title: Effects of buffering intracellular free calcium on neutrophil migration through three-dimensional matrices.
abstract: Repeated transient increases in intracellular free calcium levels ([Ca2-]i) are required for polymorphonuclear neutrophil migration on two-dimensional surfaces coated with fibronectin or vitronectin. Cells in which [Ca2+]i is buffered with quin2 become stuck on these substrates. Neutrophils migrating through the extracellular matrix in vivo encounter these and other substrates in a three-dimensional architecture that may alter the spatial distribution of adhesion receptors in contact with the matrix. In this study, we used fluorescence confocal microscopy to obtain moving three-dimensional images of neutrophils migrating through a biological tissue (human amnion) in the presence and absence of [Ca2+]i-buffering with quin2. In the absence of buffering, [Ca2+]i transients similar to those seen in cells migrating in two-dimensions were observed. [Ca2+]i-buffered neutrophils were able to migrate into the matrix, but they became attached firmly to the substrate at the rear of the cell, resulting in a drastically elongated morphology. Immunofluorescence revealed that neutrophils adhered to regions of the matrix that contained fibronectin. RGD-containing peptides and antibodies that block integrin adhesion receptors for fibronectin and vitronectin were able to rescue the migration of quin2-treated cells through three-dimensional gels containing fibronectin and vitronectin. These data show that neutrophils migrating through physiologically relevant, three-dimensional matrices undergo repetitive increases in [Ca2+]i that are required for integrin-mediated detachment from the matrix.
authors.affiliation: Department of Pathology, Columbia University College of Physicians and Surgeons, New York, New York 10021, USA.
authors.forename: J T
authors.initials: JT
authors.lastname: Mandeville
authors.forename: F R
authors.initials: FR
authors.lastname: Maxfield
mesh_terms: D000650:Amnion / Q000737:chemistry* / Q000166:cytology; D000911:Antibodies, Monoclonal; D001667:Binding, Competitive / Q000276:immunology; D002021:Buffers; D002118:Calcium / Q000378:metabolism*; D002448:Cell Adhesion / Q000502:physiology; D048429:Cell Size / Q000187:drug effects; D002634:Chemotaxis, Leukocyte / Q000502:physiology*; D005109:Extracellular Matrix / Q000737:chemistry / Q000378:metabolism; D005353:Fibronectins / Q000032:analysis / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D016023:Integrins / Q000276:immunology / Q000378:metabolism; D018613:Microscopy, Confocal; D009504:Neutrophils / Q000166:cytology* / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D011971:Receptors, Immunologic / Q000378:metabolism; D019096:Vitronectin / Q000032:analysis / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D002021:Buffers; D005353:Fibronectins; D016023:Integrins; D009842:Oligopeptides; D011971:Receptors, Immunologic; D019096:Vitronectin; C047981:arginyl-glycyl-aspartic acid; D002118:Calcium