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Oxidative stress and reduced responsiveness of challenged circulating leukocytes following pulmonary instillation of metal-rich particulate matter in rats.

Erdely A, Antonini JM, Young SH, Kashon ML, Gu JK, Hulderman T, Salmen R, Meighan T, Roberts JR, Zeidler-Erdely PC - Part Fibre Toxicol (2014)

Bottom Line: In addition, mononuclear cells were isolated 24 h post-exposure to measure oxidative stress by flow cytometry and confocal microscopy.Instillation of welding fume reduced inflammatory protein production of circulating leukocytes when challenged with the secondary stimulus LPS.The effects were not related to transcription, but were observed in conjunction with oxidative stress.

View Article: PubMed Central - HTML - PubMed

Affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown 26505, WV, USA. efi4@cdc.gov.

ABSTRACT
Welding fume is an exposure that consists of a mixture of metal-rich particulate matter with gases (ozone, carbon monoxide) and/or vapors (VOCs). Data suggests that welders are immune compromised. Given the inability of pulmonary leukocytes to properly respond to a secondary infection in animal models, the question arose whether the dysfunction persisted systemically. Our aim was to evaluate the circulating leukocyte population in terms of cellular activation, presence of oxidative stress, and functionality after a secondary challenge, following welding fume exposure. Rats were intratracheally instilled (ITI) with PBS or 2 mg of welding fume collected from a stainless steel weld. Rats were sacrificed 4 and 24 h post-exposure and whole blood was collected. Whole blood was used for cellular differential counts, RNA isolation with subsequent microarray and Ingenuity Pathway Analysis, and secondary stimulation with LPS utilizing TruCulture technology. In addition, mononuclear cells were isolated 24 h post-exposure to measure oxidative stress by flow cytometry and confocal microscopy. Welding fume exposure had rapid effects on the circulating leukocyte population as identified by relative mRNA expression changes. Instillation of welding fume reduced inflammatory protein production of circulating leukocytes when challenged with the secondary stimulus LPS. The effects were not related to transcription, but were observed in conjunction with oxidative stress. These findings support previous studies of an inadequate pulmonary immune response following a metal-rich exposure and extend those findings showing leukocyte dysfunction occurs systemically.

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Effect of MMA-SS welding fume instillation on protein production of challenged leukocytes. Anticoagulated whole blood was collected 24 h post-instillation to PBS or MMA-SS and challenged with LPS (100 ng/ml). Supernatants were collected 24 h after LPS challenge and assayed for protein concentrations. Data are represented as (concentration)/(leukocytes/ml) times a factor of 10 to obtain a whole number. CXCL10 – pg/leukocyte* 100,000; CCL4 – pg/leukocyte* 10,000; CXCL2 – pg/leukocyte* 1,000,000; TNFα – ng/leukocyte* 100,000,000. *p < 0.05 vs PBS.
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Figure 4: Effect of MMA-SS welding fume instillation on protein production of challenged leukocytes. Anticoagulated whole blood was collected 24 h post-instillation to PBS or MMA-SS and challenged with LPS (100 ng/ml). Supernatants were collected 24 h after LPS challenge and assayed for protein concentrations. Data are represented as (concentration)/(leukocytes/ml) times a factor of 10 to obtain a whole number. CXCL10 – pg/leukocyte* 100,000; CCL4 – pg/leukocyte* 10,000; CXCL2 – pg/leukocyte* 1,000,000; TNFα – ng/leukocyte* 100,000,000. *p < 0.05 vs PBS.

Mentions: Whole blood was collected 4 and 24 h after MMA-SS or PBS ITI and incubated with or without LPS for 24 h. Cxcl10, Ccl4, Cxcl2, and TNF-α concentrations were determined from collected supernatants and data collected from the 24 h time point are shown in Figure 4. Data were generated by subtracting supernatant protein concentrations of from LPS-challenged divided by the total leukocytes for each individual treated rat. It was necessary to divide by the total number of leukocytes because the TruCulture design is a specific volume of 1 mL irrespective of the number of circulating leukocytes. A significant decrease in LPS-induced protein production from circulating leukocytes harvested from rats exposed to MMA-SS fume for 24 h compared to PBS was found (Figure 4). However, despite a significant transcriptional response, there was no effect on LPS-induced protein production when comparing PBS and MMA-SS ITI stimulated whole blood collected 4 h post-exposure (data not shown).


Oxidative stress and reduced responsiveness of challenged circulating leukocytes following pulmonary instillation of metal-rich particulate matter in rats.

Erdely A, Antonini JM, Young SH, Kashon ML, Gu JK, Hulderman T, Salmen R, Meighan T, Roberts JR, Zeidler-Erdely PC - Part Fibre Toxicol (2014)

Effect of MMA-SS welding fume instillation on protein production of challenged leukocytes. Anticoagulated whole blood was collected 24 h post-instillation to PBS or MMA-SS and challenged with LPS (100 ng/ml). Supernatants were collected 24 h after LPS challenge and assayed for protein concentrations. Data are represented as (concentration)/(leukocytes/ml) times a factor of 10 to obtain a whole number. CXCL10 – pg/leukocyte* 100,000; CCL4 – pg/leukocyte* 10,000; CXCL2 – pg/leukocyte* 1,000,000; TNFα – ng/leukocyte* 100,000,000. *p < 0.05 vs PBS.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4151022&req=5

Figure 4: Effect of MMA-SS welding fume instillation on protein production of challenged leukocytes. Anticoagulated whole blood was collected 24 h post-instillation to PBS or MMA-SS and challenged with LPS (100 ng/ml). Supernatants were collected 24 h after LPS challenge and assayed for protein concentrations. Data are represented as (concentration)/(leukocytes/ml) times a factor of 10 to obtain a whole number. CXCL10 – pg/leukocyte* 100,000; CCL4 – pg/leukocyte* 10,000; CXCL2 – pg/leukocyte* 1,000,000; TNFα – ng/leukocyte* 100,000,000. *p < 0.05 vs PBS.
Mentions: Whole blood was collected 4 and 24 h after MMA-SS or PBS ITI and incubated with or without LPS for 24 h. Cxcl10, Ccl4, Cxcl2, and TNF-α concentrations were determined from collected supernatants and data collected from the 24 h time point are shown in Figure 4. Data were generated by subtracting supernatant protein concentrations of from LPS-challenged divided by the total leukocytes for each individual treated rat. It was necessary to divide by the total number of leukocytes because the TruCulture design is a specific volume of 1 mL irrespective of the number of circulating leukocytes. A significant decrease in LPS-induced protein production from circulating leukocytes harvested from rats exposed to MMA-SS fume for 24 h compared to PBS was found (Figure 4). However, despite a significant transcriptional response, there was no effect on LPS-induced protein production when comparing PBS and MMA-SS ITI stimulated whole blood collected 4 h post-exposure (data not shown).

Bottom Line: In addition, mononuclear cells were isolated 24 h post-exposure to measure oxidative stress by flow cytometry and confocal microscopy.Instillation of welding fume reduced inflammatory protein production of circulating leukocytes when challenged with the secondary stimulus LPS.The effects were not related to transcription, but were observed in conjunction with oxidative stress.

View Article: PubMed Central - HTML - PubMed

Affiliation: Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown 26505, WV, USA. efi4@cdc.gov.

ABSTRACT
Welding fume is an exposure that consists of a mixture of metal-rich particulate matter with gases (ozone, carbon monoxide) and/or vapors (VOCs). Data suggests that welders are immune compromised. Given the inability of pulmonary leukocytes to properly respond to a secondary infection in animal models, the question arose whether the dysfunction persisted systemically. Our aim was to evaluate the circulating leukocyte population in terms of cellular activation, presence of oxidative stress, and functionality after a secondary challenge, following welding fume exposure. Rats were intratracheally instilled (ITI) with PBS or 2 mg of welding fume collected from a stainless steel weld. Rats were sacrificed 4 and 24 h post-exposure and whole blood was collected. Whole blood was used for cellular differential counts, RNA isolation with subsequent microarray and Ingenuity Pathway Analysis, and secondary stimulation with LPS utilizing TruCulture technology. In addition, mononuclear cells were isolated 24 h post-exposure to measure oxidative stress by flow cytometry and confocal microscopy. Welding fume exposure had rapid effects on the circulating leukocyte population as identified by relative mRNA expression changes. Instillation of welding fume reduced inflammatory protein production of circulating leukocytes when challenged with the secondary stimulus LPS. The effects were not related to transcription, but were observed in conjunction with oxidative stress. These findings support previous studies of an inadequate pulmonary immune response following a metal-rich exposure and extend those findings showing leukocyte dysfunction occurs systemically.

Show MeSH
Related in: MedlinePlus