Rapid, Multiwell Colorimetric Assay for Measuring Neutrophil Chemoattractant Activity in Bronchoalveolar Lavage Fluid of Horses with Recurrent Airway Obstruction

Hall, Jean A.; Hoyt, Dana; Zuver, Clarissa; Skinner, Monica M.; Schlipf, John W.

doi:10.1177/104063870601800304

Cited by 3 publications

(2 citation statements)

References 28 publications

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“…TNF‐α induces an increase in the expression of the adhesion molecule ICAM‐1 in both human endothelial and bronchial epithelial cells in vitro, which may foster local neutrophil influx in vivo . Given the nature and concentration of the molecules detected in BALF from heaves‐affected horses, it is not surprising that our experiments, in agreement with previous reports, showed an increased migration of neutrophils toward BALF from heaves‐affected horses compared with healthy horses.…”

Section: Discussionsupporting

confidence: 91%

PI3K andMAPKs Regulate Neutrophil Migration Toward the Airways in Heaves

Bullone

Moran

Lavoie‐Lamoureux

et al. 2012

Veterinary Internal Medicne

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Background: Neutrophils accumulate in the airways of horses with heaves. They likely play an important role in the disease pathogenesis. Understanding the pathways regulating their migration may help identifying new therapeutic targets.Hypothesis: MAPK and PI3K pathways are involved in neutrophil migration toward the airway lumen in heaves. Animals: Twelve heaves-affected horses and 4 healthy horses.Methods: Migratory activity of bronchoalveolar lavage fluids (BALF) from horses with heaves and healthy horses was compared by means of a Boyden chamber. Involvement of MAPK and PI3K pathways in neutrophil migration was investigated by pretreating neutrophils with inhibitors of p38 MAPK, JNK, MEK1/2, and PI3K. The capacity of a p38 MAPK inhibitor at decreasing neutrophil chemotaxis toward the airways was also evaluated in vivo.Results: BALF from symptomatic heaves-affected horses induced a greater degree of chemokinesis (P = .0004) than BALF from healthy horses. Although all pathways tested were involved in neutrophil migration, inhibition of PI3K was most potent in vitro. An inhibitor of p38 MAPK administered before challenge in horses with heaves did not alter BALF chemokinetic properties. BALF neutrophil percentage and BALF migratory activity were positively correlated after 14 and 35 days of antigen challenge in healthy (P = .05; R 2 = 0.82) and heaves-affected horses (P = .03; R 2 = 0.76), respectively. Conclusions and Clinical Importance: MAPK and PI3K pathways regulate neutrophil migration induced by BALF of horses with heaves. Inhibition of multiple pathways might be required to completely abolish BALF-induced neutrophil migratory activity and possibly inflammation in heaves.

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Section: Discussionsupporting

confidence: 91%

PI3K andMAPKs Regulate Neutrophil Migration Toward the Airways in Heaves

Bullone

Moran

Lavoie‐Lamoureux

et al. 2012

Veterinary Internal Medicne

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“…Since despite much effort receptors for SCGB 1A1 have not been conclusively identified and the mechanisms for chemotaxis inhibition remain elusive. Hall and colleagues [44] reported that BAL fluid from RAO-affected animals enhanced chemotaxis. However, BAL fluid contains many mediators produced by bronchial epithelium or leaked into the airways from vasculature, and such assay does not allow dissection of individual protein effects.…”

Section: Discussionmentioning

confidence: 99%

Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation

et al. 2014

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Secretoglobin family 1A member 1 (SCGB 1A1) is a small protein mainly secreted by mucosal epithelial cells of the lungs and uterus. SCGB 1A1, also known as club (Clara) cell secretory protein, represents a major constituent of airway surface fluid. The protein has anti-inflammatory properties, and its concentration is reduced in equine recurrent airway obstruction (RAO) and human asthma. RAO is characterized by reversible airway obstruction, bronchoconstriction and neutrophilic inflammation. Direct effects of SCGB 1A1 on neutrophil functions are unknown. We have recently identified that the SCGB1A1 gene is triplicated in equids and gives rise to two distinct proteins. In this study we produced the endogenously expressed forms of SCGBs (SCGB 1A1 and 1A1A) as recombinant proteins, and analyzed their effects on reactive oxygen species production, phagocytosis, chemotaxis and neutrophil extracellular trap (NET) formation ex vivo. We further evaluated whether NETs are present in vivo in control and inflamed lungs. Our data show that SCGB 1A1A but not SCGB 1A1 increase neutrophil oxidative burst and phagocytosis; and that both proteins markedly reduce neutrophil chemotaxis. SCGB 1A1A reduced chemotaxis significantly more than SCGB 1A1. NET formation was significantly reduced in a time- and concentration-dependent manner by SCGB 1A1 and 1A1A. SCGB mRNA in bronchial biopsies, and protein concentration in bronchoalveolar lavage fluid, was lower in horses with RAO. NETs were present in bronchoalveolar lavage fluid from horses with exacerbated RAO, but not in fluid from horses with RAO in remission or in challenged healthy horses. These findings indicate that SCGB 1A1 and 1A1A have overlapping and diverging functions. Considering disparities in the relative abundance of SCGB 1A1 and 1A1A in airway secretions of animals with RAO suggests that these functional differences may contribute to the pathogenesis of RAO and other neutrophilic inflammatory lung diseases.

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Age-dependent vascular endothelial growth factor expression and angiogenic capability of bladder smooth muscle cells: implications for cell-seeded technology in bladder tissue engineering

Azzarello

Kropp

Fung

et al. 2009

J Tissue Eng Regen Med

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Cell seeding technology is commonly used in the field of tissue engineering to enhance the performance of bioscaffolds and promote tissue regeneration. The age of cells used for ex vivo seeding to achieve maximal tissue regeneration has not been defined. Since rapid angiogenesis is the most critical step for tissue graft survival and success, we evaluated passage-dependent vascular endothelial growth factor (VEGF) expression in cultured smooth muscle cells (SMCs) obtained from urinary bladder and endothelial cell response to bladder SMCs. Levels of various VEGF isoforms mRNA expression and total VEGF secretion were determined by a semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) and an enzyme-linked immunosorbent assay (ELISA) analysis, respectively. In vitro endothelial cell migration in Transwell and capillary-like tube formation in Matrigel were used to predict the ability of bladder SMCs to promote angiogenesis. VEGF produced by cultured bladder SMCs increased from passages 4 to 7, and decreased from passages 7 to 12 at both mRNA and protein levels. Endothelial cell migration as well as capillary-like tube formation correlated with levels of VEGF expression by bladder SMCs. Pre-incubation of endothelial cells with a VEGF receptor 1/2 inhibitor, SU5416, significantly reduced the number of capillary-like tubes in SMC-endothelial cell Matrigel co-culture, and confirmed the involvement of VEGF in endothelial cell tube formation. Our results demonstrate that cell passage number is related to levels of VEGF production, which may translate to angiogenesis in engineered tissues.

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Rapid, Multiwell Colorimetric Assay for Measuring Neutrophil Chemoattractant Activity in Bronchoalveolar Lavage Fluid of Horses with Recurrent Airway Obstruction

Cited by 3 publications

References 28 publications

PI3K andMAPKs Regulate Neutrophil Migration Toward the Airways in Heaves

PI3K andMAPKs Regulate Neutrophil Migration Toward the Airways in Heaves

Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation

Age-dependent vascular endothelial growth factor expression and angiogenic capability of bladder smooth muscle cells: implications for cell-seeded technology in bladder tissue engineering

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