Abstract
The CLCA gene family produces both secreted and membrane-associated proteins that modulate ion-channel function, drive mucus production and have a poorly understood pleiotropic effect on airway inflammation. The primary up-regulated human CLCA ortholog in airway inflammation is hCLCA1. Here we show that this protein can activate airway macrophages, inducing them to express cytokines and to undertake a pivotal role in airway inflammation. In a U-937 airway macrophage–monocyte cell line, conditioned media from HEK 293 cells heterologously expressing hCLCA1 (with or without fetal bovine serum) increased the levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α and IL-8). This effect was independent of the metalloprotease domain of hCLCA1. Primary porcine alveolar macrophages were similarly activated, demonstrating the effect was not cell line dependent. Similarly, immuno-purified hCLCA1 at physiologically relevant concentration of ~100 pg/mL was able to activate macrophages and induce pro-inflammatory response. This cytokine response increased with higher concentration of immuno-purified hCLCA1. These findings demonstrate the ability of hCLCA1 to function as a signaling molecule and activate macrophages, central regulators of airway inflammation.
Highlights
CLCA genes (CL stands for chloride-channel modulating and CA for calcium-activated) are induced in airway epithelial cells by inflammation [1]
The activation of macrophages by secreted hCLCA1 was investigated using the U-937 macrophage cell line treated with conditioned fetal bovine serum (FBS)-containing medium from HEK293 cells heterologously expressing hCLCA1 or eGFP
At the optimized dose of 1 mg/mL, the expression of IL-8, IL-6, IL-1β and TNF-α in response to wildtype hCLCA1 and its hydrolase-inactive E157Q mutant was followed over 24 h (Figure 2); this test revealed no difference in the mRNA expression between E157Q and wild-type hCLCA1activated macrophages
Summary
CLCA genes (CL stands for chloride-channel modulating and CA for calcium-activated) are induced in airway epithelial cells by inflammation [1]. This induced expression often exceeds that of most other inflammatory mediators [2]. CLCA proteins were originally identified as calcium activated chloride channels, we and others concluded that they only modulated channel pores (suggesting a signaling ability) [8,9,10,11,12]. How putative signaling ligands could cause a seemingly pleotropic effect on airway inflammation was unclear. One possibility was that CLCAs modified a central mediator of airway inflammation, such as the airway macrophage
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