Bacterial lipopolysaccharide rapidly inhibits expression of C-C chemokine receptors in human monocytes

Abstract

The present study was designed to investigate the effect of bacterial lipopolysaccharide (LPS) on C-C chemokine receptors (CCR) expressed in human mononuclear phagocytes. LPS caused a rapid and drastic reduction of CCR2 mRNA levels, which binds MCP-1 and -3. CCR1 and CCR5 mRNAs were also reduced, though to a lesser extent, whereas CXCR2 was unaffected. The rate of nuclear transcription of CCR2 was not affected by LPS, whereas the mRNA half life was reduced from 1.5 h to 45 min. As expected, LPS-induced inhibition of CCR2 mRNA expression was associated with a reduction of both MCP-1 binding and chemotactic responsiveness. The capacity to inhibit CCR2 expression in monocytes was shared by other microbial agents and cytokines (inactivated Streptococci, Propionibacterium acnes, and to a lesser extent, IL-1 and TNF-alpha). In contrast, IL-2 augmented CCR2 expression and MCP-1 itself had no effect. These results suggest that, regulation of receptor expression in addition to agonist production is likely a crucial point in the regulation of the chemokine system.

Figure 1

Effect of LPS on the CCR2 (A), CCR1 (B), CCR5 (C) and CXCR2 (D) mRNA expression: dose-response analysis. E shows the ethidium bromide stained ribosomal RNA. A is representative of four different donors. B and C are representative of two different donors. Total RNA was purified from fresh human monocytes incubated for 4 h as indicated.

Figure 2

Destabilization of CCR2 mRNA by LPS. (A) effect of LPS (100 ng/ml) on the CCR2B mRNA transcript stability. Total RNA was purified from fresh human monocytes incubated for 4 h as indicated. B shows the ethidium bromide stained ribosomal RNA. (C) Nuclear runoff analysis of the MCP-1, CCR1, CCR2, and CCR5 genes. Fresh human monocytes were incubated with 100 ng/ml of LPS for different periods as indicated.

Figure 3

Inhibition by LPS of MCP-1 binding and chemotaxis. (A) Binding. fresh human monocytes were incubated for 1–5 h in the presence or absence of 100 ng/ml of LPS before [¹²⁵I]MCP-1 binding assay. Results are expressed as percent of bound on total counts. (B) Chemotaxis. Fresh human monocytes were stimulated with 100 ng/ml of LPS and chemotactic response to MCP-1 (100 ng/ml) was measured after different periods as indicated.

Figure 4

Effects of microbial products (A) and cytokines (C) on CCR2 mRNA expression. B and D show the ethidium bromide stained ribosomal RNA of A and C, respectively. Total RNA was purified from fresh human monocytes incubated for 4 h as indicated. (A) lane 1, untreated; lane 2, LPS 100 ng/ml; lane 3, inactivated Streptococci OK432 (0.015 KE/ml); lane 4, P. acnes (10 μg/ml); lane 5, C. albicans (100 μg/ ml); lane 6, Glucan (100 μg/ml). (C) lane 1, untreated; lane 2, IL-2 (1,000 U/ml); lane 3, LPS (100 ng/ml); lane 4, TNF-α (500 U/ml); lane 5, IL-1-β (20 ng/ml).