BackgroundInvestigations on pulmonary macrophages (MΦ) mostly focus on alveolar MΦ (AM) as a well-defined cell population. Characteristics of MΦ in the interstitium, referred to as lung interstitial MΦ (IM), are rather ill-defined. In this study we therefore aimed to elucidate differences between AM and IM obtained from human lung tissue.MethodsHuman AM and IM were isolated from human non-tumor lung tissue from patients undergoing lung resection. Cell morphology was visualized using either light, electron or confocal microscopy. Phagocytic activity was analyzed by flow cytometry as well as confocal microscopy. Surface marker expression was measured by flow cytometry. Toll-like receptor (TLR) expression patterns as well as cytokine expression upon TLR4 or TLR9 stimulation were assessed by real time RT-PCR and cytokine protein production was measured using a fluorescent bead-based immunoassay.ResultsIM were found to be smaller and morphologically more heterogeneous than AM, whereas phagocytic activity was similar in both cell types. HLA-DR expression was markedly higher in IM compared to AM. Although analysis of TLR expression profiles revealed no differences between the two cell populations, AM and IM clearly varied in cell reaction upon activation. Both MΦ populations were markedly activated by LPS as well as DNA isolated from attenuated mycobacterial strains (M. bovis H37Ra and BCG). Whereas AM expressed higher amounts of inflammatory cytokines upon activation, IM were more efficient in producing immunoregulatory cytokines, such as IL10, IL1ra, and IL6.ConclusionAM appear to be more effective as a non-specific first line of defence against inhaled pathogens, whereas IM show a more pronounced regulatory function. These dissimilarities should be taken into consideration in future studies on the role of human lung MΦ in the inflammatory response.

Induction of the glucocorticoid-induced leucine zipper (GILZ) by glucocorticoids plays a role in their antiinflammatory action, whereas GILZ expression is reduced under inflammatory conditions. The mechanisms regulating GILZ expression during inflammation, however, have not yet been characterized. Here, we investigated GILZ expression in human alveolar macrophages (AMs) following Toll-like receptor (TLR) activation. Macrophages were shown to predominantly express GILZ transcript variant 2. Lipopolysaccharide-treated AMs, THP-1 cells, and lungs of lipopolysaccharide-exposed mice displayed decreased GILZ protein and mRNA levels. The effect was strictly dependent on the adapter molecule MyD88, as shown by using specific ligands or a knockdown strategy. Investigations on the functional significance of GILZ downregulation performed by GILZ knockdown revealed a proinflammatory response, as indicated by increased cytokine expression and NF-κB activity. We found that TLR activation reduced GILZ mRNA stability, which was mediated via the GILZ 3 -untranslated region. Finally, involvement of the mRNA-binding protein tristetraprolin (TTP) is suggested, since TTP overexpression or knockdown modulated GILZ expression and TTP was induced in a MyD88-dependent fashion. Taken together, our data show a MyD88-and TTP-dependent GILZ downregulation in human macrophages upon TLR activation. Suppression of GILZ is mediated by mRNA destabilization, which might represent a regulatory mechanism in macrophage activation.Keywords: GILZ transcript variants · Inflammation · Innate immunity · mRNA stability · Pulmonary macrophages IntroductionInflammatory processes in the lung play an important role in different diseases, such as viral and bacterial infections. Moreover, noninfectious diseases such as chronic obstructive pulmonary disCorrespondence: Prof. Alexandra K. Kiemer e-mail: pharm.bio.kiemer@mx.uni-saarland.de ease (COPD) are characterized by chronic inflammatory processes. Asthma and allergic diseases are other examples of chronic inflammatory lung diseases with major clinical relevance [1,2].Toll-like receptors (TLRs) represent an important family of innate immune receptors, which act as a first line of defense of host immunity against various pathogens. Presently, ten human TLRs are known, which recognize pathogen-associated molecular patterns including bacterial cell wall components, such as lipoproteins (TLR1/2 or TLR1/6 dimers) or lipopolysaccharide (LPS, TLR4), C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2012. 42: 1282-1293 Innate immunity 1283 bacterial flagellin (TLR5), viral RNA (TLR3, 7 and 8), as well as bacterial DNA (TLR9) [3]. Except for TLR3, all TLRs signal via the adapter molecule MyD88. The MyD88-independent pathway used by TLR3 can also be utilized by TLR4. The different signaling pathways employ different protein kinase complexes and show distinct cytokine profiles, but all activate NF-κB [4]. In addition to their role in pathogen defense, TLRs also play a dominant role in ...

Purpose: A better understanding of the vitamin D 3 metabolism is required to evaluate its potential therapeutic value for cancers. Here, we set out to contribute to the understanding of vitamin D 3 metabolism in glioblastoma multiforme. Experimental Design: We did nested touchdown reverse transcription-PCR (RT-PCR) to identify CYP27B1 splice variants and real-time RT-PCR to quantify the expression of CYP27B1. A cell line was treated with calcitriol to determine the effect on the expression of CYP27B1, 1a,25-dihydroxyvitamin D 3 -24-hydroxylase (CYP24), and vitamin D 3 receptor (VDR). We generated three antibodies for the specific detection of CYP27B1 and splice variants. HighperformanceTLC was done to determine the endogenous CYP27B1activity and the functionality of CYP27B1splice variants. Using WST-1assay, we determined the effect of vitamin D 3 metabolites on proliferation. Results: We report a total of 16 splice variants of CYP27B1 in glioblastoma multiforme and a different expression of CYP27B1 and variants between glioblastoma multiforme and normal tissues.We found preliminary evidence for enzymatic activity of endogenous CYP27B1in glioblastoma multiforme cell cultures but not for the functionality of the splice variants. By adding calcitriol, we found a proliferative effect for some cell lines depending on the dose of calcitriol. The administration of calcitriol led to an elevated expression of CYP27B1and CYP24 but left the expression of theVDR unaltered. Conclusions: Our findings show that glioblastoma multiforme cell lines metabolize calcidiol. In addition, we show various effects mediated by calcitriol. We found a special vitamin D 3 metabolism and mode of action in glioblastoma multiforme that has to be taken into account in future vitamin D 3^r elated therapies.The classic metabolism of the secosteroid hormone vitamin D 3 to its active form followed by several degradation pathways is well described. Vitamin Besides renal CYP27B1 activity, extrarenal CYP27B1 expression has been described in a variety of tissues (4). In vitro, several nonrenal cells, including microglial cells (5), produce calcitriol from its precursor. In addition, several cancer cells show CYP27B1 activity (e.g., human non -small cell lung carcinoma cells; ref. 6). Local production of calcitriol has been postulated to play an autocrine or paracrine role in vitamin Dmediated growth control (7).As shown for numerous normal and cancer cell lines, calcitriol is at high concentrations (10 À9 to 10 À6 mol/L) an antiproliferative and prodifferentiating agent that induces apoptosis and inhibits cell migration (8). The antiproliferative properties of calcitriol, as described above, were also shown for central nervous system tumors, including glioblastoma cell lines (9). Beneficial effects of the vitamin D 3 metabolite 1a-hydroxyvitamin D 3 have been reported for the treatment of glioblastoma multiforme in a small phase II clinical study (10).

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Using real-time PCR (LightCycler) and immunohistochemistry, we have analyzed expression of key components of the vitamin D system in basal cell carcinomas (BCCs) and normal human skin (NS).

Alpha-lipoic acid mediates its antiapoptotic action via activation of the insulin receptor/PI3-kinase/Akt pathway. We show for the first time a direct binding site for alpha-lipoic acid at the insulin receptor tyrosine kinase domain, which might make alpha-lipoic acid a model substance for the development of insulin mimetics.

Various human illnesses, including several types of cancer and infectious diseases, are related to changes in the cellular redox homeostasis. During the last decade, several approaches have been explored which employ such disturbed redox balances for the benefit of therapy. Compounds able to modulate the intracellular redox state of cells have been developed, which effectively, yet also selectively, appear to kill cancer cells and a range of pathogenic microorganisms. Among the various agents employed, certain redox catalysts have shown considerable promise since they are non-toxic on their own yet develop an effective, often selective cytotoxicity in the presence of the 'correct' intracellular redox partners. Aminoalkylation, amide coupling and multicomponent reactions are suitable synthetic methods to generate a vast number of such multifunctional catalysts, which are chemically diverse and, depending on their structure, exhibit various interesting biological activities.

Alveolar macrophages are the first line of host defence against mycobacteria, but an insufficient host response allows survival of bacteria within macrophages. We aimed to investigate the role of Toll-like receptor 9 (TLR9) activation in macrophage defence against mycobacteria. Human in vitrodifferentiated macrophages as well as human and mouse alveolar macrophages showed TLR9 mRNA and protein expression. The cells were markedly activated by DNA isolated from attenuated mycobacterial strains (H37Ra and Mycobacterium bovis BCG) as assessed by measuring cytokine expression by real-time PCR, whereas synthetic phosphorothioate-modified oligonucleotides had a much lower potency to activate human macrophages. Intracellular replication of H37Ra was higher in macrophages isolated from TLR9-deficient mice than in macrophages from wild-type mice, whereas H37Rv showed equal survival in cells from wild-type or mutant mice. Increased bacterial survival in mouse macrophages was accompanied by altered cytokine production as determined by Luminex bead assays. In vivo infection experiments also showed differential cytokine production in TLR9-deficient mice compared to wild-type animals. Both human monocyte-derived macrophages as well as human alveolar macrophages showed reduced activation upon treatment with DNA isolated from bacteria from virulent (M. bovis and H37Rv) compared to attenuated mycobacteria. We suggest attenuated TLR9 activation contributes to the insufficient host response against virulent mycobacteria.