Up-regulation of chemokine C-C ligand 2 (CCL2) and C-X-C chemokine 8 (CXCL8) expression by monocytes in chronic idiopathic urticaria

Abstract

The disturbed cytokine-chemokine network could play an important role in the onset of diseases with inflammatory processes such as chronic idiopathic urticaria (CIU). Our main objectives were to evaluate the relation between proinflammatory chemokine serum levels from CIU patients and their response to autologous skin test (ASST) and basophil histamine release (BHR). We also aimed to assess the chemokine secretion by peripheral blood mononuclear cells (PBMC) upon polyclonal stimulus and to evaluate chemokine C-C ligand 2/C-X-C chemokine 8 (CCL2/CXCL8) and Toll-like receptor-4 (TLR-4) expression in monocytes. We observed significantly higher serum levels of the CXCL8, CXCL9, CXCL10 and CCL2 in CIU patients compared to the healthy group, regardless of the BHR or ASST response. The basal secretion of CCL2 by PBMC or induced by Staphylococcus aureus enterotoxin A (SEA) was higher in CIU patients than in the control group, as well as for CXCL8 and CCL5 secretions upon phytohaemagglutinin stimulation. Also, up-regulation of CCL2 and CXCL8 mRNA expression was found in monocytes of patients upon SEA stimulation. The findings showed a high responsiveness of monocytes through CCL2/CXCL8 expression, contributing to the creation of a proinflammatory environment in CIU.

Fig. 1

Circulating chemokine levels in patients with chronic idiopathic urticaria (CIU). Serum samples from healthy controls (HC) and CIU patients were used to determine the presence of CXCL8, CXCL10, CCL2 and CXCL9 by cytometric bead array. Determination of CCL5 levels was performed by enzyme-linked immunosorbent assay (ELISA). Horizontal line represents the median. ***P ≤ 0·001; **P ≤ 0·01 compared with the HC group.

Fig. 2

In vitro chemokine secretion by peripheral blood mononuclear cells (PBMC) from patients with chronic idiopathic urticaria (CIU). PBMC from healthy controls (HC) and CIU patients were incubated with medium (baseline) or with phytohaemagglutinin (PHA) (2·5 µg/ml) or Staphylococcus aureus enterotoxin A (SEA) (40 ng/ml) for 24 h. Supernatants were taken to determine the presence of CCL2, CXCL8 and CCL5 by enzyme-linked immunosorbent assay (ELISA). Horizontal line represents the median. **P ≤ 0·01; *P ≤ 0·05 compared with the HC group.

Fig. 3

Up-regulation of CXCL8 and CCL2 mRNA levels in monocytes upon Staphylococcus aureus enterotoxin A (SEA) stimulation in patients with chronic idiopathic urticaria (CIU). Peripheral blood mononuclear cells (PBMC) from healthy controls (HC) (open symbol) and CIU patients (closed symbol) were incubated with medium (baseline) or with SEA and brefeldin A for 18 h; CD14⁺ cells secreting intracellular CXCL8 and CCL2 were evaluated by flow cytometry. Dot-plot graphs illustrate non-stimulated CD14⁺ cells from HC individuals expressing CXCL8 (a) or CCL2 (b). The mean fluorescence intensity (MFI) for CXCL8 (c) and CCL2 (d) expression on CD14⁺ cells is shown. CD14⁺ cells were purified from HC and CIU patients, stimulated with SEA for 2 h, and CXCL8 (e) and CCL2 (f) mRNA levels were determined by real-time polymerase chain reaction. The horizontal line represents the median. **P ≤ 0·01; *P ≤ 0·05 compared with the HC group.

Fig. 4

Toll-like receptor-4 (TLR-4) mRNA expression on monocytes and CCL5 mRNA expression on CD4⁺ T cells of patients with chronic idiopathic urticaria (CIU). Purified non-stimulated CD14⁺ cells from healthy controls (HC) (open symbol) and CIU patients (closed symbol) were evaluated for TLR-4 mRNA levels (a) or for CCL5 mRNA expression on purified CD4⁺ T cells stimulated with phytohaemagglutinin (PHA) for 2 h (b) by real-time polymerase chain reaction. The horizontal line represents the median. *P ≤ 0·05 compared with the HC group.