Intracellular Interleukin-1 Beta Research Articles

Characterizing the NLRP3 Inflammasome in Mood Disorders: Overview, Technical Development, and Measures of Peripheral Activation in Adolescent Patients.

The NOD-, LRR-, and pyrin-domain-containing protein 3 (NLRP3) inflammasome is a node of intracellular stress pathways and a druggable target which integrates mitochondrial stress and inflammatory cascades. While a body of evidence suggests the involvement of the NLRP3 inflammasome in numerous diseases, a lack of reliable measurement techniques highlights the need for a robust assay using small quantities of biological samples. We present a literature overview on peripheral activation of the NLRP3 inflammasome in mood disorders, then outline a process to develop and validate a robust assay to measure baseline and activated intracellular levels of “apoptosis-associated speck-like protein containing a CARD” (ASC) as a key component of an inflammatory profile in peripheral blood mononuclear cells (PBMC). A consistent association between high NLRP3 mRNA levels and relevant cytokines was seen in the literature. Using our method to measure ASC, stimulation of PBMC with lipopolysaccharide and nigericin or adenosine triphosphate resulted in microscopic identification of intracellular ASC specks, as well as interleukin 1 (IL-1) beta and caspase-1 p10 in the periphery. This was abolished by dose-dependent pre-treatment with 100 nM MCC950. We also report the use of this technique in a small pilot sample from patients with bipolar disorder and depressive disorders. The results show that levels of intracellular ASC and IL-1 beta are sensitive to change upon activation and maintained over time, which may be used to improve the detection of NLRP3 activation and guide personalized therapeutic strategy in the treatment of patients.

In vitro activation of human macrophages by alginate-polylysine microcapsules.

Microencapsulated islets of Langerhans have been proposed as a bioartificial pancreas. However, foreign body reaction with fibrosis has been observed around implanted microcapsules. Since macrophages are present in this reaction and interleukin-1 (IL-1), a cytokine released by activated macrophages, may induce fibrosis, we tested the capacity of alginate-polylysine microcapsules to activate macrophages. Human monocytes were isolated from whole blood of healthy donors by a Ficoll density gradient and adherence to a plastic support. Monocytes were cultured for 24 h with: (1) alginate-polylysine microcapsules; (2) lipopolysaccharide (LPS) (positive control group); and (3) alone (negative control group). Monocyte activation was evaluated by measuring the secretion of IL-1 beta and the production of intracellular IL-1 alpha and IL-1 beta. Macrophages characterization was performed by immunocytological subtyping. IL-1 beta release and intracellular IL-1 beta and IL-1 alpha production were significantly higher when macrophages were cultured with alginate-polylysine microcapsules than when macrophages were cultured alone. In conclusion, macrophages are activated in vitro by alginate-polylysine microcapsules. This effect may be involved in the fibrosis observed in vivo around implanted microcapsules. In addition, interleukin-1, released during macrophage activation, may cross the microcapsule membrane and impair islet function.

Anti-(tumor necrosis factor) alters the response of human monocytes to liposomal muramyl tripeptide.

The purpose of this study was to examine the mechanisms by which liposome-encapsulated muramyl tripeptide phosphatidylethanolamine (L-MTP-PE) stimulates monocytes to produce tumor necrosis factor (TNF) and interleukin-1 (IL-1). We have previously shown that secretion of TNF protein occurred 2-4 h following incubation of monocytes with L-MTP-PE and that this stimulation of TNF production was associated with an increase in TNF mRNA. Increased intracellular interleukin-1 alpha (IL-1 alpha) and IL-1 beta were not detected until 8 h after exposure to L-MTP-PE. To determine whether TNF played a role in the stimulation of IL-1 production by L-MTP-PE, normal human monocytes were incubated with L-MTP-PE or medium in the presence or absence of anti-TNF or anti IL-1 alpha plus anti IL-1 beta. Enhanced expression of IL-1 alpha and IL-1 beta mRNA was inhibited at 4 h but not 24 h when monocytes were incubated with L-MTP-PE plus anti-TNF compared with L-MTP-PE alone. By contrast, enhanced expression of TNF mRNA was not inhibited at any time when monocytes were incubated with L-MTP-PE and anti-IL-1 alpha plus anti-IL-1 beta. These data indicate that the up-regulation of IL-1 seen in monocytes following L-MTP-PE exposure may be due in part to the production of TNF. The up-regulation of TNF, however, appears to be independent of IL-1 production.

Effects of retinoic acid on interleukin-1 alpha and -1 beta expression by normal human keratinocytes cultured in defined medium.

In human epidermis, basal keratinocytes are the main source of interleukin-1 (IL-1), and IL-1 alpha is the predominant form, whereas IL-1 beta appears to exist predominantly in cultured keratinocytes. We investigated the effects of retinoic acid (RA) treatments on IL-1 alpha and -beta protein and mRNA expression of normal human keratinocytes cultured in low-calcium defined medium with or without hydrocortisone. Radioimmunoassay showed that after stimulation by RA, the IL-1 beta intracellular level is predominantly increased, with no significant modification of IL-1 alpha expression. The addition of hydrocortisone in the culture medium resulted in a decrease in RA-induced IL-1 beta overexpression, without notable modifications in untreated cultures. Release of both IL-1 alpha and -beta in culture supernatants was detectable only after RA treatment and in the absence of hydrocortisone. The overexpression of IL-1 beta in control and RA-treated cultures mainly concerned the 52- and 31- to 36-kD biologically inactive precursor forms. Northern blot using specific IL-1 alpha and -beta oligonucleotide probes showed that IL-1 beta mRNA predominate over IL-1 alpha mRNA and reach a maximal level 6 h before the IL-1 beta protein peak. These findings indicate that in cultured keratinocytes intracellular IL-1 beta is preferentially increased by RA but in its immature forms. The significance of this overexpression remains to be elucidated.

Modulation of lipopolysaccharide-induced production of tumor necrosis factor, interleukin 1, and interleukin 6 by synthetic precursor Ia of lipid A.

Endotoxin (lipopolysaccharide, LPS) induces the production of mediators of inflammation, which exerts pathophysiological effects such as fever or shock in mammals. In the present study we have investigated the modulation of LPS by the synthetic non-active tetraacylated precursor Ia of lipid A (compound 406) in the induction of tumor necrosis factor (TNF), interleukin 1 (IL-1) and interleukin 6 (IL-6) in human peripheral blood mononuclear cells (PBMC) and in human peripheral blood monocytes (PBMo). PBMC stimulated with LPS released TNF in a concentration dependent manner. Release of biologically active TNF, IL-1 and IL-6 was first detectable 4 h after LPS stimulation. Compound 406 alone in all concentrations tested did not induce TNF, IL-1 or IL-6 release, intracellular TNF or IL-1 beta, or mRNA for TNF or IL-1. Added to PBMC 1 h before LPS compound 406 enhanced or suppressed TNF release and suppressed IL-1 and IL-6 release depending on the ratio of concentrations between stimulator (LPS) and modulator (compound 406). In contrast to LPS stimulation alone TNF, IL-1 and IL-6 release in presence of compound 406 was delayed and first detectable after 6 to 8 h. Compound 406 was able to suppress LPS-induced intracellular TNF and IL-1 beta in PBMC. Added to PBMo 1 h before LPS it totally inhibited the production of mRNA for TNF and IL-1. When added to PBMC 1 h after LPS, TNF release was suppressed in a concentration-dependent way and release of biologically active TNF, IL-1 and IL-6 could again be detected for the first time after 4 h. Compound 406 was not able to inhibit phorbol 12-myristate 13-acetate (PMA)-induced TNF and IL-1 release in PBMo which suggests that its modulating effect is LPS-specific. This study provides evidence that the modulating effect of compound 406 on the LPS induction of TNF, IL-, 1 and IL-6 could be due to competitive binding.

Production of intracellular and extracellular interleukin-1 alpha and interleukin-1 beta by peripheral blood monocytes from patients with connective tissue diseases.

An enzyme linked immunosorbent assay (ELISA) was used to estimate the production of intracellular and extracellular interleukin-1 (IL-1) alpha and beta by peripheral blood monocytes from 26 patients with various connective tissue diseases (CTD), including 19 with systemic lupus erythematosus, four with progressive systemic sclerosis, two with mixed connective tissue disease, and one with Sjögren's syndrome. Monocytes obtained from patients with CTD with serum antibodies to nuclear ribonucleoprotein (nRNP) released significantly higher concentrations of extracellular IL-1 alpha and IL-1 beta, whereas intracellular IL-1 alpha and IL-1 beta production was similar to that by monocytes from patients with CTD without antibodies to nRNP. Furthermore, the concentrations of extracellular IL-1 alpha correlated significantly with those of extracellular IL-1 beta. There was no significant correlation between the concentrations of extracellular and intracellular IL-1 alpha, and those of extracellular and intracellular IL-1 beta, indicating that synthesis and secretion of IL-1 by human monocytes may be two distinct biological events. It seems that enhanced extracellular release of both IL-1 alpha and IL-1 beta contributes to the excessive anti-nRNP production in CTD.