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Arginine deficiency augments inflammatory mediator production by airway epithelial cells in vitro.

Fan XY, van den Berg A, Snoek M, van der Flier LG, Smids B, Jansen HM, Liu RY, Lutter R - Respir. Res. (2009)

Bottom Line: Arginine deficiency may also result from exposure to poly-L-arginine or major basic protein (MBP), which can block arginine uptake.The exaggerated IL-6 and IL-8 production due to arginine deficiency and to poly-L-arginine depend on a post-transcriptional and a transcriptional process, respectively.We conclude that both reduced arginine availability per se and the presence of polycationic proteins may promote airway inflammation by enhanced pro-inflammatory mediator production in airway epithelial cells, but due to distinct mechanisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pulmonology, The Geriatric Institute of Anhui, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, PR China. smallcloud670820@yahoo.com

ABSTRACT

Background: Previously we showed that reduced availability of the essential amino acid tryptophan per se attenuates post-transcriptional control of interleukin (IL)-6 and IL-8 leading to hyperresponsive production of these inflammatory mediators by airway epithelial cells. Availability of the non-essential amino acid arginine in the inflamed airway mucosa of patients with asthma is reduced markedly, but it is not known whether this can also lead to an exaggerated production of IL-6 and IL-8.

Methods: IL-6 and IL-8 were determined by ELISA in culture supernatants of NCI-H292 airway epithelial-like cells and normal bronchial epithelial (NHBE) cells that were exposed to TNF-alpha, LPS or no stimulus, in medium with or without arginine. Arginine deficiency may also result from exposure to poly-L-arginine or major basic protein (MBP), which can block arginine uptake. Epithelial cells were exposed to these polycationic proteins and L-(14)C-arginine uptake was assessed as well as IL-6 and IL-8 production. To determine the mode of action, IL-6 and IL-8 mRNA profiles over time were assessed as were gene transcription and post-transcriptional mRNA degradation.

Results: For both NCI-H292 and NHBE cells, low arginine concentrations enhanced basal epithelial IL-6 and IL-8 production and synergized with TNF-alpha-induced IL-6 and IL-8 production. Poly-L-arginine enhanced the stimulus-induced IL-6 and IL-8 production, however, blocking arginine uptake and the enhanced IL-6 and IL-8 production appeared unrelated. The exaggerated IL-6 and IL-8 production due to arginine deficiency and to poly-L-arginine depend on a post-transcriptional and a transcriptional process, respectively.

Conclusion: We conclude that both reduced arginine availability per se and the presence of polycationic proteins may promote airway inflammation by enhanced pro-inflammatory mediator production in airway epithelial cells, but due to distinct mechanisms.

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The effect of polycations poly-L-arginine and major basic protein on L-14C-arginine uptake and LPS-induced IL-8 production by NCI-H292 cells. Parallel cultures of NCI-H292 cells were exposed to different amounts of poly-L-arginine (A,B) or major basic protein (MBP; C,D). For arginine uptake (A, C) cells were washed with HBBS and L-14C-arginine was added for 30 min. (see Material and Methods). For IL-8 (B, D) and IL-6 (not shown) production the culture supernatants were collected after 20 h with no stimulus (white columns) or with 5 μg/ml LPS (black columns). Typical experiments are shown (triplicate samples; mean ± SEM) of 5 experiments with poly-L-arginine and 4 with MBP. *P < 0.05, **P < 0.01 and ***P < 0.001 for IL-8 production as compared to unstimulated cells; # P < 0.05, # # P < 0.01 and # # # P < 0.001 for uptake of L-14C-arginine as compared to that in the absence of poly-L-arginine or MBP.
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Figure 3: The effect of polycations poly-L-arginine and major basic protein on L-14C-arginine uptake and LPS-induced IL-8 production by NCI-H292 cells. Parallel cultures of NCI-H292 cells were exposed to different amounts of poly-L-arginine (A,B) or major basic protein (MBP; C,D). For arginine uptake (A, C) cells were washed with HBBS and L-14C-arginine was added for 30 min. (see Material and Methods). For IL-8 (B, D) and IL-6 (not shown) production the culture supernatants were collected after 20 h with no stimulus (white columns) or with 5 μg/ml LPS (black columns). Typical experiments are shown (triplicate samples; mean ± SEM) of 5 experiments with poly-L-arginine and 4 with MBP. *P < 0.05, **P < 0.01 and ***P < 0.001 for IL-8 production as compared to unstimulated cells; # P < 0.05, # # P < 0.01 and # # # P < 0.001 for uptake of L-14C-arginine as compared to that in the absence of poly-L-arginine or MBP.

Mentions: Polycations like poly-L-arginine can inhibit cationic amino acid transporter proteins, among which those transporting arginine. We reasoned that the synergy in IL-6 and IL-8 production shown in the absence of extracellular arginine may also be accomplished by inhibiting arginine uptake with poly-L-arginine. Experiments to measure the effect of poly-L-arginine on L-14C-arginine uptake and on IL-6 and IL-8 production were performed in parallel, where L-14C-arginine uptake was assessed shortly after adding poly-L-arginine and IL-6 and IL-8 were measured in culture supernatants collected at 20 h after adding poly-L-arginine. Figure 3A shows that 10 and 20 μg/ml poly-L-arginine markedly reduced L-14C-arginine uptake (P < 0.001), and even at 1.25 to 5 μg/ml poly-L-arginine, arginine-uptake was inhibited significantly (P < 0.05 and P < 0.01, respectively). Forty μg/ml poly-L-arginine and higher appeared cytotoxic with cells detaching and thus was not tested further. Interestingly, basal IL-6 and IL-8 production were not affected by poly-L-arginine itself in contrast to the enhanced basal IL-6 and IL-8 production due to reduced arginine availability. At 1.25 μg/ml and higher, poly-L-arginine significantly potentiated LPS-induced IL-8 (P < 0.001) and IL-6 production (data not shown) with maximal synergy at 5 μg/ml of poly-L-arginine. Thus, inhibition of arginine uptake and the enhanced production of IL-8 and IL-6 did not correlate strictly. Heparin (10 μg/ml) fully inhibited the effect of poly-L-arginine on the LPS-induced IL-8 and IL-6 production, indicative of a role for the positive charges of poly-L-arginine. In contrast to the findings for LPS and to that seen after stimulation with TNF-α of NCI-H292 cells in MEM-A-, TNF-α did not synergize with poly-L-arginine in IL-6 and IL-8 production (data not shown). Exposure of NCI-H292 cells to LPS and poly-L-arginine resulted in a hyperresponsive IL-6 and IL-8 production (P = 0.015 and 0.046, respectively) (Figures 4A and 4B).


Arginine deficiency augments inflammatory mediator production by airway epithelial cells in vitro.

Fan XY, van den Berg A, Snoek M, van der Flier LG, Smids B, Jansen HM, Liu RY, Lutter R - Respir. Res. (2009)

The effect of polycations poly-L-arginine and major basic protein on L-14C-arginine uptake and LPS-induced IL-8 production by NCI-H292 cells. Parallel cultures of NCI-H292 cells were exposed to different amounts of poly-L-arginine (A,B) or major basic protein (MBP; C,D). For arginine uptake (A, C) cells were washed with HBBS and L-14C-arginine was added for 30 min. (see Material and Methods). For IL-8 (B, D) and IL-6 (not shown) production the culture supernatants were collected after 20 h with no stimulus (white columns) or with 5 μg/ml LPS (black columns). Typical experiments are shown (triplicate samples; mean ± SEM) of 5 experiments with poly-L-arginine and 4 with MBP. *P < 0.05, **P < 0.01 and ***P < 0.001 for IL-8 production as compared to unstimulated cells; # P < 0.05, # # P < 0.01 and # # # P < 0.001 for uptake of L-14C-arginine as compared to that in the absence of poly-L-arginine or MBP.
© Copyright Policy - open-access
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Figure 3: The effect of polycations poly-L-arginine and major basic protein on L-14C-arginine uptake and LPS-induced IL-8 production by NCI-H292 cells. Parallel cultures of NCI-H292 cells were exposed to different amounts of poly-L-arginine (A,B) or major basic protein (MBP; C,D). For arginine uptake (A, C) cells were washed with HBBS and L-14C-arginine was added for 30 min. (see Material and Methods). For IL-8 (B, D) and IL-6 (not shown) production the culture supernatants were collected after 20 h with no stimulus (white columns) or with 5 μg/ml LPS (black columns). Typical experiments are shown (triplicate samples; mean ± SEM) of 5 experiments with poly-L-arginine and 4 with MBP. *P < 0.05, **P < 0.01 and ***P < 0.001 for IL-8 production as compared to unstimulated cells; # P < 0.05, # # P < 0.01 and # # # P < 0.001 for uptake of L-14C-arginine as compared to that in the absence of poly-L-arginine or MBP.
Mentions: Polycations like poly-L-arginine can inhibit cationic amino acid transporter proteins, among which those transporting arginine. We reasoned that the synergy in IL-6 and IL-8 production shown in the absence of extracellular arginine may also be accomplished by inhibiting arginine uptake with poly-L-arginine. Experiments to measure the effect of poly-L-arginine on L-14C-arginine uptake and on IL-6 and IL-8 production were performed in parallel, where L-14C-arginine uptake was assessed shortly after adding poly-L-arginine and IL-6 and IL-8 were measured in culture supernatants collected at 20 h after adding poly-L-arginine. Figure 3A shows that 10 and 20 μg/ml poly-L-arginine markedly reduced L-14C-arginine uptake (P < 0.001), and even at 1.25 to 5 μg/ml poly-L-arginine, arginine-uptake was inhibited significantly (P < 0.05 and P < 0.01, respectively). Forty μg/ml poly-L-arginine and higher appeared cytotoxic with cells detaching and thus was not tested further. Interestingly, basal IL-6 and IL-8 production were not affected by poly-L-arginine itself in contrast to the enhanced basal IL-6 and IL-8 production due to reduced arginine availability. At 1.25 μg/ml and higher, poly-L-arginine significantly potentiated LPS-induced IL-8 (P < 0.001) and IL-6 production (data not shown) with maximal synergy at 5 μg/ml of poly-L-arginine. Thus, inhibition of arginine uptake and the enhanced production of IL-8 and IL-6 did not correlate strictly. Heparin (10 μg/ml) fully inhibited the effect of poly-L-arginine on the LPS-induced IL-8 and IL-6 production, indicative of a role for the positive charges of poly-L-arginine. In contrast to the findings for LPS and to that seen after stimulation with TNF-α of NCI-H292 cells in MEM-A-, TNF-α did not synergize with poly-L-arginine in IL-6 and IL-8 production (data not shown). Exposure of NCI-H292 cells to LPS and poly-L-arginine resulted in a hyperresponsive IL-6 and IL-8 production (P = 0.015 and 0.046, respectively) (Figures 4A and 4B).

Bottom Line: Arginine deficiency may also result from exposure to poly-L-arginine or major basic protein (MBP), which can block arginine uptake.The exaggerated IL-6 and IL-8 production due to arginine deficiency and to poly-L-arginine depend on a post-transcriptional and a transcriptional process, respectively.We conclude that both reduced arginine availability per se and the presence of polycationic proteins may promote airway inflammation by enhanced pro-inflammatory mediator production in airway epithelial cells, but due to distinct mechanisms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pulmonology, The Geriatric Institute of Anhui, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, PR China. smallcloud670820@yahoo.com

ABSTRACT

Background: Previously we showed that reduced availability of the essential amino acid tryptophan per se attenuates post-transcriptional control of interleukin (IL)-6 and IL-8 leading to hyperresponsive production of these inflammatory mediators by airway epithelial cells. Availability of the non-essential amino acid arginine in the inflamed airway mucosa of patients with asthma is reduced markedly, but it is not known whether this can also lead to an exaggerated production of IL-6 and IL-8.

Methods: IL-6 and IL-8 were determined by ELISA in culture supernatants of NCI-H292 airway epithelial-like cells and normal bronchial epithelial (NHBE) cells that were exposed to TNF-alpha, LPS or no stimulus, in medium with or without arginine. Arginine deficiency may also result from exposure to poly-L-arginine or major basic protein (MBP), which can block arginine uptake. Epithelial cells were exposed to these polycationic proteins and L-(14)C-arginine uptake was assessed as well as IL-6 and IL-8 production. To determine the mode of action, IL-6 and IL-8 mRNA profiles over time were assessed as were gene transcription and post-transcriptional mRNA degradation.

Results: For both NCI-H292 and NHBE cells, low arginine concentrations enhanced basal epithelial IL-6 and IL-8 production and synergized with TNF-alpha-induced IL-6 and IL-8 production. Poly-L-arginine enhanced the stimulus-induced IL-6 and IL-8 production, however, blocking arginine uptake and the enhanced IL-6 and IL-8 production appeared unrelated. The exaggerated IL-6 and IL-8 production due to arginine deficiency and to poly-L-arginine depend on a post-transcriptional and a transcriptional process, respectively.

Conclusion: We conclude that both reduced arginine availability per se and the presence of polycationic proteins may promote airway inflammation by enhanced pro-inflammatory mediator production in airway epithelial cells, but due to distinct mechanisms.

Show MeSH
Related in: MedlinePlus