René C. Schweizer

Mechanisms involved in eosinophil migration. Platelet-activating factor-induced chemotaxis and interleukin-5-induced chemokinesis are mediated by different signals.

Eosinophils play an important role in the pathogenesis of allergic diseases such as allergic asthma. Eosinophil migration in vitro can be divided into directed migration, or Chemotaxis, and random migration, or chemokinesis. Here, we studied intracellular signals involved in eosinophil migration in vitro induced by platelet‐activating factor (PAF) and interleukin‐5 (IL‐5), applying a Boyden chamber assay. Migration induced by PAF (10‐11‐10‐6 M) largely consisted of Chemotaxis with some chemokinesis, whereas IL‐5 (10‐12‐10‐8 M) induced chemokinesis only. Eosinophils were depleted from intracellular and extracellular Ca2+ to study the role of Ca2+ as a second messenger. Ca2+ depletion did not change PAF‐induced Chemotaxis, however, IL‐5‐induced chemokinesis was inhibited. Interestingly, PAF, but not IL‐5, induced changes in [Ca2+]i. This rise originated mainly from internal stores. Inhibition of protein kinase A by H‐89 and protein kinase C by GF 109203X had no effect on both forms of eosinophil migration. Addition of the protein kinase inhibitor staurosporine significantly inhibited IL‐5‐induced chemokinesis. Inhibition of tyrosine kinases by herbimycin A completely blocked IL‐5‐induced chemokinesis. PAF and IL‐5‐induced actin polymerization was studied to compare migratory responses with a migration‐associated intracellular response. Ca2+ depletion significantly enhanced PAF‐induced (10‐8 M) actin polymerization, whereas IL‐5‐induced actin polymerization was not influenced. Addition of staurosporine led to an increase in F‐actin. Subsequent addition of PAF or IL‐5 resulted in an additive increase in F‐actin content. In summary, both forms of eosinophil migration are protein kinase A and protein kinase C independent. In contrast to PAF‐induced Chemotaxis, IL‐5‐induced chemokinesis was found to be completely Ca2+ and tyrosine kinase dependent.

Steroids induce a disequilibrium of secreted interleukin-1 receptor antagonist and interleukin-1β synthesis by human neutrophils

Chronic obstructive pulmonary disease (COPD) is characterised by neutrophilic inflammation in the airways and these neutrophils contribute to the production of inflammatory mediators. Dampening the production of proinflammatory mediators might be an important strategy to treat COPD and glucocorticosteroids are known to do so via inhibition of nuclear factor-&kgr;B. However, this pathway is important for the control of pro- and anti-inflammatory genes. We studied the effects of dexamethasone on production and secretion of pro-inflammatory interleukin (IL)-1&bgr; and anti-inflammatory secreted IL-1 receptor antagonist (sIL-1Ra) by human neutrophils activated with tumor necrosis factor (TNF)-&agr;. In vitro, TNF-&agr;-stimulated neutrophils produced significant amounts of IL-1&bgr; and sIL-1Ra; this production was inhibited by dexamethasone. However, synthesis and secretion of sIL-1Ra was inhibited at lower concentrations dexamethasone compared to IL-1&bgr;, which changed the IL-1&bgr;:sIL-1Ra ratio significantly. This altered ratio resulted in a more pro-inflammatory condition, as visualised by increased intercellular adhesion molecule-1 expression on human endothelial cells. In vivo, moderate-to-severe COPD patients using inhaled glucocorticosteroids have decreased plasma sIL-Ra levels compared with mild-to-moderate patients not on glucocorticosteroid treatment. In conclusion, dexamethasone induces a pro-inflammatory shift in the IL-1&bgr;:sIL-1Ra cytokine balance in neutrophils in vitro, which might contribute to a lack of endogenous anti-inflammatory signals to dampen inflammation in vivo.

Eosinophil chemotactic activity in bronchoalveolar lavage from idiopathic pulmonary fibrosis is dependent on cytokine priming of eosinophils

Increased numbers of eosinophils have been found in bronchoalveolar lavage (BAL) fluid obtained from patients with idiopathic pulmonary fibrosis (IPF). This suggests the presence of one or more cytokines in the lung tissue of patients with IPF, which are involved in the induction of migration of eosinophils towards the pulmonary compartment. To evaluate this hypothesis, we studied migratory responses of blood eosinophils towards BAL fluid. Migratory responses were tested by means of a modified Boyden chamber assay in 21 patients with IPF and 14 healthy controls. Experiments were performed with unprimed eosinophils and in vitro primed eosinophils (preincubated with 10(-11) M granulocyte macrophage-colony-stimulating factor). Changes in intracellular free Ca2+ ([Ca2+]i) in eosinophils in response to BAL fluid were also investigated, to characterize putative chemotaxins further. Chemotactic responses of eosinophils were observed towards BAL fluid from both patients with IPF and controls, provided that the eosinophils were primed. No changes in [Ca2+]i in eosinophils were detected in response to BAL fluid. Furthermore, neither a blocking antibody against interleukin-8 nor one against regulated on activation, normal T-cell, expressed and secreted (RANTES) influenced the response. Since a chemotactic response of in vitro primed eosinophils was also observed towards bronchoalveolar lavage fluid from normals, it was concluded, that in idiopathic pulmonary fibrosis, apart from the presence of a chemotactic factor in the lung tissue, other mechanisms such as priming of eosinophils in the peripheral blood are responsible for the extravasation of eosinophils into the pulmonary compartment. As no changes in [Ca2+]i were observed in the eosinophils after incubation with bronchoalveolar lavage fluid, the chemotaxin responsible for the migratory responses is probably not one of the known eosinophil-activating chemokines.

Testing bias in clinical databases: methodological considerations.

BackgroundLaboratory testing in clinical practice is never a random process. In this study we evaluated testing bias for neutrophil counts in clinical practice by using results from requested and non-requested hematological blood tests.MethodsThis study was conducted using data from the Utrecht Patient Oriented Database. This clinical database is unique, as it contains physician requested data, but also data that are not requested by the physician, but measured as result of requesting other hematological parameters. We identified adult patients, hospitalized in 2005 with at least two blood tests during admission, where requests for general blood profiles and specifically for neutrophil counts were contrasted in scenario analyses. Possible effect modifiers were diagnosis and glucocorticoid use.ResultsA total of 567 patients with requested neutrophil counts and 1,439 patients with non-requested neutrophil counts were analyzed. The absolute neutrophil count at admission differed with a mean of 7.4 × 109/l for requested counts and 8.3 × 109/l for non-requested counts (p-value < 0.001). This difference could be explained for 83.2% by the occurrence of cardiovascular disease as underlying disease and for 4.5% by glucocorticoid use.ConclusionRequests for neutrophil counts in clinical databases are associated with underlying disease and with cardiovascular disease in particular. The results from our study show the importance of evaluating testing bias in epidemiological studies obtaining data from clinical databases.

A unique protein profile of peripheral neutrophils from COPD patients does not reflect cytokine-induced protein profiles of neutrophils in vitro

BackgroundInflammation, both local and systemic, is a hallmark of chronic obstructive pulmonary disease (COPD). Inflammatory mediators such as TNFα and GM-CSF are secreted by lung epithelium, alveolar macrophages and other inflammatory cells and are thought to be important contributors in the pathogenesis of COPD. Indeed, neutrophils are activated by these cytokines and these cells are one of the major inflammatory cell types recruited to the pulmonary compartment of COPD patients. Furthermore, these inflammatory mediators are found in the peripheral blood of COPD patients and, therefore, we hypothesized that TNFα/GM-CSF-induced protein profiles can be found in peripheral neutrophils of COPD patients.MethodsUsing fluorescence 2-dimensional difference gel electrophoresis we investigated differentially regulated proteins in peripheral neutrophils from COPD patients and healthy age-matched control subjects. Furthermore, protein profiles from COPD patients were compared with those of neutrophils of healthy age-matched controls that were stimulated with TNFα and/or GM-CSF in vitro. Protein gels were compared using DeCyder 7.0 software.ResultsWe identified 7 significantly regulated protein spots between peripheral neutrophils from COPD patients and age-matched healthy control subjects. Stimulation of peripheral neutrophils with TNFα, GM-CSF or TNFα + GM-CSF in vitro resulted in 13, 20 and 22 regulated protein spots, respectively. However, these cytokine-induced protein differences did not correspond with the protein differences found in neutrophils from COPD patients.ConclusionThese results show that neutrophils from COPD patients have a unique protein profile compared to neutrophils from healthy age-matched controls. Furthermore, the neutrophil profiles of COPD patients do not reflect putative dominant signals induced by TNFα, GM-CSF or their combination. Our results indicate that systemic neutrophil responses in COPD patients are caused by a unique but subtle interplay between multiple inflammatory signals.

Hematocytometry analysis as discriminative marker for asthma phenotypes

Abstract Background: There is an increasing demand for easy to measure biomarkers in clinical practice. We created the relational database Utrecht Patient Oriented Database (UPOD) to develop tools for identifying new biomarkers for disease. In this study, we used UPOD to identify better biomarkers for discriminating different asthma phenotypes. Methods: We performed a prospective study at the University Medical Center (UMC) Utrecht using blood from patients with asthma and a healthy reference group. Since asthma is an inflammatory disease, absolute leukocyte counts and leukocyte differential parameters were analyzed using raw data files and a logistic regression model. Results: We compared 17 difficult-to-treat asthma (DTA) cases, 13 non-difficult-to-treat asthma cases, and 19 healthy volunteers. Absolute leukocyte counts and differential parameters for leukocytes were able to discriminate asthma patients from healthy volunteers. However, among patients with asthma, difficult-to-treat cases could be more accurately defined with a neutrophil morphology change (OR 8.0; 95% CI 1.5–42.0), compared to the absolute neutrophil count (OR 4.0; 95% CI 0.8–21.0). Conclusions: In this asthma patient population, we were able to define asthma phenotypes more precisely using neutrophil morphology parameters, compared to absolute counts. Using UPOD with differential parameters, it is possible to conduct larger scale biomarker studies, combining clinical, laboratory medicine, and epidemiological techniques. Clin Chem Lab Med 2009;47:573–8.

Identification of exacerbations in obstructive lung disease through biomarkers.

Inflammation has been identified as an important factor for disease exacerbation in obstructive lung disease. In this study, we used neutrophil and eosinophil counts as biomarkers for exacerbation in obstructive lung disease. We conducted a case–control study within a cohort of patients frequenting an outpatient clinic of Respiratory Medicine using data from the Utrecht Patient Oriented Database (UPOD). Cases were patients with a hospital admission for obstructive lung disease in 2005. For each case, one control patient was sampled from the same study base. We identified 143 cases (118 patients with chronic obstructive pulmonary disease and 25 asthma patients) and 143 controls. Admission was associated with both neutrophilia (adjusted odds ratio (OR) 4.3; 95% confidence interval (CI) 2.2–8.5), and eosinophilia (adjusted OR 2.6; 95% CI 1.1–6.2). The association with eosinophilia was only seen in asthma patients. In conclusion, neutrophil and eosinophil counts seem to be useful biomarkers for identifying exacerbations in pharmacoepidemiological studies on obstructive lung disease.

Medication Changes Prior to Hospitalization for Obstructive Lung Disease: A Case-Crossover Study

Background: Hospitalizations have always been seen as a solid outcome parameter in pharmacoepidemiology. However, the period leading to hospitalization and prehospital management of the patient are equally important. Objective: To evaluate medication changes in the period prior to hospitalization for obstructive lung disease and to quantify the association between medication use and the risk of hospitalization. Methods: We conducted a case-crossover study using the PHARMO record linkage system, which contains drug dispensing data from community pharmacies and hospital admission data. Patients included in the study were adults hospitalized for obstructive lung disease between 2005 and 2007. The index date of the case period was the date of hospitalization, and control moments were set at 3, 6, 9, and 12 months before admission. For each patient, all prescriptions prior to the date of hospitalization were identified. Medication use was ascertained in a 90-day time window prior to each case or control moment. Results: We identified 1481 patients who were hospitalized for obstructive lung disease. It appeared that respiratory medication use increased in the 90 days prior to hospitalization. Hospitalization was associated with the use of 3 or more respiratory drugs (OR 2.2; 95% CI 1.8 to 2.8), systemic glucocorticoids (OR 4.5; 95% CI 3.8 to 5.4), and antibiotics (OR 3.1; 95% CI 2.7 to 3.6). Conclusions: The use of systemic glucocorticoids, antibiotics, and other respiratory drugs increased prior to hospitalization for obstructive lung disease. These results could be indicative of the development and/or treatment of an exacerbation. There is a need for markers to detect exacerbations in an early phase in order to start treatment as early as possible and possibly prevent hospitalizations for obstructive lung disease.

Identification of inflammatory phenotypes of asthma by blood analysis and clinical parameters

Background Identification of an inflammatory asthma phenotype currently requires sputum induction. This technique is invasive, has a high variability, is time-consuming and a burden for patients. Therefore, there is a strong need for a routine blood test to establish the inflammatory phenotype of asthma. We designed a clinical cohort study (AIR-study, NCT01611012) in 115 asthma patients visiting the outpatient clinic to compare the results of such a blood test to sputum analysis. This abstracts focuses at the preliminary results in 21 patients.

Effects of glucocorticoids on the neutrophil count: a cohort study among hospitalized patients.

BACKGROUND Systemic glucocorticoids are often used in clinical practice for a large variety of indications. Clinical observations have shown that patients using glucocorticoids often have higher neutrophil counts. Debate remains whether this observed neutrophilia is associated with glucocorticoid use or that other factors, like disease and severity of disease, should be considered. The objective of this study was to investigate the effect of systemic glucocorticoids on the absolute neutrophil count in hospitalized patients. METHODS A cohort study was conducted using data from the Utrecht Patient Oriented Database which comprises clinical data of patients of the University Medical Center Utrecht. We identified all adult patients, hospitalized in 2005 with at least two blood samples for hematological testing during admission and compared in-hospital glucocorticoid use with non-use. RESULTS A total of 809 glucocorticoid users and 2658 non-users were included in the study with comparable neutrophil counts at admission (8.2.10(9)/l for glucocorticoid users and 8.0.10(9)/l for non-users). Overall analysis showed a slight association between glucocorticoid use and an increase in neutrophil count (RR 1.3; 95% CI 1.1-1.5). However, within diagnostic subgroups there was no increase in neutrophil count in glucocorticoid users. Furthermore, among all no dose response relationship, no effect of time between the two samples, and no effect of anti-inflammatory/sodium retaining potency was found. CONCLUSION Observed neutrophilia in users of systemic glucocorticoids is probably associated with underlying disease, rather than glucocorticoid use itself.