Elke Smits

The human homologue of Caenorhabditis elegans CED-6 specifically promotes phagocytosis of apoptotic cells

A key feature of the process of programmed cell death (apoptosis) is the efficiency with which the dying cells are recognized and engulfed by phagocytes [1]. Apoptotic cells are rapidly cleared either by neighbouring cells acting as semi-professional phagocytes or by experts of the macrophage line, so that an inflammatory response is avoided [2]. The Caenorhabditis elegans gene ced-6 is required for efficient engulfment of apoptotic cells [3] and is one of a group of genes that define two partially redundant parallel pathways for the engulfment process [4] [5]. These pathways may be conserved across evolution, as two other engulfment genes have human homologues. A CED-5 homologue is part of a human CrkII-DOCK180-Rac signaling pathway proposed to mediate cytoskeletal reorganization [6] [7] [8] and a CED-7 homologue is similar to the ABC transporters [9] [10]. Here, we report the cloning and characterization of human CED-6, a human homologue of C. elegans CED-6. The 34 kDa hCED-6 protein is expressed in most tissues, some human cancer cells, and in primary human macrophages. We developed an assay that quantitates the phagocytic activity of mammalian macrophages: the number of apoptotic cells that have been internalized is measured by the uptake of lacZ-positive apoptotic cells by adherent transgenic macrophages. The results of this assay demonstrate that overexpression of hCED-6 promotes phagocytosis only of apoptotic cells and suggest that hCED-6 is the mammalian orthologue of C. elegans CED-6 and is a part of a highly conserved pathway that specifically mediates the phagocytosis of apoptotic cells.

Simultaneous flow cytometric measurement of phagocytotic and oxidative burst activity of polymorphonuclear leukocytes in whole bovine blood.

Polymorphonuclear neutrophils (PMN) are important in host defence against bacterial infection in the bovine mammary gland and techniques are needed to evaluate their functional activities. A rapid and sensitive two-color flow cytometric method for simultaneous measurement of phagocytosis rate and oxidative burst activity of bovine PMN in small blood samples is described. The method utilizes the oxidation of intracellular dihydrorhodamine 123 to green fluorescent rhodamine 123 by oxidative burst products that were generated by incubating the PMNs with red fluorescent propidium iodide labeled Staphylococcus aureus. Phagocytosis and oxidative burst both increased with time of incubation and with increasing bacteria concentration. A 20 min phagocytosis time and a ratio of 25 bacteria to one cell were considered optimal for assaying bovine blood PMN activity. To further evaluate the proposed two-color flow cytometric method, blood samples were treated with factors known to interfere with phagocytosis and oxidative burst metabolism of human neutrophils. Incubation of whole bovine blood with cytochalasin B at 10 micrograms ml-1 resulted in an approximate 70% decrease in the phagocytosis rate with a concommitant decrease in oxidative burst activity. Staurosporine (2 micrograms ml-1) inhibited bovine neutrophil oxidative burst formation for 95% while the phagocytosis was unaffected. PMNs in whole blood samples of ten cows differed significantly in their ability to phagocytose Staphylococcus aureus and to produce reactive oxygen products. However, only a weak correlation was detected between the burst activity:phagocytosis ratio of ten individual cows as indicated by the ROD/PI index.

Diapedesis across mammary epithelium reduces phagocytic and oxidative burst of bovine neutrophils.

The effect of diapedesis on the phagocytic and oxidative burst activity of polymorphonuclear neutrophil (PMN) was examined, using an in vitro cell culture model consisting of a monolayer of primary mammary epithelial cells. Isolated blood PMN from 10 cows were added to the basal side of the epithelial cell monolayer. Diapedesis was induced by the addition of complement factor C5a to the apical side of the monolayer. PMN phagocytosis of Staphylococcus aureus and oxidative burst were measured before diapedesis on PMN that were non-activated and activated by incubation with C5a and on PMN after diapedesis, using flow cytometry. The percentages of PMN fluorescing due to phagocytosis of S. aureus and oxidative burst were reduced by 21.2 and 14.4%, respectively, after diapedesis. Pre-incubation in the presence of C5a had no effect on percentage PMN fluorescing due to phagocytosis or oxidative burst. The capacity for individual migrated PMN to phagocytose S. aureus and to produce an oxidative burst, as measured by the intensity of fluorescence, decreased by 34.2 and 30.3%. Activation of PMN with C5a increased intensity due to the oxidative burst, but had no effect on intensity due to phagocytosis. These data show that PMN diapedesis across mammary epithelium results in decreased phagocytosis and oxidative burst of the PMN.

Adhesion receptor CD11b/CD18 contributes to neutrophil diapedesis across the bovine blood-milk barrier.

epithelium. Neutrophil migration across mammary arterial endothelial cells was almost completely dependent on CD18, the beta-chain of the beta(2) integrins, and to a lesser extent on CD11b, one of the alpha-chains of the beta(2) integrins. Neutrophil migration across collagen was partially blocked by monoclonal antibodies to CD18. No inhibition was observed by monoclonal antibodies to CD11b. Conversely, neutrophil diapedesis across mammary epithelial cells was dependent to a greater extent on CD11b. These results provide evidence for different CD11b/CD18-dependent mechanisms for neutrophil diapedesis across the various cell layers of the blood-milk barrier.

Effect of antibiotic induced bacterial clearance in the udder on L-selectin shedding of blood neutrophils in cows with Escherichia coli mastitis

Bacterial clearance, L-selectin adhesion receptor expression and neutrophil recruitment during experimentally induced Escherichia coli mastitis were investigated. Acute mastitis was induced by injection of 10(4) cfu E. coli into both left quarters of 12 clinically healthy lactating cows, 2-6 weeks after parturition. Half the cows were treated intravenously 10 h after infection, and subcutaneously 30 h after infection with enrofloxacin. In both groups, bacterial count, milk serum albumin, milk somatic cell count, circulating leukocyte count and L-selectin expression on neutrophils were determined. Both groups responded to challenge with udder inflammation and fever. Treatment with enrofloxacin affected the elimination rate of bacteria at hours +14, +18, +24, +48, and +72 after challenge, but not the bacteriological cure rate (five out of six for treated and three out of six for nontreated cows). The drop in L-selectin expression found following challenge did not differ between groups throughout the study. No effect of treatment was found on SCC. Based on these findings, it may be concluded that when treatment is administered 10 h after infection, the number of bacteria in milk is not correlated with L-selectin expression on circulating PMN during experimentally induced E. coli mastitis. The initial bacterial load probably dictates the extent of the decrease in L-selectin expression and milk somatic cells.