the Synthetic and Proliferative Properties of Th1- and Th2-Type Pulmonary Granuloma Fibroblasts

Interleukin-13 (IL-13) has emerged as a major cytokine mediator of fibroblast activation and pulmonary fibrosis. Normal (from noninflamed lung), Th1-type (induced by the pulmonary embolization of purified peptide derivative-coated beads in mice sensitized to purified peptide derivative), and Th2-type (induced by the pulmonary embolization of Schistosoma mansoni egg antigen-coated beads in mice sensitized with S. mansoni eggs) primary fibroblast cell lines all exhibited constitutive gene expression of two receptor chains that bind and signal IL-13-mediated cellular events: IL-4Ralpha and IL-13Ralpha1. However, all three fibroblast cell lines exhibited divergent synthetic and proliferative responses to the exogenous addition of either recombinant IL-13 or a chimeric protein comprised of IL-13 and a truncated version of Pseudomonas exotoxin (IL13-PE), which targets and kills IL-13 receptor overexpressing cells. The exogenous addition of IL-13 to Th1-type and Th2-type fibroblast cultures significantly increased the cellular expression of IL-13Ralpha2, which may function as an IL-13 decoy receptor. After a 24-hour exposure to IL-13, the total collagen generation and cellular proliferation by Th2-type fibroblasts were significantly higher than that observed in similar numbers of normal and Th1-type fibroblasts. In addition IL13-PE, which binds with highest affinity to IL-13Ralpha2, exhibited down-regulatory effects on proliferation and matrix generation expression by Th1- and Th2-type, but not normal, fibroblasts. Thus, these data demonstrate that fibroblasts derived from murine pulmonary granulomas exhibit divergent expression of functional IL-13 receptor and this expression dictates the responsiveness and susceptibility to recombinant IL-13 and IL-13 immunotoxin, respectively.

Augmented pulmonary IL-4 and IL-13 receptor subunit expression in idiopathic interstitial pneumonia

Background: Some idiopathic interstitial pneumonias (IIPs) are characterised by fibroproliferation and deposition of extracellular matrix. Because efficacious treatment options are limited, research has been directed towards understanding the cytokine networks that may affect fibroblast activation and, hence, the progression of certain IIPs. Aims: To examine the expression of interleukin 4 (IL-4), IL-13, and their corresponding receptor subunits in the various forms of IIP and normal patient groups. Methods: Molecular and immunohistochemical analysis of IL-4, interferon γ (IFNγ), IL-13, IL-4 receptor (IL-R), and IL-13 receptor subunits in surgical lung biopsies (SLBs) from 39 patients (21 usual interstitial pneumonia (UIP), six non-specific interstitial pneumonia (NSIP), eight respiratory bronchiolitic interstitial lung disease (RBILD), and five normal controls). Results: Molecular analysis demonstrated that IL-13Rα2, IL-13Rα1, and IL-4Rα were present in a greater proportion of upper and lower lobe biopsies from patients with UIP than patients with NSIP and RBILD. Immunohistochemical analysis of patients with UIP, NSIP, and RBILD revealed interstitial staining for all three receptor subunits, whereas such staining was only seen in mononuclear cells present in normal SLBs. Fibroblastic foci in patients with UIP strongly stained for IL-4Rα and IL-13Rα2. Localised expression of IL-4Rα was also seen in SLBs from patients with NSIP but not in other groups. Conclusion: Some histological subtypes of IIP are associated with increased pulmonary expression of receptor subunits responsive to IL-4 and IL-13. These findings may be of particular importance in understanding the pathogenesis of IIP and, more importantly, may provide important novel therapeutic targets.

Focal interstitial CC chemokine receptor 7 (CCR7) expression in idiopathic interstitial pneumonia

Background/Aims: Idiopathic interstitial pneumonias (IIPs) are a diverse grouping of chronic pulmonary diseases characterised by varying degrees of pulmonary fibrosis. The triggers of the fibroproliferative process in IIP remain enigmatic but recent attention has been directed towards chemokine involvement in this process. Methods: The expression of two chemokine receptors, CCR7 and CXCR4, and their respective ligands, CCL19, CCL21, and CXCL12, were examined in surgical lung biopsies (SLBs) from patients with IIP. Transcript and protein expression of these receptors and their ligands was compared with that detected in histologically normal margin SLBs. Results: CCR7 and CXCR4 were detected by gene array and real time polymerase chain reaction analysis and CCR7, but not CXCR4, expression was significantly raised in usual interstitial pneumonia (UIP) relative to biopsies from patients diagnosed with non-specific interstitial pneumonia (NSIP) or respiratory bronchiolitis/interstitial lung disease (RBILD). CCR7 protein was expressed in interstitial areas of all upper and lower lobe UIP SLBs analysed. CCR7 expression was present in 50% of NSIP SLBs, and CCR7 was restricted to blood vessels and mononuclear cells in 75% of RBILD SLBs. Immune cell specific CXCR4 expression was seen in IIP and normal margin biopsies. CCR7 positive areas in UIP biopsies were concomitantly positive for CD45 (the leucocyte common antigen) but CCR7 positive areas in all IIP SLBs lacked the haemopoietic stem cell antigen CD34, collagen 1, and α smooth muscle actin. Conclusion: This molecular and immunohistochemical analysis showed that IIPs are associated with abnormal CCR7 transcript and protein expression.