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. 1998 Dec;153(6):1885-93.
doi: 10.1016/S0002-9440(10)65702-1.

Bradykinin stimulates type II alveolar cells to release neutrophil and monocyte chemotactic activity and inflammatory cytokines

S Koyama  1 , E Sato, H Nomura, K Kubo, M Miura, T Yamashita, S Nagai, T Izumi
Affiliations

Bradykinin stimulates type II alveolar cells to release neutrophil and monocyte chemotactic activity and inflammatory cytokines

S Koyama et al. Am J Pathol. 1998 Dec.

Abstract

In the present study, we evaluated the potential of bradykinin (BK) to induce the release of neutrophil and monocyte chemotactic activity (NCA and MCA) and cytokines from an alveolar type II epithelial cell line, A549 cells. BK stimulated A549 cells to release NCA and MCA in a dose- and time-dependent manner (P < 0.001). Checkerboard analysis revealed that both NCA and MCA involved chemotactic and chemokinetic activity. Molecular sieve column chromatography showed three molecular weight masses (near 19 kd, 8 kd, and 400 d) for NCA and several molecular weight peaks (near 66 kd, 25 kd, 19 kd, 16 kd, and 400 d) for MCA. The release of NCA and MCA was inhibited by cycloheximide and lipoxygenase inhibitors (P < 0.01). The NCA and MCA were inhibited by leukotriene B4 (LTB4) receptor antagonist (P < 0.01), and the concentration of LTB4 was high enough for NCA and MCA. Antibodies to interleukin (IL)-8 and granulocyte colony-stimulating factor (G-CSF) attenuated NCA (P < 0.01), and antibodies to monocyte chemotactic protein-1 (MCP-1), G-CSF, and transforming growth factor (TGF)-beta attenuated MCA (P < 0.01). The levels of IL-8, G-CSF, MCP-1, and TGF-beta increased time dependently (P < 0.01). BK also stimulated the release of ILeukin-6 from A549 cells (P < 0.001). The receptors responsible for the release of NCA, MCA, and individual chemokines involved both BKB1 and BKB2 receptors. These data suggest that BK may stimulate alveolar type II pneumocytes to release inflammatory cytokines, which then may modulate the lung inflammation.

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Figures

Figure 1.
Figure 1.
A: The dose-dependent release of neutrophil chemotactic activity in response to bradykinin from A549 cell monolayers after a 72-hour incubation (n = 8). Values are expressed as means ± SE. *P < 0.01 compared with medium alone. B: The time-related release of neutrophil chemotactic activity in response to 100 μmol/L bradykinin (•) from A549 cell monolayers (n = 8). ▪, baseline release of neutrophil chemotactic activity. Values are expressed as means ± SE. *P < 0.01 compared with supernatant fluids without incubation. ⋆P 0.01 compared with supernatant fluids without BK stimulation.
Figure 2.
Figure 2.
A: The dose-dependent release of monocyte chemotactic activity in response to bradykinin from A549 cell monolayers after a 72-hour incubation (n = 8). Values are expressed as means ± SE. *P < 0.01 compared with medium alone. B: The time-related release of monocyte chemotactic activity in response to 100 μmol/L bradykinin (•) from A549 cell monolayers (n = 8). ▪, baseline release of monocyte chemotactic activity. Values are expressed as means ± SE. *P < 0.01 compared with supernatant fluids without incubation. ⋆P < 0.01 compared with supernatant fluids without BK stimulation.
Figure 3.
Figure 3.
Partial characterization of the released neutrophil (A) and monocyte (B) chemotactic activity from A549 cell supernatant fluids harvested after a 72-hour incubation in response to 100 μmol/L bradykinin (n = 5). Values are expressed as means ± SE. *P < 0.01 compared with crude sample. EA, ethyl acetate.
Figure 4.
Figure 4.
The effects of nordihydroguaiaretic acid (NDGA), diethylcarbamazine (DEC), AA-861, and cycloheximide on the release of neutrophil chemotactic activity (A) and monocyte chemotactic activity (B) in response to 100 μmol/L bradykinin for a 72-hour incubation from A549 cell monolayers (n = 5). Values are expressed as means ± SE. *P < 0.01 compared with stimulus alone.
Figure 5.
Figure 5.
A: Molecular sieve column chromatographic finding of neutrophil chemotactic activity released from A549 cell monolayers in response to 100 μmol/L bradykinin for a 72-hour incubation. B: Molecular sieve column chromatographic finding of monocyte chemotactic activity released from A549 cell monolayer in response to 100 μmol/L bradykinin for a 72-hour incubation (•). □, molecular profiles of monocyte chemotactic activity under the unstimulated state. These data are the representative data of four experiments.
Figure 6.
Figure 6.
The effects of blocking antibodies on neutrophil chemotactic activity (A) and monocyte chemotactic activity (B) released from A549 cell monolayers in response to 100 μmol/L bradykinin for a 72-hour incubation (n = 6). Values are expressed as means ± SE. *P < 0.01 compared with untreated sample.
Figure 7.
Figure 7.
The individual time courses of chemokines that comprised chemotactic activity. The release of IL-8 (A), G-CSF (B), MCP-1 (C), and TGF-β (D) from A549 cell monolayer in response to 100 μmol/L bradykinin (n = 6).
Figure 8.
Figure 8.
The effects of bradykinin B1 (BKB1) and B2 (BKB2) receptor antagonists on the release of NCA (A) and MCA (B) from A549 cell monolayer after a 72-hour incubation (n = 5). *P < 0.01 compared with bradykinin alone.
Figure 9.
Figure 9.
The effects of bradykinin B1 (BKB1) and B2 (BKB2) receptor antagonists on the release of IL-8 (A), G-CSF (B), MCP-1 (C), and TGF-β (D) from A549 cell monolayer after a 72-hour incubation (n = 5). *P < 0.01 compared with bradykinin alone.
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