Lymphocyte Migration into Three-dimensional Matrices : A Quantitative Study Collagen

Lymphocytes have been plated onto the surface of three-dimensional gels of native collagen fibers, and their distribution throughout the three-dimensional collagen matrix has been determined in a quantitative fashion at various times thereafter. Information regarding the total number of applied cells may be obtained by this means. Lymphocyte penetration into the collagen gel does not appear to involve the expression of collagenolytic activity, nor does it require the presence of serum. Analysis of the kinetics of lymphocyte penetration into the gel matrix indicates that lymphocytes are migrating in a "random-walk" fashion. Our objective has been to establish a model system for studying the cell-matrix and cell-cell interactions which influence the pattern of lymphocyte recirculation in vivo and the results presented here are discussed in this context. Lymphocytes are highly mobile cells in vivo capable of moving back and forth between the peripheral blood and various tissue compartments. This active traffic of cells, referred to as lymphocyte recirculation, was first demonstrated to occur by Gowarts (7). The recirculation of lymphocytes is a complex process involving the following sequence of events (2, 5, 6): (a) the adherence of peripheral blood lymphocytes to endothelial cells in specific regions of the microvasculature, (b) the extravasation of adherent lymphocytes into the perivascular tissue space, (c) the migration and sorting out of lymphocyte subsets into defined regions of the tissue, and (d) the passage of lymphocytes through the lymphatic circulation back into the peripheral blood. The overall pattern of lymphocyte recirculation must be examined in vivo. Related studies in vitro have tended to focus attention on the effects of various soluble factors on lymphocyte migration into filter disks in order to identify putative chemotactic agents (14, 15, 27, 28). Stamper and Woodruff (24) have introduced an in vitro method for examining the specific adhesive interactions between lymphocytes and the high endothelium found in lymph nodes. Our own interest in lymphocyte recirculation is specifically concerned with identifying the various cell-matrix and cell-cell interactions which contribute to the control of lymphocyte migration and sorting out once they are within the stroma of relevant tissues. Collagen fibers are a constituent of the tissue stroma through which lymphocytes migrate in vivo (12), and three-dimensional gels of native collagen fibers have previously THE JOURNAL OF CELL BIOLOGY. VOLUME 96 APRIL 1983 1089-1096 © The Rockefeller University Press • 0021-9525/83/04/1089/08 $1.00 been used to study the migratory behavior of a number of cell types in vitro (4, 17-22). Data are presented in this communication regarding the migration of normal human peripheral blood lymphocytes into three-dimensional collagen gels. These data indicate that lymphocyte migration through this matrix proceeds in a "random-walk" fashion in a manner which does not involve the lytic degradation of collagen fibers. These results are discussed in the context of our interest in lymphocyte recirculation. MATERIALS AND METHODS Isolation o f Lymphocytes: Human peripheral blood lymphocytes were obtained from normal volunteer donors. Between 40 and 60 ml of blood were removed by venipuncture and defibrinated by shaking gently for 20 min at room temperature with glass beads in 20-ml glass beads in 20-ml glass bottles. The fibrin clot was removed and the resultant defibrinated blood mixed in 1:1 ratio with Dulbecco's A phosphate-buffered saline (PBS). 20-ml aliquots of this diluted preparation were then layered over 20-ml of Lymphocyte Separation Medium (cat. no. 16-920-54; Flow Laboratories, lrvine, Scotland) and centrifuged at 400 g for 30 min at 20°C. Leukocytes were recovered from the interface, washed 3× in PBS, and resuspended in Eagle's Minimal Essential Medium (MEM) supplemented with 15% donor calf serum and 100 U /ml penicillin and streptomycin (complete growth medium). Adherent ceils were removed by incubating the leukocyte suspension in tissue culture dishes for 60 min at 37°C. The nonadherent cells were recovered in the growth medium, washed 3x in complete growth medium, and finally suspended in complete growth medium at a concentration of 106 cells/ml. Cell numbers were routinely determined with a Neubauer hemocytometer. In a small number of experiments, cell number was measured with a Becton, Dickinson fluorescent activated cell sorter (Model 4; Becton, Dickinson & Co.). Contaminating monocytes in the final cell suspension were identified by staining 1089 on M arch 0, 2017 D ow nladed fom Published April 1, 1983