Blockade of thymic stromal lymphopoietin (TSLP) receptor inhibits TSLP-driven proliferation and signalling in lymphoblasts from a subset of B-precursor ALL patients.

PURPOSE: The cytokine thymic stromal lymphopoietin (TSLP) and its receptor TSLPR are involved in intercellular communication in the course of allergic inflammation and have recently been implicated in the development of various malignancies including B cell precursor acute lymphoblastic leukemia (BCP-ALL). We studied TSLPR expression, TSLP-induced signal transduction and its antibody-mediated inhibition in long-term cultures of primary cells derived from B-precursor ALL patients. METHODS: TSLPR expression was determined by flow cytometry and Western blot analysis, cell proliferation, signal transduction via the JAK/STAT pathway was analysed by Western blot detection of STAT tyrosine phosphorylation and by measuring TSLP-dependent activation of a STAT-specific reporter gene construct. For inhibition studies a recently introduced antagonistic antibody to the TSLPRα-subunit was used. RESULTS: TSLPR surface expression was observed in leukemic lymphoblasts from two out of ten patients with BCP-ALL. Upon TSLP stimulation, the cells with the highest TSLPR expression level showed enhanced proliferation and JAK/STAT-mediated gene regulation in a dose-dependent manner. By employment of an inhibitory antibody to the TSLPR, both TSLP-triggered cell proliferation and STAT transcription factor activation were specifically inhibited. CONCLUSIONS: These results suggest that blockade of the TSLPR might be a therapeutic option for a subset of BCP-ALL patients.

Soluble extracellular matrix metalloproteinase inducer (EMMPRIN, EMN) regulates cancer-related cellular functions by homotypic interactions with surface CD147.

EMMPRIN (extracellular matrix metalloproteinase inducer) is a widely expressed glycoprotein and a member of the immunoglobulin superfamily which exists in both a membrane-spanning and a soluble form. Homotypic interactions of EMMPRIN underlie its multiple roles in normal development and pathological situations such as viral infections, Alzheimer's disease and cancer. This study employed a recombinant soluble, fully glycosylated EMMPRIN domain (rhsEMN) as a tool to characterize the structural basis of EMMPRIN-EMMPRIN receptor (EMNR) contacts and their functional effects on MCF-7 breast carcinoma cells. rhsEMN did not form dimers in solution but bound to surface EMMPRIN (EMN) on MCF-7 cells with high affinity and was readily internalized. The interaction interface for the homotypic contact was localized to the N-terminal Ig domain. rhsEMN exerted a stimulatory effect on proliferation of MCF-7 cells whereas it reduced cell migration in a dose-dependent manner. These effects were accompanied by an upregulation of endogenous EMMPRIN as well as of matrix metalloproteinase-14 (MMP-14), a membrane-bound protease involved in the extracellular release of soluble EMMPRIN, indicating a regulatory feedback mechanism. The proliferation-promoting activity of rhsEMN was mimicked by a novel functional antibody directed to EMMPRIN, underscoring that crosslinking of cell surface EMMPRIN (EMNR) is crucial for eliciting intracellular signalling. Addressing malignancy-related signal transduction in HEK-293 cells, we could show that rhsEMN triggers the oncogenic Wnt pathway.

Impact of smoking on dendritic cell phenotypes in the airway lumen of patients with COPD.

BACKGROUND: Myeloid dendritic cells (DCs) are increased in the airway wall of patients with chronic obstructive pulmonary disease (COPD), and postulated to play a crucial role in COPD. However, DC phenotypes in COPD are poorly understood. METHODS: Function-associated surface molecules on bronchoalveolar lavage fluid (BALF) DCs were analyzed using flow cytometry in current smokers with COPD, in former smokers with COPD and in never-smoking controls. RESULTS: Myeloid DCs of current smokers with COPD displayed a significantly increased expression of receptors for antigen recognition such as BDCA-1 or Langerin, as compared with never-smoking controls. In contrast, former smokers with COPD displayed a significantly decreased expression of these receptors, as compared with never-smoking controls. A significantly reduced expression of the maturation marker CD83 on myeloid DCs was found in current smokers with COPD, but not in former smokers with COPD. The chemokine receptor CCR5 on myeloid DCs, which is also important for the uptake and procession of microbial antigens, was strongly reduced in all patients with COPD, independently of the smoking status. CONCLUSION: COPD is characterized by a strongly reduced CCR5 expression on myeloid DCs in the airway lumen, which might hamper DC interactions with microbial antigens. Further studies are needed to better understand the role of CCR5 in the pathophysiology and microbiology of COPD.

Fluticasone impact on airway dendritic cells in smokers: a randomized controlled trial.

BACKGROUND: Myeloid Dendritic cells are key drivers of inflammation in smoke-related lung diseases, whereas plasmacytoid DCs play a crucial role in the defense against infections. Effects of inhaled corticosteroids (ICS) on airway DCs in smokers are unknown. METHODS: In this randomized, double-blind, placebo-controlled clinical trial, 45 active cigarette smokers inhaled placebo, fluticasone or fluticasone plus salmeterol twice daily for 4 weeks. Bronchoalveolar lavage fluid DCs were analyzed using four-color flow cytometry before and after the inhalation period. In addition, fluticasone effects were tested on T-cell proliferation in co-cultures with blood myeloid DCs from smokers. RESULTS: Inhalation of fluticasone plus salmeterol, but not fluticasone alone or placebo, reduced endobronchial concentrations of myeloid DCs (median decrease: 24%), macrophages (median decrease: 26%) and neutrophils (median decrease: 76%). In contrast, fluticasone reduced plasmacytoid DC concentrations independently of salmeterol. There were no changes in the expression of function-associated surface molecules on myeloid DC (such as CD1a, Langerin, BDCA-1, CD83 or CCR5) in all groups after treatment. Fluticasone (either alone or in combination with salmeterol) suppressed T-cell proliferation in co-cultures with blood myeloid DCs from smokers. CONCLUSIONS: Resistance to ICS monotherapy in smokers might in part be due to lacking effects on airway myeloid DCs, whereas the increased risk for infections during ICS therapy could be attributable to a reduction in plasmacytoid DCs. Combination therapy of fluticasone with salmeterol is associated with a reduction in airway myeloid DCs, but also airway macrophages and neutrophils. TRIAL REGISTRATION: Registered at ClinicalTrials.gov (identifier: NCT00908362) and the European Clinical Trial Database, EudraCT (identifier: 2009-009459-40).

Expression analysis and specific blockade of the receptor for human thymic stromal lymphopoietin (TSLP) by novel antibodies to the human TSLPRα receptor chain.

Thymic stromal lymphopoietin (TSLP) is an interleukin-7 (IL-7)-like cytokine with a pivotal role in development and maintenance of atopic diseases such as allergic asthma and atopic dermatitis. Moreover, recent studies show an involvement of TSLP in the progression of various cancers. TSLP signaling is mediated by the TSLP receptor (TSLPR), a heterodimeric type I cytokine receptor. It consists of the IL-7 receptor alpha chain (IL-7Rα), which is shared with the IL-7 receptor, and the TSLPRα chain as a specific subunit. Blocking signal release by TSLP without affecting IL-7 function is a potentially interesting option for the treatment of atopic diseases or certain tumors. By employing the extracellular domain of human TSLPRα chain (hTSLPRα(ex)) as an antigen, we generated a set of monoclonal antibodies. Several binders to native and/or denatured receptor protein were identified and characterized by cytometry and Western blot analysis. A screen based on a STAT3-driven reporter gene assay in murine pro-B cells expressing a functional hTSLPR yielded two hybridoma clones with specific antagonistic properties towards hTSLP, but not IL-7. Kinetic studies measuring blockade of hTSLP-dependent STAT phosphorylation in a TSLP-responsive cell line revealed an inhibitory constant in the nanomolar range.

Altered phenotype of blood dendritic cells in patients with acute pneumonia.

BACKGROUND: Dendritic cells (DCs) play a key role in the host defence against inhaled pathogens. However, the phenotype of blood DCs in patients with acute respiratory infections is unknown. OBJECTIVE: To investigate the number and the expression of function-associated molecules of blood DCs in patients with acute infectious pneumonia. METHODS: Sixteen patients with acute pneumonia and 19 controls without pneumonia were included in the study. The number as well as the expression of function-associated molecules of myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) were analysed in peripheral blood using four-colour flow cytometry. RESULTS: Elevated concentrations of procalcitonin (median: 0.55 ng/ml) and the rapid response to antibiotic treatment suggested a bacterial origin of the pneumonia in the patients. Total mDC (median: 27% of the controls) and pDC counts (median: 53% of the controls) were markedly reduced in patients with pneumonia, as compared to the controls. Percentages of blood mDCs, but not pDCs, were negatively correlated with serum concentrations of C-reactive protein. Patients with pneumonia were characterised by a significantly increased expression of Fc gamma receptors (CD32 and CD64) on mDCs and the Toll-like receptor 9 (TLR9) on pDCs. CONCLUSIONS: Circulating DCs are markedly reduced in patients with pneumonia, and characterised by an up-regulation of molecules recognising pathogen-associated molecular patterns and opsonised antigens.

Impact of immunotherapy on blood dendritic cells in patients with Hymenoptera venom allergy.

BACKGROUND: Modulation of T-cell differentiation, which is controlled by dendritic cells (DCs), plays a crucial role in specific immunotherapy (SIT). However, the number and the characteristics of blood DCs before and during immunotherapy are unknown. OBJECTIVE: To analyze the number and the characteristics of blood DC subsets in patients with Hymenoptera venom allergy before and after initiation of SIT. METHODS: In this clinical trial (NCT00947908), blood myeloid and plasmacytoid DCs were analyzed in 20 patients with Hymenoptera venom allergy (bee or wasp venom) by using 4-color flow cytometry at 3 time points: directly before SIT, and 52 hours and 12 months after initiation of SIT. In addition, 20 age-matched and sex-matched controls were examined. RESULTS: In patients with Hymenoptera venom allergy, the number of plasmacytoid DCs before SIT was comparable to that of controls. Plasmacytoid DCs decreased markedly 52 hours after initiation of SIT and returned to control levels after 12 months of treatment. Myeloid DCs were elevated in patients with Hymenoptera venom allergy before, during, and after the first 12 months of SIT. In addition, there were changes in the expression of function-associated surface molecules on myeloid DCs (such as Fc γ receptor 2 and Toll-like receptor 2) during SIT. CONCLUSION: Numbers of blood myeloid DCs are elevated in patients with Hymenoptera venom allergy, and there are specific changes in the expression of function-associated surface molecules on these cells during SIT. Numbers of plasmacytoid DCs in blood are profoundly but are only transiently decreased after initiation of SIT.

Interleukin-4 suppresses the cytotoxic potential of in vitro generated, adaptive regulatory CD4 T cells by down-regulation of granzyme B.

Regulatory CD4+ T cells (Tregs) control immune responses using secretion of anti-inflammatory cytokines and/or cytotoxic mechanisms and play a central role in the outcomes of several immune pathologies. Previous studies suggest an impaired function of Tregs in allergy, especially during allergen seasons, but the underlying mechanism is not known. Therefore, we analysed the impact of the T helper type 2 cytokine interleukin (IL)-4 on in vitro generated adaptive Tregs (aTregs), which have been reported to use the granzyme B (GrB)/perforin pathway to kill autologous immune cells. aTregs were generated by co-ligation of CD3 and CD46 on CD4+ T lymphocytes and granzyme expression was analysed using flow cytometry. To quantify GrB and perforin expression as well as IL-10 secretion in response to IL-4, specific enzyme-linked immunosorbent assays were performed in cell lysates and/or culture supernatants. Using a flow cytometry-based cytotoxicity assay the impact of IL-4 on the cytotoxic potential of aTregs was investigated. While IL-4 did not affect IL-10 secretion and perforin expression in aTregs, a significant suppression of GrB synthesis was detected in the presence of IL-4. In addition, IL-4-mediated suppression of GrB led to impaired cytotoxicity of aTregs against K562 target cells. In conclusion, our data suggest that IL-4 might play a role in impaired aTreg function in allergy.

Signal transduction around thymic stromal lymphopoietin (TSLP) in atopic asthma.

Thymic stromal lymphopoietin (TSLP), a novel interleukin-7-like cytokine, triggers dendritic cell-mediated inflammatory responses ultimately executed by T helper cells of the Th2 subtype. TSLP emerged as a central player in the development of allergic symptoms, especially in the airways, and is a prime regulatory cytokine at the interface of virus- or antigen-exposed epithelial cells and dendritic cells (DCs). DCs activated by epithelium-derived TSLP can promote naïve CD4+ T cells to adopt a Th2 phenotype, which in turn recruite eosinophilic and basophilic granulocytes as well as mast cells into the airway mucosa. These different cells secrete inflammatory cytokines and chemokines operative in inducing an allergic inflammation and atopic asthma. TSLP is, thus, involved in the control of both an innate and an adaptive immune response. Since TSLP links contact of allergen with the airway epithelium to the onset and maintainance of the asthmatic syndrome, defining the signal transduction underlying TSLP expression and function is of profound interest for a better understandimg of the disease and for the development of new therapeutics.

Activation of human alveolar macrophages via P2 receptors: coupling to intracellular Ca2+ increases and cytokine secretion.

Alveolar macrophages play a crucial role in the pathogenesis of inflammatory airway diseases. By the generation and release of different inflammatory mediators they contribute to both recruitment of different leukocytes into the lung and to airway remodeling. A potent stimulus for the release of inflammatory cytokines is ATP, which mediates its cellular effects through the interaction with different membrane receptors, belonging to the P2X and P2Y families. The aim of this study was to characterize the biological properties of purinoceptors in human alveolar macrophages obtained from bronchoalveolar lavages in the context of inflammatory airway diseases. The present study is the first showing that human alveolar macrophages express mRNA for different P2 subtypes, namely P2X(1), P2X(4), P2X(5), P2X(7), P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(11), P2Y(13), and P2Y(14). We also showed that extracellular ATP induced Ca(2+) transients and increased IL-1beta secretion via P2X receptors. Furthermore, extracellular nucleotides inhibited production of IL-12p40 and TNF-alpha, whereas IL-6 secretion was up-regulated. In summary, our data further support the hypothesis that purinoceptors are involved in the pathogenesis of inflammatory lung diseases.

Function-associated surface molecules on airway dendritic cells in cigarette smokers.

Airway dendritic cells (DCs) control pulmonary immune responses to inhaled particles. However, the profile of function-associated surface molecules on airway DCs in smokers is unknown. In this study, function-associated surface molecules were analyzed using four-color flow cytometry on myeloid DCs (mDCs) in bronchoalveolar lavage fluid (BALF) of cigarette smokers and never-smokers. Furthermore, the lung function was assessed directly before bronchoscopy in all participants. There was a 7-fold increase in total cell numbers in BALF of smokers, as compared with never-smokers. The percentage of mDCs among BALF cells and the expression of the maturation marker CD83 on mDCs did not differ between smokers and never-smokers. However, there was a strong increase in the expression of Langerin and CD1a (markers of Langerhans cells) on mDCs of smokers. Furthermore, mDCs of smokers were characterized by an increased expression of antigen presentation markers such as CD80 and CD86. By contrast, mDCs of smokers displayed a decreased expression of the lymph node homing receptor CCR7, as compared with mDCs of never-smokers. Decreased expression of CCR7 on mDCs, but not any of the other surface molecules studied, was specifically associated with airway obstruction and pulmonary hyperinflation in smokers. In conclusion, our data suggest that smoking affects the expression profile of function-associated surface molecules on airway mDCs. We provide the first evidence that a reduced CCR7 expression on airway mDCs is associated with airflow limitation in smokers.

Safety of segmental allergen challenge in human allergic asthma.

BACKGROUND: Segmental allergen challenge is widely used to study mechanisms of human allergic asthma. Despite the relatively large dissemination, limited information is available about the safety of this method. OBJECTIVE: Observational, retrospective study to report the adverse events of segmental allergen challenge in a large group of volunteers with asthma. METHODS: In total, 78 cases from several studies performed between 1994 and 2007 were pooled for this analysis. Volunteers underwent allergen challenge using either a fixed dose of allergen (7 cases) or an individually standardized allergen dose defined by an inhaled allergen test before the challenge (71 cases). A subgroup of 13 volunteers underwent repeated challenges, with more than 6 months between the challenges. RESULTS: With a fixed dose instilled during bronchoscopy, 43% of the participants developed wheezing and coughing, requiring 2-6 puffs of a ss(2)-agonist after segmental allergen challenge. In volunteers with individually standardized doses, a ss(2)-agonist was required in only 19% of the cases. No severe adverse events occurred in all cases studied. Volunteers who underwent repeated challenges did not develop more adverse events than those who underwent 1 challenge. CONCLUSIONS: Segmental allergen challenge is a safe tool to study the mechanisms of human allergic asthma, even when repeated challenges are performed in the same patient. It is associated with only a few, tolerable adverse events, especially when the dose of allergen is standardized individually.

A role for the neurotrophin receptor TrkB on maturing dendritic cells.

BACKGROUND: Neurotrophins are involved in neuroimmune interactions. However, the expression and role of neurotrophin receptors on dendritic cells have not been systematically studied. METHODS: The neurotrophin receptors p75NTR, TrkA, TrkB and TrkC were analyzed on immature and mature Human Monocyte-derived Dendritic Cells (HMDCs) using flow cytometry. In addition, we compared the impact of five different maturing protocols on the expression of neurotrophin receptors on HMDCs. Finally, the effect of neurotrophins on surface molecule expression and the survival of HMDCs was investigated. RESULTS: There was a low expression of p75NTR, TrkA and TrkB on immature HMDCs. HMDCs at different maturation stages did not show a significantly different expression of TrkA, as compared to immature HMDCs. By contrast, there was an upregulation of TrkB on suboptimally, but not optimally matured HMDC. In addition, there was a non-significant trend to a parallel increase of p75NTR expression on these cells. Functional experiments with maturing HMDCs revealed that both ligands of the TrkB receptor, Brain-derived neurotrophic factor (BDNF) and Neurotrophin 4 (NT-4), significantly increased the expression of markers of antigen presentation (HLA-DR and CD80) and the survival of maturing HMDCs. CONCLUSION: These data suggest that the TrkB ligands BDNF and NT-4 can directly influence human dendritic cells during their maturation.

Dendritic cell subsets in human bronchoalveolar lavage fluid after segmental allergen challenge.

BACKGROUND: Dendritic cells control pulmonary immune reactions. Characteristics of dendritic cells in human bronchoalveolar lavage fluid (BALF) after allergen challenge are unknown. METHODS: 7 patients with allergic asthma (median 23 years, range 19-25 years) underwent segmental challenge and were lavaged 10 min and 24 h after challenge. Dendritic cell subsets and surface markers in BALF and in peripheral blood were analysed using four-colour flow cytometry. RESULTS: Plasmacytoid dendritic cells (pDCs, median 0.06%, range 0.01-0.08%) and myeloid dendritic cells (mDCs, median 0.47%, range 0.27-0.87%) were detectable in BALF from control segments. CD1a-positive dendritic cells in BALF were identified as a subpopulation of mDCs. Both pDCs (median 0.56%, range 0.09-1.83%) and mDCs (median 1.82%, range 0.95-2.29%) increased significantly in BALF 24 h (p = 0.018 compared with the control segments for pDCs and mDCs), but not 10 min, after allergen challenge. The percentage increase in pDCs was higher than that of mDCs after allergen challenge, as reflected by an enhanced pDC:mDC ratio after allergen challenge. In peripheral blood, there was a significant decrease in mDCs (p = 0.038) and a trend to a decrease in pDCs (p = 0.068) 24 h after allergen challenge. Analysis of dendritic cell surface molecules showed that after allergen challenge, BALF dendritic cells have a less mature phenotype compared with BALF dendritic cells from control segments. CONCLUSION: Using a comprehensive strategy to analyse dendritic cell subsets in human BALF, we have shown for the first time that both myeloid and plasmacytoid dendritic cells accumulate in the airway lumen after allergen challenge in patients with asthma.

The course of allergen-induced leukocyte infiltration in human and experimental asthma.

BACKGROUND: Although the timing of allergen-induced bronchoconstriction is well defined, there is little information about the kinetics of allergen-induced leukocyte infiltration in asthma and its comparability between human and animal models of asthma. OBJECTIVE: To investigate systematically allergen-induced leukocyte infiltration into the airway lumen in human and experimental asthma by using bronchoalveolar lavage. METHODS: Patients with allergic asthma were lavaged at different time points as long as 1 week after segmental allergen challenge. Allergen-sensitized mice were lavaged as long as 3 weeks after allergen challenge. Differential cell counts, lymphocyte subsets, and cytokines were assessed in bronchoalveolar lavage fluid. RESULTS: In both models, neutrophil infiltration was a relatively early event (maximum: 18 hours after challenge). In contrast, eosinophil infiltration peaked 42 hours (human model) to 4 days (mouse model) after allergen challenge, paralleled by an IL-5 peak in this period. There were elevated macrophage counts over a period of several days after allergen challenge in both models. Lymphocytes (predominantly CD4+ T cells) peaked 18 hours after challenge in the human model, but not until 2 weeks after challenge in the murine model. CONCLUSION: Early neutrophil accumulation (within hours after challenge) and delayed eosinophil accumulation (within days after challenge) in the airway lumen are common features of allergen-induced airway inflammation, whereas lymphocyte kinetics are dependent on the asthma model. CLINICAL IMPLICATIONS: Similarities in the infiltration kinetics of granulocytes after allergen challenge suggest a common role for these cells in asthma, whereas the presumed orchestration of allergic inflammation by lymphocytes appears to differ between the models.

Differential expression of human granzymes A, B, and K in natural killer cells and during CD8+ T cell differentiation in peripheral blood.

NK cells and cytotoxic T lymphocytes can induce apoptosis in virus-infected and transformed target cells via the granule exocytosis pathway. The key components of the cytolytic granules are perforin and several serine esterases, termed granzymes. While the cellular distribution of human granzymes A (GrA) and B (GrB) has been well characterized much less is known about the expression pattern of human granzyme K (GrK). In this study GrA, GrB, and GrK expression was analyzed in human peripheral blood lymphocytes using flow cytometry. There was a distinct population of GrK expressing CD8+ T cells with a CD27+/CD28+/CCR5high/CCR7-/perforin-/low/IFN-gamma+ memory-like phenotype, while all CD56bright NK cells were also positive for GrK. In addition, GrK was also expressed in subpopulations of CD56+ T cells, CD4+ T cells, and TCRgammadelta+ T cells. In contrast, GrB was primarily expressed in CD56dim NK cells and differentiated memory CD8+ T cells with the CD27-/low/CD28-/low/CCR5-/low/CCR7-/CD11b+/perforinhigh phenotype. Only few CD8+ T cells expressed both GrB and GrK. GrA was found to be co-expressed in all GrB- and GrK-expressing T cells. Our findings suggest that granzyme expression during the differentiation process of memory CD8+ T cells might be as follows: GrA+/GrB-/GrK+ --> GrA+/GrB+/GrK+ --> GrA+/GrB+/GrK-.

Generation of human antibody fragments against Streptococcus mutans using a phage display chain shuffling approach.

BACKGROUND: Common oral diseases and dental caries can be prevented effectively by passive immunization. In humans, passive immunotherapy may require the use of humanized or human antibodies to prevent adverse immune responses against murine epitopes. Therefore we generated human single chain and diabody antibody derivatives based on the binding characteristics of the murine monoclonal antibody Guy's 13. The murine form of this antibody has been used successfully to prevent Streptococcus mutans colonization and the development of dental caries in non-human primates, and to prevent bacterial colonization in human clinical trials. RESULTS: The antibody derivatives were generated using a chain-shuffling approach based on human antibody variable gene phage-display libraries. Like the parent antibody, these derivatives bound specifically to SAI/II, the surface adhesin of the oral pathogen S. mutans. CONCLUSIONS: Humanization of murine antibodies can be easily achieved using phage display libraries. The human antibody fragments bind the antigen as well as the causative agent of dental caries. In addition the human diabody derivative is capable of aggregating S. mutans in vitro, making it a useful candidate passive immunotherapeutic agent for oral diseases.