The cystatin M/E-controlled pathway of skin barrier formation: expression of its key components in psoriasis and atopic dermatitis.

BACKGROUND: The antiprotease activity of cystatin M/E regulates skin barrier formation, as it inhibits the activity of cathepsin V, cathepsin L and legumain, thereby controlling the processing of transglutaminase 3. Misregulation of this pathway by unrestrained protease activity, as seen in cystatin M/E-deficient mice, leads to abnormal stratum corneum and hair follicle formation, and severe disturbance of skin barrier function. OBJECTIVES: Our major aim was to make a quantitative analysis of the expression of all players of this pathway in the epidermis of patients with inflammatory skin diseases. A second aim was to determine if reconstructed human skin could be used as an in vitro model system to investigate this pathway. METHODS: Autopsy material from normal human tissues, biopsies from normal skin of healthy volunteers, and lesional skin from patients with atopic dermatitis and psoriasis were used to study the expression of the above-mentioned molecules at the mRNA level by quantitative real-time polymerase chain reaction. Localization of the protein was performed by immunofluorescence microscopy, and expression was quantitated by image analysis. RESULTS: In skin, cystatin M/E is expressed at relatively higher levels than its target proteases, when compared with other tissues, which emphasizes its prominent role in cutaneous biology. We found decreased expression of cystatin M/E and cathepsin V in lesional atopic dermatitis and psoriasis epidermis at the mRNA level as well as the protein level. Cathepsin L and transglutaminase 3 were increased at the transcriptional level; however, this was not reflected by higher protein levels. Interestingly, the expression of all these molecules in reconstructed skin was qualitatively and quantitatively similar to the in vivo situation. CONCLUSIONS: Disturbance of the cystatin M/E-cathepsin pathway could contribute to the dysregulated skin barrier function observed in inflammatory dermatoses. Human reconstructed skin appears to be a valuable model to study this novel biochemical pathway in vitro.

Plasma secretory leukocyte protease inhibitor in febrile patients.

OBJECTIVES: Secretory leukocyte protease inhibitor (SLPI) forms an integral part of the lung's defence, by its antimicrobial activity and by its ability to neutralize serine proteases that are released by granulocytes into the inflammatory exudate. Here, we investigate in febrile patients admitted to hospital whether plasma SLPI can serve as a marker of lung infection. METHODS: We prospectively determined the SLPI concentration in 152 febrile patients (median 73 [inter-quantile range (IQR): 58-82] year; 50% male) admitted to hospital because of infection of the airways (n = 44) or pneumonia (n = 108; i.e. consolidation on chest X-ray), and in 48 febrile patients (78 [IQR: 71-85] year; 52% male) admitted because of pyelonephritis, as well as afebrile age-matched controls (n = 38). In addition, erythrocyte sedimentation rate (ESR), peripheral blood leukocytes, plasma TNFalpha and IL-10, and parameters of the APACHE-II score were determined on admission. RESULTS: In febrile patients, SLPI was significantly increased (P < 0.001) compared with afebrile controls (63 [IQR: 50-76] ng/mL): plasma SLPI (113 [IQR: 83-176] ng/mL) was highest (P < 0.005) in patients with pneumonia compared with other groups (88 [IQR: 70-118] ng/mL). Only in patients with pneumonia, bacteremia significantly increased (P < 0.01) SLPI concentrations. Using a radiological classification of pulmonary infiltrates based on their size, it was found that plasma SLPI was proportional to the extent of lung tissue involved: the median concentration increased from 95 [IQR: 74-139] ng/mL in unilateral segmental consolidation up to 271 [IQR: 180-460] ng/mL in bilateral lobar consolidations. In a multivariate analysis, the association between SLPI and extent of consolidation was about two-fold stronger than, and independent of, the association between SLPI and erythrocyte sedimentation rate, TNFalpha, and parameters of the composite APACHE-II score, such as heart rate and blood pressure, that reflect severity of illness. CONCLUSION: SLPI is an indicator of the presence and extent of pneumonia in febrile patients admitted to hospital. In patients with an infection with its primary source located outside the lung, plasma SLPI likely reflects the mucosal response to circulating inflammatory mediators reflecting severity of illness.

A molecularly defined array based on native fibrillar collagen for the assessment of skin tissue engineering biomaterials.

Large-scale in vivo evaluation of biomaterials is time-consuming and limited by ethical considerations. The availability of a library of biomaterials would allow a fast and rational in vitro selection of those biomaterials to be evaluated in vivo. For this reason, we developed an array of 48 different, molecularly-defined films based on native fibrillar collagen. The films differed in the type and amount of extracellular matrix components (type I/IV collagens, fibrous/solubilised elastin, glycosaminoglycans, heparin, chondroitin sulfate or dermatan sulfate), method of preparation (homogenisation) and method and extent of crosslinking (carbodiimide (EDC/NHS) or glutaraldehyde). The array was evaluated by studying morphology, proliferation and differentiation of primary human keratinocytes/fibroblasts. Major differences were observed. Only a small selection of films (especially those containing elastin fibres) specifically stimulated the proliferation of keratinocytes, but not fibroblasts. Such films may be the biomaterials of choice for in vivo evaluation for skin tissue engineering and regenerative medicine.

The polymine spermine regulates osteogenic differentiation in adipose stem cells.

For bone tissue engineering, it is important that mesenchymal stem cells (MSCs) differentiate into osteoblasts. To develop a method for differentiation of adipose tissue-derived mesenchymal stem cells (AT-MSCs) along the osteogenic lineage, we studied the effect of polyamines, which are organic cations implicated in bone growth and development, on differentiation of AT-MSCs. Treatment of goat-derived AT-MSCs with 1,25-dihydroxyvitamin-D3 (1,25(OH)(2)D(3)), which stimulates osteogenic differentiation, for 7 days induced gene expression of the polyamine-modulated transcription factor-1 (PMF-1) and spermidine/spermine N (1)-acetyltransferase (SSAT), which are both involved in polyamine metabolism, suggesting that polyamines are involved in osteogenic differentiation of AT-MSCs. Furthermore, treatment of AT-MSCs with the polyamine spermine-regulated gene expression of runx-2, a transcription factor involved in early stages of osteogenic differentiation, and that of osteopontin, a bone matrix protein expressed in later stages of osteogenic differentiation. Runx-2 gene expression was increased 4 and 14 days after a short 30 min. treatment with spermine, while osteopontin gene expression was only increased 4 days after spermine treatment. Finally, alkaline phosphatase activity, which is intimately involved in the formation of extracellular matrix of bone, was increased 4 weeks after the 30 min.-spermine treatment of AT-MSCs. In conclusion, this study shows for the first time that the polyamine spermine regulates differentiation of AT-MSCs along the osteogenic lineage, which can be used as a new method for differentiation of AT-MSCs along the osteogenic lineage. Therefore, polyamines may constitute a promising tool for bone tissue engineering approaches using AT-MSCs, such as a one-step surgical procedure for spinal interbody fusion.

Mechanisms of cell death induced by the neutrophil antimicrobial peptides alpha-defensins and LL-37.

OBJECTIVE: The aim of this study was to investigate the mechanisms of cell death mediated by the antimicrobial peptides neutrophil defensins (human neutrophil peptides 1-3 [HNP1-3]) and LL-37. MATERIALS AND METHODS: HNP1-3- and LL-37-mediated cell death was assessed in human lung epithelial cells and Jurkat T-cells in serum-free culture media. RESULTS: Both HNP1-3 and LL-37 induced cell death in Jurkat T-cells and A549 cells. HNP1-3 but not LL-37 induced caspase-3/-7 activity and caused cleavage of [ADP-ribose] polymerase (PARP) in Jurkat cells, while in A549 cells neither peptides induced caspase-3/-7 activation. Furthermore, both peptides increased mitochondrial cytochrome c release in A549 and Jurkat cells. Our observation that over-expression of the anti-apoptotic protein Bcl-2 in Jurkat cells did not affect HNP1-3- or LL-37-induced cell death indicates that antimicrobial peptide-induced cytochrome c release is not involved in peptide-induced cell death. Finally, in A549 cells and in primary bronchial epithelial cells, both HNP1-3 and LL-37 induced DNA breaks as demonstrated by increased TUNEL labelling. CONCLUSIONS: The results from this study suggest that the antimicrobial peptides HNP1-3 and LL-37 induce cell death, which is associated with mitochondrial injury and mediated via different intracellular pathways.

Human cathelicidin, hCAP-18, is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3.

Cathelicidins are a family of antimicrobial proteins found in the peroxidase-negative granules of neutrophils. The known biologic functions reside in the C-terminus, which must be cleaved from the holoprotein to become active. Bovine and porcine cathelicidins are cleaved by elastase from the azurophil granules to yield the active antimicrobial peptides. The aim of this study was to identify the physiological setting for cleavage of the only human cathelicidin, hCAP-18, to liberate the antibacterial and cytotoxic peptide LL-37 and to identify the protease responsible for this cleavage. Immunoelectron microscopy demonstrated that both hCAP-18 and azurophil granule proteins were present in the phagolysosome. Immunoblotting revealed no detectable cleavage of hCAP-18 in cells after phagocytosis. In contrast, hCAP-18 was cleaved to generate LL-37 in exocytosed material. Of the 3 known serine proteases from azurophil granules, proteinase 3 was solely responsible for cleavage of hCAP-18 after exocytosis. This is the first detailed study describing the generation of a human antimicrobial peptide from a promicrobicidal protein, and it demonstrates that the generation of active antimicrobial peptides from common proproteins occurs differently in related species. (Blood. 2001;97:3951-3959)