The protease inhibitor cystatin C downregulates the release of IL-β and TNF-α in lipopolysaccharide activated monocytes

Human cystatin C, a member of the cysteine proteinaseinhibitory family, is produced by all nucleated cells and has important roles in regulating natural immunity. Nematode homologs to human cystatin C have been shown to have anti-inflammatory effects on monocytes and to reduce colitis in mice. In Crohn’s disease, pathogenic activated monocytes help drive inflammatory processes via the release of proinflammatory cytokines and chemokines. In particular, tumor necrosis factora-producing inflammatory monocytes have a central role in the intestinal inflammation in patients with Crohn’s disease. We investigated the potential of human cystatin C to regulate pathogenic activated monocytes and its potential as an Immunomodulator in Crohn’s disease.We found that cystatin C significantly decreased the lipopolysaccharide-stimulated release and expression of interleukin-1b and tumor necrosis factor-α in monocyte and peripheral blood mononuclear cell cultures from healthy donors, whereas interleukin-6 and interleukin-8 levels were unchanged. A similar reduction of interleukin-1b and tumor necrosis factor-α was also seen in peripheral bloodmononuclear cell cultures from patientswith Crohn’s disease, and in particular, tumor necrosis factor-α was reduced in supernatants from lamina propria cell cultures from patients with Crohn’s disease. Further investigation revealed that cystatin C was internalized by monocytes via an active endocytic process, decreased phosphorylation of the mitogen-αctivated protein kinase pathway extracellular signal-regulated kinase-1/2, and altered surface marker expression. The ability of cystatin C to modulate the cytokine expression of monocytes, together with its protease-inhibitory function, indicates that modulation of the local cystatin C expression could be an option in future Crohn’s disease therapy

Why do some adults with PiMZ α1-antitrypsin develop bronchiectasis? [corrected]

Recurrent infections of the upper airways in early life may be a warning sign of inherited α1-antitrypsin deficiency http://ow.ly/iJsF300kbyV.S

Exogenous alpha 1-antitrypsin down-regulates SERPINA1 expression.

The main goal of the therapy with purified human plasma alpha1-antitrypsin (A1AT) is to increase A1AT levels and to prevent lungs from elastolytic activity in patients with PiZZ (Glu342Lys) A1AT deficiency-related emphysema. Potential hepatic gains of this therapy are unknown. Herein, we investigated the effect of A1AT therapy on SERPINA1 (gene encoding A1AT) expression. The expression of SERPINA1 was determined in A1AT or A1AT plus Oncostatin M (OSM) treated primary human hepatocytes isolated from liver tissues from A1AT deficient patients and control liver tissues. In addition, SERPINA1 mRNA was assessed in lung tissues from PiZZ emphysema patients with and without A1AT therapy, and in adherent human peripheral blood mononuclear cells (PBMC) isolated from healthy PiMM donors. In a dose-dependent manner purified A1AT lowered SERPINA1 expression in hepatocytes. This latter effect was more prominent in hepatocytes stimulated with OSM. Although it did not reach statistical significance (P = 0.0539)-analysis of lung tissues showed lower SERPINA1 expression in PiZZ emphysema patients receiving augmentation therapy relative to those without therapy. Finally, exogenously added purified A1AT (1mg/ml) reduced SERPINA1 expression in naïve as well as in lipopolysaccharide (LPS)-stimulated human adherent PBMCs. Exogenous A1AT protein reduces its own endogenous expression. Hence, augmentation with native M-A1AT protein and a parallel reduction in expression of dysfunctional mutant Z-A1AT may be beneficial for PiZZ liver, and this motivates further studies

Characteristics of liver tissues used for cell isolation.

<p>Characteristics of liver tissues used for cell isolation.</p

Purified A1AT reduces SERPINA1 expression in a dose-dependent manner in primary human hepatocytes.

<p>(<b>A</b>) Exogenously added purified A1AT reduced SERPINA1 expression in primary human hepatocytes isolated from both proficient and deficient liver tissue. The reduction was more prominent following Oncostatin M (10ng/ml) stimulation (Kruskal-Wallis test on ranks). SERPINA1 gene expression was reduced in a dose-dependent manner with marked decrease at A1AT levels of >1 mg/ml (<b>B</b>) Oncostatin M showed increased expression of SERPINA1 in primary human hepatocytes (Mann-Whitney <i>U</i> test), * P<0.05, ** P <.001, *** P<0.001, **** P<0.0001).</p

Purified A1AT reduces SERPINA1 expression in adherent PBMCs <i>in vitro</i>.

<p>Adherent PBMCs isolated from healthy donors showed a reduction in SERPINA1 expression following purified A1AT protein treatment. The reduction could be observed in both naïve and LPS-stimulated cells and was in coherence with the effects seen in hepatocytes (n = 7, Wilcoxon test), * P<0.05.</p

Increased rate of proliferation in the PiZZ liver.

<p>Liver tissue from PiZZ patients (n = 6) was stained for proliferation using Ki67 antibody and visualized by diaminobenzidine. Liver tissue from A1AT proficient individuals (n = 13) was used as reference. PiZZ livers show increased frequency of Ki67 positive (proliferating) cells compared to the proficient group. No differences could be observed in the non-parenchymal cells (NPC). (Mann-Whitney <i>U</i> test) * P<0.05, ns not significant. Scale bars: 100μm.</p

Effect of purified A1AT therapy on SERPINA1 gene expression in the PiZZ lung.

<p>SERPINA1 gene expression in lung tissue from PiZZ patients receiving purified A1AT (Prolastin) therapy (n = 10) compared to PiZZ patients without therapy (n = 4) showed similar tendencies to other cell types investigated. There might be reduced SERPINA1 expression in treated PiZZ patients, the difference did however not reach statistical significance (P = 0.0539, Mann-Whitney <i>U</i> test).</p

Proficient versus deficient primary human hepatocytes.

<p>RT-PCR <b>e</b>xpression of several genes showed no remarkable difference between proficient and deficient primary human hepatocytes. SERPINA1 expression was at equal levels in both deficient and proficient hepatocytes, Ki67 levels differed between the groups, although this was not significant. However, expression of CASP3 was increased in A1AT deficient hepatocytes (Mann-Whitney <i>U</i> test) * P<0.05, ns not significant.</p