Uteroglobin, a possible ligand of the lipoxin receptor inhibits serum amyloid A-driven inflammation

Abstract

Serum amyloid A (SAA) production is increased by inflamed arthritic synovial tissue, where it acts as a cytokine/chemoattractant for inflammatory and immune cells and as an inducer of matrix degrading enzymes. SAA has been shown to bind lipoxin A4 receptor, a member of the formyl-peptide related 2 G-protein coupled receptor family (ALX) and elicit proinflammatory activities in human primary fibroblast-like synoviocytes (FLS). We report on the identification of uteroglobin, a small globular protein with potent anti-inflammatory activities, as a possible ligand of ALX. Uteroglobin-specific association with ALX was demonstrated by an enzyme immunoassay experiment employing a cell line engineered to express the human ALX receptor. Uteroglobin's interaction with ALX resulted in the inhibition of SAA responses, such as attenuation of phospholipase A2 activation and cellular chemotaxis. In FLS, uteroglobin showed an antagonism against SAA-induced interleukin-8 release and decreased cell migration. These novel roles described for uteroglobin via ALX may help elucidate genetic and clinical observations indicating that a polymorphism in the uteroglobin promoter is linked to disease outcome, specifically prediction of bone erosion in patients with rheumatoid arthritis or severity of IgA glomerulonephritis and sarcoidosis.

Figure 1

Exogenous hrUG binding by CHO cells expressing ALX shown by modified enzyme immunoassay. CHO cells expressing either the empty vector pcDNA3 vector (CHO^pINF) or containing the full ALX open reading frame (CHO^ALX) were exposed to increasing hrUG concentrations. The asterix notation indicates data points with a P value of <0.02 when comparing CHO^ALX to CHO^pINF data series. Results are plotted as a mean ± SD of quadruplicate determinations performed in three separate experiments. Reverse transcription-PCR was used to monitor the ALX mRNA expression in CHO cells stably transfected with ALX cDNA (inset). Control represents no template added to reaction. ALX is lipoxin A₄ receptor or FPR2/ALX. The result is from a representative gel of two performed.

Figure 2

SAA-induced phospholipase A₂ activity is attenuated by UG in CHO cells overexpressing ALX. Time response of SAA-induced PLA₂ activity in CHO^ALX cells (a) over a 5-minute monitoring period in the presence and absence of UG, as compared to mock transfected CHO cells (b). Significance is noted by Student's t-test of SAA treatment versus SAA + UG treatment. ∗ for P < 0.005 and ∗∗ for P < 0.001. Results represent the mean ± SD of four separate experiments with triplicate determinations.

Figure 3

SAA-induced IL-8 levels were significantly downregulated in HEC-1A cells overexpressing UG. (a) Reverse transcription-PCR of ALX mRNA in HEC-1A cells and ALX-antisense (AS-ALX) oligo-treated HEC-1A cells. The result is from a representative gel of two performed. (b) SAA-dependent IL-8 release as measured by ELISA, in HEC-1A cells, is inhibited when pretreated with ALX-antisense oligo. In HEC-1A^UG cells SAA-induced IL-8 release is significantly lower in comparison to HEC-1A. Student's t-test for SAA versus AS-ALX treated HEC-1A cells or HEC-1A^UG cells was calculated, P < 0.0001 (∗∗). Data were normalized using number of cells. Results are the mean ± SD of three separate experiments with quadruplicate determinations.

Figure 4

SAA-stimulated migration of HL-60 is mediated by ALX while being inhibited by UG. (a) Migration of HL60^ALX and HL60 into the lower chamber when SAA at two different concentrations (100 and 500 nM) or 5% FBS (positive control) are added. (b) Migration of HL60, HL60^ALX, and UG-antisense treated HL60 cells when SAA (500 nM) or 5% FBS were added. Results are the mean ± SD of four separate experiments with triplicate determinations.

Figure 5

Attenuation of SAA induced cell migration and released IL-8 by hrUG in FLS. (a) Migration of FLS exposed to FBS, SAA (100 nM), or SAA with UG (100 nM), shown as % variation above control (basal growth medium). Results are the mean ± SD of three separate experiments with duplicate determinations. (b) Released IL-8 levels as measured by ELISA in the lower and upper chamber medium following FLS treatment with 5% FBS, SAA (100 nM) alone or in combination with UG or LXA₄ (both at 100 nM). Results are the mean ± SD of duplicate determinations from two experiments. Data are normalized using cell numbers. (c) Migration of FLS following SAA (100 nM) treatment in lower chambers when IL-8 blocking antibody was added to lower, upper, or both compartments of the transwell system. Student's t-test SAA versus control P values of <0.005 (∗∗) and SAA versus SAA + UG treatments P values of <0.01 (¶) (a) and <0.002 (¶¶) (b). Results are the mean ± SD from quadruple determinations of two experiments performed.