Function of liver activation-regulated chemokine/CC chemokine ligand 20 is differently affected by cathepsin B and cathepsin D processing

Chemokine processing by proteases is emerging as an important regulatory mechanism of leukocyte functions and possibly also of cancer progression. We screened a large panel of chemokines for degradation by cathepsins B and D, two proteases involved in tumor progression. Among the few substrates processed by both proteases, we focused on CCL20, the unique chemokine ligand of CCR6 that is expressed on immature dendritic cells and subtypes of memory lymphocytes. Analysis of the cleavage sites demonstrate that cathepsin B specifically cleaves off four C-terminally located amino acids and generates a CCL20(1-66) isoform with full functional activity. By contrast, cathepsin D totally inactivates the chemotactic potency of CCL20 by generating CCL20(1-55), CCL20(1-52), and a 12-aa C-terminal peptide CCL20(59-70). Proteolytic cleavage of CCL20 occurs also with chemokine bound to glycosaminoglycans. In addition, we characterized human melanoma cells as a novel CCL20 source and as cathepsin producers. CCL20 production was up-regulated by IL-1alpha and TNF-alpha in all cell lines tested, and in human metastatic melanoma cells. Whereas cathepsin D is secreted in the extracellular milieu, cathepsin B activity is confined to cytosol and cellular membranes. Our studies suggest that CCL20 processing in the extracellular environment of melanoma cells is exclusively mediated by cathepsin D. Thus, we propose a model where cathepsin D inactivates CCL20 and possibly prevents the establishment of an effective antitumoral immune response in melanomas.

Poor sleep is associated with higher plasma proinflammatory cytokine interleukin-6 and procoagulant marker fibrin D-dimer in older caregivers of people with Alzheimer's disease

OBJECTIVES: To determine whether objective measures of sleep correlate with plasma levels of the proinflammatory cytokine interleukin (IL)-6 and the procoagulant marker fibrin D-dimer in caregivers of patients with dementia. DESIGN: Cross-sectional study. SETTING: Subjects' homes. PARTICIPANTS: Sixty-four community-dwelling spousal caregivers (69% women, mean age+/-standard deviation 72+/-9) and 36 sex-matched noncaregiving controls. MEASUREMENTS: All participants underwent in-home full-night polysomnography. Demographic and lifestyle factors, depression, diseases, and medication that could affect inflammation, coagulation, and sleep were controlled for in analyses regressing sleep variables and caregiver status and their interaction on plasma levels of IL-6 and D-dimer. RESULTS: Caregivers had higher levels of D-dimer (781+/-591 vs 463+/-214 ng/mL, P=.001) and IL-6 (1.42+/-1.52 vs 0.99+/-0.86 pg/mL, P<.06) and lower levels of total sleep time (369+/-70 vs 393+/-51 minutes, P=.049) and sleep efficiency (77+/-11 vs 82+/-9%, P=.04) than controls. After controlling for age and body mass index, longer wake time after sleep onset (change in coefficient of determination (DeltaR2)=0.039, P=.04) and the interaction between caregiver status and higher apnea-hypopnea index (DeltaR2=0.054, P=.01) were predictors of IL-6. Controlling for age, caregiver status independently predicted D-dimer levels (DeltaR2=0.047, P=.01). Controlling for age and caregiver status, lower sleep efficiency (DeltaR2=0.032, P=.03) and the interaction between caregiver status and more Stage 2 sleep (DeltaR2=0.037, P=.02) independently predicted plasma D-dimer levels. CONCLUSION: Poor sleep was associated with higher plasma IL-6 and D-dimer levels. These effects were most pronounced in caregivers of subjects with Alzheimer's disease. The findings suggest a mechanism that may explain how disturbed sleep might be associated downstream with cardiovascular risk, particularly in older people under chronic stress.

Heterologous expression from the human D-Loop in organello

We report the expression of a linear reporter construct in isolated human mitochondria. The reporter construct contained the entire human D-Loop with adjacent tRNA (MTT) genes (mt.15956-647), the human ND1 gene with an in frame GFP gene and adjacent endogenous MTT genes and heterologous rat MTT genes. Natural competence of isolated human mitochondria of HepG2 cells was used to import reporter constructs. The import efficiency of various fluorescently labelled PCR-generated import substrates in the range of 250bp up to 3.5kb was assessed by quantitative PCR and evaluated by confocal microscopy. Heterologous expression of the imported construct was confirmed at RNA level by a circular RNA (cRNA)-RT-PCR assay for the expression of tRNAs and by in organello [α-(32)P]-UTP labelling and subsequent hybridisation to reporter-specific sequences for monitoring mRNA expression. Heterologous expression of rat mitochondrial tRNA(Leu(UUR)) (rMT-TL1) was confirmed by co-/post-transcriptional trinucleotide (CCA) addition. Interestingly, the rat-specific MT-TL1 was correctly processed in isolated human mitochondria at the 3' end, but showed an aberrant 5' end processing. Correct 3' end processing of the heterologous expressed mitochondrial rat tRNA(Ser2) (MT-TS2) was detected. These findings demonstrate the feasibility of genetic manipulation of human mitochondria, providing a tool for characterisation of cis-acting elements of the human mitochondrial genome and for the study of human mitochondrial tRNA processing in organello.

The role of inducible nitric oxide synthase for interstitial remodeling of alveolar septa in surfactant protein D-deficient mice

Surfactant protein D (SP-D) modulates the lung's immune system. Its absence leads to NOS2-independent alveolar lipoproteinosis and NOS2-dependent chronic inflammation, which is critical for early emphysematous remodeling. With aging, SP-D knockout mice develop an additional interstitial fibrotic component. We hypothesize that this age-related interstitial septal wall remodeling is mediated by NOS2. Using invasive pulmonary function testing such as the forced oscillation technique and quasistatic pressure-volume perturbation and design-based stereology, we compared 29-wk-old SP-D knockout (Sftpd(-/-)) mice, SP-D/NOS2 double-knockout (DiNOS) mice, and wild-type mice (WT). Structural changes, including alveolar epithelial surface area, distribution of septal wall thickness, and volumes of septal wall components (alveolar epithelium, interstitial tissue, and endothelium) were quantified. Twenty-nine-week-old Sftpd(-/-) mice had preserved lung mechanics at the organ level, whereas elastance was increased in DiNOS. Airspace enlargement and loss of surface area of alveolar epithelium coexist with increased septal wall thickness in Sftpd(-/-) mice. These changes were reduced in DiNOS, and compared with Sftpd(-/-) mice a decrease in volumes of interstitial tissue and alveolar epithelium was found. To understand the effects of lung pathology on measured lung mechanics, structural data were used to inform a computational model, simulating lung mechanics as a function of airspace derecruitment, septal wall destruction (loss of surface area), and septal wall thickening. In conclusion, NOS2 mediates remodeling of septal walls, resulting in deposition of interstitial tissue in Sftpd(-/-). Forward modeling linking structure and lung mechanics describes the complex mechanical properties by parenchymatous destruction (emphysema), interstitial remodeling (septal wall thickening), and altered recruitability of acinar airspaces.