OBJECTIVE To investigate the effects of recombinant equine IL-1β 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-1β were assessed. RESULTS Challenge of cultured ECFCs with IL-1β for 48 hours inhibited tubule formation. Continuous challenge (54 hours) with IL-1β in the wound healing assay reduced gap closure. The IL-1β 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-1β in the inhibition of ECFC function in vitro. Functional changes in ECFCs following challenge with IL-1β 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.

The lung is a complex organ, and its physiology and immunology are regulated by various immune molecules and cells. Lung surfactant, a mixture of phospholipids and proteins produced by the bronchiolar and type II alveolar epithelial cells, is one such important player in lung physiology. Compared to knowledge about the biology of the surfactant in rodents and humans, only limited data are available on the surfactant in the horse. Although there are data linking levels of surfactant proteins with respiratory disease in the horse, there are no data on the cellular localization of surfactant protein A (SP-A) and surfactant protein D (SP-D). A member of the tetraspanin family of proteins, CD9 is a cell-signaling and adhesion protein and its expression has been detected in both normal and cancer cells, including those in the lung. Because there are no immunolocalization data on SP-A, SP-D, and CD9 in the normal lungs of the horse, our objective was to conduct a light and electron microscopic immunocytochemical study on normal lungs of the horse. The data showed SP-A and SP-D in bronchiolar epithelial and type II alveolar epithelial cells. These proteins were also localized in type I alveolar epithelial cells, pulmonary intravascular macrophages, and neutrophils, which is likely an outcome of endocytosis of the proteins by these cells. CD9 was present in the airway and vascular smooth muscle cells, endothelium, and blood cells, but not in the airway epithelium. These new data provide a baseline to further examine the expression and functions of SP-A, SP-D, and CD9 proteins in inflammation associated with respiratory diseases in the horse.

The hypothesis that an altered expression of CD11/CD18 on bovine circulating monocytes, polymorphonuclear leukocytes (PMN), or both, contributes to an increased mastitis susceptibility in periparturient cows was tested. Expression of CD18 and CD11a, -b, -c on bovine monocytes and PMN were assessed in 8 Friesian-Holstein cows by flow cytometry from 2 wk before calving to 5 wk after calving. Minor changes in adhesion molecule expression levels were detected throughout the experimental period. Compared with PMN, monocytes exhibited an expression level that was similar for CD18, higher for CD11a and CD11c, but lower for CD11b. Differences in density may reflect the relative importance of these adhesion molecules on both leukocyte types. In this study, the decreased number of milk resident macrophages and PMN observed during the periparturient period could not be attributed to changes of CD11/CD18 levels on circulating leukocytes.

Marked differences in bone marrow cellularity were observed between cattle affected with leukocyte adhesion deficiency (LAD) and control cattle. The number of nucleated cells in bone marrow was 2.9 to 8.8 times higher in cattle affected with LAD, compared with controls. The myeloid-to-erythroid ratio of bone marrow from 3 cattle affected with LAD ranged from 2.4 to 12. Deficient CD18 expression on neutrophils isolated from bone marrow of cattle with LAD was clearly detected by flow cytometric analysis. Neutrophils from bone marrow of cattle affected with LAD appeared round and not flat, after adherence to plastic wells under agarose, whereas neutrophils from bone marrow of clinically normal cattle were firmly spread on the surface of plastic wells. In the chemotaxis under-agarose assay, many pseudopodia were detected on bone marrow neutrophils from clinically normal cattle, but were not detected on bone marrow neutrophils from cattle with LAD. Activities of chemotactic movements and phagocytosis of neutrophils isolated from bone marrow of cattle affected with LAD were documented to be severely impaired.

To study increase of the Salmonella population in the cecum of chickens infected with Eimeria tenella, quantitative changes in mannose residues on the cecal mucosa were investigated. Inhibition of S typhimurium adherence to the cecum by a 2% carbohydrate (D-mannose, D-galactose, L-fucose, alpha-methyl-D-glucoside) in phosphate-buffered saline solution was examined. Only D-mannose had inhibitory effects. Whereas, D-galactose had somewhat enhancing effects on adherence of S typhimurium to the cecal mucosa of uninfected germ-free chickens. In infected and uninfected chickens, D-mannose inhibited adherence of S typhimurium. D-mannose significantly (P < 0.05) increased adherence of Bacteroides sp. In infected and uninfected chickens, D-mannose did not have any effect on adherence of Clostridium perfringens and Bifidobacterium thermophilum. Under microscopic observation, only concanavalin A and Lens culinaris agglutinin, of 8 lectins examined, were recognized as lectin-positive staining lines or spots in the cecal mucosa, indicating presence of mannose residues on the cecal mucosa. In E tenella-infected chickens, lectin-positive staining was seen strongly on the coarse surface of damaged cells and at the bottom of the crypts. These results indicate that coccidial infection may induce increase of mannose residues on the intestinal surface and allow adhesion of more salmonellae to the intestine.

When Salmonella typhimurium and Clostridium perfringens were tested in conventional chickens, larger numbers of S typhimurium and C perfringens adhered to Eimeria tenella-infected ceca than to uninfected ceca. In germ-free chickens, S typhimurium and C perfringens adhered to the E tenella-infected cecal mucosa more than to the uninfected cecal mucosa, but fewer Bacteroides vulgatus and Bifidobacterium thermophilum adhered to the E tenella-infected ceca than to the uninfected ceca. Many bacteria adhered to the lesions caused by E tenella as observed by scanning electron microscopy. On the basis of our findings, we suggest that infection with E tenella upsets the balance of competitive adherence of bacteria, allowing more colonization of S typhimurium and C perfringens.

This work was an attempt to develop an in vitro adherence model for Mycoplasma hyopneumoniae, using monolayers of human and porcine lung fibroblasts and porcine kidney cells. Mycoplasma hyopneumoniae grown in Friis mycoplasma broth was radiolabeled with 35[S]-methionine, washed, concentrated, and inoculated on the monolayers. After 15 minutes of centrifugation to facilitate adherence, monolayers were washed 3 times, dissolved with 0.1N NaOH, and suspended in scintillation liquid, and the radioactivity was determined in a liquid scintillation counter. Adherence, measured as a percentage of counts added, varied according to the mycoplasma strain and the cell line used. Comparison of strains J, 144L, and 232 of M hyopneumoniae revealed 7.5 +/- 5.9, 31.9 +/- 13, and 9.6 +/- 5% adherence to porcine kidney cells, respectively. Slightly different, but proportionally the same relationships were obtained with swine or human fibroblasts. Adherence was decreased slightly by repeated washings of the mycoplasma-treated cell monolayers; however, a plateau was reached, indicating irreversibility of the adherence process. Pretreatment of cell monolayers with nonlabeled organisms substantially blocked adherence by labeled organisms. Dilution of labeled organisms resulted in an increased proportion adhering. Therefore, it appears that the adherence was a receptor-dependent event. Treatment of the mycoplasmas with trypsin prior to the inoculation of monolayers resulted in a marked reduction in adherence. Treatment of the mycoplasmas with hyperimmune swine serum against M hyopneumoniae or normal swine serum resulted in 80 to 90% reduction of adherence; however, no inhibition occurred when mycoplasmas were treated with purified IgG from the hyperimmune serum.

The binding of bovine complement S protein (vitronectin) to Streptococcus dysgalactiae isolates from cattle with mastitis and the S protein's role in streptococcal adherence to bovine epithelial cells were investigated. All 25 clinical isolates of S dysgalactiae interacted with bovine S protein. None of the other streptococcal species tested bound to bovine S protein. The S protein-binding sites were saturable and highly sensitive to trypsin. The binding of bovine S protein to S dysgalactiae isolates was specific and could not be inhibited by other plasma proteins, such as fibronectin, albumin, fibrinogen, alpha 2-macroglobulin, or IgG. Similarly, streptococcal binding of bovine S protein was not influenced by the synthetic peptide Gly-Arg-Gly-Asp-Ser, which constituted the host cell attachment sequence of S protein. In adherence experiments, prior binding of bovine S protein to S dysgalactiae enhanced streptococcal adherence to bovine epithelial cells. The enhancing effects by bovine S protein were abolished when the respective binding sites on the streptococci were digested by trypsin. Thus, bovine S protein could be an important mediator of adherence of S dysgalactiae to bovine epithelial cells.