Background: The inflammatory process in chronic obstructive pulmonary disease (COPD) is characterised by the presence of neutrophils in the lung that are able to synthesise de novo several inflammatory mediators. The local chronic persistent inflammatory response is accompanied by systemic effects such as cytokine induced priming of peripheral leucocytes and muscle wasting. The preactivation or priming of peripheral blood neutrophils was used to gain more insight into the mechanisms of this systemic inflammatory response. Methods: Gene arrays were performed on peripheral blood neutrophils obtained from healthy donors after stimulation in vitro with tumour necrosis factor (TNF)-a, granulocyte-macrophage colony stimulating factor (GM-CSF), or both. The expression of many inflammatory genes was regulated in these cells following stimulation. The expression of inflammatory genes in peripheral blood neutrophils in healthy subjects and those with COPD was measured by real time RT-PCR after stimulation with TNFa, GM-CSF, interleukin (IL)-8, fMLP, TNFa + GM-CSF, and lipopolysaccharide (LPS). Conclusions: These data are consistent with the hypothesis that progression of COPD is associated with the activation of neutrophils in the systemic compartment. De novo expression of inflammatory mediators by peripheral blood neutrophils suggests a pro-inflammatory role for these cells in the pathogenesis of COPD.
Assaying activation of signal transduction is laborious and does not allow the study of large numbers of samples, essential for high-throughput drug screens or for large groups of patients. Using phosphospecific antibodies, we have developed ELISA techniques enabling non-radioactive semi-quantitative assessment of the activation state of p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, protein kinase B and the transcription factor cAMP-response-element-binding protein (CREB) in 96-well plates. This assay has been termed PACE (phosphospecific antibody cell-based ELISA) and was used successfully for both adherent and suspension cells. Various stimuli induced dose-dependent enzymic activity of which the kinetics closely correlated with those measured via classical methodology. Using PACE we have now characterized for the first time the concentration-dependent effects of various inflammatory prostaglandins on CREB phosphorylation in macrophages. PACE is a straightforward and novel technique enabling the large-scale analysis of signal transduction.
UV radiation, in particular UVB, suppresses the skin immune response. In patients with polymorphous light eruption (PLE) the skin immune response seems activated after UV exposure. Typical PLE skin lesions can occur as early as several hours after UV exposure. In healthy volunteers, neutrophils infiltrate the skin shortly after UV exposure. The kinetics and mechanisms of neutrophil infiltration in the skin of PLE patients after UVB exposure was studied. Skin biopsies at 0, 3, 6, and 18 h were taken from five PLE patients and six healthy controls after irradiation with three minimal erythema dose UVB. Furthermore, neutrophils were isolated from blood of five PLE patients and six healthy controls to test their chemotactic activity. Immunohistochemical analysis showed a significant decreased neutrophil infiltration in PLE skin after UVB irradiation compared with healthy controls (p<0.05). In both healthy controls and PLE patients, after UVB irradiation, ICAM-1 and E-selectin expression on endothelial cells increased at 6 h after irradiation. Blood neutrophil chemotactic response towards IL-8 and C5a, as well as the expression of cell surface markers involved in adhesion and chemotaxis, was not different between PLE patients and healthy controls. In conclusion, PLE is marked by a decreased skin infiltration of neutrophils after UVB irradiation, possibly leading to a diminished neutrophil-induced suppression.
Assaying activation of signal transduction is laborious and does not allow the study of large numbers of samples, essential for high-throughput drug screens or for large groups of patients. Using phosphospecific antibodies, we have developed ELISA techniques enabling non-radioactive semi-quantitative assessment of the activation state of p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, protein kinase B and the transcription factor cAMP-response-element-binding protein (CREB) in 96-well plates. This assay has been termed PACE (phosphospecific antibody cell-based ELISA) and was used successfully for both adherent and suspension cells. Various stimuli induced dose-dependent enzymic activity of which the kinetics closely correlated with those measured via classical methodology. Using PACE we have now characterized for the first time the concentration-dependent effects of various inflammatory prostaglandins on CREB phosphorylation in macrophages. PACE is a straightforward and novel technique enabling the large-scale analysis of signal transduction.
CD4+ T cells play a critical role in allergic diseases, both in the affected tissue as well as systemically. Our objective was to investigate the in vivo activation state of peripheral blood CD4+ T cells of atopic dermatitis (AD) patients by analyzing gene expression profiles of unstimulated CD4+ T cells. mRNA samples from blood CD4+ T cells, isolated from five AD patients and seven healthy controls (HC), were analyzed using oligonucleotide arrays. Differentially regulated genes were validated by quantitative PCR (Q-PCR) in a larger group of patients with AD, in a group of patients with allergic asthma (AA), and HC subjects. In addition, "typical" T helper type 1 (Th1)- and Th2-related genes were analyzed by Q-PCR. Microarray analysis revealed differential expression of 52 genes in AD patients. Q-PCR confirmed several differentially regulated genes in AD, including CCR10, CRTH2, C-JUN, and NR4A2. Two groups of genes with highly correlating gene expression levels involved in tissue homing and proliferation or apoptosis, respectively, were identified. No marked differences were found in gene expression levels of typical Th1 or Th2 genes in AD or in AA patients. This study demonstrates that peripheral blood, unstimulated CD4+ T cells in AD patients show differentially expressed genes involved in tissue homing, proliferation, and apoptosis. No marked expression differences of "typical" atopy genes were found.
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