Increased High Molecular Weight IL-5 Complex After Anti-IL-5 (Mepolizumab) Therapy

S118 Abstracts

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J ALLERGY CLIN IMMUNOL FEBRUARY 2008

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Increased High Molecular Weight IL-5 Complex After Anti-IL5 (Mepolizumab) Therapy M. L. Stein, A. Munitz, M. E. Rothenberg; Cincinnati Children’s Hospital Medical Center. Division of Allergy and Immunology, Cincinnati, OH. RATIONALE: High levels of IL-5 have been found after anti-IL-5 therapy despite the marked decreases in eosinophilia. We hypothesized that increased levels of IL-5 may reflect the presence of an IL-5/ mepolizumab complex. METHODS: Patients (n 5 21) with eosinophilic-disorders received antiIL-5 therapy (three monthly doses of 750 mg i.v.). Samples with increased plasma IL-5 (ELISA) after therapy were analyzed by an exclusion filtration of 100 kDa (microconÒ); low and high molecular weight (MW) IL-5 were then measured by ELISA. Immunoreactive IL-5 fractions and plasma samples were subsequently precipitated with increasing concentrations of protein A/G. RESULTS: Patients (15/21) had >9-fold mean increase in immunoreactive plasma IL-5 after anti-IL-5 therapy. Exclusion filtration revealed that IL-5 was completely detected in the high MW fraction. As a control, recombinant IL-5 was completely present in the filtered fraction consistent with its MW (26 and 52 kDa for monomer and dimer, respectively). Representative samples of the high MW fraction were significantly precipitated with protein A/G. As a control, protein A/G did not precipitate IL-5 derived from stimulated PBMC. Protein A/G induced a concentrationdependent decrease in plasma IL-5 and the original samples were reduced to undetectable levels after precipitation with saturating amounts. CONCLUSIONS: Increased IL-5 levels in plasma samples from patients with eosinophilic disorders following anti-IL-5 therapy appear to be the result of a high molecular weight IL-5 bound complex. The precipitation of the complex by protein A/G identifies the complex as IL-5Immunoglobulin, formed by mepolizumab treatment, and likely prolonging non-active IL-5 half-life. Funding: Food and Drug Administration Grant # FD-R 002313

class switch recombination and immunoglobulin synthesis by B cells. Funding: NIH and the Ernest S. Bazley Trust

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Release of B cell-activating Factor of the TNF Family (BAFF) after Segmental Allergen Challenge of Allergic Subjects A. Kato1, H. Xiao2, M. C. Liu2, R. P. Schleimer1; 1Allergy-Immunology Division, Northwestern University Feinberg School of Medicine, Chicago, IL, 2Divisions of Allergy and Clinical Immunology, Pulmonary and Critical Care Medicine, Johns Hopkins Asthma and Allergy Center, Baltimore, MD. RATIONALE: Local production of IgA and IgE in the airways has been proposed to be an important event in both the protection from pathogens and the pathogenesis of airway allergic diseases. BAFF is a cytokine that plays important roles in B cell survival and immunoglobulin class switch recombination. We report increased production of BAFF in bronchoalveolar lavage (BAL) fluid after segmental allergen challenge (SAC) of allergic subjects. METHODS: SAC with saline or allergen (ragweed or dust mite) was performed in 16 adult allergic subjects. BAL was performed at both salineand allergen-challenged sites 20-24 hr after challenge. Concentrations of B cell-active cytokines including BAFF, IL-6 and IL-13 were measured using specific ELISA and cytometric bead array assays. RESULTS: BAFF protein was significantly elevated in BAL fluid after allergen challenge (90.4 6 29.5 pg/ml, p 5 0.001) compared with those at saline sites (2.2 6 2.2 pg/ml). In the BAL fluid after allergen challenge, BAFF levels were strongly correlated with absolute numbers of total cells (r 5 0.779, p < 0.001), lymphocytes (r 5 0.842, p < 0.001), neutrophils (r 5 0.809, p < 0.001) and eosinophils (r 5 0.621, p 5 0.010) but did not correlate with macrophages. Normalization to albumin indicated that BAFF production occurred locally in the airways. BAFF levels were also strongly correlated with other B cell-activating cytokines, IL-6 (r 5 0.875, p < 0.001) and IL-13 (r 5 0.812, p < 0.001). CONCLUSIONS: BAFF is up-regulated in the airway of allergic subjects following allergen exposure and levels were highly correlated with IL-13 and IL-6. The production of BAFF in the airway may contribute to local

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IL-25 Induces Type 2 Immune Responses By Lineage-Negative Undifferentiated Cells K. R. Bartemes, H. Kita; Mayo Clinic, Rochester, MN. RATIONALE: IL-25, a member of the IL-17 cytokine family, plays a critical role in Th2-mediated immunity during helminthic infection in mice; however, the cellular source and targets of IL-25 are not fully understood. The goal of this project was to characterize the effects of IL-25 in humans and to identify its cellular target. METHODS: Peripheral blood mononuclear cells (PBMCs) from healthy individuals were cultured with IL-25 in the presence or absence of IL-2 for up to 14 days. Cytokine levels in the supernatants were measured by ELISA. The immunomagnetic cell sorting system and FACS were used to isolate specific immune cell subsets. RESULTS: Incubation of human PBMCs with IL-25 induced marked production of IL-5 and IL-13; IL-4 and IFN-g were undetectable. IL-25mediated IL-5 production was dependent on the presence of IL-2 and reached a peak between days 9 and 13 in culture. IL-25 did not induce IL-5 production from isolated conventional T cells or NK cells, but did induce IL-5 from PBMCs depleted of these cell types. Indeed, FACS-sorted CD7positive, lineage-negative (i.e. CD3, CD16, and CD19 negative) cells responded vigorously to IL-25; CD7-negative, lineage-negative cells did not respond. Furthermore, mRNA transcription analyses of IL-25-stimulated CD7-positive, lineage-negative cells verified increases in IL-5 and IL-13, a marginal change in IFN-g, and a decrease in IL-4. CONCLUSION: IL-25 induces Th2-like cytokine production from nonconventional immune cells, namely lineage-negative undifferentiated cells. Thus, IL-25 may play a pivotal role in the early initiation phase of the innate and adaptive Th2 immune responses. Funding: NIH Effect of IL-13 on Thymic Stromal Lymphopoietin in Human Bronchial Epithelial Cells J. Y. Zhang, K. Chibana, J. B. Trudeau, S. E. Wenzel; University of Pittsburgh Medical Center, Pittsburgh, PA. RATIONALE: Thymic stromal lymphopoietin (TSLP), a cytokine produced by epithelial cells, may induce Th2 inflammatory responses. Few studies have evaluated its expression in primary human cells ex vivo, but it has been reported to be increased in asthma. We hypothesized IL-13 would increase expression of TSLP in primary human airway epithelial cells in Air Liquid Interface (ALI) culture. METHODS: Human bronchial epithelial cells were obtained from endobronchial brushings from 6 normal, 3 mild/moderate, and 5 severe asthmatics. Fresh epithelial cells were placed in TriZol for mRNA analysis. Cultured epithelial cells in ALI were stimulated (or not) with IL-13 (10 ng/ ml) for 10 days. mRNA (by quantitative real time PCR) and total protein levels (by ELISA) were analyzed. RESULTS: In small numbers, TSLP mRNA in fresh epithelial cells did not differ among the subject groups. In contrast, cultured asthmatic epithelial cells in ALI had higher baseline expression of TSLP mRNA (0.67 arbitrary units (AU), SEM from 0.48 to 0.94, compared to normal 0.24 AU, SEM from 0.18 to 0.32) which was highest in those with ongoing eosinophilic inflammation. Interestingly, IL-13, significantly decreased TSLP mRNA/protein levels to near undetectable levels. CONCLUSIONS: TSLP expression appears increased in asthmatic cells in vitro and decreases with IL-13 stimulation. This elevated baseline TSLP may indicate a genetic or epigenetic predisposition to increased TSLP expression in asthmatic subjects. While TSLP may initiate a Th2 response, persistent IL-13 activity could down-regulate production of TSLP dampening the effect. Funding: NIH