Background: Mechanisms governing the normal resolution processes of inflammation are poorly understood, yet their elucidation may lead to a greater understanding of the pathogenesis of chronic inflammation. The removal of apoptotic cell material and their potentially histotoxic contents is a prerequisite of resolution. Engulfment by macrophages is an important disposal route, and changes in the apoptotic cells that are associated with their recognition by macrophages are the subject of this report. Methods: Apoptosis and necrosis in primary cells and cell lines were induced by various stimuli. The binding profile of 23 different lectins for vital, apoptotic, and necrotic cells were analyzed by flow cytometry. Results: We observed that lectins were able to attach to the cell surfaces of vital and dying cells. Some lectins exhibited membrane destructive properties and, consecutively, changed the morphology of the cells as detected by flow cytometry. Other lectins did not show differences in their binding to viable and apoptotic cells. Those lectins were, therefore, not used for analyses of surface changes. The lectins Griffonia

Changes in the glycomic profile can significantly affect the cells' communication with the environment. Plant lectins have so far been used to address the issue as to whether the courses of apoptosis or necrosis are associated with such alterations. We, here, initiate the study of members of the family of functionally pleiotropic human galectins in this respect. Established protocols for the induction of apoptosis/necrosis of blood cells and for flow cytometry using annexin V/propidium iodide were combined with cell surface staining using biotinylated galectins at a nontoxic concentration. The galectin panel covered members from all three subfamilies. Flow cytometry revealed specific binding of galectins to viable control cells and conspicuous staining differences when testing apoptotic or necrotic cells. Onset and especially progression of cell death led to pronounced reactivity with the proto-type galectins-1, -2, and -7 and tandem-repeattype galectin-4. Extent of staining depended on the nature and stage of cell death, type of dying cell, and type of galectin. Galectins act as sensors for cell-death-associated surface changes. Staining of late-apoptotic polymorphonuclear cells was particularly strong. Examining the functional significance of this result may reveal a new aspect within the surveillance system to protect against autoinflammation. ' 2008 International Society for Analytical Cytology Key terms apoptosis; flow cytometry; galectin; glycosylation; lectin; necrosis; phagocytosis; sialylation THE safe disposal of apoptotic and necrotic cell material will limit the hazard to develop autoimmunity (1). In its initial phase, endogenous safeguard systems trace biochemical deviations on the cell surface, which signal the onset of cell death. Consecutive steps toward late stages are accompanied by further surface alterations. They are supposed to mark dying cells for elimination but their biochemical nature is not yet fully characterized. To explain efficient clearance, probing into distinct surface characteristics is a means to delineate operative routes of dying-self recognition. That said that the analysis of cell surface glycans is an obvious choice, because the glycomic profile is known to undergo disease-associated changes and to present a large panel of sugar-encoded signals to the environment (2-4). Using plant lectins as marker for glycan determinants (5), the hypothesis that their profile and/or mode of presentation are altered by cell death was already tested, and changes of lectin binding to dying and dead leukemic cells had been reported (6-9). These results obtained with plant proteins support the concept that glycan recognition by endogenous receptors can figure as a way to identify and clear nonviable cells. Focusing on spatially accessible branch-end determinants of glycan antennae, case studies with hepatic, and macrophage C-type asialoglycoprotein receptors already illustrated their

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Glycans cover the surfaces of all mammalian cells. Their structural variety provides enormous potential for information storage and transfer. According to the concept of the sugar code, they act as biochemical signals decoded by a large number of lectins which are defined as sugar binding proteins. The importance of glycan-lectin interaction in diverse immune system functions becomes increasingly apparent. Here, we review apoptotic cell clearance and especially focus on modifications of glycans on apoptotic cells.

The ultrastructural morphometric quantification of cytoplasmic microtubular distribution densities requires electron micro­graph magnifications whereby only small cytoplasmic areas are included per negative. Consecutive electron micrographs can be composed in montages to attain complete profiles of high magnification cell sections. Conventional morphometric methods are not suitable for application on areas that may be as great or even greater than 1 m² in size. Modifications of conventional morphometric technology to facilitate investigations of this nature are described. Statistical comparison of results obtained with this method and a standard method reflect the soundness of the modifications.