Helicobacter pylori lipopolysaccharide binds to CD14 and stimulates release of interleukin-8, epithelial neutrophil-activating peptide 78, and monocyte chemotactic protein 1 by human monocytes

Abstract

Helicobacter pylori gastritis is characterized by leukocyte infiltration of the gastric mucosa. The aims of this study were to determine whether H. pylori-derived factors stimulate chemokine release from human monocytes and to ascertain whether H. pylori lipopolysaccharide (LPS) may be responsible for this effect. Human peripheral blood monocytes were exposed to an H. pylori water extract (HPE) or to purified H. pylori LPS. Levels of the chemokines interleukin-8 (IL-8), epithelial neutrophil-activating peptide 78 (ENA-78), and monocyte chemotactic protein 1 (MCP-1) were measured by enzyme-linked immunosorbent assay. The contribution of H. pylori LPS to monocyte activation was determined by using the LPS antagonist Rhodobacter sphaeroides lipid A (RSLA) and a blocking monoclonal antibody to CD14 (60bca). HPE increased monocyte secretion of IL-8, ENA-78, and MCP-1. Heat treatment of HPE did not reduce its ability to activate monocytes. Purified H. pylori LPS also stimulated monocyte chemokine production but was 1,000-fold less potent than Salmonella minnesota lipid A. RSLA blocked H. pylori LPS-induced monocyte IL-8 release in a dose-dependent fashion (maximal inhibition 82%, P < 0.001). RSLA also inhibited HPE-induced IL-8 release (by 93%, P < 0.001). The anti-CD14 monoclonal antibody 60bca substantially inhibited IL-8 release from HPE-stimulated monocytes (by 88%, P < 0.01), whereas the nonblocking anti-CD14 monoclonal antibody did not. These experiments with potent and specific LPS inhibitors indicate that the main monocyte-stimulating factor in HPE is LPS. H. pylori LPS, acting through CD14, stimulates human monocytes to release the neutrophil-activating chemokines IL-8 and ENA-78 and the monocyte-activating chemokine MCP-1. Despite its low relative potency, H. pylori LPS may play an important role in the pathogenesis of H. pylori gastritis.

FIG. 1

HPE stimulates IL-8 production by human monocytes. Human monocytes were exposed to various concentrations of HPE (see Materials and Methods), and IL-8 levels were measured by ELISA in conditioned media harvested after 18 h. In this representative experiment, n = 4 for each dose. Results are expressed as means ± the standard errors (SE). The IL-8 levels were 0.07 ± 0.01 ng/ml in control, unstimulated monocytes and 2.70 ± 0.35 ng/ml in positive control monocytes exposed to 0.1 ng of S. minnesota lipid A per ml. ∗, P < 0.01 versus control, unstimulated monocytes.

FIG. 2

HPE stimulates MCP-1 production by human monocytes. Human monocytes were exposed to various concentrations of HPE, and MCP-1 levels were measured by ELISA in conditioned media harvested after 18 h. In this representative experiment, n ≥ 3 for each dose. Results are expressed as means ± SE. The MCP-1 levels were 8 ± 1 pg/ml in control monocytes and 75 ± 13 pg/ml in monocytes exposed to 0.1 ng of S. minnesota lipid A per ml. ∗, P < 0.01 versus control monocytes.

FIG. 3

HPE and H. pylori LPS stimulate ENA-78 production by human monocytes. Human monocytes were exposed to HPE (Hp extract; 10% concentration) or to H. pylori LPS (Hp LPS; 100 ng/ml), and the ENA-78 levels were measured by ELISA in conditioned media harvested after 18 h. In this representative experiment, n = 8 for each condition. Results are expressed as the means ± SE. ∗, P < 0.01 versus control unstimulated monocytes.

FIG. 4

H. pylori LPS has low potency in stimulating monocyte IL-8 release. Human monocytes were exposed to various concentrations of H. pylori LPS or to S. minnesota lipid A, and the IL-8 levels were measured by ELISA in conditioned media harvested after 18 h. In this representative experiment, n = 3 for each condition. Results are expressed as means ± SE. The IL-8 levels were 0.15 ± 0.02 ng/ml in control monocytes.

FIG. 5

H. pylori LPS and HPE do not directly upregulate endothelial-cell ICAM-1 expression. Human endothelial cells were exposed to IL-1β (25 ng/ml), H. pylori LPS (100 ng/ml), HPE (30% concentration), and to conditioned medium from either control or HPE-stimulated monocytes. Endothelial-cell ICAM-1 surface expression was evaluated after 18 h by cell-ELISA. In this representative experiment, n was ≥3 for each condition. The results are expressed as means ± SE. ∗, P < 0.001 versus control.

FIG. 6

RSLA blocks the ability of H. pylori LPS to stimulate monocyte IL-8 production. Human monocytes were exposed to various concentrations of RSLA in the presence or absence of H. pylori LPS (100 ng/ml) or S. minnesota lipid A (1 ng/ml). The IL-8 levels were measured by ELISA in the conditioned media harvested after 18 h. In this representative experiment, n = 3 for each condition. The results are expressed as means ± SE. For the H. pylori LPS series, ∗ denotes P < 0.01 compared to H. pylori LPS alone (i.e., 0 μg of RSLA per ml).

FIG. 7

The LPS inhibitor RSLA blocks the ability of HPE to stimulate monocyte IL-8 production. Human monocytes were exposed to various concentrations of HPE alone or with RSLA (1 μg/ml). The IL-8 levels were measured by ELISA in conditioned media harvested after 18 h. In this representative experiment, n = 4 for each condition. The results are expressed as means ± SE. ∗, P < 0.01 compared to the same concentration of HPE alone (Student’s t test).

FIG. 8

The monocyte-activating factor in HPE is heat stable. Human monocytes were exposed to HPE (10% concentration) or to the same concentration of heat-treated HPE (100°C for 15 min) in the presence or absence of RSLA (1 μg/ml). The IL-8 levels were measured by ELISA in conditioned media harvested after 18 h. In this representative experiment, n = 4 for each condition. The results are expressed as means ± SE. ∗, P < 0.05 compared to monocyte IL-8 release in the absence of RSLA (Student’s t test).

FIG. 9

The monocyte-activating factor in HPE uses the CD14 receptor. Human monocytes were exposed to S. minnesota lipid A (Sal LA), H. pylori LPS (Hp LPS; 10 μg/ml), or HPE (Hp extract; 10% concentration) alone or in the presence of either 26iC (a nonblocking anti-CD14 monoclonal antibody, 10 μg/ml) or 60bca (a blocking anti-CD14 monoclonal antibody, 10 μg/ml). The IL-8 levels were measured by ELISA in conditioned media harvested after 18 h. In this representative experiment, n = 4 for each condition. The results are expressed as means ± SE. ∗, P < 0.05; ∗∗, P < 0.01 (versus the corresponding no antibody group).