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. 2011 Jul;79(7):2567-77.
doi: 10.1128/IAI.00067-11. Epub 2011 Apr 25.

Monocyte chemoattractant protein 1 regulates pulmonary host defense via neutrophil recruitment during Escherichia coli infection

Gayathriy Balamayooran  1 , Sanjay Batra, Theivanthiran Balamayooran, Shanshan Cai, Samithamby Jeyaseelan
Affiliations

Monocyte chemoattractant protein 1 regulates pulmonary host defense via neutrophil recruitment during Escherichia coli infection

Gayathriy Balamayooran et al. Infect Immun. 2011 Jul.

Erratum in

  • Infect Immun. 2013 Nov;81(11):4323

Abstract

Neutrophil accumulation is a critical event to clear bacteria. Since uncontrolled neutrophil recruitment can cause severe lung damage, understanding neutrophil trafficking mechanisms is important to attenuate neutrophil-mediated damage. While monocyte chemoattractant protein 1 (MCP-1) is known to be a monocyte chemoattractant, its role in pulmonary neutrophil-mediated host defense against Gram-negative bacterial infection is not understood. We hypothesized that MCP-1/chemokine (C-C motif) ligand 2 is important for neutrophil-mediated host defense. Reduced bacterial clearance in the lungs was observed in MCP-1(-/-) mice following Escherichia coli infection. Neutrophil influx, along with cytokines/chemokines, leukotriene B(4) (LTB(4)), and vascular cell adhesion molecule 1 levels in the lungs, was reduced in MCP-1(-/-) mice after infection. E. coli-induced activation of NF-κB and mitogen-activated protein kinases in the lung was also reduced in MCP-1(-/-) mice. Administration of intratracheal recombinant MCP-1 (rMCP-1) to MCP-1(-/-) mice induced pulmonary neutrophil influx and cytokine/chemokine responses in the presence or absence of E. coli infection. Our in vitro migration experiment demonstrates MCP-1-mediated neutrophil chemotaxis. Notably, chemokine receptor 2 is expressed on lung and blood neutrophils, which are increased upon E. coli infection. Furthermore, our findings show that neutrophil depletion impairs E. coli clearance and that exogenous rMCP-1 after infection improves bacterial clearance in the lungs. Overall, these new findings demonstrate that E. coli-induced MCP-1 causes neutrophil recruitment directly via chemotaxis as well as indirectly via modulation of keratinocyte cell-derived chemokine, macrophage inflammatory protein 2, and LTB(4).

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Figures

Fig. 1.
Fig. 1.
(A and B) Kinetics of MCP-1 production. The MCP-1 concentration was measured in BALF (A) and lung homogenate (B) from WT mice by ELISA after i.t. infection with E. coli (106/mouse). *, significant difference between MCP-1−/− and WT mice (P < 0.005; n = 4 to 5 mice/group/time point; the figure is a representation of 3 individual experiments). (C) MCP-1 expression in mice transplanted with bone marrow following E. coli infection. *, significant difference between knockout and WT mice (P < 0.005). Data shown are a representation of 3 individual experiments.
Fig. 2.
Fig. 2.
(A) Bacterial burden in the lungs of MCP-1−/− mice following E. coli infection (106/mouse). Lungs were collected from control and infected groups of mice at the designated times and homogenized, and the bacteria were enumerated (n = 5 to 6 mice/group). *, significant difference between knockout and WT mice (P < 0.005). Data shown are a representation of 3 individual experiments. (B and C) Cellular infiltration in the lung in MCP-1−/− mice against E. coli. Mice were inoculated with E. coli (106 CFU/mouse), BALF was obtained at 6 and 24 h postinfection, and cell enumeration was performed to determine neutrophil and macrophage infiltration to the lung (n = 4 to 5 mice/group; P < 0.005; data are a representation of 3 individual experiments). (D) Lung histology in MCP-1−/− mice following E. coli infection. Mice were inoculated with E. coli (106 CFU/mouse), and lungs were obtained at 24 h postinfection. This picture is representative of 3 separate mice with identical results.
Fig. 3.
Fig. 3.
(A to E) Cytokine, chemokine, and chemotactic lipid levels in the lung following E. coli infection. Mice were infected by intratracheal instillation of E. coli (106 CFU/mouse), and BALF was collected from the lungs at designated time points. Concentrations (pg/ml) of TNF-α (A), IL-6 (B), KC (C), and MIP-2 (D) in BALF were quantified by sandwich ELISA. *, significant difference between MCP-1−/− and WT mice (P < 0.005; n = 4 to 6 mice in each group at each time point; data are a representation of 3 separate experiments). (E) Expression of LTB4 in the lung following E. coli infection. Lung homogenates were prepared after E. coli infection, and the levels of LTB4 were measured in homogenates and were normalized against total protein concentration. (F) Expression of ICAM-1 and VCAM-1 in the lung in response to E. coli challenge. Infected lungs were homogenized, and total proteins were isolated, resolved on an SDS-polyacrylamide gel, and transferred onto a nitrocellulose membrane. The membrane was blotted with Abs for ICAM-1, VCAM-1, and GAPDH. This is a representative blot of 3 independent experiments with identical results. (G and H) Densitometric analysis was performed in 3 blots to demonstrate the expression of ICAM-1 and VCAM-1 in the lung following E. coli infection.
Fig. 4.
Fig. 4.
(A and B) Activation of NF-κB in the lung following infection with E. coli. Lung homogenates and nuclear lysates from MCP-1−/− mice and their controls were prepared at 6 and 24 h after infection with E. coli. NF-κB binding assay was performed in nuclear lysates from lung (A), and expression and phosphorylation of NF-κB pathway members were determined using Western blots of lung homogenates (B). The blots are representative of 3 independent experiments with identical results. (C to G) Relative densities normalized against GAPDH are representatives of 3 independent experiments. OD, optical density; encircled P, phosphorylated form; *, P < 0.005.
Fig. 5.
Fig. 5.
(A) Activation of MAPKs in the lung following E. coli infection. Total proteins in the lung were isolated from MCP-1−/− and control mice at 6 and 24 h after infection with E. coli and resolved on an SDS-polyacrylamide gel, and the membrane was blotted with the Abs against the activated/phosphorylated (encircled P) form of MAPKs as described in Materials and Methods. This is a representative of 3 separate experiments with identical results. (B) Densitometric analysis of MAPK activation was performed from 3 separate blots. *, P < 0.05 for difference between MCP-1−/− mice and their WT controls.
Fig. 6.
Fig. 6.
(A and B) Cellular infiltration in BALF at 24 h after i.t. treatment with rMCP-1 (50 μg) and E. coli infection. Fifty micrograms of rMCP-1 was i.t. administered at 1 h after E. coli infection, and BALF was collected 24 h after infection. (C to F) Cytokine (C and D) and chemokine (E and F) production in the lungs of infected animals after rMCP-1 treatment. For the experiments indicated in panels A to F, n = 4 to 6 mice/group and data are representative of 3 experimental repeats. WBC, white blood cells; SAL, saline. (G) Numbers of CFU in lungs of WT and MCP-1−/− mice administered rMCP-1 following i.t. E. coli infection. The controls were treated with PBS. Each group had 5 to 6 mice, and the result shown is representative of 3 independent experiments. (H) Numbers of CFU in lungs of neutrophil-depleted mice at 24 h after E. coli infection. Neutrophils were depleted by using anti-Ly6G Ab intraperitoneally at 12 and 2 h prior to infection, and control mice were treated with isotype Ab prior to infection. Lung samples were homogenized, diluted, and plated to enumerate bacterial CFU (n = 5 to 6 mice/group/time point, and data are from 3 separate experiments). *, P < 0.05.
Fig. 7.
Fig. 7.
(A) Activation of NF-κB and MAPKs, and expression of cellular adhesion molecules in lungs of mice administered rMCP-1 (10 μg) at 24 h after E. coli infection. (B and C) Densitometric analysis of expression/phosphorylation levels of identified proteins normalized with GAPDH. The results are representative of 3 independent experiments with identical results. *, P < 0.05; encircled P, phosphorylated form.
Fig. 8.
Fig. 8.
(A) Cellular recruitment into the alveoli after i.t. treatment with rMCP-1 (10 μg and 50 μg) alone. After 12 h, BALF was processed for cellular count (n = 5 to 6 mice/group; *, P < 0.005; data are a representation of 3 individual experiments). MPO, myeloperoxidase. (B) CCR2 expression in neutrophils. Flow cytometric analysis of BALF and blood from WT mice at 24 h after i.t. E. coli (1 × 106 CFU/mouse) infection. Data are representative of 3 independent experiments with identical results. (D) Picture demonstrating the number of PMNs in the lower chamber of a transwell plate after incubation with chemoattractants rMCP-1 and KC. The figure is representative of 20 random fields from 3 separate experiments. (E) Chemotaxis of neutrophils toward MCP-1. PMN numbers in the lower chamber of a transwell after 3 h of incubation with rMCP-1 and KC. Data shown here are a representation of 3 individual experiments (n = 3 to 5; *, P < 0.05). (F) Bone marrow neutrophils produce MCP-1 at 2 h after E. coli infection (n = 4 to 6 mice/group from 3 separate experiments).
Fig. 9.
Fig. 9.
(A and B) Activation of NF-κB and MAPKs in alveolar macrophages obtained from WT and MCP-1−/− mice following infection with E. coli. Representative Western blots from 3 separate experiments are shown. (B) Relative densities normalized against GAPDH are representatives of 3 independent experiments (n = 4 to 5 mice/group; *, P < 0.05). Encircled P, phosphorylated form.
Fig. 10.
Fig. 10.
Proposed model of MCP-1-mediated neutrophil recruitment to the lung during E. coli infection. E. coli induces MCP-1 through the MD-2 signaling pathway, which involves Toll-like receptors. MCP-1 in turn regulates neutrophil recruitment directly via chemotaxis and indirectly through neutrophil chemokines, such as KC, MIP-2, as well as the cytokine TNF-α.
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