Inhibition of ATP-Binding Cassette Transporter Downregulates Interleukin-1β-Mediated Autocrine Activation of Human Dermal Fibroblasts

Abstract

Fibroblasts constitute an important source of cytokines during inflammatory processes in the skin. Interleukin-1 is a potent, pleiotropic cytokine that is induced in activated human dermal fibroblasts. Interleukin-1 further induces many inflammatory mediators, including the chemokine interleukin-8. As fibroblasts express both interleukin-1 and the interleukin-1 receptor complex, the cellular response may be enhanced by autocrine activation. Interleukin-1alpha and interleukin-1beta lack a signal peptide and are translocated at the plasma membrane using an alternative secretory pathway, which may involve ATP-binding cassette transporter proteins. We hypothesize that inhibition of this pathway prevents secretion of interleukin-1, thereby downregulating interleukin-1-dependent autocrine induction of interleukin-8. We used the ATP-binding cassette 1 transporter inhibitor glybenclamide, which has been previously shown to block interleukin-1beta secretion in human monocytes. Using enzyme-linked immunosorbent assay, we assessed the effect of glybenclamide on interleukin-8 production in human dermal fibroblasts. In interleukin-1beta-transfected human dermal fibroblasts, interleukin-8 was induced through an autocrine activity of interleukin-1beta. Glybenclamide disabled this activation loop and significantly reduced interleukin-8. In human dermal fibroblasts that were stimulated with tumor necrosis factor alpha to reach high interleukin-1 expression levels, glybenclamide similarly suppressed interleukin-8. In contrast, glybenclamide did not affect interleukin-8 production in cells stimulated with interleukin-1 only. Glybenclamide did not affect caspase-1 in fibroblasts, which was expressed as an inactive precursor form, irrespective of treatments with tumor necrosis factor alpha and/or glybenclamide. Using overexpressing, interleukin-1-transfected COS-1 cells, inhibition of interleukin-1alpha and interleukin-1beta secretion was directly demonstrated on Western blots. These results are consistent with glybenclamide preventing externalization of interleukin-1 and subsequent autocrine induction of interleukin-8 in human dermal fibroblasts. Acting through such a mechanism, ATP-binding cassette transporter inhibitors may downregulate inflammation locally.

Immunologic properties of human dermal fibroblasts

“…The absence of keratinocytes in the passage 4 fibroblasts was confirmed by absence of interleukin-1␣ expression (Fig. 2B), as keratinocytes constitutively express this gene [14] whereas dermal fibroblasts do not [15]. The absence of Langerhans cells, which are HLA-DR-positive cells from the epidermis, was confirmed by the absence of expression of Langerin, which is a specific marker for these cells [16] (Fig.…”

Interleukin‐1β and interleukin‐18: regulation and activity in local inflammation

“…Drugs blocking the secretory pathway through the endoplasmic reticulum and Golgi apparatus do not affect IL‐1β secretion, suggesting a rather unusual, non‐classical secretion pathway (3, 112). A recently suggested transport mechanism to release IL‐1β into the extracellular compartment may be the adenosine‐5′‐trisphosphate‐binding cassette transporter system (47, 69). In contrast to IL‐1β, IL‐1Ra is synthesized and secreted via the endoplasmic reticulum and the Golgi apparatus.…”

Inflammasome Activation in Chronic Glomerular Diseases

“…In addition, glyburide, commonly used to treat type II diabetes, was found to block chloride and potassium channels in pancreatic β cells to regulate insulin release [9] . This compound is able to inhibit IL-1β release upon stimulation with LPS in human monocytes [75] , but it does not block capsase-1 activation unless Nlrp3, NF-κB or P2X7 genes are knocked out in mice [50, 54, 57, 63] . It is expected that more therapeutic strategies will be forthcoming, which include the use of NLRP3 inhibitors, the P2X7 receptor blockers, IL-1 and IL-18 receptor blockers, caspase-1 inhibitors, alpha-1 antitrypsin, adenosine A2B receptor blockers, miRNAs, H 2 S donor Na2S, lysosome stabilizer, cathepsin-B inhibitors, and milk fat globule EGF-8 as an endogenous inhibitor of inflammasome-induced IL-1β production.…”