Endothelin-1 production by the canine macrophage cell line DH82: enhanced production in response to microbial challenge

Abstract

Endothelin-1 (ET-1) is a potent vasoconstrictive peptide which plays an important role in regulating mammalian cardiovascular development and homeostasis. Originally identified as a factor released by vascular endothelial cells, ET-1 is now recognized as a product of numerous cells and tissues with demonstrated involvement in an array of physiological and pathological processes. An area of great interest is the production of ET-1 by mononuclear cells (monocytes and macrophages) and its role in inflammation. We report that the canine macrophage cell line, DH82, constitutively secretes both ET-1 and its biologically inactive precursor big ET-1. The production of both peptides was increased following stimulation with lipopolysaccharide (endotoxin) from gram-negative bacteria. ET-1 production was also increased in response to stimulation with intact and viable gram-positive and gram-negative bacteria. In addition to producing ET-1, DH82 cells express transcripts encoding two receptors for the ET-1 peptide (ET(A) and ET(B) receptors) and an enzyme involved in the conversion of big ET-1 to ET-1. The constitutive secretion of ET-1 and the expression of ET(A) and ET(B) receptors may be related to the malignant origin of this cell line. Our results are the first report of ET-1 production by a canine cell line and provide the basis for further investigation into the role of ET-1 during infection and inflammation.

Fig.1

The constitutive and LPS-induced production of ET-1 by DH82 cells. (A) DH82 cells were incubated in medium alone or in the presence of 100 ng / ml LPS for increasing periods of time. (B) DH82 cells were incubated for 6 hrs in medium alone, or increasing concentrations of LPS. At the indicated times, culture supernatants were collected and assayed for ET-1 peptide concentration using a commercially available EIA kit. Peptide values represent the mean ( ± SD ) of triplicate determinations. Each figure is representative of three individual experiments, each performed in triplicate.

Fig. 2

The effect of microbial challenge on DH82 ET-1 production. DH82 cells were incubated for 6 hours in either medium alone (“Control”), 100 ng / ml of LPS, or the indicated bacteria. Culture supernatants were collected and assayed for ET-1 peptide concentration using a commercially available EIA kit. Peptide values represent the mean ( ± SD ) of triplicate determinations. This figure is representative of three individual experiments, each performed in triplicate. The symbol “ * “ indicates significant changes (P < 0.004) in ET-1 production relative to the control.

Fig. 3

The secretion of big ET-1 by DH82 cells. DH82 cells were cultured in medium alone or 1000 ng / ml of LPS, in the presence or absence of the ECE-1 inhibitor SM-19712. The concentrations of ET-1 (A) and big ET-1 (B) were determined using commercially available EIA kits. Peptide values represent the mean (± SD ) of a single experiment performed in triplicate. The symbols “ * “ and “**” indicate significant changes (P < 0.001) in ET-1 or big ET-1 production relative to cells receiving no LPS or SM-19712 or cells receiving LPS, but no SM-19712, respectively.

Fig. 4

Expression of mRNAs encoding the endothelin axis by DH82 cells. DH82 cells were cultured for 6 hours in medium alone or 1000 ng / ml of LPS. Monolayers were washed and total cellular RNA harvested. Reverse transcriptase PCR analysis was performed to amplify transcripts encoding preproendothelin-1 (ET-1), endothelin-converting enzyme-1 (ECE-1), endothelin receptor A (ETR-A), endothelin receptor B (ETR-B), and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The PCR primers used are described in Table I. Identical expression patterns were observed following RNA extraction from LPS treated and untreated cultures on three separate occasions.