Concentration-dependent effects of native and polymerised alpha1-antitrypsin on primary human monocytes, in vitro - PubMed (original) (raw)

Concentration-dependent effects of native and polymerised alpha1-antitrypsin on primary human monocytes, in vitro

Ruta Aldonyte et al. BMC Cell Biol. 2004.

Abstract

Background: Alpha1-antitrypsin (AAT) is one of the major serine proteinase inhibitors controlling proteinases in many biological pathways. There is increasing evidence that AAT is able to exert other than antiproteolytic effects. To further examine this question we compared how various doses of the native (inhibitory) and the polymerised (non-inhibitory) molecular form of AAT affect pro-inflammatory responses in human monocytes, in vitro. Human monocytes isolated from different donors were exposed to the native or polymerised form of AAT at concentrations of 0.01, 0.02, 0.05, 0.1, 0.5 and 1 mg/ml for 18 h, and analysed to determine the release of cytokines and to detect the activity of NF-kappaB.

Results: We found that native and polymerised AAT at lower concentrations, such as 0.1 mg/ml, enhance expression of TNFalpha (10.9- and 4.8-fold, p < 0.001), IL-6 (22.8- and 23.4-fold, p < 0.001), IL-8 (2.4- and 5.5-fold, p < 0.001) and MCP-1 (8.3- and 7.7-fold, p < 0.001), respectively, compared to buffer exposed cells or cells treated with higher doses of AAT (0.5 and 1 mg/ml). In parallel to increased cytokine levels, low concentrations of either conformation of AAT (0.02-0.1 mg/ml) induced NF-kappaB p50 activation, while 1 mg/ml of either conformation of AAT suppressed the activity of NF-kappaB, compared to controls.

Conclusions: The observations reported here provide further support for a central role of AAT in inflammation, both as a regulator of proteinase activity, and as a signalling molecule for the expression of pro-inflammatory molecules. This latter role is dependent on the concentration of AAT, rather than on its proteinase inhibitory activity.

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Figures

Figure 1

Figure 1

Native and polymerised AAT (10 μg/ml) analysed by 7.5% native PAGE. Lane M, molecular size markers, lane 1, native AAT and lane 2, polymerised AAT (incubated at 60°C, 3 h).

Figure 2

Figure 2

Cytokines released from monocytes stimulated with various concentrations of native (A) or polymerized (B) form of AAT. Each point represents the mean ± SD of six separate experiments. Significantly high up-regulation of the pro-inflammatory cytokines (TNF-α and IL-6) was found when cells were treated with low doses (0.05 and 0.1 mg/ml) of native or polymerized AAT, respectively, compared to control treated with equivalent amounts of buffer alone. The buffer control levels are indicated on the graphs (TNFα in blue and IL-6 in red).

Figure 3

Figure 3

Chemokines released from monocytes stimulated with various concentrations of native (A) or polymerized (B) form of AAT. Each point represents the mean ± SD of six separate experiments. Significantly high up-regulation of the pro-inflammatory chemokines (IL-8 and MCP-1) was found when cells were treated with low doses (0.05 and 0.1 mg/ml) of native or polymerized AAT, respectively, compared to controls treated with equivalent amounts of buffer alone. The buffer control levels are indicated on the graphs (MCP-1 in blue and IL-8 in red).

Figure 4

Figure 4

A and B. Monitoring of NFκB p50 activation in monocytes after 18 h of exposure to different concentrations of native or polymerized AAT by ELISA-based TransAM kitAM. Cell nuclear extracts were prepared from monocytes alone or treated with different concentrations of AAT. NFκB activity assay was performed in 96-well plates with 10 μg of cell extract per well. Absorbance was measured by spectrophotometry at 450 nm. Specificity controls were performed by adding a molar excess (20 pmol/well) of mutant NFκB oligonucleotide (the positive signal remained unaffected) and wild-type NFκB oligonucleotide (a signal was abolished). A and B represent results from two independent experiments. Each point represents the mean of two experiments.

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