Metformin: A potential adjunct for treatment of systemic mastocytosis.

Background: Systemic mastocytosis (SM) is a clonal disorder of mast cells in which the KIT Asp816Val mutation can be detected not only in mature mast cells but also in the hematopoietic stem cell and in non-mast cell lineages. Current treatment with tyrosine kinase inhibitors provides improved clinical responses in patients with advanced mastocytosis but no cures. Targeting of cancer stem cells (CSCs) resistant to chemotherapy and radiation therapy potentially could improve clinical outcomes in mastocytosis. In recent years, nonchemotherapeutic medications such as metformin have been repurposed for this role because of their ability to destroy CSCs from both solid tumors and leukemias and also because of their ability to act as chemosensitizers. Objective: We sought to determine whether those patients with both type 2 diabetes mellitus (DM2) and SM who were receiving metformin, which has been reported to inhibit CSCs, experienced clinical or laboratory benefit to their SM from this agent. Methods: Mayo Clinic databases were searched for patients with diagnoses of DM plus SM. The clinical courses of mastocytosis for patients with DM2 were compared among patients treated with metformin or by other means. Effects of metformin on human mast cell (HMC) leukemia line (HMC-1.1 and HMC-1.2) cell proliferation were tested in vitro. Results: No patient treated with metformin before SM was diagnosed developed advanced forms of disease. A lower percentage of these patients had splenomegaly compared with other groups not treated with metformin, and none of these patients developed Janus kinase 2, tet methylcytosine dioxygenase 2, or serine and arginine-rich splicing factor 2 mutations. In vitro results showed that metformin inhibited the proliferation of both cell lines; HMC-1.1 cells were more sensitive to metformin. Conclusions: These preliminary findings suggest that early use of metformin to target CSCs has the possibility to complement current treatments available for SM.

Assessing the capacity of methylated DNA markers of cervical squamous cell carcinoma to discriminate oropharyngeal squamous cell carcinoma in human papillomavirus mediated disease.

OBJECTIVE: Early identification of human papillomavirus associated oropharyngeal squamous cell carcinoma (HPV(+)OPSCC) is challenging and novel biomarkers are needed. We hypothesized that a panel of methylated DNA markers (MDMs) found in HPV(+) cervical squamous cell carcinoma (CSCC) will have similar discrimination in HPV(+)OPSCC tissues. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded tissues were obtained from patients with primary HPV(+)OPSCC or HPV(+)CSCC; control tissues included normal oropharynx palatine tonsil (NOP) and cervix (NCS). Using a methylation-specific polymerase chain reaction, 21 previously validated cervical MDMs were evaluated on tissue-extracted DNA. Discrimination between case and control cervical and oropharynx tissue was assessed using area under the curve (AUC). RESULTS: 34 HPV(+)OPSCC, 36 HPV(+)CSCC, 26 NOP, and 24 NCS patients met inclusion criteria. Within HPV(+)CSCC, 18/21 (86%) of MDMs achieved an AUC ≥ 0.9 and all MDMs exhibited better than chance classifications relative to control cervical tissue (all p < 0.001). In contrast, within HPV(+)OPSCC only 5/21 (24%) MDMs achieved an AUC ≥ 0.90 but 19/21 (90%) exhibited better than chance classifications relative to control tonsil tissue (all p < 0.001). Overall, 13/21 MDMs had statistically significant lower AUCs in the oropharyngeal cohort compared to the cervical cohort, and only 1 MDM exhibited a statistically significant increase in AUC. CONCLUSIONS: Previously validated MDMs exhibited robust performance in independent HPV(+)CSCC patients. However, most of these MDMs exhibited higher discrimination for HPV(+)CSCC than for HPV(+)OPSCC. This suggests that each SCC subtype requires a unique set of MDMs for optimal discrimination. Future studies are necessary to establish an MDM panel for HPV(+)OPSCC.

Impact of Tumor-Infiltrating Lymphocytes on Disease Progression in Human Papillomavirus-Related Oropharyngeal Carcinoma.

OBJECTIVE: We aim to explore the prognostic value of tumor-infiltrating lymphocytes (TILs) in the primary tumor and metastatic lymph nodes of patients with HPV(+)OPSCC. We hypothesize that TILS density at both sites is associated with disease-free survival in HPV(+)OPSCC. STUDY DESIGN: Matched case-control study among HPV(+)OPSCC patients who underwent intent-to-cure surgery. Cases developed locoregional or distant recurrence. Controls were matched based on age, sex, pathologic T, N, and overall stage, year of surgery, type of adjuvant treatment received, and the Adult Comorbidity Evaluation-27 (ACE-27) score. SETTING: Single tertiary care center, May 2007 to December 2016. METHODS: Tumoral TILs (tTILs) density was defined as % TILs; stromal TILs (sTILs) density was defined as absent/sparse or moderate/dense crowding. Associations between TILs and time to disease progression were assessed using Cox regression models. RESULTS: Forty-four case-control pairs (N = 88) were included: 42 (48%) AJCC pStage I, 39 (44%) pStage II, and 7 (8%) pStage III. tTILs density ≥10% (hazard ratio [HR] 0.41, 95% confidence interval [CI] 0.17-0.99, p = .048) and a moderate/dense sTILs density (HR 0.21, 95% CI 0.06-0.75, p = .016) in the primary tumor were significantly associated with decreased risk of progression. TILs density in the lymph node was associated with decreased risk of progression but did not reach statistical significance. The tTILs and sTILs density correlated strongly between the primary tumor and lymph node. Concordance between the pathologists' was moderate (60%-70%). CONCLUSIONS: In HPV(+)OPSCC, a higher density of tumoral and stromal TILs in the primary tumor and possibly the lymph node may predict a lower risk of disease progression.

Larynx proteomics after jellyfish collagen IL: Increased ECM/collagen and suppressed inflammation.

Objectives/Hypothesis: Compare proteomic profiles of rabbit vocal folds (VFs) injected with micronized cross-linked jellyfish collagen "collagen Type 0" (MX-JC) against two clinical products for injection medialization laryngoplasty (IL). Study Design: Animal model. Methods: Left recurrent laryngeal nerve sectioning and IL were performed in New Zealand White rabbits (N = 6/group). Group 1 received (MX-JC) and adipose-derived stem cells (ADSCs), Group 2, MX-JC alone; Group 3, cross-linked hyaluronic acid; and Group 4, micronized acellular dermis. Animals were sacrificed at 4 and 12 weeks. Proteomic profiling of injected versus noninjected VFs by nano-liquid chromatography, tandem mass spectrometry, and reactome gene ontology analysis was performed. Results: Overall, 37-61 proteins were found to be upregulated and 60-284 downregulated in injected versus non-injected VFs (>1.5 fold, false discovery rate-adjusted p < .05). Over-representation analysis (% of total) revealed top up-regulated pathways at 4 and 12 weeks, respectively: Group 1, keratan sulfate metabolism (46%) and cellular processes (29%); Group 2, extracellular matrix (ECM)/collagen processes (33%) and beta oxidation (39%); Group 3, cellular processes (50%) and energy metabolism (100%); and Group 4, keratan sulfate metabolism (31%) and inflammation (50%). Top downregulated pathways were: Group 1, Inflammation (36%) and glucose/citric acid metabolism (42%); Group 2, cell signaling (38%) and glucose/citric acid metabolism (35%); Group 3, keratan sulfate metabolism (31%) and ECM/collagen processes (48%); and Group 4, glucose/citric acid metabolism (33%) and ECM/collagen processes (43%). Conclusions: MX-JC "collagen Type 0" upregulates pathways related to ECM/collagen formation and downregulates pathways related to inflammation suggesting that it is promising biomaterial for IL. Level of Evidence: NA.

Low postoperative lymphocyte count increases risk of progression in human papillomavirus associated oropharyngeal cancer.

BACKGROUND: We aim to explore the prognostic role of absolute lymphocyte count (ALC) before, during, and after treatment on oncologic outcomes in human papillomavirus associated oropharyngeal cancer (HPV(+)OPSCC). METHODS: Retrospective cohort at a tertiary center, 2006-2018. Multivariable Cox regressions were used to determine the effect of ALC on risk of progression. Univariate linear regression was performed to determine clinical factors associated with lower ALC. RESULTS: All 197 patients underwent primary surgery. Mean (SD) ALC nadirs (×109  cells/L) were: baseline (N = 149): 1.69 (0.56); postoperative (N = 126): 1.58 (0.59); post-RT (N = 141): 0.68 (0.35) and long-term (N = 105): 0.88 (0.37). Lower baseline ALC nadir was associated with worse overall survival (HR 3.85, 95%CI: 1.03-14.29, p = 0.04). Lower postoperative ALC nadir was associated with higher risk of progression (HR 2.63, 95%CI: 1.04-6.67, p = 0.04). CONCLUSIONS: Lower baseline ALC is associated with worse survival, whereas lower postoperative ALC is associated with increased risk of progression in surgically treated HPV(+)OPSCC.

MRI imaging versus histologic volumetric estimation of residual injection laryngoplasty material.

Objectives: To examine the degree of agreement between MRI and histologically generated volumetric measurements of residual injection laryngoplasty material. Methods: Following left recurrent laryngeal nerve transection, rabbit vocal cords were injected with jellyfish collagen, Cymetra®, or Restylane®. Laryngeal tissue was harvested 4 or 12 weeks post injection followed by MRI imaging and histologic cross-sectioning. Two raters estimated the volume of remaining injection material in specimens within MRI and histologic axial cross sections. Wilcoxon signed rank tests were employed to detect gross differences between inter-rater measurements and between imaging modalities across time. Agreement between rater measurements and imaging (histology and MRI) was assessed using intra-class correlation coefficients. Results: Data was available from 16 rabbits sacrificed at 4 weeks (n = 8) and 12 weeks (n = 8). Inter-rater testing of MRI imaging revealed no significant differences (p > .05) between rater measurements across time points, and excellent agreement (0.93; 95% confidence interval 0.80-0.98) while histologically estimated volumes demonstrated a significant difference at 4 weeks (p < .05) and overall good agreement (0.89; 95% confidence interval 0.59-0.97). Comparison of MRI and histologically estimated volume measurements revealed significant differences at the 4-week time point (p < .05) but not at 12 weeks (p > .05). Overall, there is only moderate agreement between MRI and histology estimates (0.72; 95% confidence interval 0.22-0.90). Conclusions: MRI imaging demonstrates good reliability and similar estimates of volume to histologically estimated measurements of residual injection laryngoplasty material at time points clinically relevant for future injection laryngoplasty experiments. Level of Evidence: NA.

Roles of innate lymphoid cells (ILCs) in allergic diseases: The 10-year anniversary for ILC2s.

In the 12 years since the discovery of innate lymphoid cells (ILCs), our knowledge of their immunobiology has expanded rapidly. Group 2 ILCs (ILC2s) respond rapidly to allergen exposure and environmental insults in mucosal organs, producing type 2 cytokines. Early studies showed that epithelium-derived cytokines activate ILC2s, resulting in eosinophilia, mucus hypersecretion, and remodeling of mucosal tissues. We now know that ILC2s are regulated by other cytokines, eicosanoids, and neuropeptides as well, and interact with both immune and stromal cells. Furthermore, ILC2s exhibit plasticity by adjusting their functions depending on their tissue environment and may consist of several heterogeneous subpopulations. Clinical studies show that ILC2s are involved in asthma, allergic rhinitis, chronic rhinosinusitis, food allergy, and eosinophilic esophagitis. However, much remains unknown about the immunologic mechanisms involved. Beneficial functions of ILCs in maintenance or restoration of tissue well-being and human health also need to be clarified. As our understanding of the crucial functions ILCs play in both homeostasis and disease pathology expands, we are poised to make tremendous strides in diagnostic and therapeutic options for patients with allergic diseases. This review summarizes discoveries in immunobiology of ILCs and their roles in allergic diseases in the past 5 years, discusses controversies and gaps in our knowledge, and suggests future research directions.

In vitro Culture with Cytokines Provides a Tool to Assess the Effector Functions of ILC2s in Peripheral Blood in Asthma.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) play crucial roles in type 2 immunity and asthma development. While ILC2s are resident in mucosal tissues, they also circulate in peripheral blood. It remains controversial whether ILC2s are increased in the peripheral blood of patients with asthma. PURPOSE: The goal of this project was to study the effector functions of ILC2s in peripheral blood samples by in vitro culture with cytokines. PATIENTS AND METHODS: Peripheral blood mononuclear cells (PBMCs) were collected from 11 adult patients with mild asthma and 12 healthy control subjects. The number of peripheral blood ILC2s in PBMCs was analyzed by flow cytometry. PBMCs were cultured with IL-33 and IL-25 without any antigens, and the amounts of type 2 cytokines in cell-free supernatants were analyzed by ELISA. In selected experiments, production of cytokines by ILC2s was analyzed by intracellular cytokine staining and flow cytometry. RESULTS: In response to either IL-33 or IL-25 stimulation, PBMCs from patients with mild asthma produced larger amounts of IL-5 and IL-13 than PBMCs from healthy control subjects. However, ILC2 numbers or proportions were not significantly different between these two groups. Flow cytometric analysis confirmed production of IL-5 by ILCs when stimulated with IL-33. CONCLUSION: In vitro culture of PBMCs with a cocktail of cytokines, such as either IL-33 or IL-25 plus IL-2, may provide a valuable tool to assess the effector functions of ILC2s and may serve as a biomarker for human asthma.

T cell fraction impacts oncologic outcomes in human papillomavirus associated oropharyngeal squamous cell carcinoma.

BACKGROUND: We investigated T cell clonality (TCC) and T cell fraction (TCF) in human papilloma virus associated oropharyngeal squamous cell carcinoma (HPV(+)OPSCC) progressors [cases] vs. non-progressors [controls]. METHODS: This nested case-control study included patients undergoing intent-to-cure surgery ± adjuvant therapy from 6/1/2007-10/3/2016. Patients experiencing local/regional/distant disease (progressors), and a consecutive sample of non-progressors were matched (2 controls: 1 case) on tumor subsite, T-stage and number of metastatic lymph nodes. We performed imunosequencing of the CDR3 regions of human TCRß chains. RESULTS: 34 progressors and 65 non-progressors were included. There was no statistically significant difference in baseline TCF (range: 0.039-1.084) and TCC (range: 0.007-0.240) (p > 0.05). Female sex was associated with higher TCF (p = 0.03), while extranodal extension (ENE) was associated with lower TCF (p = 0.01). There was a positive correlation between tumor size and clonality (R = 0.34, p < 0.01). The strongest predictor of progression-free survival (PFS) was TCF (HR 0.80, 95%CI 0.66-0.96, p = 0.02). The strongest predictors of cancer specific survival (CSS) were TCF (HR0.69, 95%CI 0.47-1.00, p < 0.05) and Adult Comorbidity Evaluation-27 (ACE-27) score (p < 0.05). Similarly, the strongest predictors of overall survival (OS) were TCF (HR 0.62, 95%CI 0.43-0.91, p = 0.01) and ACE-27 score (p = 0.03). On multivariable modeling, TCF ≥ 0.4 was independently associated with PFS (HR 0.34, 95%CI 0.14-0.85, p = 0.02) while an ACE-27 score of ≥ 2 independently predicted CSS (HR 3.85, 95%CI 1.07-13.85, p = 0.04) and OS (HR 3.51, 95%CI 1.10-11.20, p = 0.03). CONCLUSIONS: In patients with HPV(+)OPSCC, TCF was higher in female patients and those without ENE, suggesting differential immune responses. Lower TCF was significantly and independently associated with disease progression. Better ACE-27 scores appear to predict improved oncologic control.

Innate and adaptive immune responses to fungi in the airway.

Fungi are ubiquitous outdoors and indoors. Exposure, sensitization, or both to fungi are strongly associated with development of asthma and allergic airway diseases. Furthermore, global climate change will likely increase the prevalence of fungi and enhance their antigenicity. Major progress has been made during the past several years regarding our understanding of antifungal immunity. Fungi contain cell-wall molecules, such as ß-glucan and chitin, and secrete biologically active proteases and glycosidases. Airway epithelial cells and innate immune cells, such as dendritic cells, are equipped with cell-surface molecules that react to these fungal products, resulting in production of cytokines and proinflammatory mediators. As a result, the adaptive arm of antifungal immunity, including TH1-, TH2-, and TH17-type CD4+ T cells, is established, reinforcing protection against fungal infection and causing detrimental immunopathology in certain subjects. We are only in the beginning stages of understanding the complex biology of fungi and detailed mechanisms of how they activate the immune response that can protect against or drive diseases in human subjects. Here we describe our current understanding with an emphasis on airway allergic immune responses. The gaps in our knowledge and desirable future directions are also discussed.

IL-33-Responsive Group 2 Innate Lymphoid Cells Are Regulated by Female Sex Hormones in the Uterus.

Group 2 innate lymphoid cells (ILC2s) reside in multiple organs in the body, where they play roles in immunity, tissue homeostasis, and metabolic regulation. However, little is known about the regulatory mechanisms of ILC2s in different organs. Here, we identified ILC2s in the mouse uterus and found that they express cell surface molecules, including the IL-33 receptor, ST2, that are roughly comparable to those expressed by lung ILC2s. Both in vivo and in vitro treatment with IL-33 induced type 2 cytokine production in uterine ILC2s, suggesting that they respond to IL-33 in a manner similar to ILC2s in other organs. Importantly, uterine ILC2s were nearly absent in ovariectomized mice and were increased in wild-type mice by estrogen administration, whereas lung ILC2s were unaffected by both ovariectomy and estrogen administration. Likewise, a marked reduction in uterine ILC2s was observed in mice deficient in estrogen receptor α or estrogen receptor ß. Furthermore, uterine ILC2s highly expressed estrogen receptor α, and in vitro culture of isolated uterine ILC2s with 17ß-estradiol modified expression of a number of genes. Finally, an increased prevalence in neonatal mortality was observed in litters from dams lacking the IL-33 receptor, ST2. Taken together, our findings indicate that unlike lung IL2Cs, uterine ILC2s are regulated by female sex hormones, which may specialize them for specific physiological functions.

Group 2 Innate Lymphoid Cells Promote an Early Antibody Response to a Respiratory Antigen in Mice.

Innate lymphoid cells (ILCs) are a new family of immune cells that play important roles in innate immunity in mucosal tissues, and in the maintenance of tissue and metabolic homeostasis. Recently, group 2 ILCs (ILC2s) were found to promote the development and effector functions of Th2-type CD4(+) T cells by interacting directly with T cells or by activating dendritic cells, suggesting a role for ILC2s in regulating adaptive immunity. However, our current knowledge on the role of ILCs in humoral immunity is limited. In this study, we found that ILC2s isolated from the lungs of naive BALB/c mice enhanced the proliferation of B1- as well as B2-type B cells and promoted the production of IgM, IgG1, IgA, and IgE by these cells in vitro. Soluble factors secreted by ILC2s were sufficient to enhance B cell Ig production. By using blocking Abs and ILC2s isolated from IL-5-deficient mice, we found that ILC2-derived IL-5 is critically involved in the enhanced production of IgM. Furthermore, when adoptively transferred to Il7r(-/-) mice, which lack ILC2s and mature T cells, lung ILC2s promoted the production of IgM Abs to a polysaccharide Ag, 4-hydroxy-3-nitrophenylacetyl Ficoll, within 7 d of airway exposure in vivo. These findings add to the growing body of literature regarding the regulatory functions of ILCs in adaptive immunity, and suggest that lung ILC2s promote B cell production of early Abs to a respiratory Ag even in the absence of T cells.

Enhanced innate type 2 immune response in peripheral blood from patients with asthma.

BACKGROUND: In mice, group 2 innate lymphoid cells (ILC2s) likely mediate helminth immunity, inflammation, and tissue repair and remodeling. However, the involvement of ILC2s in human diseases, such as asthma, is not well understood. OBJECTIVES: The goals of this study were to investigate whether peripheral blood specimens can be used to monitor innate type 2 immunity in human subjects and to examine whether ILC2s are involved in human asthma. METHODS: PBMCs from subjects with allergic asthma (AA), subjects with allergic rhinitis (AR), or healthy control (HC) subjects were cultured in vitro with IL-25 or IL-33. Flow cytometry and cell sorting were used to identify, isolate, and quantitate ILC2s in PBMCs. RESULTS: Human PBMCs produced IL-5 and IL-13 when stimulated with IL-33 or IL-25 in the presence of IL-2 without antigens. In addition, IL-7 or thymic stromal lymphopoietin were able to replace IL-2. The cell population with phenotypic ILC2 characteristics, lineage(-)CD127(+)CRTH2(+) cells, responded to IL-33 and produced large quantities of IL-5 and IL-13 but undetectable levels of IL-4. PBMCs from subjects with AA produced significantly larger amounts of IL-5 and IL-13 in response to IL-25 or IL-33 than from subjects with AR or HC. The prevalence of ILC2s in blood was greater in the AA group than in the AR group or the HC group. CONCLUSIONS: Innate type 2 immune responses are increased in asthma but not in AR, suggesting potential differences in the immunopathogenesis of these diseases. Peripheral blood is useful for evaluating innate type 2 immunity in humans.

Dynamic role of epithelium-derived cytokines in asthma.

Asthma is an inflammatory disorder of the airways, characterized by infiltration of mast cells, eosinophils, and Th2-type CD4+ T cells in the airway wall. Airway epithelium constitutes the first line of interaction with our atmospheric environment. The protective barrier function of the airway epithelium is likely impaired in asthma. Furthermore, recent studies suggest critical immunogenic and immunomodulatory functions of airway epithelium. In particular, a triad of cytokines, including IL-25, IL-33 and TSLP, is produced and released by airway epithelial cells in response to various environmental and microbial stimuli or by cellular damage. These cytokines induce and promote Th2-type airway inflammation and cause remodeling and pathological changes in the airway walls, suggesting their pivotal roles in the pathophysiology of asthma. Thus, the airway epithelium can no longer be regarded as a mere structural barrier, but must be considered an active player in the pathogenesis of asthma and other allergic disorders.

IL-33-responsive lineage- CD25+ CD44(hi) lymphoid cells mediate innate type 2 immunity and allergic inflammation in the lungs.

Innate immunity provides the first line of response to invading pathogens and a variety of environmental insults. Recent studies identified novel subsets of innate lymphoid cells that are capable of mediating immune responses in mucosal organs. In this paper, we describe a subset of lymphoid cells that is involved in innate type 2 immunity in the lungs. Airway exposure of naive BALB/c or C57BL/6J mice to IL-33 results in a rapid (<12 h) production of IL-5 and IL-13 and marked airway eosinophilia independently of adaptive immunity. In the lungs of nonsensitized naive mice, IL-33-responsive cells were identified that have a lymphoid morphology, lack lineage markers, highly express CD25, CD44, Thy1.2, ICOS, Sca-1, and IL-7Rα (i.e., Lin(-)CD25(+)CD44(hi) lymphoid cells), and require IL-7Rα for their development. Airway exposure of naive mice to a clinically relevant ubiquitous fungal allergen, Alternaria alternata, increases bronchoalveolar lavage levels of IL-33, followed by IL-5 and IL-13 production and airway eosinophilia without T or B cells. This innate type 2 response to the allergen is nearly abolished in mice deficient in IL-33R (i.e., ST2), and the Lin(-)CD25(+)CD44(hi) lymphoid cells in the lungs are required and sufficient to mediate the response. Thus, a subset of innate immune cells that responds to IL-33 and vigorously produces Th2-type cytokines is present in mouse lungs. These cells may provide a novel mechanism for type 2 immunity in the airways and induction of allergic airway diseases such as asthma.

IL-33-activated dendritic cells induce an atypical TH2-type response.

BACKGROUND: IL-33, a recently discovered IL-1 family cytokine, is implicated in the development of T(H)2-type responses in vivo. However, the cellular targets for IL-33 are poorly understood. OBJECTIVE: We tested the hypotheses that dendritic cells (DCs) respond to IL-33 and that IL-33-activated DCs prime naive CD4(+) T cells to produce T(H)2-type cytokines. METHODS: Dendritic cells were derived from mouse bone marrow, and their expression of the IL-33 receptor, ST2, was examined by fluorescence-activated cell sorting and real-time RT-PCR. The DCs' responses to IL-33 were examined by fluorescence-activated cell sorting (MHC-II and CD86 expression) and by ELISA (IL-6 and IL-12 production). The ability of IL-33-activated DCs to prime naive T cells was assessed by coculture with isolated CD4(+) T cells and by measuring cytokines in the supernatants. RESULTS: ST2 mRNA was detectable in highly purified DCs. ST2 protein was abundant within DCs, but was barely detectable on their cell surfaces. Incubation of DCs with IL-33 increased their expression of MHC-II and CD86 and production of IL-6, but IL-12 was not produced. Anti-ST2 antibody inhibited IL-6 production from IL-33-activated DCs by approximately 60%; anti-ST2 did not affect IL-6 production from LPS-activated DCs. When incubated with naive CD4(+) T cells alone, IL-33 failed to stimulate cytokine production. In contrast, naive CD4(+) T cells incubated with IL-33-activated DCs showed robust production of IL-5 and IL-13, but IL-4 and IFN-gamma were undetectable. CONCLUSION: Dendritic cells respond directly to IL-33 through ST2. The IL-33 and DC interaction may represent a new pathway to initiate T(H)2-type immune responses.

Beta2-integrin-mediated adhesion and intracellular Ca2+ release in human eosinophils.

Human eosinophils spontaneously adhere to various substrates in the absence of exogenously added activators. In the present study a method was developed for characterizing eosinophil adhesion by measuring changes in impedance. Impedance measurements were performed in HCO(3)-buffered HybriCare medium maintained in a humidified 5% CO(2) incubator at 37 degrees C. Impedance increased by more than 1 kOmega within minutes after eosinophils made contact with the substrate, reaching a peak within 20 min. Blocking mobilization of intracellular [Ca(2+)] that precedes adhesion with BAPTA-AM (10 microM) completely inhibited the rise in impedance as well as the changes in cell shape typically observed in adherent cells. However, lowering the extracellular [Ca(2+)] with 2.5 mM EGTA did not inhibit the increase in impedance. Pretreatment with anti-CD18 antibody to block substrate interactions with beta(2)-integrins, or jasplakinolide (2 microM) to block actin reorganization, abolished the increase in impedance and adherent morphology of the cells. Exposure of eosinophils to the phosphatidylinositol 3 kinase inhibitor LY294002 (5 microM) or treatment with protein kinase C zeta pseudosubstrate to competitively inhibit activity of the enzyme significantly reduced the increase in impedance and inhibited the cell spreading associated with adhesion. These results demonstrate a novel method for measuring eosinophil adhesion and showed that, following formation of a tethered attachment, a rapid increase in intracellular [Ca(2+)] precedes the cytoskeletal rearrangements required for cell shape changes and plasma membrane-substrate interactions associated with adhesion.

A novel IL-1 family cytokine, IL-33, potently activates human eosinophils.

BACKGROUND: Eosinophils are likely key cells involved in the pathogenesis of asthma and allergic diseases; however, the mechanisms that regulate eosinophil dynamics and functions in mucosal tissues are incompletely understood. IL-33, which is produced by mucosal cells, is a new member of the IL-1 cytokine family. Mice injected with IL-33 display profound mucosal eosinophilia with associated pathologic changes. Although mast cells and T(H)2 cells express the IL-33 receptor, ST2, the roles of IL-33 and ST2 in eosinophil biology are unknown. OBJECTIVES: We investigated the effects of IL-33 on human eosinophils in vitro. METHODS: Eosinophils and neutrophils were isolated from blood of normal individuals and mildly atopic patients. Real-time RT-PCR and flow cytometry were used to detect ST2. Granulocyte responses to IL-33 were monitored by superoxide anion production and by degranulation; IL-5, IL-1beta, and TNF-alpha served as controls. Eosinophil survival and cytokine production were assessed by flow cytometry and ELISA, respectively. RESULTS: ST2 mRNA and protein were detected on eosinophils. IL-33 induced eosinophil superoxide anion production and degranulation as potently as IL-5. IL-33 also increased eosinophil survival and induced production of IL-8. Anti-ST2 inhibited eosinophil responses to IL-33. Neutrophils did not express ST2, nor did they respond to IL-33. CONCLUSION: IL-33 and its receptor, ST2, may play important roles in eosinophil-mediated inflammation; they may provide new therapeutic targets for controlling mucosal eosinophilic inflammation.

Secretory IgA induces antigen-independent eosinophil survival and cytokine production without inducing effector functions.

BACKGROUND: Eosinophils in human beings reside in tissues, especially the mucosal tissues of the gastrointestinal tract and inflamed airways. Secretory IgA (S-IgA) is the predominant antibody secreted by these tissues and likely plays a role in the innate immune response. OBJECTIVE: Because eosinophils and S-IgA are often colocalized in mucosal tissues, we examined the potential regulatory effects of S-IgA without antigens on survival, gene expression, and effector functions of human eosinophils. METHODS: Eosinophils were incubated with S-IgA in solution without antigens (soluble S-IgA) or with S-IgA immobilized to mimic multivalent antigen cross-linking. Eosinophil activation was monitored by superoxide anion generation and degranulation. Survival was assessed between 24 and 96 hours. Gene and protein expression were examined by microarray and ELISA. Eosinophil lysates were examined by immunoblot for extracellular signal-regulated kinase (ERK) phosphorylation. RESULTS: Immobilized S-IgA stimulated eosinophil superoxide production and degranulation; soluble S-IgA did not. Although immobilized S-IgA inhibited eosinophil survival in vitro, soluble S-IgA enhanced survival; this involved autocrine production of GM-CSF. Soluble S-IgA without antigens induced increases in mRNA levels of various cytokines, chemokines, signal transduction molecules, antiapoptotic factors, and cell surface markers. By using ELISA, we confirmed protein expression of selected mediators. Eosinophil interaction with soluble S-IgA likely involves FcalphaRI (CD89) and ERK pathway activation. CONCLUSION: Secretory IgA without multivalent antigens may regulate survival and gene expression of eosinophils. Eosinophils in mucosal tissues can be either primed for action (cytokine production and survival) or fully activated (degranulation and superoxide release) by different forms of S-IgA.