Effect of recombinant equine interleukin-1ß on function of equine endothelial colony-forming cells in vitro.

OBJECTIVE: To investigate the effects of recombinant equine IL-1ß on function of equine endothelial colony-forming cells (ECFCs) in vitro. SAMPLE: ECFCs derived from peripheral blood samples of 3 healthy adult geldings. PROCEDURES: Function testing was performed to assess in vitro wound healing, tubule formation, cell adhesion, and uptake of 1,1'-dioctadecyl-3,3,3',3' tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) by cultured ECFCs. Cell proliferation was determined by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay. Effects on function test results of different concentrations and exposure times of recombinant equine IL-1ß were assessed. RESULTS: Challenge of cultured ECFCs with IL-1ß for 48 hours inhibited tubule formation. Continuous challenge (54 hours) with IL-1ß in the wound healing assay reduced gap closure. The IL-1ß exposure did not significantly affect ECFC adhesion, DiI-Ac-LDL uptake, or ECFC proliferation. CONCLUSIONS AND CLINICAL RELEVANCE: These results suggested a role for IL-1ß in the inhibition of ECFC function in vitro. Functional changes in ECFCs following challenge with IL-1ß did not appear to be due to changes in cell proliferative capacity. These findings have implications for designing microenvironments for and optimizing therapeutic effects of ECFCs used to treat ischemic diseases in horses.

Effects of in vitro exposure to autologous blood and serum on expression of interleukin-8, interleukin-1ß, and chemokine (C-X-C motif) ligand 2 in equine primary bronchial epithelial cell cultures.

OBJECTIVE: To examine the effects of in vitro exposure to solutions of autologous horse blood (AHB) and autologous horse serum (AHS) on expressions of selected cytokine genes in equine primary bronchial epithelial cell (BEC) cultures and to contrast these responses to those induced in BEC cultures by endotoxin and hay dust. SAMPLE: BEC cultures established from bronchi of 6 healthy horses. PROCEDURES: 5-day-old BEC cultures were treated with PBS solution, AHB (2 concentrations), AHS, hay dust solution, and lipopolysaccharide solution for 24 hours. Gene expressions of interleukin (IL)-8, IL-1ß, chemokine (C-X-C motif) ligand 2 (CXCL2), and glyceralde-hyde-3-phosphate dehydrogenase were subsequently measured with a kinetic PCR assay. RESULTS: With the exception of AHS, all treatments of the BECs resulted in upregulation of each target gene expression relative to its expression in cultures exposed to PBS solution. Treatment with AHB induced a dose-dependent increase of each target gene, with IL-1ß expression increasing the most (> 1,200-fold increase). Lipopolysaccharide and hay dust solution treatments each resulted in 20-fold increases in IL-8 and IL-1ß gene expressions. Lipopolysaccharide and hay dust solution treatments also resulted in a 7- and 8-fold increase in CXCL2 gene expression, respectively. The increases in IL-8 and CXCL2 gene expressions following treatment with the higher concentration of blood were equivalent to those associated with hay dust solution or lipopolysaccharide. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that chemokine expression by cultured equine BECs following exposure to pulmonary hemorrhage conditions may contribute to the development of inflammatory airway disease in horses.

Effects of in vitro exposure to hay dust on expression of interleukin-23, -17, -8, and -1beta and chemokine (C-X-C motif) ligand 2 by pulmonary mononuclear cells from horses susceptible to recurrent airway obstruction.

OBJECTIVE: To examine gene expression of selected cytokines in pulmonary mononuclear cells isolated from healthy horses and horses susceptible to recurrent airway obstruction (RAO), and to determine whether interleukin (IL)-17 and IL-23 were associated with pulmonary inflammation. ANIMALS: 6 RAO-susceptible and 5 healthy horses. PROCEDURES: Bronchoalveolar lavage cells were retrieved from horses that were stabled and fed dusty hay for 24 hours. Lavage cells devoid of neutrophils were incubated for 24 hours with solutions of PBS, hay dust, lipopolysaccharide, or B-glucan. Gene expression of IL-17, IL-23 (p19 and p40 subunits), IL-8, IL-1B, chemokine (C-X-C motif) ligand 2 (CXCL2), and B-actin was measured by use of real-time reverse transcription PCR assays. RESULTS: The degree of inherent expression of target genes in bronchoalveolar lavage cells treated with PBSS was not different between the 2 groups of horses. Relative to exposure to PBSS, exposure to the hay dust solution increased gene expression of all cytokines more than 2-fold in cells from both groups of horses, but the magnitudes of these increases were not different between the groups. Exposure to lipopolysaccharide solution increased gene expression of IL-8, CXCL2, and IL-1B in cells from RAO-susceptible horses, but this increase was not significantly different from that in cells from control horses. Exposure to B-glucan solution failed to increase gene expression in cells from either horse group, compared with gene expression when cells were exposed to PBSS. CONCLUSIONS AND CLINICAL RELEVANCE: The acute pulmonary neutrophilia characteristic of RAO was not associated with an increase in upregulation of gene expression of chemokines in pulmonary mononuclear cells from disease-susceptible horses.

Effects of in vitro exposure to hay dust on the gene expression of chemokines and cell-surface receptors in primary bronchial epithelial cell cultures established from horses with chronic recurrent airway obstruction.

OBJECTIVE: To examine effects of in vitro exposure to solutions of hay dust, lipopolysaccharide (LPS), or beta-glucan on chemokine and cell-surface receptor (CSR) gene expression in primary bronchial epithelial cell cultures (BECCs) established from healthy horses and horses with recurrent airway obstruction (RAO). SAMPLE POPULATION: BECCs established from bronchial biopsy specimens of 6 RAO-affected horses and 6 healthy horses. PROCEDURES: 5-day-old BECCs were treated with PBS solution, hay dust solutions, LPS, or beta-glucan for 6 or 24 hours. Gene expression of interleukin (IL)-8, chemokine (C-X-C motif) ligand 2 (CXCL2), IL-1beta, toll-like receptor 2, toll-like receptor 4, IL-1 receptor 1, and glyceraldehyde 3-phosphate dehydrogenase was measured with a kinetic PCR assay. RESULTS: Treatment with PBS solution for 6 or 24 hours was not associated with a significant difference in chemokine or CSR expression between BECCs from either group of horses. In all BECCs, treatment with hay dust or LPS for 6 hours increased IL-8, CXCL2, and IL-1beta gene expression > 3-fold; at 24 hours, only IL-1beta expression was upregulated by > 3-fold. In all BECCs, CSR gene expression was not increased following any treatment. With the exception of a 3.7-fold upregulation of CXCL2 in BECCs from RAO-affected horses (following 6-hour hay dust treatment), no differences in chemokine or CSR gene expression were detected between the 2 groups. At 24 hours, CXCL2 gene expression in all BECCs was downregulated. CONCLUSIONS AND CLINICAL RELEVANCE: Epithelial CXCL2 upregulation in response to hay dust particulates may incite early airway neutrophilia in horses with RAO.